Podcast appearances and mentions of Ruth Williams

In today's episode of The Pelvic Floor Connection, I'm joined by the deeply insightful Natalie Nuttall; wellbeing coach, writer, and co-founder of Smile Group, a perinatal mental health charity based in Cheshire.Together, we dive into how our personal and professional experiences, helped guide us to the knowledge that midlife and menopause can become an invitation to come home to yourself—not something to endure or "bounce back". It can be a powerful opportunity for reconnection, reflection, and renewal.Key Themes:         

In today's episode, we sit down with Dr. Ruth Williams, CEO emeritus of Wheaton Eye Clinic, to discuss private practice leadership and the challenges of private equity. Plus, we hear about her role as the editor of EyeNet Magazine. Subscribe to the podcast: https://mayoclinicophthalmology.podbean.com  Follow and reach out to us on IG and X: @mayocliniceye   

Of the five ‘giant evils' William Beveridge identified, the Attlee government set out to deal with want through social security, squalor through better housing, ignorance through more schooling and disease through the National Health Service. When it came to the fifth giant, idleness, the government's tackeld unemployment by setting out to rebuild the British economy and, overall, that didn't go too badly. Unemployment was kept to 2% of the workforce despite the return of two and a half million men to the employment market from the army, and a massive trade deficit was wiped out. But the price was a tough economy with rationing still in place and little in the way of luxury to make life more pleasurable. Survival had been made easier, but living was short of joy in a rather dour postwar Britain.Greyness at home was reflected in continuing decline abroad. This episode traces the loss of status and, indeed, of value of the pound, once the world's reserve currency, now forced aside by the dollar. It also looks at the sad story of how Britain handled, or rather mishandled, its territory of Bechuanaland in Southern Africa, behaving shamefully towards its hereditary ruler Seretse Khama and his white wife Ruth Williams, to accommodate the growing racism of South Africa, source of the uranium Britain needed for its A-bomb.Things went no more smoothly in Palestine, where Britain simply abandoned its mandate, leaving Jews and Arabs to sort out their differences themselves, kicking off the long series of repeating wars that have poisoned the existence of Israel ever since. To cap the episode off, we talk about the start of the Malaya emergency, a counter-insurgency war as ugly and as strewn with atrocities as any other. It underlines the lesson that it isn't government intentions that matter in such conflicts, it's the nature of colonial war itself that makes it vile.Illustration: Seretse Khama, first President of Botswana, and the first First Lady, Ruth WilliamsMusic: Bach Partita #2c by J Bu licensed under an Attribution-NonCommercial-No Derivatives (aka Music Sharing) 3.0 International License

Die Geschäftsführerin des Fundraising Verbandes, über die Spendenkultur in Österreich.

Gerade rund um Weihnachten stehen Spenden hoch im Kurs. Die Österreicherinnen und Österreicher greifen aber auch über das Jahr hinweg immer wieder für den guten Zweck in die Tasche. Wie die beiden Jahre zuvor wurden auch heuer wieder mehr als eine Milliarde Euro gespendet. Große Organisationen oder direkt über Crowdfundingplattformen: Wie kann man mit den eigenen finaziellen Mitteln möglichst effizient Gutes tun? Was passiert dann mit unseren Spendengeldern – und wer kontrolliert das? Diese und weitere Fragen beantwortet Ruth Williams, Geschäftsführerin des Fundraising Verband Austria, im Gespräch mit Moderatorin Anika Dang.

An einem heißen Juniabend im Jahr 1947 kreuzen sich die Wege von Seretse Khama und Ruth Williams bei einer Jazz-Veranstaltung in London. Für Seretse gibt es keinen Zweifel: Er hat gerade die Liebe seines Lebens kennengelernt. Auch Ruth ist von Seretse fasziniert. Was sie noch nicht ahnt: Seretse soll eines Tages König seines Volkes in Betchuanaland, dem heutigen Botswana, werden. Je tiefer ihre Liebe wird, desto größer werden die Widerstände. Eine weiße Engländerin und ein schwarzer Thronfolger? Die ganze Welt scheint sich gegen ihre Verbindung zu stellen. Ihre Familien sind entsetzt, Regierungen mischen sich ein, Politiker spinnen Intrigen, um die beiden zu trennen. Doch ihre Liebe wird nicht nur ihr Leben, sondern das eines ganzen Landes verändern. "True Love" ist ein Podcast über wahre Liebesgeschichten. Eine Produktion von Auf Ex Productions. Hosts: Leonie Bartsch & Linn Schütze Recherche: Anna Busse, Linn Schütze Redaktion: Antonia Fischer Produktion: Lorenz Schütze Expertin: Miss History Gästin: Tahlia Naledi Khama Für Hintergrundinformationen, Bilder und Videos könnt ihr uns auf Instagram oder TikTok unter @true.lovepodcast folgen. Oder auf unseren privaten Profilen unter @leonie_bartsch & @linnschuetze. Wir würden uns riesig freuen, wenn ihr den Podcast bewertet und teilt. Haben euch lieb & bis in zwei Wochen! Du möchtest mehr über unsere Werbepartner erfahren? [**Hier findest du alle Infos & Rabatte!**](https://linktr.ee/truelove_podcast) Du möchtest Werbung in diesem Podcast schalten? [**Dann erfahre hier mehr über die Werbemöglichkeiten bei Seven.One Audio!**](https://www.seven.one/portfolio/sevenone-audio)

Episode 6: In Jungian psychology (one of the branches of depth psychology), individuation is the life long process of becoming yourself - as a unique individual. This process of becoming often has a feeling tone of moving toward what feels purposeful for you, maybe even elements of destiny or fate. The astrology birth chart is well suited to be a guide and connector to help us explore the multitude of potentials when we feel a lack of purpose, potential, or simply stuck. In this episode, I share some thoughts about individuation and the birth chart in the context of working with astrology in psychotherapy. In this episode, I mention the book, "C.G. Jung: The Basics" by Jungian analyst Ruth Williams. Enjoy! ... Shawna McGrath, AMFT is a psychotherapist and astrologer. She offers depth-oriented psychotherapy with astrology to those seeking healing, insight, new possibilities, and deeper meaning in life. ⁠⁠⁠Find more here.

Ruth Williams ist ab kommenden Jahr die neue Geschäftsführerin des FVA und ist daher genau die richtige Gesprächspartnerin für unsere Fragen.

On the Breakfast show, Gary talks to Ruth Williams of the Smile Group who are reaching out to those that maybe suffering from both pre and post-natal depression. In today's interview Ruth tells us all about the new group the are forming in Crewe.

Part 2 catalogs Smiley's journey out of high school and into the world of jobs and being a young adult. Then we get to her time at BVOH, which began about four years ago. It started with an entry-level job, which she didn't mind doing because it got her foot in the door. Built in 1888 by the Masons, the then-designated "South San Francisco Opera House" was a spot for them to meet and host performances. Acoustically, that intention shows to this day. The opera house also has dressing rooms with direct access to the stage (which Smiley told me is not a common feature in buildings of this kind). With the Great Migration and its influx of Black shipyard workers here in San Francisco, the building became more of a community center. It also changed demographically, from white folks to members of that Black community. But, in Smiley's words, over the years, the building "wasn't loved on," and fell into some dilapidation. In the 1950s, The City wanted to tear the opera house down and build apartment buildings ... until Ruth Williams said "no." Black Panther free breakfasts happened there. People met each other there. Performances like plays and dance classes happened on the regular. Voters cast ballots there. In 1966, SFPD officers shot and killed a young Black boy named Matthew "Peanut" Johnson, sparking a riot. The opera house was hit with several hundred bullets in the ensuing melee. Things were so bad for Black folks in SF, James Baldwin filmed Take This Hammer just outside the opera house in 1964. As Smiley puts it, Bayview Opera House "has always been a place for history-making and Black culture." In 1989, an organization was formed through the SF Arts Commission to manage programs at the building. The opera house was established as San Francisco Historical Landmark No. 8. Over the years, it changed hands several times, and in 2015, a multimillion dollar grant was secured to do much-needed renovations, including a new outdoor amphitheater that came in handy during the pandemic. 2023 has been all about re-inviting people inside. There are world premieres of operas and films. The calendar is full. See for yourself. We end the podcast hearing from two folks who've been through BVOH's Tools of the Trade program: Joe Kool and Miss White, the First Lady of Frisco. Photography by Jeff Hunt

Civil right Activist Mr. H.B. Williams Mrs. Nanny Ruth Williams W/ Host #HOWCEE 5/30/23 #HOWCEE PRODUCTIONS. https://www.facebook.com/search/top?q=h%20b%20williams Former Principal Mr. H.B Williams spoiled many of us by giving snacks and hugs! Thanks for your services at Monroe Senior High School   A Powerful interview With Civil Right Activist"  A Conversation with Civil Rights Activitist Homer Beecher Williams “H.B.” to his friends & wife Nannie Ruth Williams https://www.blogtalkradio.com/howcee-productions-gospel/2017/08/21/conversation-homer-beecher-williamshb-to-his-friends-nannie-ruth-williams Nannie Ruth Williams her father a sharecropper as well as her grandfather ., talks about her role as family protector vs wife/supporter for Civil Rights Activitist Homer Beecher Williams “H.B.” H.B., Williams then a school principal life changing/rights began when was offered the job of assistant principal, and when he refused, pointing out that it was a demotion, he was fired. He spent years fighting for his reinstatement. Durning this battle about for justic about ten years, was his awaking to real life "Civil Rights" on a personal scale set in motion by the Monroe County Board of Education. Real life injustic at the hands of segregated school system where black educators role of leadership were set in stone in class rooms, but not as administrators

A mulher do primeiro presidente do Botswana morreu faz hoje 21 anos.

This month on Episode 48 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the April 28th issue of Circulation Research. This Episode also includes a discussion between Dr Mina Chung, Dr DeLisa Fairweather and Dr Milka Koupenova, who all contributed to manuscripts to the May 12th Compendium on Covid-19 and the Cardiovascular System.     Article highlights:   Heijman, et al. Mechanisms of Enhanced SK-Channel Current in AF   Chen, et al. IL-37 Attenuates Platelet Activation   Enzan, et al. ZBP1 Protects Against Myocardial Inflammation   Compendium on Covid-19 and the Cardiovascular System.   Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, from the Vascular Medicine Institute at the University of Pittsburgh. Today, I'm going to be highlighting articles from our April 28th and May 12th issues of Circulation Research. I'm also going to have a chat with Dr Mina Chung, Dr DeLisa Fairweather and Dr Milka Koupenova, who all contributed to articles in the May 12th COVID Compendium. But before we have that interview, let's first talk about some highlights.   The first article I want to present is titled Enhanced Calcium-Dependent SK-Channel Gating and Membrane Trafficking in Human Atrial Fibrillation. This article is coming from the University of Essen by Heijman and Zhou, et al. Atrial fibrillation is one of the most common forms of heart arrhythmia in humans and is characterized by irregular, often rapid heartbeats that can cause palpitations, dizziness and extreme fatigue. Atrial fibrillation can increase a person's risk of heart failure, and though treatments exist such as beta blockers, blood thinners and antiarrhythmia medications, they can have limited efficacy and side effects. A new family of drugs in development are those blocking small-conductance calcium-activated potassium channels called SK channels, which exhibit increased activity in animal models of AF and suppression of which attenuates the arrhythmia. In humans however, the relationship between SK channels and atrial fibrillation is less clear, at least in terms of SK channel mRNA levels. Because mRNA might not reflect actual channel activity, this group looked at just that and they found indeed that channel activity was increased in cardiomyocytes from atrial fibrillation patients compared to those from controls even though the mRNA and protein levels themselves were similar. The altered currents were instead due to changes in SK channel trafficking and membrane targeting. By confirming that SK channels play a role in human atrial fibrillation, this work supports the pursuit of SK channel inhibitors as possible new atrial fibrillation treatments.   The next article I want to present is titled IL-37 Attenuates Platelet Activation and Thrombosis Through IL-1R8 Pathway. This article comes from Fudan University by Chen and Hong, et al. Thrombus formation followed by the rupture of a coronary plaque is a major pathophysiological step in the development of a myocardial infarction. Understanding the endogenous antithrombotic factors at play could provide insights and opportunities for developing treatments. With this in mind, Chen and Hong, et al. investigated the role of interleukin-1 receptor 8, or IL-1R8, which suppresses platelet aggregation in mice, and of IL-37, a newly discovered human interleukin that forms a complex with IL-1R8 and is found at increased levels in the blood of patients with myocardial infarction. Indeed, the amount of IL-37 in myocardial infarction patients negatively correlates with platelet aggregation. They also show that treatment of human platelets in vitro with IL-37 suppresses the cell's aggregation and does so in a concentration-dependent manner. Moreover, injection of the protein into the veins of mice inhibits thrombus development and better preserves heart function even after myocardial infarction. Such effects were not seen in mice lacking IL-1R8. This suggests IL-37's antithrombotic action depends on its interaction with the receptor. Together, the results suggest IL-37 could be developed as a antithrombotic agent for use in MI patients or indeed perhaps other thrombotic conditions.   The last article I want to present before our interview is titled ZBP1 Protects Against Mitochondrial DNA-Induced Myocardial Inflammation in Failing Hearts. This article is coming from Kyushu University and is by Enzan, et al. Myocardial inflammation is a key factor in the pathological progression of heart failure and occurs when damaged mitochondria within the stricken cardiomyocyte release their DNA, triggering an innate inflammatory reaction. In a variety of cells, DNA sensors such as Z-DNA-binding protein 1 or ZBP1 are responsible for such mitochondrial DNA-induced inflammation. In theory then, it's conceivable that therapeutic suppression of ZBP1 might reduce myocardial inflammation in heart failure and preserve function. But as Enzan and colleagues have now discovered to their surprise, mice lacking ZBP1 exhibited worse, not better heart inflammation and more failure after induced myocardial infarction. Indeed, the test animals' hearts had increased infiltration of immune cells, production of inflammatory cytokines and fibrosis together with decreased function compared with the hearts of mice with normal ZBP1 levels. Experiments in rodent cardiomyocytes further confirmed that loss of ZBP1 exacerbated mitochondrial DNA-induced inflammatory cytokine production while overexpression of ZBP1 had the opposite effect. While the reason behind ZBP1's opposing roles in different cells is not yet clear, the finding suggests that boosting ZBP1 activity in the heart might be a strategy for mitigating heart inflammation after infarction.   Cindy St. Hilaire:         The May 12th issue of Circulation Research is our COVID compendium, which consists of a series of 10 reviews on all angles of COVID-19 as it relates to cardiovascular health and disease. Today, three of the authors of the articles in this series are here with me. Dr Mina Chung is a professor of medicine at the Cleveland Clinic. She and Dr Tamanna Singh and their colleagues wrote the article, A Post Pandemic Enigma: The Cardiovascular Impact of Post-Acute Sequelae of SARS-CoV-2. Dr DeLisa Fairweather, professor of medicine, immunology and clinical and translational science at the Mayo Clinic, and she and her colleagues penned the article, COVID-19 Myocarditis and Pericarditis. Dr Milka Koupenova is an assistant professor of medicine at the UMass Chan School of Medical and she led the group writing the article, Platelets and SARS-CoV-2 During COVID-19: Immunity, Thrombosis, and Beyond. Thank you all for joining me today.   DeLisa Fairweather:    Thank you so much for having us.   Mina Chung:   Thank you.   Milka Koupenova:       Thank you for having us, Cindy.   Cindy St. Hilaire:         In addition to these three articles, we have another seven that are on all different aspects of COVID. Dr Messinger's group wrote the article, Interaction of COVID-19 With Common Cardiovascular Disorders. Emily Tsai covered cell-specific mechanisms in the heart of COVID-19 patients. Mark Chappell and colleagues wrote about the renin-angiotensin system and sex differences in COVID-19. Michael Bristow covered vaccination-associated myocarditis and myocardial injury. Jow Loacalzo and colleagues covered repurposing drugs for the treatment of COVID-19 and its cardiovascular manifestations. Dr Stephen Holby covered multimodality cardiac imaging in COVID, and Arun Sharma covered microfluidic organ chips in stem cell models in the fight against COVID-19.   Cindy St. Hilaire          As of today, worldwide, there have been over six hundred million individuals infected with the virus and more than six and a half million have died from COVID-19. In the US, we are about a sixth of all of those deaths. Obviously now we're in 2023, the numbers of individuals getting infected and dying are much, much lower. As my husband read to me this morning, one doctor in Boston was quoted saying, "People are still getting wicked sick." In 75% of deaths, people have had underlying conditions and cardiovascular disease is found in about 60% of all those deaths. In the introduction to the compendium, you mentioned that the remarkable COVID-19 rapid response initiative released by the AHA, which again is the parent organization of Circ Research and this podcast, if I were to guess when that rapid response initiative started, I would've guessed well into the pandemic, but it was actually March 26th, 2020. I know in Pittsburgh, our labs have barely shut down. So how soon after we knew of SARS-CoV-2 and COVID, how soon after that did we know that there were cardiovascular complications?   Mina Chung:               I think we saw cardiovascular complications happening pretty early. We saw troponin increases very early. It was really amazing what AHA did in terms of this rapid response grant mechanism. You mentioned that the RFA was announced, first of all, putting it together by March 26th when we were just shutting down in March was pretty incredible to get even the RFA out. Then the grants were supposed to be submitted by April 6th and there were 750 grants that were put together and submitted. They were all reviewed within 10 days from 150 volunteer reviewers. The notices were distributed April 23rd, less than a month out.   Cindy St. Hilaire:         Amazing.   Mina Chung:               So this is an amazing, you're right, paradigm for grant requests and submissions and reviews.   DeLisa Fairweather:    For myocarditis, reports of that occurred almost immediately coming out of China, so it was incredibly rapid.   Cindy St. Hilaire:         Yeah, and that was a perfect lead up to my next question. Was myocarditis, I guess, the first link or the first clue that this was not just going to be a respiratory infection?   DeLisa Fairweather:    I think myocarditis appearing very early, especially it has a history both of being induced by viruses, but being strongly an autoimmune disease, the combination of both of those, I think, started to hint that something different was going to happen, although a lot of people probably didn't realize the significance of that right away.   Cindy St. Hilaire:         What other disease states, I guess I'm thinking viruses, but anything, what causes myocarditis and pericarditis normally and how unique is it that we are seeing this as a sequelae of COVID?   DeLisa Fairweather:    I think it's not surprising that we find it. Viruses around the world are the primary cause of myocarditis, although in South America, it's the parasite Trypanosoma cruzi. Really, many viruses that also we think target mitochondria, including SARS-CoV-2, have an important role in driving myocarditis. Also, we know that SARS-CoV-1 and MERS also reported myocarditis in those previous infections. We knew about it beforehand that they could cause myocarditis.   Cindy St. Hilaire:        Is it presenting differently in a COVID patient than say those South American patients with the... I forget the name of the organism you said, but does it come quickly or get worse quickly or is it all once you get it, it's the same progression?   DeLisa Fairweather:    Yeah. That's a good question. Basically, what we find is that no matter what the viral infection is, that myocarditis really appears for signs and symptoms and how we treat it identically and we see that with COVID-19. So that really isn't any different.   Cindy St. Hilaire:         Another huge observation that we noticed in COVID-19 patients, which was the increased risk of thrombic outcomes in the patients. Dr Koupenova, Milka, you are a world expert in platelets and viruses and so you and your team were leading the writing of that article. My guess is knowing what you know about platelets and viruses, this wasn't so surprising to you, but could you at least tell us the state of the field in terms of what we knew about viruses and platelets before COVID, before Feb 2020?   Milka Koupenova:       Before Feb 2020, we actually knew that influenza gets inside in platelets. It leads to not directly prothrombotic events, but it would lead to release of complement 3 from them. That complement 3 would actually increase the immunothrombosis by pushing neutrophils to release their DNA, forming aggregates. In cases when you have compromised endothelium and people with underlying conditions, you would expect certain thrombotic outcomes. That, we actually published 2019 and then 2020 hit. The difference between influenza and SARS-CoV-2, they're different viruses. They carry their genome in a different RNA strand. I remember thinking perhaps viruses are getting inside in platelets, but perhaps they do not. So we went through surprising discoveries that it seemed like it is another RNA virus. It also got into platelets. It was a bit hard to tweak things surrounding BSL-3 to tell you if the response was the same. It is still not very clear how much SARS or rather what receptor, particularly when it gets inside would induce an immune response. There are some literature showing the MDA5, but not for sure, may be responsible. But what we found is that once it gets in platelets, it just induces this profound activation of programmed cell death pathways and release of extracellular vesicles and all these prothrombotic, procoagulant form of content that can induce damage around, because platelets are everywhere. So that how it started in 2019 and surprisingly progressed to 2021 or 2020 without the plan of really studying this virus.   Cindy St. Hilaire:         How similar and how different is what you observe in platelets infected, obviously in the lab, so I know it's not exactly the same, but how similar and how different is it between the flu? Do you know all the differences yet?   Milka Koupenova:       No offense here, they don't get infected.   Cindy St. Hilaire:         Okay.   Milka Koupenova:       Done the proper research. The virus does not impact platelets, but induces the response.   Cindy St. Hilaire:         Okay.   Milka Koupenova:       That goes back to sensing mechanism. Thank goodness platelets don't get infected because we would be in a particularly bad situation, but they remove the infectious virus from the plasma from what we can see with function.   Cindy St. Hilaire:         Got it. So they're helping the cleanup process and in that cleaning up is where the virus within them activates. That is a really complicated mechanism.    Milka Koupenova:       Oh, they're sensing it in some form to alert the environment. It's hard to say how similar and how different they are unless you study them hint by hint next to each other. All I can tell is that particularly with SARS-C, you definitely see a lot more various kinds of extracellular vesicles coming out of them that you don't see the same way or rather through the same proportion with influenza. But what that means in how platelet activates the immune system with one versus the other, and that goes back to the prothrombotic mechanisms. That is exactly what needs to be studied and that was the call for this COVID compendium is to point out how much we have done as a team. As scientists who put heads together, as Mina said, superfast response, it's an amazing going back and looking at what happened to think of what we achieved. There is so much more, so much more that we do not understand how one contributes to all of these profound responses in the organs themselves, such as myocarditis. We see it's important and that will be the problem that we're dealing from here on trying to figure it out and then long COVID, right?   Cindy St. Hilaire:         Yeah. Related to what you just said about the mechanism, this cleanup by the platelets or the act of cleaning up helps trigger their activation, is that partly why the antiplatelet and anticoagulant therapies failed in patients? Can you speculate on that? I know the jury's still out and there's a lot of work to be done, but is that part of why those therapies weren't beneficial?   Milka Koupenova:       The answer to that in my personally biased opinion is yes. Clearly, the antiplatelet therapies couldn't really control the classical activation of a platelet. So what I think we need to do from here on is to look at things that we don't understand that non-classically contribute to the thrombotic response downstream. If we manage to control the immune response in some way or the inflammation of the infection or how a platelet responds to a virus, then perhaps we can ameliorate a little bit of the downstream prothrombotic effect. So it's a lot more for us to trickle down and to understand in my personal opinion.   DeLisa Fairweather:    There is one thing that was really remarkable to me in hearing your experience, Milka, is that I had developed an autoimmune viral model of myocarditis in mice during my postdoc. So I've been studying that for the last 20 years. What is unique about that model is rather than using an adjuvant, we use a mild viral infection so it doesn't take very much virus at all going to the heart to induce it. I also, more recently, started studying extracellular vesicles really as a therapy, and in doing that, inadvertently found out that actually, the model that I'd created where we passage the virus through the heart to induce this autoimmune model, we were actually injecting extracellular vesicles into the mice and that's what was really driving the disease. This is really brought out. So from early days, I did my postdoc with Dr Noel Rose. If you've heard of him, he came up with the idea of autoimmune disease in the '50s. We had always, in that environment, really believed that viruses were triggering autoimmune disease and yet it took COVID before we could really prove that because no one could identify them. Here we have an example and I think the incidence rates with COVID were so high for myocarditis because for the first time, we had distinguished symptoms of patients going to the doctor right at the beginning of their infection having an actual test to examine the virus, knowing whether it's present or not, whether PCR or antibody test, and then being able to see when myocarditis happened.   Cindy St. Hilaire:         Yeah. I think one thing we can all appreciate now is just some of the basic biology we've learned on the backend of this. Actually, those last comments really led well to the article that your team led, Dr Chung, about what we call long COVID, which I guess I didn't realize has an actual name, post-acute sequelae of SARS-CoV-2 or PASC is the now more formal name for long COVID. But what is it? We hinted at it that there's these bits about autoimmune and things like that. What counts as long COVID?   Mina Chung:   Yeah. Our article was led by Tamanna Singh. She did a fantastic job of putting this together. We've had, and others, theorized that the huge palette of symptoms that you can experience post-COVID, they can affect all these organ systems with brain fog, these atypical chest pains, postural orthostatic tachycardia, a lot of palpitations, atrial fibrillation, many weakness and fatigue. To us, really, you can get GI symptoms. We've been very interested in, is this an autoimmune phenomenon directed against nerves and all those things. It's also very interesting because many of the non-COVID syndromes that existed pre-COVID like POTS and chronic fatigue syndrome and a lot of other syndromes are associated with autoantibodies. So that is a very interesting area to explore. Is there a persistence of viral fragments. Is there autoimmunity? Is it also a component of persistence of the damage from the initial infection? So it's an area that still needs a lot of work and a lot of work is going into it, but this is like a post or inter pandemic of itself, so hopefully we'll get more insights into that.   Cindy St. Hilaire:         Yeah, it's really interesting. I have a friend who has very debilitating long COVID and one of her doctors had said, "If I didn't know any better, I would just describe this as a autoimmune type X." What do we know, I guess, about the current hypothesis of the pathogenesis of PASC? Are there any prevailing theories right now as to why it's occurring? Is the virus still active or is it these domino effects that are leading to multi-organ collapse of some sort?   Mina Chung:   Yeah. In some people, persistent viral particles can be identified for months, but whether or not that's what's triggering it, it's hard to know. We see more autoimmune disease that's been reported and various antibodies being reported. So those are clearly processes to be investigated. The microthrombosis is still up there in terms of potentially playing a role in long COVID.   Milka Koupenova:       Mina, you probably know better because you see patients, but to all I have been exposed to, long COVID does not really have a homogeneous symptom presentation and then a few theories as to what may be going on in these patients. Not everybody has a microthrombosis. Not everybody have a D-dimer elevated, but some people do. Some people have, as you pointed out, these spectacularly profound brain fog. People can't function. It's probably your friend, Cindy, right?   Cindy St. Hilaire:         Yeah.   Milka Koupenova:       So one of the theories that I have been, from a viral perspective, very interested in is that a lot of the symptoms in certain individuals such as fatigue, brain fog, sensitivity to light and skin can very well be explained by a flare-up of Epstein-Barr virus that may be what SARS-CoV-2 somehow is inducing. I don't know, DeLisa, what your experience with long COVID is as a scientist. I hope only. But I would like to hear your perspective too because it's so heterogeneous and it is amazing what happens.   DeLisa Fairweather:    I have a very interesting perspective from a number of different directions. One, as I mentioned before, my long history with Dr Rose and I've written many articles theorizing how viruses could cause autoimmune disease. This has grown and really, I think this has been extremely revealing during COVID for many of those theories. One thing that I write about in the review for this article is that mast cells, from all the research I've done with myocarditis in our model, mast cells are central to what is driving everything. We show they're the first innate immune cell acting as an antigen-presenting cell, completely driving the response in a susceptible pattern. One of the things that's very important in autoimmune disease is both sex and race. I'd say one of the big weaknesses we have in myocarditis pre-COVID and post-COVID has been ignoring what's going on with race. In the United States, myocarditis is 90%, 95% white men that are under 50 years of age and most of the cases are under 40 or some of the ones really associated with sudden cardiac death are under 30. So it's very specific. I've been studying sex and race differences and we see those exact differences in our animal models. In animal models, whether you're susceptible or not depends on how many mast cells you have. Well, I've proposed from the beginning, looking, I've written a lot of different sex difference reviews looking at viruses and autoimmune disease with different autoimmune diseases and hypothesizing and really seeing that mast cells do a lot of the things we're talking about. They have all of the receptors, the whole group of them that have been related to SARS-CoV-2 so they can be activated or stimulated by the virus itself. They act as a antigen-presenting cell. They're critical in the complement pathway as well as macrophages. We see the dominant immune phenotype really being macrophages. Mast cells just are usually not counted anywhere. And of course, these receptors, a lot of them have to do with enzymes and things that are all related to mast cells pathways. Then how they activate the immune response and lead it towards the pathway that leads to chronic autoimmune disease with increased autoantibodies in females, mast cells are very different by sex. This has to do also when we talked in the Review about myocarditis and pericarditis. It's both those appearing. Although clinically, we have really boxed them as separate things, because there is some definite clinical pericarditis phenotypes that are different, myocarditis in animal models is always myopericarditis. It always then, in that outer pericardial areas where mast cells sit, they sit around the vascular area in most concentrated. So when they degranulate, we see inflammation coming in the vessel, but really concentrated with fibrosis there and along the pericardium. So that's very typical of what's going on. When we shift anything that shifts that, it changes whether you have more pericarditis or less pericarditis and the vascular inflammation by altering anything that affects the mast cells. I talk a little bit about in the review, I think there's only been a few recent things looking at it in COVID, but I think mast cells and certain susceptibility to autoimmune diseases that occur more often in women can really predispose.We need to pay more attention to mast cells and what they might indicate for all these pathways.   Milka Koupenova:       I think we should study the platelet mast cell access at this point.   DeLisa Fairweather:    Yes.   Milka Koupenova:       Because as you're talking about these sex differences, which is spectacular, these things to me are so mind-boggling how one, the infection itself would be more prevalent in men, but then long COVID is more prevalent in women. All of these things and why we understand so very little, what we found about a few years ago in the Framingham Heart Study in the platelets from those people is that all toll-like receptors are expressed at the higher level in women and they associate with different things between men and female. For instance, toll-like receptors in women will associate more with a prothrombotic response while in male with pro-inflammatory response. I think they grossly underestimate the amount of our sex differences from cell to cell.   DeLisa Fairweather:    It is, yeah.   Mina Chung:   One other thing that I learned about the sex differences from this compendium is Mark Chappell also notes, you mentioned TLR and TLR7 and ACE2 are X chromosome in an area that he says escapes X-linked inactivation. So it could very well be involved in further.   DeLisa Fairweather: Further, yeah. And ACE2 is expressed more highly in male cells for what's been researched because of the sex difference in COVID, both the COVID infection   Cindy St. Hilaire:         So a variety of organ systems are impacted in patients with PASC, also referred to as long COVID, the lungs, the heart, the pancreas, the GI system, pretty much any system, the brain, nervous system. We've just been talking about the mast cell impact. I was really thinking in my head, well, the one thing that connects all of it is the vasculature. I'm a vascular biologist, so I have certain biases, I'm sure, but how much of the sequelae that we see is a function of vascular phenotypes?       Milka Koupenova:       I do think the vasculature is super important. It's clear that not all endothelial cells, for instance, will pick up the virus and respond to it. That's why you have this patchy breakage when you look at autopsies. Hence, platelets will respond according to what's local. That's why you find these micro thrombotic events at certain places. Why does it happen in each organ? How does the virus get to each organ to respond? Or is it just inflammation, but why is it in specific places? That's what we don't understand. That's where we need to go. Perhaps, as DeLisa points out, perhaps it's a lot more complicated than how we traditionally think of thrombosis. Actually, my personal bias, again 100% sure that it is a lot more complicated than the traditional mechanisms that we have understood, and that's where the immune system comes and autoimmunity perhaps stems from and they probably speak to each other, right? It's not just one thing.   DeLisa Fairweather:    Yeah. I think really, EVs are bringing lots of understanding. A lot of things we used to just think were maybe free-floating and the serum are inside EVs. I think that the immune response is perhaps even more specific than we ever thought and more regulated than we ever understood.   When an EV comes through a cardiomyocyte, whether it's from the mitochondria or through a lysosome, is part of what goes into its outer membrane, something that tells the immune system that that came from the heart, so it knows to go. This will solve a lot of our questions with autoimmune disease if it's very specific like that. It doesn't just have to be the release of free-floating cardiac myosin. We know cardiac myosin is the driver of the autoimmune response in myocarditis, but they're probably  much more fine-tuned.   Cindy St. Hilaire:         Yeah. I just would love to end with hearing from each of you. You each have your own domain of specialty. If I gave you a massive pot of money, what would be the question you would want to tackle? What's the gap you would love to answer?   Milka Koupenova:       We still don't understand specifically what kind of vesicles are coming out, what are their contents in addition to those vesicles. We don't understand. When it comes to platelets, what comes from their granules? We see these breakages of the membrane. Those are non-granule proteins, and non-granule proteins, they serve as dangerous associated molecular pattern signals and can be profoundly inflammatory to the surrounding environment, can be procoagulant. What are those? How are they affecting the surrounding environment? Ultimately, why is there a microthrombi? Why is there not a profound thrombosis everywhere? Thank goodness there isn't, but why isn't? That's what I would do with my money.   DeLisa Fairweather:    I think I would do something very similar. All of our research in our animal model, on the one side, we are looking in this viral myocarditis animal model and finding the EVs that come from that are driving myocarditis. On the other hand, we're using EVs that come from healthy human plasma or fat, and we're seeing a profound downregulation of everything if you give it early and we're trying to see how late you can give it and still get an effect. So looking at those and really understanding the components in the context of COVID and COVID vaccines to understand those components, I really think that's the future of where we're going to find what's causing disease and also how we can find therapies. They may be able to reverse this.   Mina Chung:   Yeah, I'm interested very much in the autoimmunity and the autoantibodies that are    and how they may react with those microthrombi. Perhaps there's autoantibodies within a lot of that material. We're looking at using human and pluripotent stem cell-derived cell models to study the effects of those. That is what I would use our money for.   Cindy St. Hilaire:        Well, Dr Mina Chung, Dr DeLisa Fairweather, Dr Milka Koupenova, thank you all so much for joining me today and talking about not only the articles that you wrote and with your colleagues, but also other articles in this amazing compendium. I do think this is one of the first all-encompassing compendiums or group of articles that focus specifically on COVID and cardiovascular disease. So thank you all so much.   Mina Chung:   Thank you.   DeLisa Fairweather:    Thank you.   Milka Koupenova:       You're welcome.   Cindy St. Hilaire:         That's it for highlights from the April 28th and May 12th issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @circres and #DiscoverCircRes. Thank you to our guests, Dr Mina Chung, Dr DeLisa Fairweather and Dr Milka Koupenova. This podcast is produced by Ishara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on-the-go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2023. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.    

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This month on Episode 46 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the March 3 and March 17th issues of Circulation Research. This episode also features an interview with Dr Andrew Hughes and Dr Jessilyn Dunn about their review, Wearable Devices in Cardiovascular Medicine.   Article highlights:   Delgobo, et al. Deep Phenotyping Heart-Specific Tregs   Sun, et al. Inhibition of Fap Promotes Cardiac Repair After MI   Sun, et al. Endosomal PI3Kγ Regulates Hypoxia Sensing   Johnson, et al. Hypoxemia Induces Minimal Cardiomyocyte Division   Cindy St. Hilaire:        Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's Journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to share four articles selected from the March 3rd and March 17th issues of CircRes. I'm also going to have a discussion with Dr Andrew Hughes and Dr Jessilyn Dunn about their review, Wearable Devices in Cardiovascular Medicine. And the Review is also featured in our March 3rd issue.   Cindy St. Hilaire:        First, the highlights. The first article I'm going to present is Myocardial Milieu Favors Local Differentiation of Regulatory T-Cells. The first author is Murilo Delgobo and the corresponding author is Gustavo Campos Ramos. After myocardial infarction, the release of autoantigens from the damaged heart cells activates local and infiltrating immune cells such as the T-cell. Studies in mice have shown that fragments of the muscle protein myosin can act as autoantigens, and these myosin fragments are the dominant driver of the T-cell response.   But how do these myosin specific T-cells behave in the damaged heart to drive inflammation and repair is unknown. To find out, Delgobo and colleagues studied endogenous myosin specific T-cells, as well as those transferred into recipient mice. They found, whether exogenously supplied or endogenously created, the myosin specific T-cells that accumulated in the animals' infarcted hearts tended to adopt an immunosuppressive T-regulatory phenotype.   Strikingly, even if the exogenous cells were differentiated into inflammatory TH-17 cells prior to transfer, a significant proportion of them were still reprogrammed into T-regs within the heart. Although cells pre-differentiated into an inflammatory TH-17 phenotype were less inclined to change after the transfer, the results nevertheless indicate that, by and large, the infarcted heart promotes T-cell reprogramming to quell inflammation and drive repair. Yet exactly how the heart does this is a question for future studies.   Cindy St. Hilaire:        The next article I'm going to present is titled Inhibition of FAP Promotes Cardiac Repair by Stabilizing BNP. The first authors of the study are Yuxi Sun and Mengqiu Ma, and the corresponding author is Rui Yue, and they are from Tongji University. After myocardial infarction, there needs to be a balance of recovery processes to protect the tissue. Fibrosis, for example, acts like an immediate bandaid to hold the damaged heart muscle together, but fibrosis can limit contractile function.   Similarly, angiogenesis and sufficient revascularization is required to promote survival of cardiomyocytes within the ischemic tissue and protect heart function. To better understand the balance between fibrotic and angiogenic responses, Sun and colleagues examined the role of fibroblasts activated protein, or FAP, which is dramatically upregulated in damaged hearts, and brain natriuretic peptide, or BNP, which promotes angiogenesis in the heart.   In this study, they found that genetic deletion or pharmacological inhibition of FAP in mice reduces cardiac fibrosis and improves angiogenesis and heart function after MI. Such benefits are not seen if BNP or its receptor, NRP-1, are lacking. The in vitro experiments revealed that FAP's protease activity degrades BNP, thus inhibiting the latter's angiogenic activity. Interestingly, while FAP is upregulated in the heart, its levels drop in the blood, showing that BNP inhibition is localized. Together, these results suggest that blocking FAP's activity in the heart after MI could be a possible strategy for protecting the muscle's function.   Cindy St. Hilaire:        The next article I want to present is Hypoxia Sensing of Beta-Adrenergic Receptor is Regulated by Endosomal PI-3 Kinase Gamma. The first author of this study is Yu Sun, and the corresponding author is Sathyamangla Naga Prasad. Hypoxia is the most proximate acute stress encountered by the heart during an ischemic event. Hypoxia triggers dysfunction of the beta-adrenergic receptors, beta-1AR and beta-2AR, which are critical regulators of cardiac function.   Under normoxic conditions, activation of PI3K-gamma by beta-adrenergic receptors leads to feedback regulation of the receptor by hindering its dephosphorylation through inhibition of protein phosphatase 2A or PP2A. Although it is known that ischemia reduces beta-adrenergic receptor function, the impact of hypoxia on interfering with this PI3K feedback loop was unknown.   Using in vitro and in vivo techniques, this group found that activation of PI3K-gamma underlies hypoxia sensing mechanisms in the heart. Exposing PI3K-gamma knockout mice to acute hypoxia resulted in preserved cardiac function and reduced beta-adrenergic receptor phosphorylation. And this was due to a normalized beta-2AR associated PP2A activity, thus uncovering a unique role for PI3K-gamma in hypoxia sensing and cardiac function.   Similarly, challenging wild-type mice post hypoxia with dobutamine resulted in an impaired cardiac response that was normalized in the PI3K-gamma knockout mice. These data suggests that preserving beta-adrenergic resensitization by targeting the PI3K-gamma pathway would maintain beta-adrenergic signaling and cardiac function, thereby permitting the heart to meet the metabolic demands of the body following ischemia.   Cindy St. Hilaire:        The last article I want to highlight is Systemic Hypoxia Induces Cardiomyocyte Hypertrophy and Right Ventricle Specific Induction of Proliferation. First author of this study is Jaslyn Johnson, and the corresponding author is Steven Houser, and they're at Temple University.   The cardiac hypoxia created by myocardial infarction leads to the death of the heart tissue, including the cardiomyocytes. While some procedures such as reperfusion therapy prevent some cardiomyocyte death, true repair of the infarcted heart requires that dead cells be replaced. There have been many studies that have attempted new approaches to repopulate the heart with new myocytes. However, these approaches have had only marginal success.   A recent study suggested that systemic hypoxemia in adult male mice could induce cardiac monocytes to proliferate. Building on this observation, Johnson and colleagues wanted to identify the mechanisms that induced adult cardiomyocyte cell cycle reentry and wanted to determine whether this hypoxemia could also induce cardiomyocyte proliferation in female mice.   Mice were kept in hypoxic conditions for two weeks, and using methods to trace cell proliferation in-vivo, the group found that hypoxia induced cardiac hypertrophy in both the left ventricle and the right ventricle in the myocytes of the left ventricle and of the right ventricle. However, the left ventricle monocytes lengthened while the RV monocytes widened and lengthened.   Hypoxia induced an increase in the number of right ventricular cardiomyocytes, but did not affect left ventricular monocyte proliferation in male or in female mice. RNA sequencing showed upregulation of cell cycle genes which promote the G1 to S phase transition in hypoxic mice, as well as a downregulation of cullen genes, which are the scaffold proteins related to the ubiquitin ligase complexes. There was significant proliferation of non monocytes in mild cardiac fibrosis in the hypoxic mice that did not disrupt cardiac function.   Male and female mice exhibited similar gene expression patterns following hypoxia. Thus, systemic hypoxia induced a global hypertrophic stress response that was associated with increased RV proliferation, while LV monocytes did not show increased proliferation. These results confirm previous reports that hypoxia can induce cardiomyocyte cell cycle activity in-vivo, and also show that this hypoxia induced proliferation also occurs in the female mice.   Cindy St. Hilaire:        With me today for our interview, I have Dr Andrew Hughes and Dr Jessilyn Dunn, and they're from Vanderbilt University Medical Center. And they're here to discuss the review article that they helped co-author called Wearable Devices in Cardiovascular Medicine. And just as a side note, the corresponding author, Evan Brittain, unfortunately just wasn't able to join us due to clinical service, but they're going to help dissect and discuss this Review with us. Thank you both so much for joining me today. Andy, can you just tell us a little bit about yourself?   Andy Hughes:             Yeah, thank you, Cindy. I'm Andy Hughes. I'm a third year medicine resident at Vanderbilt University who is currently on an NIH supported research year this year. And then will be applying to cardiology fellowships coming up in the upcoming cycle.   Cindy St. Hilaire:        Great, thank you. And Jessilyn, I said you are from Vanderbilt. I know you're from Duke. It was Evan and Andy at Vanderbilt. Jessilyn, tell us about yourself.   Jessilyn Dunn:             Thanks. I am an Assistant Professor at Duke. I have a joint appointment between biomedical engineering and biostatistics and bioinformatics. The work that my lab does is mainly centered on digital health technologies in developing what we call digital biomarkers, using data from often consumer wearables to try to detect early signs of health abnormalities and ultimately try to develop interventions.   Cindy St. Hilaire:        Thank you. We're talking about wearable devices today, and obviously the first thing I think most of us think about are the watch-like ones, the ones you wear on your wrists. But there's really a whole lot more out there. It's not just Apple Watches and Fitbits and the like. Can you just give us a quick summary of all these different types of devices and how they're classified?   Jessilyn Dunn:             Yeah, absolutely. We have a wide variety of different sensors that can be useful. A lot of times, we like to think about them in terms of the types of properties that they measure. So mechanical properties like movement, electrical properties like electrical activity of the heart. We have optical sensors. And so, a lot of the common consumer wearables that we think about contain these different types of sensors.   A good example that we can think about is your consumer smartwatch, like an Apple Watch or a Fitbit or a Garmin device where it has something called an accelerometer that can measure movement. And oftentimes, that gets converted into step counts. And then it may also have an optical sensor that can be used to measure heart rate in a particular method called PPG, or photoplethysmography. And then some of the newer devices also have the ability to take an ECG, so you can actually measure electrical activity as well as the optical based PPG heart rate measurement. These are some of the simpler components that make up the more complex devices that we call wearables.   Cindy St. Hilaire:        And how accurate are the measurements? You did mention three of the companies, and I know there's probably even more, and there's also the clinical grade at-home ECG machines versus the one in the smartwatch. How accurate are the measurements between companies? And we also hear recent stories about somebody's Apple Watch calling 911 because they think they're dead, things like that. Obviously, there's proprietary information involved, but how accurate are these devices and how accurate are they between each other?   Jessilyn Dunn:             This is a really interesting question and we've done quite a bit of work in my lab on this very topic, all the way from what does it mean for something to be accurate? Because we might say, "Well, the more accurate, the better," but then we can start to think about, "Well, how accurate do we need something to be in order to make a clinical decision based off of that?" And if it costs significantly more to make a device super, super accurate, but we don't need it to be that accurate to make useful decisions, then it actually might not be serving people well to try to get it to that extreme level of accuracy.   So there are a lot of trade-offs, and I think that's a tough thing to think about in the circumstances, is these trade-offs between the accuracy and, I don't know, the generalizability or being able to apply this to a lot of people. That being said, it also depends on the circumstances of use. When we think about something like step counts, for example, if you're off by a hundred step counts and you're just trying to get a general view of your step counts, it's not that much of a problem.   But if we're talking about trying to detect an irregular heart rhythm, it can be very bad to either miss something that's abnormal or to call something abnormal that's not and have people worried. We've been working with the Digital Medicine Society to develop this framework that we call V3, which is verification, analytic validation and clinical validation. And these are the different levels of analysis or evaluation that you can do on these devices to determine how fit for purpose are they.   Given the population we're trying to measure in and given what the goal of the measurement is, does the device do the job? And what's also interesting about this topic is that the FDA has been evolving how they think about these types of devices because there's, in the past, been this very clear distinction between wellness devices and medical devices. But the problem is that a lot of these devices blur that line. And so, I think we're going to see more changes in the way that the FDA is overseeing and potentially regulating things like this as well.   Cindy St. Hilaire:        These consumer-based devices have started early on as the step counters. When did they start to bridge into the medical sphere? When did that start to peak the interest of clinicians and researchers?   Jessilyn Dunn:             Yeah, sure. What's interesting is if we think back to accelerometers, these have been used prior to the existence of mobile phones. These really are mechanical sensors that could be used to count steps. And when we think about the smartwatch in the form that we most commonly think of today, probably looking back to about 2014 is when ... maybe between 2012, 2014 is when we saw these devices really hitting the market more ... Timing for when the devices that we know as our typical consumer smartwatch today was around 2012 to 2014.   And those were things that were counting steps and then the next generation of that added in the PPG or photoplethysmography sensor. That's that green light when we look on the back of our watch that measures heart rate. And so, thinking back to the early days, probably Jawbone, there was a watch called Basis, the Intel Basis watch. Well, it was Basis and then got acquired by Intel. Fitbit was also an early joining the market, but that was really the timing.   Cindy St. Hilaire:        How good are these devices at actually changing behavior? We know we're really good at tracking our steps now and maybe monitoring our heartbeat or our oxygen levels. How good are they at changing behavior though? Do we know yet?   Andy Hughes:             Yeah, that's a great question and certainly a significant area of ongoing research right now with physical activity interventions. Things that we've seen right now is that simple interventions that use the wearable devices alone may not be as effective as multifaceted interventions. And what I mean by that is interventions that use the smartwatch but may be coupled with another component, whether that is health education or counseling or more complex interventions that use gamification or just in time adaptive interventions.   And gamification really takes things to another level because that integrates components, competition or support or collaboration and really helps to build upon features of behaviors that we know have an increased likelihood of sustaining activity. With that being said, that is one of the challenges of physical activity interventions, is the sustainability of their improvements over the course of months to years.   And something that we have seen is the effects do typically decrease over time, but there is work on how do we integrate all of these features to develop interventions that can help to sustain the results more effectively. So we have seen some improvement, but finding ways to sustain the effects of physical activity is certainly an area of ongoing research.   Cindy St. Hilaire:        I know it's funny that even as adults we love getting those gold stars or the circle completions. All of these devices, whether it's smartwatches like we're just talking about, or the other things for cardiac rehabilitation, they're generating a ton of data. What is happening with all this data? Who's actually analyzing it? How is it stored and what's that flow through from getting from the patient's body to the room where their physician is looking at it?   Andy Hughes:             And that is certainly a challenge right now that is limiting the widespread adoption of these devices into routine clinical care is, as Jessilyn mentioned. The wearables generate a vast amount of data, and right now, we need to identify and develop a way as clinicians to sort through all of the noise in order to be able to identify the information that is clinically meaningful and worthy of action without significantly increasing the workload.   And a few of the barriers that will be necessary in order to reach that point is, one, finding ways to integrate the wearables' data into the electronic health record and also developing some machine learning algorithms or ways with which we can use the computational power of those technologies to be able to identify when there is meaningful data within all of the vast data that comes from wearables. So it's somewhere that certainly we need to get to for these devices to reach their full clinical potential, but we are limited right now by a few of those challenges.   Jessilyn Dunn:             I was just going to say, I will add on to what Andy was saying about this idea behind digital biomarkers because this fits really nicely with this idea that giving people this huge data deluge is not helpful, but if we had a single metric where we can say, "Here's the digital biomarker of step count, and if you're above some threshold, you're good to go. And if you're below some threshold, some intervention is needed." That's a lot of the work that we've been doing, is trying to develop what are these digital biomarkers and how can they be ingested in a really digestible way?   Cindy St. Hilaire:        Yeah, that's great. Regarding the clinical and the research grade devices, I know a Fitbit or Apple Watch can sometimes be used for those, but I guess I'm talking also about the other kind of more clinically oriented devices, how good is compliance and how trustable is that data? Everybody's on probably their best behavior when they're in the office with the physician or if they're on the treadmill in the cardiac lab, but home is a different story. And what don't we know about compliance when people are out of the office and the reliability of that data that's generated in that space?   Andy Hughes:             I think you touched on a really important point right here, and one of the potential advantages of these wearable devices is that they provide continuous long-term monitoring over the course of weeks to months to years as opposed to those erratic measurements that we get from the traditional office visits or hospitalizations where, for example, the measurements we're taking are either in a supervised environment with a six-minute walk distance, for example, or self-reported or questionnaires.   So we build upon that information, but then additionally, we go beyond the observer effect where many individuals, the first week or two that you're wearing this new device, you may be more prone to increase your activity because you know that you're being monitored or you have this novel technology, but as you wear it for months to years, you outgrow those potential biases and you really can garner more comprehensive information.   In terms of compliance, we can speak to some of the research studies that have either really struggled with compliance and that limits the interpretability of their results and something we'll need to address in the future, but I think that's something that can be addressed with future studies keeping in mind all of the advantages that these devices offer compared to some of the traditional measures that we have used in the past.   Cindy St. Hilaire:        With all this data we're collecting, whether it be biological data or even just behavioral data, have we actually learned anything new? And I mean that in terms of All Of Us study this, I don't know, it was like 5,000 patients I think, and lo and behold, it found out that higher step count correlated with lower risk for a ton of diseases, which is not exactly groundbreaking. So are we, at this point in time, learning anything new from the use of these at-home devices, or are they really just able to help us enforce what we thought we knew regarding behavior?   Andy Hughes:             I think these devices have certainly provided some novel insights that build upon our understanding of physical activity. Many of us can hypothesize that decreased activity would have poor outcomes on health, which the studies have demonstrated in many facets. But in reference to All Of Us study that you mentioned, I think it's interesting to look as well at some of the diagnoses or conditions that were associated with decreased activity.   For example, reflux disease was also highlighted in that study, which may not have been identified if we didn't have the vast data and ability to really look for associations with diseases that have not been previously studied or thought to be related to physical activity. So I think that's one of the strong features of that database, is the wealth of knowledge that really will be hypothesis generating and help to inform future studies as we look even beyond cardiovascular conditions.   Cindy St. Hilaire:        One question, and you did bring it up in a bit of the discussion in your piece, is the bias that is in these devices. We know from COVID at-home pulse oximeters do not work as efficiently on darker skin. We actually know that going into bathrooms with the hand sensors that spit out the paper towels. So what kind of disparities or biases do these devices create or reinforce in the population?   Jessilyn Dunn:             This is such a critical topic because a lot of these issues had been discovered retrospectively because the people who were developing the technologies were not the representative of the people who were using the technologies. I think that's something that across the board we've been looking at from device development to AI implementation, which is having people who are going to be using the devices in the process of developing the technology and having voices heard from across the board.   We did a detailed look when we were evaluating devices for their accuracy at this exact question of where the heart rate sensors in smartwatches use optical based technology. And there was some evidence that was also an issue for people with varying skin tones, for people with wrist tattoos or more hair or freckles. And so, we did a deep dive and the generation of devices that we looked at which would meet this study was probably about three years ago.   We didn't see any discrepancies. And so, that's just one study and there are many more to be done, but I think prior to the technology development as well as once the technology comes out, keeping an eye on how that technology is doing, whether there are continued reports of failure of the technologies is really important. And there are a lot of ways that we can be vigilant about that.   Cindy St. Hilaire:        Yeah, that's great. And so, Andy, regarding patient populations, I can also see perhaps socioeconomic implications of this because smartwatches are not cheap. So how do we see that in terms of helping our patients? Are we going to be able to get a smartwatch through our insurance company?   Andy Hughes:             I think that's one of the really important next steps, is finding ways to make sure that as we advance the field of wearable devices in clinical care, that we recognize some of the existing inequities in terms of access to care, access to digital technologies that currently exist, and find ways by partnering with health insurance companies and the industry and providers and members of that community, finding ways to not only advance wearables, but use it in a way that we can decrease health disparities by really helping to increase access for these digital technologies to the underserved communities.   Jessilyn Dunn:             Yeah, the beauty of these technologies is that truthfully, at their core, they're very cheap. They're not difficult to develop, they're not difficult to build and disseminate. So a lot of what we think about is the infrastructure that goes around these devices. Does it require a smartphone to transfer data? Does it require internet access? What are the other pieces that need to be in place for these devices to work within an ecosystem? So this starts to get to questions beyond the devices themselves, but there's certainly a lot to think about and be done in the area of equity and ensuring that these devices can help everyone.   Cindy St. Hilaire:        And there's also the, I guess, ethical considerations of who owns this data. Obviously, if it's a consumable that you went and bought at Target, that's probably different than the one you're getting from your cardiologist. But who owns the data? Who has access to it? And are there any cases in the literature where an individual who's had certain measurements taken, have those measurements come back to bite them?   And I guess I'm thinking of something like cardiac rehab. If a patient doesn't get up and move enough or doesn't follow their physical therapy enough or lose weight quick enough, could their insurance coverage get cut? Could their premiums go up? What safeguards are in place for these very tricky situations? Are there safeguards in place?   Andy Hughes:             And on the clinical side, I think it will be important to treat this information just like any other protected health information that we have as part of the electronic health record. And so, there will be inherently safeguards around that in a similar manner for how we treat other protected health information.   But I think another important component of that will be a very clear consent policy when we reach the point that patients are consenting to include this information and their electronic health record, in terms of what the proposed benefits are and the potential risks associated with it, because it really is a vast amount of unique data that needs to be protected and safeguarded. And part of that comes by treating it as protected health information, but we will also need to make sure that there's a very clear consent policy that goes with it.   Cindy St. Hilaire:        Yeah. What do we see as the next steps in wearable devices? What do you guys see as the next big thing? I know one's coming from the actual AI and device side of things, and the other one is coming from the clinical side of things. What do each of you see as the next thing in this field?   Jessilyn Dunn:             I think on the device and AI side of things, I think we're thinking toward improving battery life, increasing the suite of sensors that are being added to these devices so we have a wider variety of measurements that are more representative of physiology, and then better algorithms to have better detection of sleep or activity or certain types of activity or certain types of arrhythmias. This combination of hardware and software and algorithms, I think coming together as all of these different pieces evolve will show us some really cool technology in the years to come.   Andy Hughes:             And I think from a clinical side, it's really twofold moving forward. I think as Jessilyn mentioned, there's a lot of novel sensor technologies that have a lot of exciting and evolving potential that we can hopefully integrate into the clinical space, but on the other hand, it's how can we use these wearable devices to enhance traditional therapies that we're already using?   For example, if we take the heart failure population, is there a way that we can use the wearable devices and the existing measurements with heart rate and physical activity and blood pressure to find a way to improve remote management and safely up-titrate guideline directed medical therapy, which are medications that we know have clinical benefit. But can we augment their clinical benefit and their utility by using some of the existing technologies that we already have?   And then lastly, building upon the initial studies with larger trials in more diverse generalizable populations to really enhance our understanding of the benefits that these devices may have for different cardiovascular conditions.   Cindy St. Hilaire:        Well, this was wonderful. Dr Andrew Hughes and Dr Jessilyn Dunn, thank you so much for joining me. The review, Wearable Devices in Cardiovascular Medicine, will be out in our March 3rd issue of Circulation Research. I forget which one, so I'll have to edit that out. Thank you so much for joining us, and I learned a ton. This was great.   Jessilyn Dunn:             Thank you.   Andy Hughes:             Thank you.   Cindy St. Hilaire:        That's it for our highlights from the March 3rd and March 17th issues of Circulation Research. Thank you for listening. Please check out the Circulation Research Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Dr Andrew Hughes and Dr Jessilyn Dunn.   This podcast is produced by Ishara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy texts for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, you're on-the-go Source for the most exciting discoveries in basic cardiovascular research.   This program is copyright of the American Heart Association, 2023. The opinions expressed by speakers in this podcast are their own, and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.  

This month on Episode 45 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the February 3rd and February 17th issues of Circulation Research. This episode also features an interview with Dr Hind Lal and Dr Tousif Sultan from the University of Alabama at Birmingham about their study Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation.   Article highlights:   Pi, et al. Metabolomic Signatures in PAH   Carnevale, et al. Thrombosis TLR4-Mediated in SARS-CoV-2 Infection   Cai, et al. Macrophage ADAR1 in AAA   Koide, et al. sEVs Accelerate Vascular Calcification in CKD   Cindy St. Hilaire:        Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cynthia St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to be highlighting the articles from our February 3rd and 17th issues of Circulation Research. I'm also going to have a chat with Dr Hind Lal and Dr Tousif Sultan from the University of Alabama at Birmingham about their study, Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation. But before I get to the interviews, here are a few article highlights.   Cindy St. Hilaire:        The first article I want to highlight comes from the laboratory of Dr Peter Leary at the University of Washington, and the title is Metabolomic Signatures Associated With Pulmonary Arterial Hypertension Outcomes. Pulmonary Arterial Hypertension or PAH is a rare but life-threatening disease in which progressive thickening of the walls of the lung's blood vessels causes increased blood pressure and that increased blood pressure ultimately damages the heart's right ventricle.   Interestingly, progression to heart failure varies considerably among patients, but the reasons why there is variability are not well understood. To find out, this group turned their attention to patient metabolomes, which differ significantly from those of healthy people and thus may also change with severity. Blood samples from 117 PAH patients were analyzed for more than a thousand metabolites by mass spectrometry and the patient's progress was followed for the next three years. 22 patients died within a three-year period and 27 developed significant right ventricle dilation. Other measures of severity included pulmonary vascular resistance, exercise capacity and levels of BNP, which is a metric of heart health. Two metabolic pathways, those relating to polyamine and histidine metabolism, were found to be linked with all measures of severity suggesting a key role for them in disease pathology. While determining how these pathways influence disease as a subject for further study, the current findings may nevertheless lead to new prognostic indicators to inform patient care.   Cindy St. Hilaire:        The next article I want to discuss is coming from our February 3rd issue of Circulation Research and this is coming from the laboratory of Dr Francisco Violi at the University of Rome and the title is Toll-Like Receptor 4-Dependent Platelet-Related Thrombosis in SARS-CoV-2 Infection. Thrombosis can be a complication of COVID-19 and it is associated with poor outcomes, including death. However, the exact mechanism by which the virus activates platelets, which are the cells that drive thrombosis, is not clear. For one thing, platelets do not appear to express the receptor for SARS-CoV-2. They do however, express the TLR4 receptor and that's a receptor that mediates entry of other viruses as part of the immune response. And TLR4 is ramped up in COVID-19 patient platelets. This group now confirms that, indeed, SARS-CoV-2 interacts with TLR4, which in turn triggers thrombosis.   The team analyzed platelets from 25 patients and 10 healthy controls and they found that the platelet activation and thrombic activity were both boosted in the patient samples and could not be blocked using a TLR4 inhibitor. Additionally, immunoprecipitation and immunofluorescent experiments further revealed colocalization between the virus protein and the TLR4 receptor on patient platelets. The team went on to show that the signaling pathway involved reactive oxygen species producing factors p47phox and Nox2, and that inhibition of phox 47, like that of the TLR4 receptor itsel,f could prevent platelet activation. As such, this study suggests that inhibiting either of these proteins may form the basis of an antithrombotic treatment for COVID-19.   Cindy St. Hilaire:        The third article I want to highlight is coming from the lab of Shi-You Chen at University of Missouri and the title of this article is ADAR1 Non-Editing Function in Macrophage Activation and Abdominal Aortic Aneurysm. Macrophage activation plays a critical role in abdominal aortic aneurysm development, or AAA development. Inflammation is a component of this pathology; however, the mechanisms controlling macrophage activation and vascular inflammation in AAA are largely unknown. The ADAR1 enzyme catalyzes the conversion of adenosine to inosine in RNA molecules and thus this conversion can serve as a rheostat to regulate RNA structure or the gene coding sequence of proteins. Several studies have explored the role of ADAR1 in inflammation, but its precise contribution is not fully understood, so the objective of this group was to study the role of ADAR1 in macrophage activation and AAA formation.   Aortic transplantation was conducted to determine the importance of nonvascular ADAR1 in AAA development and dissection and angiotensin II infusion of ApoE knockout mice combined with a macrophage specific knockout of ADAR1 was used to study the role of ADAR1 macrophage specific contributions to AAA formation and dissection. Allograft transplantation of wild type abdominal aortas to ADAR1 haploinsufficient recipient mice significantly attenuated AAA formation. ADAR1 deficiency in hematopoietic stem cells also decreased the prevalence and the severity of AAA and it also inhibited macrophage infiltration into the aortic wall. ADAR1 deletion blocked the classic macrophage activation pathway. It diminished NF-κB signaling and it enhanced the expression of a number of anti-inflammatory microRNAs. Reconstitution of ADAR1 deficient but not wild type human monocytes to immunodeficient mice blocked the aneurysm formation in transplanted human arteries. Together these results suggest that macrophage ADAR1 promotes aneurysm formation in both mouse and human arteries through a novel mechanism of editing the microRNAs that target NF-κB signaling, which ultimately promotes vascular inflammation in AAA.     Cindy St. Hilaire:        The last article I want to highlight is also from our February 17th issue of Circulation Research and it is coming from the lab of Shintaro Mandai at Tokyo Medical and Dental University and the title of the article is Circulating Extracellular Vesicle Propagated MicroRNA signatures as a Vascular Calcification Factor in Chronic Kidney Disease. Chronic Kidney Disease or CKD accelerates vascular calcification in part by promoting the phenotypic switching of vascular smooth muscle cells to osteoblast like cells. This study investigated the role of circulating small extracellular vesicles or SUVs from the kidneys in promoting this osteogenic switch. CKD was induced in rats and in mice by an adenine induced tubular interstitial fibrosis and serum from these animals induced calcification in in vitro cultures of A-10 embryonic rat smooth muscle cells. Intraperitoneal administration of a compound that prevents SEV biosynthesis and release inhibited thoracic aortic calcification in CKD mice under a high phosphorus diet. In Chronic Kidney Disease, the microRNA transcriptome of SUVs revealed a depletion of four microRNAs and the expression of the microRNAs inversely correlated with kidney function in CKD patients.   In vitro studies found that transected microRNA mimics prevented smooth muscle cell calcification in vitro. In silico analyses revealed that VEGF-A was a convergent target of all four microRNAs and leveraging this, the group used in vitro and in vivo models of calcification to show the inhibition of the VEGF-A, VEGFR-2 signaling pathway mitigated calcification. So in addition to identifying a new potential therapeutic target, these SUV propagated microRNAs are a potential biomarker that can be used for screening patients to determine the severity of CKD and possibly even vascular calcification.   Cindy St. Hilaire:        Today I have with me Dr Hind Lal who's an associate professor of medicine at the University of Alabama Birmingham and his post-doctoral fellow and the lead author of the study Dr Tousif Sultan. And their manuscript is titled Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation. And this article is in our February 3rd issue of Circulation Research. So thank you both so much for joining me today.   Tousif Sultan:              Thank you.   Hind Lal:                     Thank you for taking time.   Cindy St. Hilaire:        So ponatinib, it's a tyrosine kinase inhibitor and from my understanding it's the only treatment option for a specific group of patients who have chronic myelogenous leukemia and they have to harbor a specific mutation. And while this drug helps to keep these patients alive essentially, it's extremely cardiotoxic. So cardiotoxicity is somewhat of a new field. So Dr Lal, I was wondering how did you get into this line of research?    Hind Lal:                    So I was fortunate enough to be in the lab of Dr Tom Force and he was kind of father of this new area, now is very developed, it's called cardio-oncology. On those days there were basically everything started in cardio-oncology. So I just recall the first tyrosine kinase approved by FDA was in 2000 and that was... Imagine and our paper came in Nature Medicine 2005 and discovering there is... so to elaborate it a little bit, the cancer therapy broadly divided in two parts. One is called non-targeted therapy like chemotherapy, radiations, et cetera, and then there are cytotoxic drugs. So those cytotoxic drugs because they do not have any targeted name on it so they are, cardiotoxic are toxic to any organ was very obvious and understanding. When these targeted therapy came, which is mainly kinase inhibitor are monoclonal antibodies. So these are targeted to a specific pathway that is activated only in the cancer cells but not in any other cells in the body so they were proposed as like magic bullets that can take off the cancer without any cardiotoxity or minimal side effects. But even in the early phase like 2005 to 2010, these came out, these so-called targeted, they are not very targeted and they are not also the magic bullets and they have serious cardiotoxicity.   Cindy St. Hilaire:        And so what's the mechanism of action of ponatinib in the leukemia and how does that intersect with the cardiovascular system?   Hind Lal:                     Yeah, so this is very good question I must say. So what we believe at this point because, so leukemia if you know is driven by the famous Philadelphia chromosome, which is a translicational gene, one part of human chromosome nine and one part of human chromosome 22 and they translocate make a new gene which is BCR-ABL gene. And because it was discovered in Philadelphia UPENN, is named that Philadelphia chromosome, which is very established mechanism, that's how CML is driven. But what we have discovered that the cardiotoxicity driven by totally, totally different from the ponatinib is one of the inflammatory So it's kind of goodening. So this question is so good. One kind of toxicity is called on-target, when toxicity is mediated by the same mechanism, what is the mechanism of the drug to cure the cancer? So in that case your absolute is minimal because if you manipulate that, the drug's ability to cure the cancer will be affected but if the toxicity and the efficacy is driven by two different mechanism, then as in case of ponatinib seems like it's NLRP3 and inflammasome related mechanism. So this can be managed by manipulating this pathway without hampering the drug efficacy on the cancer.   Cindy St. Hilaire:        So what exactly is cardiotoxicity and how does it present itself in these patients?   Hind Lal:                     So these drugs like ponatinib, they call broader CVD effects. So it's not just cardiac, so they also in hypertensives and atherosclerosis and thrombosis, those kind of thing. But our lab is primarily focused on the heart. So that's why in this paper we have given impresses on the heart. So what we believe at this point that ponatinib lead to this proinflammatory pathway described in this paper, which is just 108A9-NLRP3-IL-1β and this inflammatory pathway lead to a cytokine storm very much like in the COVID-19 and these cytokine storms lead to excessive myocarditis and then finally cardiac dysfunction.   Cindy St. Hilaire:        Is the cytokine storm just local in the cardiac tissue or is it also systemic in the patients? Is cardiotoxicity localized only or is it a more systemic problem?   Tousif Sultan:              I would like to add in this paper we have included that we look this cytokine things and explain blood circulation, bone marrow. So the effect is everywhere, it's not local. So we didn't check other organs, maybe other organs also being affected with the ponatinib treatment.   Cindy St. Hilaire:        And what's the initial phenotype of a patient has when they start to get cardiotoxicity, what's kind of like a telltale symptom?   Hind Lal:                     So good thing that in recent years cardio-oncology developed. So initially the patient that were going for cancer treatment, they were not monitored very closely. So they only end up in cardiology clinic when they are having some cardiac events already. So thanks to the lot of development and growth in the cardio-oncology field, now most patients who going for a long-term cancer treatment, they are closely monitored by cardiology clinics.   Cindy St. Hilaire:        Got it. So they can often catch it before a symptom or an event. That's wonderful.   Hind Lal:                     Yeah, so there's a lot of development in monitoring.   Cindy St. Hilaire:        Wonderful. So you were really interested in figuring out why ponatinib induces cardiotoxicity and you mentioned that really up until now it's been very difficult to study and that's because of the limitation of available murine models. If you just inject a wild type mouse with ponatinib, nothing happens really. So what was your approach to finding relatively good murine models? How did you go about that?   Hind Lal:                     So this is the top scientific question you can ask. So like science, the field is try and try again. So initially this is the first paper with the ponatinib toxicity using the real in vivo models. Any paper before this including ours studies published, they were done on the cellular model in hiPSC, that isolated cardiomyocytes. So you directly putting the ponatinib directly the isolated cells. So this is first case when we were trying to do in vivo, maybe other attempt in vivo but at least not published. So first we also treated the animals with ponatinib and that failed, we don't see any cardiotoxic effect. And then when we going back to the literature, the clinical data is very, very clear from pharmacovigilance that ponatinib is cardiotoxic in humans. So when we're not able to see any phenotype in mouse, we realize that we are not mimicking what's happening in the humans.   So we certainly missing something. Now once again I quote this COVID-19, so many people get infected with COVID-19 but people are having preexisting conditions are on high risk to developing CVD. So there was some literature on that line. So we use this very, very same concept that if there is preexisting conditions, so likely who'd have developing future cardiac event will be more. So we use two model in this paper one atherosclerosis model which is APoE null mice mice, another is tag branding which is pressure overload model for the heart and as soon as we start using what we call comorbidity model like patient is having some preexisting conditions and we very clearly see the robust defect of ponatinib on cardiac dysfunction.   Cindy St. Hilaire:        Yeah, it's really, really well done and I really like that you use kind of two different models of this. Do you think it's also going to be operative in maybe like the diabetic mirroring models? Do you think if we expand to other comorbidities, you might also recapitulate the cardiotoxicity?   Hind Lal:                     So you got all the best questions.   Cindy St. Hilaire:        Thank you. I try.   Hind Lal:                     So because this is CML drug and lot of the risk factor for cardiovascular and cancer are common and even metabolic disease. So most of the time these patients are elderly patients and they're having metabolic conditions and most of the time they have blood pressure or something CVD risk factors. So I agree with you, it'll be very relevant to expand this to the diabetes or metabolic models, but these were the first study, we put all our focus to get this one out so news is there then we can expand the field adding additional models et cetera. But I agree with you that will be very logical next step to do.   Cindy St. Hilaire:        Yeah. And so I guess going back to what you know from the human study or the clinical trials or the human observations, are different populations of patients with CML more predisposed to cardio toxicity than others or is that not known yet?   Hind Lal:                     So one other area called pharmacovigilance. So what pharmacovigilance does patient all over the world taking these drugs. So WHO have their own vigilance system and FDA have their own, so it's called BG-Base for the WHO and it's called the FAERS for the FDA. So one can go back in those data sets and see if X patient taking this Y drug and what kind of symptoms or adverse effect they are seeing and if these symptoms are associated with something else. So there is data that if patients having CVD risk factor, they are more prone to develop ponatinib induced cardiac events. But it needs more polish like you asked the just previous question, diabetes versus maybe blood pressure means hypertension, atherosclerosis, or thrombosis. So it has not been delineated further but in a one big bucket if patients are having CVD risk factor before they are more prone and more likely to develop the cardiac events.   Cindy St. Hilaire:        So after you established that these two murine models could pretty robustly recapitulate the human phenotype, what did you do next? How did you come upon the S100A8/A9-NLRP3-IL-1β signaling circuit? How did you get to that?   Hind Lal:                     So in basic science work, whenever we do mouse is called until we get there is cardiac dysfunction, it's called phenotype, right? So mouse had a cardiac phenotype. So next step is, "Why? What is leading to that phenotype?" That's what we call mechanism. So there the best idea to fit the mechanism is using one of the unbiased approaches like you do unbiased proteomics, unbiased RNC analysis, something like this that will analyze the entire transcript like RNC and say, "Okay, these pathway are," then you can do further analysis that will indicate these pathway are different, are altered. So in this case we used RNC analysis and it came out that this yes A8 and yes A9, 100A8 and nine, they were the most upregulated in this whole set. And thereafter we were very lucky. So we started this study at Vanderbilt, where my lab was and thereafter we very lucky to move here and found Sultan who had a lot of experience with this inflammation and immune system and then Sultan may add something on this so he'll be the better person to say something on this.   Tousif Sultan:              So after our RNC analysis, so we got this S100A8 and nine as top hit with the ponatinib treatment. So then we validated this finding with our flow cytometric, qRT PCR aand then we started which pathway is going to release cytokine and all that. So we found that is NLRP3 inflammasome.   Cindy St. Hilaire:        Yeah and well and I guess maybe step back, what is S100A8/A9? What are those? Tousif Sultan:              Yeah, S10A8/A9 is a calcium binding protein. So that's also called alarmin and they basically binds with the pathogen associated pattern and other TLR2 like receptors and then start inflammatory pathway to release cytokine and all that and it's stable in heterodimer form. So S100A8 heterodimer with A9 and then bind with TLR and a start in this inflammatory pathway.   Cindy St. Hilaire:        And what type of cell is that happening in? Is that happening in the immune cells only or is it also in the cardiomyocyte, or...?   Tousif Sultan:              Yeah, we have included all this data. So from where this alarmin is coming with ponatinib treatment, so literature also suggested that neutrophils and monocytes, those cells are the potential to release the alarmin. So here we also found these two type of cells, neutrophils and monocytes. They release huge alarmin with the treatment of ponatinib.   Cindy St. Hilaire:        And so really taking this really neat mechanism to the next level, you then tried attenuating it by using broad anti-inflammatory steroid dexamethasone but also by targeting these specific components, the NLRP and the S100A specific inhibitors and they worked well. It worked really nicely. Does your data show that any of these therapies work better than the other and then are these viable options to use in humans?   Hind Lal:                     Yeah, we have some data in the paper. Are very broad which help a lot in COVID patients, far very acute infections. So in this case, situation is very different cause most of CML patients will going to take ponatinib for lifelong, there is no remission, right? So in those case, its certainly not a very attractive option. We have shown data in the paper that dexamethasone help with the heart but lead to some metabolic changes. So we have compared those with the NLRP3 inhibitors, those metabolic alterations, dexa versus the NLRP3 inhibitors, CY-09. And we demonstrated that targeting is specifically with paquinimod, our NLRP3 inhibitor CY-09, feel better. It can still rescue the cardiac phenotype without having those adverse effect on metabolic parameters.   Cindy St. Hilaire:        That's wonderful. Do you think though that because you have to take ponatinib for life, that long-term NLRP inhibition would also cause problems or...?   Hind Lal:                     So because not every patient who taking ponatinib would develop the cardiac phenotype, right? Which is like a 10%, 12%, patient developing cardiac dysfunction. So I think someone like I strongly believe paquinimod, which is inhibitor of S100A9, will be really good option or at least we have enough data that make us nail for at least a small clinical trial. And we quickly moving on that. At UAB we have our clinical cardio-oncology program and we are already in touch with the director for the clinical cardio-oncology program. So what we trying to do in that small trial is if one of the standard therapy for heart like beta blocker or ARBs inhibitor, is there any preference like one work better than the other in the standard care? So first we doing that project, then we obviously looking forward if one small clinical trial can be done with paquinimod. I strongly believe it should be helpful.   Cindy St. Hilaire:        That is wonderful. And so do you think... There's other chemotherapeutic agents or probably even other non-cancer drugs that cause cardiotoxicity, do you think this mechanism, this pathway, this S100A-NLRP-IL-1β axis is operative in all cardiotoxicities or do you think it's going to be very specific to the ponatinib?   Hind Lal:                     So it's certainly not all, but it'll be certainly more than ponatinib. So in our lab we are using another kinase inhibitor, which is osimertinib and it's not published yet, but now we know that it's also cardiotoxic because it's taking metabolic root or energetics disruption but not this pro-inflammatory part, but we're doing another project which is strep pneumonia induced cardiac dysfunction, which is called pneumonia. So strep pneumoniae, which leads to the pneumonia ,and lot patient die because of the failing heart we see here in the hospitals and we see these pathways operational over there and we gearing up to do clinical trial on that aspect as well, but it's not generalized like all kind of heart will have the same mechanism.   Cindy St. Hilaire:        It's wonderful to see you're already taking those next steps towards really kind of bringing this to a translational/clinical study. So what was the most challenging aspect of this study?   Tousif Sultan:              The challenging aspect, ponatinib is a kinase inhibitor and that was surprising for us how it's activating immune cells. Generally kinase inhibitors, inhibits all the cells like that. So that was challenging. So we repeated it many times did in vitro experiment to confirm that. So we just added, just treated in vitro immune cells with the ponatinib and confirmed it. So that was little challenging.   Cindy St. Hilaire:        So what's next? You mentioned you're going to try some clinical trials, early stage clinical trials. What's next mechanistically, what do you want to go after?   Hind Lal:                     So what we are doing next and we are very, very eagerly trying to do that. So what it was done, we used the cardiac comorbidity models, but as you know, anybody who will take ponatinib will have cancer, right? So we strongly believe that we miss one factor. There was no cancer on these. So that is very logical next step. What that will allow us to do, what rescue experiment we'll have done in this paper. So we saw, "Okay, this rescue the cardiac phenotype, which is taken care of now," but very same time, we not able to demonstrate that this is happening without hurting the cancer efficacy. So if we have the dual comorbid mouse, which have CML a real thing and we have cardiac thing, then that will allow us to demonstrate, "Okay, we got something that can take care of the cardiac problem without hurting the efficacy on the cancer." And it will be best if you also help little bit to more potentiate the cancer efficacy.   Cindy St. Hilaire:        Yes. Excellent. Well, congratulations on a beautiful study, really exciting findings. Dr Lal and Dr Sultan, thank you so much for taking the time to talk with me today.   Tousif Sultan:              Thank you so much.   Hind Lal:                     Well thank you, Cynthia. We really appreciate your time. Thank you for having us.   Cindy St. Hilaire:        Yeah, it was great.   Cindy St. Hilaire:        That's it for our highlights from the February 3rd and February 17th issues of Circulation Research. Thank you so much for listening. Please check out the Circulation Research Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Dr Hind Lal and Dr Tousif Sultan. This podcast is produced by Ishara Ratnayake, edited by Melissa Stoner and supported by the editorial team at Circulation Research. Some of the copy text for the highlighted articles was provided by Ruth Williams. I'm your host, Dr Cynthia St. Hilaire, and this is Discover CircRes, you're on-the-go source for most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2023. And the opinions expressed by the speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahajournals.org.  

We'll be nicer to the Bravermans next week, we promise.

This month on Episode 44 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the January 6th and January 20th issue of Circulation Research. This episode also features an interview with Dr Timothy McKinsey and Dr Marcello Rubino about their study, Inhibition of Eicosanoid Degradation Mitigates Fibrosis of the Heart.   Article highlights:   Prasad, et al. ACE2 in Gut Integrity and Diabetic Retinopathy   Cui, et al. Epsins Regulate Lipid Metabolism and Transport   Li, et al. Endothelial H2S modulates EndoMT in HF   Luo, et al. F. plautii Attenuates Arterial Stiffness   Cindy St. Hilaire:        Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's Journal Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh. And today I'm going to be highlighting articles from our January 6th and January 20th issues of Circulation Research. I'm also going to have a chat with Dr Timothy McKinsey and Dr Marcello Rubino about their study, Inhibition of Eicosanoid Degradation Mitigates Fibrosis of the Heart. But before the interview, I want to get to a few articles to highlight.   Cindy St. Hilaire:        The first article is titled, Maintenance of Enteral ACE2 Prevents Diabetic Retinopathy in Type 1 Diabetes. The first authors are Ram Prasad and Jason Floyd, and the corresponding author is Maria Grant, and they are from the University of Alabama.   Type 1 Diabetes has a complex etiology and pathology that are not entirely understood. In addition to the destruction of insulin-producing cells, a recently discovered feature of the disease in both humans and in rodent models is that the levels of angiotensin converting enzyme 2 or ACE2 can be unusually low in certain tissues. ACE2 is a component of the renin angiotensin system controlling hemodynamics and interestingly, genetic deficiency of ACE2 in rodents exacerbates aspects of diabetes such as gut permeability, systemic inflammation and diabetic retinopathy, while boosting ACE2 has been shown to ameliorate diabetic retinopathy in mice. This study shows that ACE2 treatment also improves gut integrity and systemic inflammation as well as retinopathy. Six months after the onset of diabetes in a mouse model, oral doses of a bacteria engineered to express humanized ACE2 led to a reversal of the animal's gut barrier dysfunction and its retinopathy. Humans with diabetic retinopathy also displayed evidence of increased gut permeability in low levels of ACE2. This study suggests they may benefit from a similar probiotic treatment.   Cindy St. Hilaire:        The next article I want to highlight is titled, Epsin Nanotherapy Regulates Cholesterol Transport to Fortify Atheroma Regression. The first authors are Kui Cui, Xinlei Gao and Beibei Wang, and the corresponding authors are Hong Chen and Kaifu Chen and they're from Boston Children's Hospital. Epsins are a family of plasma membrane proteins that drive endocytosis. They're expressed at varying levels throughout the tissues of the body, and recent research shows that they are unusually abundant on macrophages within atherosclerotic lesions. In mice, macrophage specific Epsin loss results in a reduction in foam cell formation and atherosclerotic plaque development. This study now shows that this effect on foam cells is because Epsins normally promote the internalization of lipids into macrophages through their endosytic activity.   But that's not all. The proteins also impede cholesterol efflux from macrophages to further exacerbate lipid retention. It turns out out Epsins regulate the endocytosis and the degradation of a cholesterol efflux factor called ABCG1. Importantly, these pro atrogenic activities of Epsins can be stopped. Using macrophage targeted nanoparticles carrying Epson specific silencing RNA, the team could suppress reduction of the protein in cultured macrophages and could reduce the size and number of plaques in atherosclerosis prone mice. Together these results suggest blocking Epsins via nanotherapy or other means could be a therapeutic approach to stopping or slowing atherosclerotic plaque progression.   Cindy St. Hilaire:        The third article I want to highlight is coming from our January 20th issue of Circ Res and is titled, Hydrogen Sulfide Modulates Endothelial-Mesenchymal Transition in Heart Failure. The first author is Zhen Li, and the corresponding author is David Lefer and they're from Cedars-Sinai. Hydrogen sulfide is a critical endogenous signaling molecule that exerts protective effects in the setting of heart failure. Cystathionine γ-lyase, or CSE, is one of the three hydrogen sulfide producing enzymes, and it's predominantly localized in the vascular endothelium. Genetic deletion of CSE, specifically in the endothelium, leads to reduced nitric oxide bioavailability, impaired vascular relaxation and impaired exercise capacity, while genetic over-expression of PSE in endothelial cells improves endothelial cell dysfunction, and attenuates myocardial infarction following myocardial ischemia-reperfusion injury.   In this study, endothelial cell specific CSE knockout mice and endothelial cell specific CSE overexpressing transgenic mice were subjected to transverse aortic constriction to induce heart failure with reduced ejection fraction. And the goal was to investigate the contribution of the CSE hydrogen sulfide access in heart failure. Endothelial specific CSE knockout mice exhibited increased endothelial to mesenchymal transition and reduced nitric oxide bioavailability in the myocardium. And this was associated with increased cardiac fibrosis, impaired cardiac and vascular function, and it worsened the vascular performance of these animals. In contrast, genetic overexpression of CSE in endothelial cells led to increased myocardial nitric oxide, decreased EndoMT and decreased cardiac fibrosis. It also improved exercise capacity. These data demonstrate that endothelial CSE modulates endothelial mesenchymal transition and ameliorated the severity of pressure overload induced heart failure , in part through nitric oxide related mechanisms. This data further suggests that endothelium derived hydrogen sulfide is a potential therapeutic for the treatment of heart failure with reduced ejection fraction.   Cindy St. Hilaire         The last article I want to highlight is titled, Flavonifractor plautii Protects Against Elevated Arterial Stiffness. The first authors are Shiyun Luo and Yawen Zhao, and the corresponding author is Min Xia, and they are at Sun Yat-sen University. Dysbiosis of gut microbiota contributes to vascular dysfunction and gut microbial diversity has been reported to be inversely correlated with arterial stiffness. However, the causal role of gut microbiota in the progression of arterial stiffness and the specific species along with the molecular mechanisms underlying this change remain largely unknown. In this study, the microbial composition in metabolic capacities were compared in participants with elevated arterial stiffness and in normal controls free of medication. And these groups were age and sex match.   Human fecal metagenomic sequencing identified a significant presence of Flavonifractor plautii or F. plautii in normal controls, which was absent in the subjects with elevated arterial stiffness. The microbiome of normal controls exhibited an enhanced capacity for glycolysis and polysaccharide degradation, whereas individuals with increased arterial stiffness exhibited increased biosynthesis of fatty acids and aromatic amino acids. Additionally, experiments in the angiotensin II induced and humanized mouse model show that replenishment with F. plautii or its main effector cis-aconitic acid or CCA improved elastic fiber network and reversed increased pulse wave velocity through the suppression of matrix metalloproteinase-2 and through the inhibition of monocyte chemoattractant protein-1. And this was seen in both the angiotensin II induced and humanized models of arterial stiffness. This study now identifies a novel link between F. plautii and arterial function and raises the possibility of sustaining vascular health by targeting the gut microbiota.   Cindy St. Hilaire:        Today with me I have Dr Tim McKinsey and Dr Marcello Rubino from the University of Colorado Anschutz Medical Campus, and we're here to talk about their paper Inhibition of Eicosanoid Degradati`on Mitigates Fibrosis of the Heart. And this article is in our January 6th issue of Circulation Research, so thank you both so much for joining me today.   Timothy McKinsey:    Thank you for inviting us.   Marcello Rubino:        Yeah, thank you for the opportunity.   Cindy St. Hilaire:        And so Dr McKinsey, you're a professor at the University of Colorado. How long have you been investigating cardiac fibrosis?   Timothy McKinsey:    Oh, a long time. Before I started the lab here in 2010, I was in industry working in biotech with Myogenic Gilead, and we were very interested in cardiac fibrosis all the way back then.   Cindy St. Hilaire:        Oh wow, so you actually made an industry to academia transfer.   Timothy McKinsey:    Yes.   Cindy St. Hilaire:        Good topic for another podcast. That is really great.   Timothy McKinsey:    Yeah, it's of interest to a lot of people, including trainees.   Cindy St. Hilaire:        Yeah, I bet. Dr Rubino, you were or are a postdoc in the McKinsey lab? Marcello Rubino:        Yeah, I was a postdoc in Timothy McKinsey lab. I spent four years in Tim's lab. It was my first time studying cardio fibrosis, so it was a little bit difficult at the end, but I think I was right choosing Tim, so I'm really happy now.   Cindy St. Hilaire:        Nice and are you sticking with fibrosis or are you moving on?   Marcello Rubino:        Yeah, so now I'm back in Milan where I did my PhD student and postdoc. I am like an independent researcher, but it's still not a principal investigator, so I want to become one of the that, studying cardiac fibrosis. Yeah. And inflammation and epigenetics, so yeah, I'm going try to go to my way, thanks to Tim, I think that I find my own way.   Cindy St. Hilaire:        I'm sure you will. I mean, based on the great work in this study, right. Building upon that, I'm sure you'll be a success.   Timothy McKinsey:    No doubt about it.   Cindy St. Hilaire:        So your manuscript, this study, it's investigating whether eicosanoid availability can attenuate fibrosis in the heart. But before we kind of jump into this study, why is fibrosis in the heart a bad thing? Is it always detrimental? Is there some level of fibrosis that's necessary or even helpful?   Timothy McKinsey:    I mean, a certain level of extracellular matrix is deposited in your heart and that maintains the structure of the heart. Fibrosis can also be good after you have a myocardial infarction and a big piece of the muscle of your heart has died, it needs to be replaced with a fibrotic scar, essentially to prevent rupture of the ventricle. So fibrosis isn't always bad, but chronic fibrosis can be really deleterious to the heart and contribute to stiffening of the heart and cause diastolic dysfunction. It can create substrates for arrhythmias and sudden cardiac death. So we're really trying to block the maladaptive fibrosis that occurs in response to chronic stress.   Cindy St. Hilaire:        Yeah, yeah. And what about eicosanoids? What are they and what role do they play in cardiac fibrosis or what was known about their role in this process before your study?   Timothy McKinsey:    Eicosanoids are lipids, they're basically fatty acids, 20 carbon in length and a lot is known about them. It's a very complex system. There are many different eicosanoids, but they're produced from arachidonic acid through the action of cyclooxygenase enzymes like COX-2. And so you're probably familiar with the literature showing that non-steroidal anti-inflammatory drugs that target the COX enzymes can actually increase the risk of cardiac disease, so there was a lot known about what produces eicosanoids in the heart, but our study is really the first to address how they're degraded and how that controls cardiac fibrosis.   Cindy St. Hilaire:        What I thought you did really well in the introduction and what I guess I didn't really fully appreciate until I had read your study, was that your goal was to identify compounds that could attenuate fibrosis. And you spent some time emphasizing the differences between a targeted small molecule screen and a phenotype based screen. And I was wondering if you could just expand on this difference for the audience and maybe just explain why in your case you went with the latter.   Timothy McKinsey:    Well, we wanted to use an unbiased approach and some people call this a chemical biology approach where we took a targeted library, meaning we took compounds with known activities, meaning compounds that with known targets and we screened that library using a phenotypic assays that we developed in the lab. And the phenotypic assay is an unbiased assay, right? We're just screening for compounds that have the ability to block the activation of fibroblasts. And we monitor activation by looking at markers of fibroblast activation such as alpha smooth muscle Actin. And we can do this in a very quantitative and high throughput manner using this imaging system, high content imaging system that we have in the lab.   It was an unbiased screen looking for inhibitors of fibroblasts activation across organ systems. We not only studied cardiac fibroblasts, but we also studied lung and renal fibroblasts looking for compounds with a common ability to block the activation state of each of those cell types.   One of the things that I get asked frequently is how do we maintain the cardiac fibroblasts in a quiescent state? Because you may know this, but when fibroblasts are plated on cell culture plastic, which has a very high 10 cell strength, they tend to spontaneously activate, so we actually spent a couple of years working out the conditions to maintain the cells in quiescent state, and I think that will also be of great interest to the field.   Cindy St. Hilaire:        Probably even the smooth muscle cell biology field where I hang out and even valve interstitial cells that we study. All of those, I guess basic things related to cell culture, we have taken for granted that plastic is not physiological.   Timothy McKinsey:    Right.   Cindy St. Hilaire:        And so I think with this really nice phenotypic or chemical screen that you conducted, you first identified nine compounds, but what made you zero in on this one, SW033291?   Timothy McKinsey:    When we got the hits, we were intrigued by the SW compound SW033291 because there was only one paper describing its action and there was a paper published in Science showing that SW or inhibition of this enzyme 15-PGDH could enhance organ regeneration.   Cindy St. Hilaire:        Oh, okay.   Timothy McKinsey:    And there's a very interesting interplay between fibrosis and organ regeneration where fibrosis inhibits regeneration and if you can stimulate regenerative pathways, they can actually block fibrosis, so there's this back and forth. And so that's really the main reason we were interested in pursuing SW just because of the novelty and the potential. And also it was a compound that behaved beautifully in our cell culture models with beautiful dose-dependent inhibition of each of the fibroblast types.   Cindy St. Hilaire:        It's kind of like the cleanest thing to start with. Also, if there's nothing known, it's ripe for investigation, so that's great. You just said this SW compound acts on 15-PGDH, so what is the role of that protein in fibroblasts and what if any known effects are there on this protein's inhibition in other cell types or disease states?   Marcello Rubino:        In fibroblasts team, I would like to say that this was really the first article that was published. Maybe there was just one published in Pulmonary Fibrosis, but like last year, but I didn't really talk about 15-PGDH, so you need to consider that 15-PGDH is an inhibitor, an enzyme that degrades prostaglandin, so if you inhibit the inhibitor, the release increase production, a lot of prostaglandin. And so a lot of paper were talking about this effect, so they will see we are just using SW in order to increase Prostaglandin E2 level and that was why we had this like anti-inflammatory or whatever effect. I would like to say that until now, maybe this can be the first really paper talking about no more than not just prostaglandin but 15-PGDH. Its action total level, a global level at particularly on fibroblasts.   To answer your question, I would like to say that this was also our question first and we checked by level other browser to try to find the answer to your question. We figured out that it was known that 15-PGDH was increasing a pathology condition in different organ, not just related by fibroblasts, not just related to cardiac disease, about the function with discover a function in macrophage that interested us because it can regulate maybe the polarization macrophage, so still involving the prostaglandin production inflammation, so that's why also we decide to take a look because it was still novel in fibrolbasts and we still know that it was doing something important and we were trying not to put the piece together and find something new in that we were lucky for this.   Timothy McKinsey:    15-PGDH is actually expressed at very low levels in fibroblasts. It's much more highly expressed in macrophage, just as Marcello pointed out, so in the future we're very interested in knocking out or inhibiting 15-PGDH in different cell types to see how that contributes to inhibition of cardiac fibrosis.   Cindy St. Hilaire:        Really interesting. Related to that, you used a couple different animal models for fibrosis. They're all different or special in their own way. How well did these recapitulate what we observe in humans. Are there any limitations of benefits?   Timothy McKinsey:    They're always limitations to animal models. We started out with a very robust commonly used model of cardiac fibrosis, which relies on Angiotensin II infusion in mice. We like that model because it's robust and quick so we can get answers quickly. And then we transitioned into a model of diastolic dysfunction that we've been working with in a lab where we remove a kidney from a mouse and we implant something called DOCA, which is an aldosterone memetic. And so the animals develop hypertension that leads to a mild but significant diastolic dysfunction with preserved ejection fraction.   And that's a model that we like a lot. It has something that we call hidden fibrosis, so if you just do standard histochemical staining of the hearts from the DOCA unit, nephrectomy model, that diastolic dysfunction model, you really can't see robust fibrosis. It's only when you dive more deeply with more sensitive assays like mass spectrometry or atomic force microscopy that you can detect this fibrosis and stiffening of the heart, so we usually lead with a robust model of fibrosis, cardiac fibrosis, and then transition into a slightly more complex model but more physiologically relevant model or disease relevant model.   Cindy St. Hilaire:        Obviously you showed some really nice robust results with this SW compound. So in the continuum of heart failure in human, what do you think or what would you speculate would be the ideal timeframe for administration of this compound?   Timothy McKinsey:    Wouldn't want to give it immediately after someone's had a heart attack. As we discussed earlier, you need that reparative scar to form so you don't want to block that fibrotic remodeling. We believe that there's kind of smoldering fibroblast activation in the heart, even in someone who's had heart disease for many, many years. And if we can dampen that, we can either prevent further progression of heart failure or perhaps reverse it. We don't really know if we can reverse really established fibrosis in the heart yet. But I would want to try to catch fibrosis fairly early on in the disease process in someone who has chronic hypertension or obesity or a variety of different comorbidities and then start delivering an antifibrotic therapy at that point.   Marcello Rubino:        I would like to add that, so it is really tricky when we talk about clinical trials because a lot of molecules that maybe they can work hopefully in a preclinical model don't work at the end in the clinical model. That's because can be some off target also like you just asked what is really important is when you do the administration of the molecule and talk about this in SW, like things say we don't want to prevent the fibrosis because there is something like called a kneeling at the beginning, so it is the good fibrosis we like to say, but the good thing of SW compound is that is affecting in a good way the proliferation of fibroblast that is different for all the other. I would like to say all the other inhibitor that we saw so far, because I remember the first time that I presented this work, there was an expert told me that he didn't believe that all my data because the compound was inhibiting fibrosis, it was inhibiting proliferation.   And I show him, no, this is contrary, so oh okay, I like it. We need to consider this that the action seems to be not like the retire for the cell, so because the cells continue to proliferate, they can proliferate more. But the good thing and we need to investigate more is that SW action seems to increase when the cell are more fibrotic, because we show just few human fibroblasts isolating from a human patient and we saw a higher positive effect of SW compound when the cell were more fibrotic. That can be interesting. I think that it's worth to try to test in the future like in different preclinical models and maybe in patients at the end because if we really can find something like maybe SW that can be specific for the state of pathology, that will be wonderful. I don't really know if we can really do it, but we need some therapy like this, so that's why we were really excited about what we discovered for this compound.   Timothy McKinsey:    We have a lot more to learn about this pathway and about fibrosis in general.   Cindy St. Hilaire:        Yeah.   Timothy McKinsey:    It's a very exciting time to be doing science because of the amazing technologies that we have at our disposal to address detailed mechanisms of disease.   Cindy St. Hilaire:        What was the most challenging aspect of the study?   Timothy McKinsey:    This was an incredibly difficult study. I can't even stress to you how much work went into this. Spearheaded by Marcello's awesome leadership. There was huge input from a big team. Keith Cook and I worked together in industry and we were able to recruit him over here for a few years as part of our fibrosis center called the CFReT. It's an advertisement. And Keith was able to implement some of the drug discovery approaches that we used in biotech and create this imaging system that we initially employed for the screens. That was challenging. Maintaining the cells in a quiescent state was very challenging as I mentioned. That took a couple of years and then just following up on SW and trying to figure out its mechanism of action was really challenging as well because as Marcello mentioned, most people have attributed SW's effects to an increase in PGE2 levels, so PGE2 is an eicosanoid that is degraded by 15-PGDH.   And definitely when you inhibit 15-PGDH with SW, you see increased PGE2. But surprisingly we couldn't find that PGE2 was doing anything in our cell culture systems, meaning when we added it exogenously it was not blocking fibroblast activation, so then Marcello set out to identify which eicosanoid that is regulated by 15-PGDH is actually the antifibrotic eicosanoid. And that led him to something called 12(S)-HETE. That was challenging. And then just determining at the molecular level what was going on was also challenging. And that led Marcello to this kind of paradoxical discovery that it activating ERK signaling was actually blocking fibroblast activation.   Cindy St. Hilaire:        And of course ERK does everything right?   Marcello Rubino:        It does. Everything.   Timothy McKinsey:    And sort of the dogma is that ERK is promoting fibrosis in the heart, but Marcello's data suggests otherwise.   Timothy McKinsey:    And then other shout outs, Josh Travers, who's the second author of the paper provided huge input, especially after Marcello left. Josh helped get this across the finish line. We have an amazing in vivo team conducting the animal model studies. Maria Cavasin and Elizabeth Hardy. I could go on and on. There are a lot of authors and if I didn't mention one of them, it doesn't mean that they weren't key contributors. I just wanted to throw that out there. We also had great collaborators. I think another component of this paper that is of great interest to us, and initially I was against doing any of this, is that Marcello and Josh created this biobank of human cardiac fibroblasts that we obtained from explanted hearts from individuals undergoing heart transplantation.   And initially I thought it was going to be a waste of time and money for Marcello and Josh to do that, but they were persistent and they started isolating these cells. And the cells are really fascinating because even after you take them out of that failed human heart and culture them, they maintain this constituently active state, which is different than the cells we were using for screening where we kept them quiescent and then we stimulated them with TGF-β to activate them. These human cardiac fibroblasts from the failed human hearts are just on all the time.   Cindy St. Hilaire:        Wow.   Timothy McKinsey:    And SW does a really amazing job of reversing that activated state.   Cindy St. Hilaire:        Very cool and excellent resource I'm sure for future studies. So my last question is what's next? You know, you discovered a lot in this paper. What's the next thing you want to tackle?   Timothy McKinsey:    Cell type specific roles for 15-PGDH in the heart, in the control of cardiac homeostasis and disease. Basically we want to knock it out in fibroblasts. We want to knock it out in our macrophages and see what the consequences are. That's one thing. We want to really pursue the whole GPR31 12(S)-HETE pathway in the heart. That's something that has never been studied. And so GPR31 is a G protein coupled receptor that is bound by this eicosanoid called 12(S)-HETE. And that seems to be blocking fibroblast activation, so we're going to further pursue that pathway. And then we think that this paradoxical finding related to ERK signaling in the heart is also worthy of pursuit. Why is it that stimulating ERK in a cardiac fibroblast is actually blocking the activation state of that cell?   Marcello Rubino:        I'm interested in this like Tim says, but also interested in the role of the interaction of the cell because it's important to study like a specific gene inhibitor, whatever role in a specific cell, but what happened to the other cell, the interaction the other cell when you do knocking in some specific cell, so that's what I'm trying to do in general. Now I move back in Italy, like I told you, I'm like a kind of independent research and I'm studying a lot single cell sequencing right now. Try to do also try to see what happened to interaction, understand during pathology.   The idea is to study like inhibitor treatment and to see what really happened because gene expression is important, but we need to consider also of course the protein shape, the protein interaction, the cell interaction, so I try to grow in this field and see what really happened because the problem of the cell, they're just cell in vitro. They can mimic what happened, but it's not what really happened in vivo, so can we use this novel technology to improve our knowledge, that's what I want to try to do.   Cindy St. Hilaire:        Well that's great. Dr McKinsey, Dr Rubino, thank you so much for taking the time to speak with me today. Title of their article was Inhibition of Eicosanoid Degradation Mitigates Fibrosis of the Heart. It's in our January 6th issue of Circ Res. And thank you both so much for joining me today and thank you to you and all of your colleagues who worked so hard on this for this amazing study.   Timothy McKinsey:    Thank you. We really enjoyed this visit and we're grateful to have our work published in Circulation Research.   Cindy St. Hilaire:        That's it for highlights from the January 6th and 20th issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes or #DiscoverCircRes. Thank you to our guests, Dr Tim McKinsey and Dr Marcello Rubino. This podcast is produced by Ishara Rantayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on-the-go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2022. The opinions expressed by the speakers of this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahajournals.org.  

This month on Episode 43 of Discover CircRes, guest host Nicole Purcell highlights two original research articles featured in the December 2 issue of Circulation Research. This episode also features an interview with Drs Aaron Phillips and Kevin O'Gallagher about their study, The Effect of a Neuronal Nitric Oxide Synthase Inhibitor on Neurovascular Regulation in Humans.   Article highlights:   Akerberg, et al. RBPMS2 Regulates RNA Splicing in Cardiomyocytes   Lv, et al. Cardiac Protection by MG53-S255A Mutant   Nicole Purcell:             Hi and welcome to Discover CircRes, the podcast of the American Heart Association's Journal, Circulation Research. I am your host, Dr Nicole Purcell, from the Huntington Medical Research Institutes in Pasadena, California, and today I will be highlighting two articles from our December 2 issue of Circulation Research. I'll also have a chat with Drs Aaron Phillips and Kevin O'Gallagher about their study, The Effect of a Neuronal Nitric Oxide Synthase Inhibitor on Neurovascular Regulation in Humans.   Nicole Purcell:             But before I get to the interview, here are a few article highlights. The first article we're going to highlight is RBPMS2 Is a Myocardial Enriched Splicing Regulator Required for Cardiac Function. This comes from Boston Children's Hospital with first author Dr Alexander Akerberg, and corresponding author Dr Jeffrey Burns. RNA splicing, along with transcription control and post-translational modifications, is a mechanism for fine tuning the expression of a gene for a particular purpose in a particular tissue. Factors that control splicing are thus often enriched in certain cell types. The factor, RBPMS2, for example, is enriched in the myocytes of amphibians, fish, birds and mammals.  This conserve tissue specificity suggesting essential role of RBPMS2 in heart function.   Akerberg and colleagues now confirm this is indeed the case. They generated zebra fish embryos and human cardiomyocytes lacking RBPMS2, and found the fish suffered early cardiac dysfunction by 48 hours post fertilization. The animal's hearts had reduced ejection fractions, compared with the hearts of controlled fish. At the cellular level, the RBPMS2 lacking fish cardiomyocytes displayed malformed sarcomere fibers and disrupted calcium handling, both of which were also seen in the RBPMS2 deficient human cardiomyocytes. Furthermore, RNA sequencing experiments revealed a conserve set of 29 genes in the RBPMS2-lacking fish and human cells that were incorrectly spliced. In revealing the essential cardiac role of RBPMS2 and its RNA targets, the work provides new molecular details for understanding vertebrate heart function and disease, say the team.   Nicole Purcell:             Our second article being highlighted is Blocking MG53 Serine 255 Phosphorylation Protects Diabetic Heart from Ischemic Injury. This comes from Peking University with first authors, Fengxiang L, Yingfan Wang and Dan Shan, as well as corresponding author Dr Rui-Ping Xiao. Midsegment 53, or MG53, is a recently discovered muscle-specific protein that is an essential component of the cell membrane repair machinery with cardioprotective effects. MG53 thus has therapeutic potential, but for patients whose heart disease is linked to type 2 diabetes, there's a problem. MG53 also tags certain cellular proteins for destruction, including the insulin receptor and the insulin signaling factor, IRS1. Loss of these factors could worsen insulin resistance. lev and colleagues therefore investigate whether MG53 could be tweaked to provide protection without the diabetes downside.   Nicole Purcell:             They discovered the phosphorylation of MG53 at serine 255 is required for its role in protein destruction, and that a mutant version of MG53, incapable of this phosphorylation, MG53 serine to 255 alanine mutant, could still promote cardiomyocyte survival, and protect the cells from membrane damaging insults. Importantly, when a diabetic mouse model was injected with MG53 serine 255 to alanine mutant, the protein better protected the animals against myocardial infarction than injection with the wild type MG53, recipients of which had poor insulin sensitivity. Based on these findings, the authors suggest MG53 serine 255 alanine mutant could be developed into a heart protective drug, for use in diabetic and non-diabetic patients alike.   Nicole Purcell:             Today, Dr Aaron Phillips and Dr Kevin O'Gallagher from University of Calgary are with me to discuss their study, the Effect of a Neuronal Nitric Oxide Synthase Inhibitor on Neurovascular Regulation in Humans in our December 2 issue of Circulation Research. Thank you for joining me today.   Kevin O'Gallagher:    Hello, my name's Dr Kevin O'Gallagher. I'm a British Heart Foundation clinician scientist and interventional cardiologist at Kings College London and Kings College Hospital NHS Foundation Trust.   Aaron Phillips:            Hello, my name's Dr Aaron Phillips. I'm an associate professor in physiology, pharmacology, cardiac sciences, biomedical engineering and clinical neurosciences at the University of Calgary in the Hotchkiss Brain Institute and Libin Cardiovascular Institute. I am also the director of the Restore Network, which is a large platform at the University of Calgary spanning all these groups, developing new tools and techniques for translational research into neurological conditions.   Nicole Purcell:            There are a lot of authors involved in this study. While all could not join us, I appreciate you taking the time to discuss your findings today. Your paper deals with looking at neurovascular control in humans. Two primary regulatory pathways are neurovascular coupling, or NVC, and dynamic cerebral autoregulation. Dr Phillips, can you explain what NVC to our audience, and what does dysregulation lead to?   Aaron Phillips:            Yeah, thanks Nicole and I'm happy to be here. Thank you for the invitation. NVC, or neurovascular coupling, we've been studying it for about 15 years. At its fundamental level, it's kind of this elegant interplay between neurons, which unfortunately have very limited capacity for substrate storage. The brain has very limited substrate storage capacity, and so neurons need to very rapidly match their metabolic activity to the blood flow that's being delivered to them, and that needs to happen locally, for areas of the brain that have greater metabolic needs as opposed to other areas.   What happens, in terms of dysregulation or conditions that are associated with dysregulation, it's an interesting story because we still really need to understand the mechanisms fully, in order to suss out what clinical conditions should have dysfunction of this unit. We know that certain conditions, such as vascular cognitive impairment, even spinal cord injury, we've done some work in stroke patients, it seems to be dysfunctional in all of these conditions, but understanding exactly why it's dysfunctional, we're still establishing that.   Nicole Purcell:             Great. You were talking about how it's the connection or interplay between blood flow, so we're talking about altered blood pressure seems to play a key role in neurovascular coupling. So, for those listeners not familiar with this field, can you explain how nitric oxide synthase and its isoforms, how this relates to NVC?   Aaron Phillips:            Well, nitric oxide synthase is an enzyme that produces nitric oxide that's expressed primarily in neurons. Nitric oxide is a powerful vasodilator. It actually works on quite a rapid time course. So, we surmised, we suspected, and there were some preclinical work before our human study, that neuronal sources of nitric oxide, being that nitric oxide is a potent vasodilator, we thought that would be likely to be mediating a large part of the neurovascular coupling response.   Nicole Purcell:             Great. So, Dr O'Gallagher, based on that, what was your main objective or hypothesis of this study, and how is your study novel from those that have already just suggested, looked at NOS regulation for cerebral blood flow?   Kevin O'Gallagher:    Thanks very much for the invite to talk. I mean, we hypothesized that nNOS would have a role in regulating neurovascular coupling. I think the novelty of our study is that although people have been interested in NOS and its regulation of cerebral vascular and cardiovascular blood flow, it's only relatively recently that there has become an agent available that will specifically inhibit nNOS, and therefore give us an idea of what it is doing, rather than previous inhibitors which just inhibit all of the three NOS isoforms. It was really that the development of the agent was what allowed us to do this study. I think it was really through that, that makes this an interesting finding that nNOS does play a role in neurovascular coupling, and really pushes the field forward ever so slightly.   Nicole Purcell:             Great. So, as you pointed out, this is a specific nNOS inhibitor, which is known as SMTC. It's a synthetic L-Arginine analog, right? That's really what sets your study apart. Can you tell us a little bit the audience, whether that be you, Dr Phillips or Dr O'Gallagher, about what your study was and what did you find, and how did an ambition of using this SMTC to inhibit nNOS affect systemic hemodynamic changes and NVC?   Aaron Phillips:            Yeah, I think both of us can probably speak to this interchangeably and add in different elements of the experiment. This is kind of a summary of the study, I guess. In advance of this, adding on what Kevin had just said in terms of the novelty of the study and the importance, we had done a lot of work previous to this paper where we were one of the groups that helped establish neurovascular coupling as a measure that could be tested in humans. This involved kind of understanding metabolism of the eye, how that's coupled to the visual cortex, and how to measure blood flow on a high temporal resolution in the visual cortex in response to visual input. That's why we used very well standardized perturbations involving tracking an eye, tracking a dot on a screen at a known one rate and a known one amplitude of movement, while also measuring the hyperemic response in the posterior brain.   Then we kind of went on and developed some new measures, developed some software that we're now proud is used in a few different labs around the world, that kind of automatically takes that input of repetitive eyes opening and closing and that hyperemic response, and it breaks it down into a single wave form. A single hyperemic response is superimposed of 10, 15, 20 cycles of those eyes open and eyes closed, and then when we superimpose all the wave forms together, we can generate different metrics from that hyperemic response that correspond to different elements.   One of the ways where software can, I guess dice out the hyperemic response, is by timing. We can look at very specific unique time windows over that 30 seconds of eyes open, and we can also look at the slope of the response, as well as we recently did some dimensionality reduction techniques and looked at specific computed measures of that hyperemic response. We published that a few years ago. Those were some of the tools that enabled this study, along with a fantastically unique drug that really could isolate that neuron expression of NOS and the capacity of nNOS to mediate neurovascular coupling.   Kevin O'Gallagher:    Obviously, we're going to use a systemic infusion of SMTC, the study drug, and we've used that before and shown it to be safe. But because a systemic infusion of SMTC through peripheral and systemic nNOS inhibition does cause an increase in systemic vascular resistance, and therefore an increase in mean arterial pressure of around about 7 mm of mercury, in addition to a cline placebo control condition, we also felt the need to have a pressure control condition. For that, we used phenylephrine to match the rise in mean arterial pressure that we anticipated we'd see with SMTC. We ended up with 12 healthy volunteers who attended on three separate visits, and so we had a party randomized double blinded intervention study where we measured the neurovascular coupling metrics, both before and after an infusion of one of the three conditions on each particular visit.   Aaron Phillips:            I just wanted to add into that, we had found previously that mean arterial pressure does have an effect on the hyperemic response. This was actually classically found by 1960s by Harper and Glass in a dog study, but we've repeated that in humans and kind of found that the ability of the brain to kind of... It's reserve for further vasodilation is dependent on pressure. As you drop it, neurovascular coupling will go away, and as you increase it, neurovascular coupling will increase partially, so it's important to standardize the mean arterial pressure levels. I always liken it to your water pressure in your house. You can't turn on a faucet with a given pressure unless you have that in the system upstream. That was a really important aspect of the study.   Nicole Purcell:             That was quite unique for your study, too. Not a lot of people have control for pressure.   Aaron Phillips:            Correct.     Kevin O'Gallagher:    I think it reflects the challenges of these healthy volunteer studies where you're trying to look at one particular part of the cardiovascular system, because as a cardiologist, if we were doing a study like this, looking at cardiovascular regulation, we would put a catheter into the coronary arteries in patients who had come for angiograms, and we'd give a local infusion of SMTC, as we've done in studies before. But with healthy volunteers, and ethically it really demanded a systemic infusion, so it was a really nice workaround to have that pressure control condition.   Nicole Purcell:             So, can you tell us a little bit about what your findings were?   Kevin O'Gallagher:    I think testament to the study design and the rigorous methodology that we employed, we did find with the resting steady state hemodynamics that SMTC condition performed as we would expect, and as we've seen in prior studies where we've given a systemic dose in that compared to both placebo and pressure control conditions, SMTC decreased cardiac output, and it decreased stroke volume, and also increased systemic vascular resistance, so very much as expected the resting hemodynamic conditions.   Aaron Phillips:            Yeah, thanks. Just adding onto that, moving on into some of the cerebral vascular measures. So again, we were measuring posterior cerebral artery velocity, blood velocity and specific responsiveness that it has to a visual stimuli. Between conditions, we didn't see a change in resting posterior cerebral artery velocity, so that was consistent between the conditions. Where we saw most of our change actually was in this very early period, the first five seconds of what we're going to call the hyperemic response, or the first five seconds of the neurovascular coupling response. That's where we saw our primary effect. We didn't see an effect in almost any of the neurovascular coupling measures that we generated in the actual sustained period after that initial rise, so that's where we saw our key inhibition with nNOS inhibition. What permitted that was the phenylephrine control group, again, allowing us to really look at apples and apples, not apples and oranges.   Nicole Purcell:             Great. So that early transient change that you saw, that as you said, hyperemic response, what therapeutic implications does this have for the field?   Kevin O'Gallagher:    Well, certainly there are conditions in which nNOS dysfunction, nNOS may be implicated, we mentioned a couple in the paper, some neurodegenerative diseases. But also, I think the field is now open for any vascular mediated headache syndrome, such as migraine, to investigate the potential role of nNOS from that angle. Then we haven't touched on already, but as well as dysfunctional, so decreased nNOS activity, there's also some conditions in which there's dysregulation or abnormally increased nNOS function. Again, we've highlighted this kind of study methodology is a tool that could be used to investigate those types of conditions.   Aaron Phillips:            These are all terrific points, and I think there's a lot of conditions where neurovascular coupling is impaired, and it's worth exploring them and understanding the specific role where nNOS might be a part of it. I also think there's a lot of interesting basic science surrounding this, in terms of the mechanisms. What was really interesting in this study, which is still kind of wracking my brain, is why didn't more of the neurovascular coupling response go away? This is a highly selective inhibitor for what was potentially thought by some groups to be a large mediator, this response. It was a relatively small inhibitory effect, and isolated to a small part of the neurovascular coupling response, just that early phase. So, still lots of work to do to kind of dice out the other pathways. They're probably highly redundant. This is such a critical mechanism in the central nervous system. Getting at it and humans is going to be tricky, but we're excited about the future and exploring some of those other avenues on the mechanistic cascade.   Nicole Purcell:             Based on the fact that you just had 12 healthy individuals, what do you see as some of the limitations of your study going forward, thinking about what you did?   Kevin O'Gallagher:    I think you've just hit on a key limitation. It was a small number of volunteers. They were all healthy, so we can't extrapolate these findings to conditions such as hypertension, where we know from other studies that cardiovascular responses, nNOS responses are impaired Also, this was a noninvasive study. We looked at the blood flow through Doppler, but we don't really know the effect of SMTC on cerebral artery diameter or other markers like that, so I think those are important limitations to mention.   Nicole Purcell:             I know I didn't ask this, and I know it was mentioned in the paper, but for our audience, and it was a small sample size, but did you see any sex differences between your male and female cohort?   Kevin O'Gallagher:    No. We did analyze for that and there were no sex differences. But again, it's an important limitation in that we didn't control for things like phase of the menstrual cycle. And again, with those limitations, all the results should be interpreted with those in mind.   Nicole Purcell:             Were there any challenges to the study that you found?   Kevin O'Gallagher:    I work in London in the UK, where we performed this study related protocols, and Professor Phillips from University of Calgary, his team flew over to perform the studies. I think there was a real organizational challenge because we had a relatively small time window in which to get all of the volunteers and their three study visits done. But I think it's testament to just how well Professor Phillips runs his team, and how fantastic a team they are in working together that all of those challenges were minimized and everything. It ran fairly smoothly, and certainly, the data was connected back in early 2020. I think we all retrospectively breathed a sigh of relief when the Covid pandemic started and we realized that had we had to reschedule another set of visits, we would've then knocked the study back a couple of years. So yeah, there were organizational challenges, but it was an absolute pleasure to work with Professor Phillips's and his team in this.   Aaron Phillips:            To add to that, I mean, it's not really related to necessarily the challenges, but I was going to list kind of the exact same thing. In the background. Kevin, and Professor Shaw, and Dr Gallagher were a tour de force on organizing quite a complicated study that involves some invasive protocols and unique experimental drug infusion. Getting all of that ethically approved, and organized, and structured, that was probably one of the biggest challenges of pulling this study off. Nicole Purcell:            Great. It was a very nice study. So lastly, what future studies are needed or have come out of this work that you'd like to tell us about?   Aaron Phillips:            Mechanistically, I would still like to explore why nNOS inhibition doesn't seem to affect the sustained elevation in blood flow. This maybe means going back to some of the astrocyte mediated mechanisms, and understanding knocking out, knocking in, exploring some of those. I'd also like to continue to study the neurovascular cupping response itself in clinical conditions. This may be a tool for helping to characterize the severity of a given neurovascular condition over time, and kind of validating this outcome measure as potentially a clinical tool and further expanding its research application.   Kevin O'Gallagher:    I would just add to that, that I tend to come to all of these things from a cardiologist light, and there are some conditions in cardiology where the microvascular is involved, and so the interest is then to see whether there's a linkage between the dysfunctional coronary microvascular responses with then cerebral microvascular responses. So again, I think there's plenty of future work to be done in that sphere.   Nicole Purcell:             Well, I want to thank you so much for joining me today, Dr Kevin O'Gallagher and Dr Aaron Phillips, for discussing your exciting findings with me today, and I look forward to seeing your future work. Thank you.   Aaron Phillips:            Thank you so much.   Kevin O'Gallagher:    Thank you so much.   Nicole Purcell:            That's it for highlights from the December 2 issue of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Drs Aaron Phillips and Kevin O'Gallagher. This podcast is produced by Ishara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy texts for highlighted articles provided by Ruth Williams.   I am your host, Dr Nicole Purcell, filling in for Dr Cindy St. Hilaire, and this is Discover CircRes, your on-the-go source for the most up-to-date and exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2022. The opinions expressed by speakers in this podcast are their own, and not necessarily those of the editors or of the American Heart Association. For more, visit ahajournals.org.  

Dr. Ruth Williams, renowned leader within ophthalmology, sits down with us to talk about leadership and how the roles of leaders have changed over the years. We discuss her vision for leadership within her practice and within ophthalmology at large. We also touch on how the pandemic has shaped goals and priorities for ophthalmologists, work-life balance, and the concept of burnout. This is a can't miss discussion with an incredible thought leader in our field.  Subscribe to the podcast:  https://MayoClinicOphthalmology.podbean.com  Follow and reach out to us on Twitter: @mayocliniceye 

This month on Episode 42 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the October 28 and November 11th  issues of Circulation Research. This episode also features an interview with Dr Miguel Lopez-Ramirez and undergraduate student Bliss Nelson from University of California San Diego about their study, Neuroinflammation Plays a Critical Role in Cerebral Cavernous Malformations.   Article highlights:   Jia, et al. Prohibitin2 Maintains VSMC Contractile Phenotype   Rammah, et al. PPARg and Non-Canonical NOTCH Signaling in the OFT   Wang, et al. Histone Lactylation in Myocardial Infarction   Katsuki, et al. PCSK9 Promotes Vein Graft Lesion Development   Cindy St. Hilaire:        Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's Journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today, I'm going to be highlighting articles from our October 28th and our November 11th issues of Circ Res. I'm also going to have a chat with Dr Miguel Lopez-Ramirez and undergraduate student Bliss Nelson, about their study, Neuroinflammation Plays a Critical Role in Cerebral Cavernous Malformations. But, before I get into the interviews, here are a few article highlights.   Cindy St. Hilaire:        The first article is from our October 28th issue, and the title is, PHB2 Maintains the Contractile Phenotype of Smooth Muscle Cells by Counteracting PKM Splicing. The corresponding author is Wei Kong, and the first authors are Yiting Jia and Chengfeng Mao, and they are all from Peking University. Insults to blood vessels, whether in the form of atherosclerosis, physical injury, or inflammation, can trigger vascular smooth muscle cells to transition from a contractile state to a proliferative and migratory one. Accompanying this conversion is a switch in the cells' metabolism from the mitochondria to glycolysis. But what controls this switch? To investigate, this group compared the transcriptomes of contractile and proliferative smooth muscle cells.   Among the differentially expressed genes, more than 1800 were reciprocally up and down regulated. Of those, six were associated with glucose metabolism, including one called Prohibitin-2, or PHB2, which the team showed localized to the artery wall. In cultured smooth muscle cells, suppression of PHB2 reduced expression of several contractile genes. While in rat arteries, injury caused a decrease in production of PHB2 itself, and of contractile markers.   Furthermore, expression of PHB2 in proliferative smooth muscle cells could revert these cells to a contractile phenotype. Further experiments revealed PHB2 controlled the splicing of the metabolic enzyme to up-regulate the phenotypic switch. Regardless of mechanism, the results suggest that boosting PHB2 might be a way to reduce adverse smooth muscle cell overgrowth and conditions such as atherosclerosis and restenosis.   Cindy St. Hilaire:        The second article I'm going to highlight is also from our October 28th issue, and the first authors are Mayassa Rammah and Magali Theveniau-Ruissy. And the corresponding authors are Francesca Rochais and Robert Kelly. And they are all from Marseille University. Abnormal development of the heart's outflow track, which ultimately forms the bases of the aorta and the pulmonary artery, accounts for more than 30% of all human congenital heart defects. To gain a better understanding of outflow tract development, and thus the origins of such defects, this group investigated the role of transcription factors thought to be involved in specifying the superior outflow tract, or SOFT, which gives rise to the subaortic myocardium, and the inferior outflow tract, which gives rise to the subpulmonary myocardium. Transcription factor S1 is over-expressed in superior outflow tract cells and the transcription factors, TBX1 and PPAR gamma, are expressed in inferior outflow tract cells.   And now this group has shown that TBX1 drives PPAR gamma expression in the inferior outflow tract, while Hess-1 surpasses PPAR gamma expression in the superior outflow tract. Indeed, in mouse embryos lacking TBX1, PPAR gamma expression was absent in the outflow tract. While in mouse embryos lacking Hess-1, PPAR gamma expression was increased and PPAR gamma positive cells were more widespread in the outflow tract.   The team also identified that signaling kinase DLK is an upstream activator of Hess-1 and a suppressor of PPAR gamma. In further detailing the molecular interplay regulating outflow tract patterning, the work will shed light on congenital heart disease etiologies, and inform potential interventions for future therapies.   Cindy St. Hilaire:        The third article I want to highlight is from our November 11th issue of Circulation Research, and the title is Histone Lactylation Boosts Reparative Gene Activation Post Myocardial Infarction. The first author is Jinjin Wang and the corresponding author is Maomao Zhang, and they're from Harbin Medical University. Lactylation of histones is a recently discovered epigenetic modification that regulates gene expression in a variety of biological processes. In inflammation, for example, a significant increase in histone lactylation is responsible for switching on reparative genes and macrophages when pro-inflammatory processes give way to pro-resolvin ones.   The role of histone lactylation in inflammation resolution has been shown in a variety of pathologies, but has not been examined in myocardial infarction. Wang and colleagues have now done just that. They isolated monocytes from the bone marrow and the circulation of mice at various time points after induced myocardial infarctions, and examined the cells' gene expression patterns. Within a day of myocardial infarction, monocytes from both bone marrow and the blood had begun upregulating genes involved in inflammation resolution. And, concordant with this, histone lactylation was dramatically increased in the cells, specifically at genes involved in repair processes.   The team went on to show that injection of sodium lactate into mice boosted monocyte histone lactylation and improved heart function after myocardial infarction, findings that suggest further studies of lactylation's pro-resolving benefits are warranted. Cindy St. Hilaire:        The last article I want to highlight is titled, PCSK9 Promotes Macrophage Activation via LDL Receptor Independent Mechanisms. The first authors are Shunsuke Katsuki and Prabhash Kumar Jha, and the corresponding author is Masanori Aikawa, and they are from Brigham and Women's Hospital in Harvard. Statins are the go-to drug for lowering cholesterol in atherosclerosis patients. But the more recently approved PCSK9 inhibitors also lower cholesterol and can be used to augment or replace statins in patients where these drugs are insufficient.   PCSK9 is an enzyme that circulates in the blood and destroys the LDL receptor, thereby impeding the removal of bad cholesterol. The enzyme also appears to promote inflammation, thus potentially contributing to atherosclerosis in two ways. This group now confirms that PCSK9 does indeed promote pro-inflammatory macrophage activation and lesion development, and does so independent of its actions on the LDL receptor.   The team assessed PCSK9-induced lesions in animals with saphenous vein grafts, which are commonly used in bypass surgery but are prone to lesion regrowth. They found that LDL receptor lacking graft containing mice had greater graft macrophage accumulation and lesion development when PCSK9 activity was boosted than when it was not. The animal's macrophages also had higher levels of the pro-inflammatory factor expression. Together, this work shows that PCSK9 inhibitors provide a double punch against atherosclerosis and might be effective drugs for preventing the all too common failure of saphenous vein grafts.   Cindy St. Hilaire:        So, today with me I have Dr Miguel Lopez-Ramirez and undergraduate student Bliss Nelson from the University of California in San Diego, and we're going to talk about their study, Neuroinflammation Plays a Critical Role in Cerebral Cavernous Malformation Disease, and this article is in our November 11th  issue of Circulation Research. Thank you both so much for joining me today. Before we talk about the science, want to just maybe tell me a little bit about yourselves?   Bliss Nelson:                My name is Bliss Nelson. I'm a member of Miguel Lopez-Ramirez's lab here at UC San Diego at the School of Medicine. I'm an undergraduate student here at UC San Diego. I'm actually a transfer student. I went to a community college here in California and I got involved in research after I transferred.   Cindy St. Hilaire:        What's your major?   Bliss Nelson:                I'm a cognitive science major.   Cindy St. Hilaire:        Excellent. You might be the first undergrad on the podcast, which is exciting.   Bliss Nelson:                Wow. What an honor. Thank so much.   Cindy St. Hilaire:        And Miguel, how about you?   Miguel Lopez-Ramirez: Yes, thank you. Well, first thank you very much for the opportunity to present our work through this media. It's very exciting for us. My name is Miguel Alejandro Lopez-Ramirez, and I'm an assistant professor in the Department of Medicine and Pharmacology here at UCSD. Cindy St. Hilaire:        Wonderful. I loved your paper, because, well, first, I don't think I've talked about cerebral cavernous malformations. So what are CCMs, and why are they so bad?   Bliss Nelson:                Cerebral cavernous malformations, or CCMs for short, are common neurovascular lesions caused by a loss of function mutation in one of three genes. These genes are KRIT1, or CCM1, CCM2 and PDCD10, or CCM3, and generally regarded as an endothelial cell autonomous disease found in the central nervous system, so the brain and the spinal cord.   The relevance of CCMs is that it affects about one in every 200 children and adults, and this causes a lifelong risk of chronic and acute hemorrhaging. CCMs can be quiescent or dynamic lesions. If they are dynamic, they can enlarge, regress, or behave progressively, producing repetitive hemorrhaging and exacerbations of the disease.   Other side effects of the disease could be chronic bleedings, focal neurological deficits, headaches, epileptic seizures and, in some cases, death. There's no pharmacological treatment for CCMs. There's only one type of option some patients may have, which would be to have surgery to cut out the lesions. But of course this depends on where the lesion or lesions are in the central nervous system, if that's even an option. So sometimes there's no option these patients have, there's no treatment, which is what propels our lab to towards finding a pharmacological treatment or uncovering some of the mechanisms behind that.   Cindy St. Hilaire:        Do people who have CCM know that they have them or sometimes it not detected? And when it is detected, what are the symptoms?   Bliss Nelson:                Sometimes patients who have them may not show any symptoms either ever in their lifetime or until a certain point, so really the only way to find out if you were to have them is if you went to go get a brain scan, if you went to go see a doctor, or if you started having symptoms. But also, one of the issues with CCMs is that they're very hard to diagnose, and in the medical community there's a lack of knowledge for CCMs, so sometimes you may not get directed to the right specialist in time, or even ever, and be diagnosed.   Miguel Lopez-Ramirez: I will just add a little bit. It is fabulous, what you're doing. I think this is very, very good. But yes, that's why they're considered rare disease, because it's not obvious disease, so sometimes most of the patient, they go asymptomatic even when they have one lesions, but there's still no answers of why patients that are asymptomatics can become symptomatics. And there is a lot in neuro study, this study that we will start mentioning a little bit more in detail. We try to explain these transitions from silent or, quiescent, lesion, into a more active lesion that gives the disability to the patient.   Some of the symptoms, it can start even with headaches, or, in some cases, they have more neurological deficits that could be like weakness in the arms or loss of vision. In many cases also problems with the speech or balance. So it depends where the lesion is present, in the brain or in the spinal cord, the symptoms that the patient will experience. And some of the most, I will say, severe symptoms is the hemorrhagic stroke and the vascular thrombosis and seizure that the patients can present. Those would be the most significant symptoms that the patient will experience.   Cindy St. Hilaire:        What have been some limitations in the study of CCMs? What have been limitations in trying to figure out what's going on here?   Bliss Nelson:                The limitations to the disease is that, well, one, the propensity for lesions, or the disease, to come about, isn't known, so a lot of the labs that work on it, just going down to the basic building blocks of what's even happening in the disease is a major problem, because until that's well established, it's really hard to go over to the pharmacological side of treating the disease or helping patients with the disease, without knowing what's going on at the molecular level.   Cindy St. Hilaire:        You just mentioned molecular level. Maybe let's take a step back. What's actually going on at the cellular level in CCMs? What are the major cell types that are not happy, that shift and become unhappy cells? Which are the key players?   Bliss Nelson:                That's a great question and a great part of this paper. So when we're talking about the neuroinflammation in the disease, our paper, we're reporting the interactions between the endothelium, the astrocytes, leukocytes, microglia and neutrophils, and we've actually coined this term as the CaLM interaction.   Cindy St. Hilaire:        Great name, by the way.   Bliss Nelson:                Thank you. All props to Miguel. And if you look at our paper, in figure seven we actually have a great graphic that's showing this interaction in play, showing the different components happening and the different cell types involved in the CaLM interaction that's happening within or around the CCM lesions.   Cindy St. Hilaire:        What does a astrocyte normally do? I think our podcast listening base is definitely well versed in probably endothelial and smooth muscle cell and pericyte, but not many of us, not going to lie, including me, really know what a astrocyte does. So what does that cell do and why do we care about its interaction with the endothelium?   Miguel Lopez-Ramirez: Well, the astrocytes play a very important role. Actually, there are more astrocytes than any other cells in the central nervous system, so that can tell you how important they are. Obviously play a very important role maintaining the neurological synapses, maintaining also the hemostasis of the central nervous system by supporting not only the neurons during the neural communication, but also by supporting the blood vessels of the brain.   All this is telling us that also another important role is the inflammation, or the response to damage. So in this case, what also this study proposed, is that new signature for these reactive astrocytes during cerebral malformation disease. So understanding better how the vasculature with malformations can activate the astrocytes, and how the astrocytes can contribute back to these developing of malformations. It will teach us a lot of how new therapeutic targets can be implemented for the disease.   This is part of this work, and now we extend it to see how it can also contribute to the communication with immune cells as Bliss already mentioned.   Cindy St. Hilaire:        Is it a fair analogy to say that a astrocyte is more similar to a pericyte in the periphery? Is that accurate?   Miguel Lopez-Ramirez: No, actually there are pericytes in the central nervous system as well. They have different roles. The pericyte is still a neuron cell that give the shape, plays a role in the contractility and maintains the integrity of the vessels, while the astrocyte is more like part of the immune system, but also part of the supporting of growth factors or maintaining if something leaks out of the vasculature to be able to capture that.   Cindy St. Hilaire:        You used a handful of really interesting mouse models to conduct this study. Can you tell us a little bit about, I guess, the base model for CCM and then some of the unique tools that you used to study the cells specifically?   Bliss Nelson:                Yeah, of course. I do a lot of the animal work in the lab. I'd love to tell you about the mouse model. So to this study we use the animal model with CCM3 mutation. We use this one because it is the most aggressive form of CCM and it really gives us a wide range of options to study the disease super intricately. We use tamoxifen-regulated Cre recombinase under the control of brain endothelial specific promoter, driving the silencing of the gene CCM3, which we call the PDCD10 betco animal, as you can see in our manuscript. To this, the animal without the Cre system, that does not develop any lesions, that we use as a control, we call the PDCD10 plox. And these animals are injected with the tamoxifen postnatally day one, and then for brain collection to investigate, wcollected at different stages. So we do P15, which we call the acute stage, P50, which we term the progressive stage, and then P80, which is the chronocytes stage. And after enough brain collections, we use them for histology, gene expression, RNA analysis, flow cytometry, and different imaging to help us further look into CCMs.   Cindy St. Hilaire:        How similar is a murine CCM to a human CCM? Is there really good overlap or are there some differences?   Miguel Lopez-Ramirez: Yes. So, actually, that's a very good question, and that's part of the work that we are doing. This model definitely has advantages in which the lesions of the vascular formations are in an adult and juvenile animals, which represent an advantage for the field in which now we will be able to test pharmacological therapies in a more meaningful, way where we can test different doses, different, again, approaches. But definitely, I mean, I think I cannot say that it's only one perfect model for to mimic the human disease. It's the complementary of multiple models that give us certain advantages in another, so the integration of this knowledge is what will help us to understand better the disease.   Cindy St. Hilaire:        That's great. I now want to hear a little bit about your findings, because they're really cool. So you took two approaches to study this, and the first was looking at the astrocytes and how they become these, what you're calling reactive astrocytes, and then you look specifically at the brain endothelium. So could you maybe just summarize those two big findings for us?   Miguel Lopez-Ramirez: Yeah, so, basically by doing these studies we use trangenic animal in this case that they give us the visibility to obtain the transcripts in the astrocytes. And basically this is very important because we don't need to isolate the cells, we don't need to manipulate anything, we just took all the ribosomes that were basically capturing the mRNAs and we profile those RNAs that are specifically expressed in the astrocytes.   By doing this, we actually went into looking at in depth the transcripts that were altered in the animals that developed the disease, in this case the cerebral cavernous malformation disease, and what we look at is multiple genes that were changing. Many of them were already described in our previous work, which were associated with hypoxia and angiogenesis. But what we found in this work is that now there were a lot of genes associated with inflammation and coagulation actually, which were not identified before.   What we notice is that now these astrocytes, during the initial phase of the vascular malformation, may play a more important role in angiogenesis or the degradation of the vessels. Later during the stage of the malformation, they play a more important role in the thrombosis, in the inflammation, and recruitment of leukocyte   That was a great advantage in this work by using this approach and looking in detail, these astrocytes. Also, we identified there were very important signature in these astrocytes that we refer as a reactive astrocytes with neuroinflammatory properties. In the same animals, basically, not in the same animal, but in the same basically the experimental approach, we isolated brain vasculature. And by doing the same, we actually identified not only the astrocyte but also the endothelium was quite a different pattern that we were not seeing before. And this pattern was also associated with inflammation, hypoxia and coagulation pathways.   That lead us to go into more detail of what was relevant in this vascular malformations. And one additional part that in the paper this is novel and very impactful, is that we identify inflammasome as a one important component, and particularly in those lesions that are multi-cavernous.   Now we have two different approaches. One, we see this temporality in which the lesions forms different patterns in which the initial phase maybe is more aneugenic, but as they become more progressive in chronocytes, inflammation and hypoxy pathways are more relevant for the recruitment of the inflammatory cells and also the precipitation of immunothrombosis.   But also what we notice is that inflammasome in endothelial and in the leukocytes may play an important role in the multi-cavernous formation, and that's something that we are looking in more detail, if therapeutics or also interventions in these pathways could ameliorate the transition of phases between single lesions into a more aggressive lesions.   Cindy St. Hilaire:        That's kind of one of the follow up questions I was thinking about too is, from looking at the data that you have, obviously to get a CCM, there's a physical issue in the vessel, right? It's not formed properly. Does that form influence the activation of the astrocyte, and then the astrocytes, I guess, secrete inflammatory factors, target more inflammation in the vessel? Or is there something coming from the CCM initially that's then activating the astrocyte? It's kind of a chicken and the egg question, but do you have a sense of secondary to the malformation, what is the initial trigger?   Miguel Lopez-Ramirez: The malformations in our model, and this is important in our model, definitely start by producing changes in the brain endothelial. And as you mention it, these endothelium start secreting molecules that actually directly affect the neighboring cells.   One of the first neighboring cells that at least we have identified to be affected is the astrocytes, but clearly could be also pericytes or other cells that are in the neurovascular unit or form part of the neurovascular unit. But what we have seen now is that this interaction gets extended into more robust interactions that what you were referring as the CaLM interactions.   Definitely I think during the vascular malformations maybe is the discommunication that we identify already few of those very strong iteration that is part of the follow up manuscript that we have. But also it could be the blood brain barrier breakdown and other changes in the endothelium could also trigger the activation of the astrocytes and brain cells.   Cindy St. Hilaire:        What does your data suggest about potential future therapies of CCM? I know you have a really intriguing statement or data that showed targeting NF-kappa B isn't likely going to be a good therapeutic strategy. So maybe tell us just a little bit about that, but also, what does that imply, perhaps, of what a therapeutic strategy could be?   Bliss Nelson:                Originally we did think that the inhibition of NF-kappa B would cause an improvement potentially downstream of the CCMs. And unexpectedly, to our surprise, the partial or total loss of the brain endothelial NF-kappa B activity in the chronic model of the mice, it didn't prevent or cause any improvement in the lesion genesis or neuroinflammation, but instead it resulted in a trend to increase the number of lesions and immunothrombosis, suggesting that the inhibition of it is actually worsening the disease and shouldn't be used as a target for therapeutical approaches.   Miguel Lopez-Ramirez: Yes, particularly that's also part of the work that we have ongoing in which NF-kappa B may also play a role in preventing the further increase of inflammation. So that is something that it can also be very important. And this is very particular for certain cell types. It's very little known what the NF-kappa B actually is doing in the brain endothelial during malformations or inflammation per se. So now it's telling us that this is something that we have to consider for the future.   Also, our future therapeutics of what we propose are two main therapeutic targets. One is the harmful hypoxia pathway, which involves activation, again, of the population pathway inflammation, but also the inflammasomes. So these two venues are part of our ongoing work in trying to see if we have a way to target with a more safe and basically efficient way this inflammation.   However, knowing the mechanisms of how these neuroinflammation take place is what is the key for understanding the disease. And maybe even that inflammatory and inflammatory compounds may not be the direct therapeutic approach, but by understanding these mechanisms, we may come with  new approaches that will help for safe and effective therapies.   Cindy St. Hilaire:        What was the most challenging part of this study? I'm going to guess it has something to do with the mice, but in terms of collecting the data or figure out what's going on, what was the most challenging?   Bliss Nelson:                To this, I'd like to say that I think our team is very strong. We work very well together, so I think even the most challenging part of completing this paper wasn't so challenging because we have a really strong support system among ourselves, with Miguel as a great mentor. And then there's also two postdocs in the lab who are also first authors that contributed a lot to it.   Cindy St. Hilaire:        Great. Well, I just want to commend both of you on an amazing, beautiful story. I loved a lot of the imaging in it, really well done, very technically challenging, I think, pulling out these specific sets of cells and investigating what's happening in them. Really well done study. And Bliss, as an undergraduate student, quite an impressive amount of work. And I congratulate both you and your team on such a wonderful story.   Bliss Nelson:                Thank you very much.   Miguel Lopez-Ramirez: Thank you for Bliss and also Elios and Edo and Katrine, who all contributed      enormously to the completion of this project.   Cindy St. Hilaire:        It always takes a team.   Miguel Lopez-Ramirez: Yes.   Cindy St. Hilaire:        Great. Well, thank you so much, and I can't wait to see what's next for this story.   Cindy St. Hilaire:        That's it for the highlights from October 28th and November 11th issues of Circulation Research. Thank you so much for listening. Please check out the Circ Res Facebook page and follow us on Twitter and Instagram with the handle @circres and #discovercircres. Thank you to our guests, Dr Miguel Lopez-Ramirez and Bliss Nelson. This podcast is produced by Ashara Retniyaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for our highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, you're on the go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2022. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahagenerals.org.

This month on Episode 41 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the September 30 and October 14 issues of Circulation Research. This episode also features an interview with Dr Kory Lavine and Dr Chieh-Yu Lin from Washington University St. Louis, to discuss their study, Transcriptional and Immune Landscape of Cardiac Sarcoidosis.   Article highlights:   Tian, et al. EV-Mediated Heart Brain Communication in CHF   Wleklinski, et al.  Impaired Dynamic SR Ca Buffering Causes AD-CPVT2   Masson, et al. Orai1 Inhibition as a Treatment for PAH   Li, et al. F. Prausnitzii Ameliorates Chronic Kidney Disease   Cindy St. Hilaire:        Hi, and welcome to Discover Circ Res, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cynthia St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to highlight articles from our September 30th and October 14th issues of Circulation Research.                                           I'm also going to have a chat with Dr Kory Lavine and Dr Chieh-Yu Lin from Washington University St. Louis, and we're going to discuss their study Transcriptional and Immune Landscape of Cardiac Sarcoidosis. But before I get to the interview, I'm going to highlight a few articles.   Cindy St. Hilaire: The first article I'm going to share is Extracellular Vesicles Regulate Sympathoexcitation by Nrf2 in Heart Failure. The first author of this study is Changhai Tian, and the corresponding author is Irving Zucker, and they are at University of Nebraska. After a myocardial infarction, increased oxidative stress in the heart can contribute to adverse cardiac remodeling, and ultimately, heart failure. Nrf2 is a master activator of antioxidant genes, suggesting a protective role, but studies in rats have shown its expression to be suppressed after MI, likely due to upregulation of Nrf2-targeting microRNAs. These microRNAs can also be packaged into vesicles and released from stressed heart cells.   Now, this group has shown that rats and humans with chronic heart failure have an abundance of these microRNA-containing EVs in their blood. In the rats with chronic heart failure, these extracellular vesicles were found to be taken up by neurons of the rostral ventrolateral medulla, RVLM, wherein the microRNA suppressed Nrf2 expression. The RVLM is a brain region that controls the sympathetic nervous system, and in the presence of EVs, it is ramped up by sympathetic excitation. Because such elevated sympathetic activity can induce the fight or flight response, including increased heart rate and blood pressure, this would likely worsen heart failure progression. The team, however, found that inhibiting microRNAs in the extracellular vesicles prevented Nrf2 suppression in the RVLM and sympathetic activation, suggesting the pathway could be targeted therapeutically.   Cindy St. Hilaire:        The next article I want to highlight is titled, Impaired Dynamic Sarcoplasmic Reticulum Calcium Buffering in Autosomal Dominant CPVT2. The first author of this study is Matthew Wleklinski, and the corresponding author is Bjӧrn Knollmann, and they are at Vanderbilt University.   Exercise or emotional stress can prompt the release of catecholamine hormones, which induce a fast heart rate, increased blood pressure, and other features of the fight or flight response. For people with catecholaminergic polymorphic ventricular tachycardia, or CPVT, physical activity or stress can cause potentially lethal arrhythmias. Mutations of calsequestrin-2, or CASQ2, which is a sarcoplasmic reticulum calcium-binding protein, is a major cause of CPVT, and can be recessive or dominant in nature.   For many recessive mutations, disease occurs due to loss of CASQ2 protein. This group investigated a dominant lysine to arginine mutation in this protein, and found by contrast, protein levels remain normal. In mice carrying the mutation, not only was the level of CASQ2 comparable to that in control animals, but so, too, was the protein's subcellular localization. The mutation instead interfered with CASQ2's calcium binding or buffering capability within the sarcoplasmic reticulum. The result was that upon catecholamine injection or exercise, the unbound calcium released prematurely from the sarcoplasmic reticulum, triggering spontaneous cell contractions. In uncovering this novel molecular etiology of CPVT, the work provides a basis for studying the consequences of other dominant CASQ2 mutations.   Cindy St. Hilaire:        The next article I want to highlight is from our October 14th issue of Circulation Research, and the title of the article is ORAI1 Inhibitors as Potential Treatments for Pulmonary Arterial Hypertension. The first author is Bastien Masson, and the corresponding author is Fabrice Antigny, and they're from Inserm in France. In pulmonary arterial hypertension, the arteries of the lungs become progressively obstructed, making it harder for the heart to pump blood through them, ultimately leading to right ventricular hypertrophy and heart failure. A contributing factor in the molecular pathology of pulmonary arterial hypertension is abnormal calcium handling within the pulmonary artery smooth muscle cells. Indeed, excess calcium signaling causes these cells to proliferate, migrate, and become resistant to apoptotic death, thus leading to narrowing of the vessel.   This group now identified the calcium channel ORAI1 as a major culprit behind this excess signaling. Samples of lung tissue from pulmonary arterial hypertension patients and a pulmonary arterial hypertension rat model had significantly upregulated expression of this channel compared with controls. And in patient pulmonary arterial smooth muscle cells, the high ORAI1 levels resulted in heightened calcium influx, heightened proliferation, heightened migration and reduced apoptosis. Inhibition of ORAI1 reversed these effects. Furthermore, in pulmonary hypertension model rats, ORAI1 inhibition reduced right ventricle systolic pressure and attenuated right ventricle hypertrophy when compared with untreated controls. This study indicates that ORAI1 inhibitors could be a new potential target for treating this incurable condition.   Cindy St. Hilaire:        The last article I want to share is titled Faecalibacterium Prausnitzii Attenuates CKD via Butyrate-Renal GPR43 Axis. The first author of this study is Hong-Bao Li, and the corresponding author is Tao Yang, and they are from the University of Toledo.   Progressive renal inflammation and fibrosis accompanied by hypertension are hallmarks of chronic kidney disease, which is an incurable condition affecting a significant chunk of the world's population. Studies indicate that chronic kidney disease is linked to gut dysbiosis. Specifically, depletion of lactobacillus bifidobacterium and faecalibacterium, prompting investigations into the use of probiotics. While supplements including lactobacillus and bifidobacterium have shown little effectiveness in chronic kidney disease, supplementations with F. prausnitzii have not been investigated.   Now, this group has shown in a mouse model of chronic kidney disease that oral administration of F. prausnitzii has beneficial effects on renal function, reducing renal fibrosis and inflammation. This bacterial supplementation also produced the short chain fatty acid butyrate, which was found to be at unusually low levels in the blood samples from the CKD model mice and from chronic kidney disease patients. Oral supplementation with this bacterium boosted butyrate levels in the mice, and in fact, oral administration of butyrate itself mimicked the effects of the bacteria. These findings suggest that supplementation with F. prausnitzii or, indeed, butyrate could be worth investigating as a treatment for chronic kidney disease.   Cindy St. Hilaire:        Today I have with me Dr Kory Lavine and Dr Chieh-Yu Lin from Washington University St. Louis, and we're going to talk about their paper, Transcriptional and Immune Landscape of Cardiac Sarcoidosis. This is in our September 30th issue of Circulation Research. Welcome, and thank you for taking the time to speak with me today.   Chieh-Yu Lin:             Thank you for inviting us. It's a great honor to be here today.   Kory Lavine:               Thank you.   Cindy St. Hilaire:        Really great paper, ton of data, and hopefully, we can pick some of it apart. But before we get into it, I actually want to just talk about sarcoidosis generally. I know it's a systemic inflammatory disease that has this kind of aggregation of immune cells as its culprit, and it can happen in a bunch of different organs. It's mostly in the lung, but it's also, like you're studying, in the heart. Can you just give us a little bit of background? What is sarcoidosis, and how common is cardiac sarcoidosis?   Chieh-Yu Lin:             Well, this is actually a great question, and I'll try to answer it. You actually capture one of the most important kind of features for sarcoidosis. It happens in all kind of organ system, mostly commonly in lung, in lymph nodes, but also in heart, spleen, even in brain, or even orbit, like eyes. It's really a truly multisystemic disease that has been characterized by this aggregate of macrophages, or myeloid cells, with scattered multinucleated giant cells, as the name implies, have multiple nuclear big, chunky, cells that form an aggregate. That's kind of like a pathognomonic feature for sarcoidosis, whether it's happening in lung, in the heart. When any organ system, a lot of studies has been done, but as of now, a very clear pathogenesis or mechanism has been, I would say, still pretty elusive, or still remain quite unclear, despite all the great effort has been made in this field. The other thing is that a lot of the studies actually focusing on pulmonary sarcoidosis for good reasons. Actually, that's one of the most common manifestations. For cardiac sarcoidosis, although it's only effect in probably, I would say depends on the data, 20% to 30% of the outpatient that with sarcoidosis, with or without lung involvement. It's actually carry a very significant clinical implications as of matter that the presentation of cardiac sarcoidosis can be devastating and sometimes actually fatal. Some of the study actually show that cardiac sarcoidosis actually higher, up to 80%, just because the first presentation's actually, unfortunately, sudden cardiac death. That's why Kory and I, we teamed up. I'm a cardiothoracic pathologist, so in my clinical practice I see specimens and samples from human body, from patient suffer from sarcoidosis, both in lung, lymph node, and heart. Kory is an outstanding heart failure, heart transplant cardiologist, see the other end, which is the patient care. This disease, specifically in heart, its presentation and its pathogens in heart, really attracts our attention.   Cindy St. Hilaire:        Do we know any or some of the potential causes? Why it would start, maybe in a different patient population, but also in the heart versus the lung? Do we know anything about that process?   Kory Lavine:              We know nothing about it. Sarcoid has no known etiology. There's been thoughts in the past that it may be driven by infection, the typical pathogens or autoimmune ideologies, but really, there's little data out there to support those possibilities. Right now, the field's wide open. The other challenge is we don't really have a good way to treat this disease, so a lot of the therapies available are things like steroids, which can have some effect on the disease but carry a lot of risk of complications. The other agents that we sometimes use to lower the doses of steroids, things like methotrexate and azathioprine, are only modestly effective.   These are really the motivation for Chieh-Yu and myself to pursue this. We don't really know what causes the disease, and we don't really have very good treatments. We really wanted to take the first step, that's to study the real disease, and understand what are the pathologic cell types that are present within the granuloma, which is these aggregation of immune cells that Chieh-Yu was speaking about.   Cindy St. Hilaire:        What is actually happening at the beginning of this disease? These granulomas form, and then what is the pathological progression in the heart? What goes on there?   Chieh-Yu Lin:             This is actually another great question that I will say there's not much that has been discovered because, especially in human tissue, every time we have a sample, it's actually a kind of time point. We cannot do a longitudinal study. But in general speaking, very little is known about how it's initiated because it will need to accumulate to a certain disease burden for this to have a clinical symptom sign and be manifested, and then being clinically studied. We do know that in both heart and lung after treatment of progressions, it's usually in, a general speaking, going through a phase from a more proliferative means that it's creating more granulomas, more  inflammatory cell aggregate, to a more fibrotic phase. Means that sometimes you actually see the granuloma start to disappear or dissipate, and then showing this kind of dense collagen and fibrosis. That has been commonly documented in both lung and heart sarcoidosis. The other things is that very difficult to study this disease that we do not have a great animal model, so we cannot use animal model to try to approximate or really study the disease pathogenesis. There are several animal models they try to use microbacteria or infectious agents, and these infectious agents can create morphologically similar granuloma, per se, but just like in human body. For instance, patients suffer from TB in their lung, biopsy will show this. But clinically, these are two very distinct disease entities, even though they look alike. Even in the heart, one of the conditions that we study in our paper is giant cell myocarditis, as the name implying having multinucleated giant cells granuloma. It looks really alike under microscopy for pathologists like me, but their clinical course in response to treatment is drastically different. This type of barriers and in the current limitations of our study tool makes, as Kory just said, this is really a wide open. We just know so little despite all the effort.   Cindy St. Hilaire:        Yeah. I'm guessing based on this granuloma information, to start with, the obvious question you went after is going after the immune cell populations that possibly contribute to sarcoidosis. To do this, because you have the human tissue, you went for single cell transcriptional profiling, which is a great use of the technology. But what biological sources did you use, and how did you go about choosing patient? Because the great thing about single cell is you can do just that, you can look at however many thousands of cells in one patient. But how do you make sure or check that that is broadly seen versus just a co-founding observation in that patient?   Kory Lavine:               We use explanted hearts and heart tissue from patients that underwent either heart transplantation or implementation of LVADs. It's a pretty big hunk of myocardium, and we're lucky to work with outstanding pathologists both at WashU, JU, as well as our collaborators at Duke. Between the two institutions, we're able to pull together a collection of tissues where we knew there were granulomas within that piece of tissue we analyzed. You bring up an important challenge. You need to make sure the disease and cause of the disease is present in the tissue that you're analyzing, otherwise you'll not come up with the data that really is informative.   Chieh-Yu Lin:             Kory beautifully answered the question, but I just wanted to add one little thing, and that's also why we use various different modalities. Some of them is more inside you, like the NanoString Technologies' spatial transcriptomic. You can visualize and confirm that we are studying the phenomenon that has been described for sarcoidosis, and then using multichannel immunofluorescence to validate our sequencing data, to complement such limitations of certain technology.   Cindy St. Hilaire:        Especially, I feel like with this diseased tissue that it's such a large tissue, there's so much information, it's really hard to dig in and figure out where the signal is. This was a wonderful paper for kind of highlighting, integrating all these new technologies with also just classical staining. Makes for great pictures as well. How does this cellular landscape of cardiac sarcoidosis compare to a normal heart? What'd you find?   Chieh-Yu Lin:             This is a great question. Compared to normal heart, we have been talking about this accumulation of macrophages with scattered multinucleated giant cells. For the similar landscape, first and foremost, you do not see those type of accumulations in brain microscopy or by myeloid markers in the heart. Although, indeed, in even normal heart tissue we have rest and macrophages. It just doesn't form such morphological alterations. But then we dive deep into it, and then we found that from a different cell type perspective, we realized that the granuloma is composed by several different type of inflammatory cells, with most of the T cells and NKT cells kind of adding periphery. The myeloid cells, including the multinucleated giant cells also, are kind of in the center of the granuloma of the sarcoidosis. Then, we further dive in and realize that there are at least six different subtype of myeloid cells that is contributing to the formation of this very eye-catching distinctive granular malformations, and to just never feel first off and foremost, of course, is those multinucleated giant cells that is really distinct, even on the line microscopy] routine change stand.   And then we have a typical monocyte that's more like a precursor being recently recruited to the heart, and we finally sent the other four different type of myeloid cell that carry different markers, and then improving the resident macrophages. Especially for me as a pathologist, I'm using my eye and looking at stand every day, is actually these six type of cells, myeloid cells, actually form a very beautiful special kind of distribution with the connections or special arrangement with all different type, kind of like multinucleated giant cell in the middle, flanked by HLA-DR positive epithelioid macrophages, kind of scatter, and then with dendritic cells and a typical monocyte at the peripheral, and then resident macrophage kind of like in the mix of the seas of granuloma information. All these are distinct from normal heart tissues that does carry a certain amount of macrophages, but just don't form this orchestrated architectural distinct structure that's composed of this very complicated landscape.   Cindy St. Hilaire:        Those images, I think it was figure six, it's just gorgeous to look at, the model you made. One of the questions I was thinking is there must be a significance between these cells that are on the periphery and those that are in the center of this granuloma. Do you have an idea or can we speculate as to are some more cause and some more consequence of the granuloma? Were you able to capture any more information about maybe the initiating steps of these from your study?   Kory Lavine:              That's a great question, and a question the field has had for a long time. Now, we know there's different populations of cells. The single cell data allows us to understand what are the transcriptional differences and distinctions between them to gain some insights. One thing that we do know from the field is that disease activity correlates with mTOR activity within these granulomas. We took advantage of phospho-S6 kinase staining as a downstream marker of mTOR activity, and Ki-67 is a marker of self proliferation.   Which of these populations within the granuloma might be most active with respect to mTOR and respect to proliferation? If you ask most people in the field, they would jump up and say, "It's the giant cell in the middle." We found that that's not actually the case at all. It's the macrophages that surround the giant cell, the ones that are HLA-DR positive, the epithelioid macrophages, and the ones that are SYLT-3 positive that are scattered around them. That's really interesting and could make a lot of sense, and leads to hypothesis that perhaps activation mTOR signaling within certain parts of the granuloma might be sufficient to set up the rest of the architecture. That's something that we can explore in animal models, and are doing so to try to create a cause and effect relationship. Cindy St. Hilaire:        Yeah, and I was actually thinking about this, too, in relation to kind of the resident macrophages versus infiltrating macrophages or even just infiltrating immune cells. Do you know the original source of the cells that make up the granuloma? Is it mostly resident immune, or are they recruited in?   Kory Lavine:               We can make predictions from the single cell data where you can use trajectory analysis to make strong predictions about what the origin of different populations might be. What those analyses predicted is that the giant cells and the cells that surround the giant cells, the HLA-DR positive and SYLT-3 positive macrophages, come from monocytes. That's the prediction, and, of course, resident macrophages do not. However, that prediction has to be tested, and that's the beauty and importance of developing animal models. The wonderful thing today is we now have genetic tools to do that. We can ask that question.   Cindy St. Hilaire:        I don't know. Maybe you don't want to spoil the lead of the next paper, but what kind of mouse model are you thinking about trying?   Kory Lavine:               Yeah. First of all, let me talk about the tools that are available, because they're published in Circulation Research, of course. We have a nice tool to specifically mark, track and delete in tissue resident macrophages using a CX3CR1 ERT pre-mouse, and taking advantage of the concept that tissue macrophages don't turn over from monocytes and turn over from themselves. We can give tamoxifen to label all monocytes macrophages in Dcs with that CRE, and then wait a period of time where only the resident macrophages remain labeled. We can use that trick to modulate mTOR signaling as a first step, and ask whether mTOR signaling is required in that population. We've now developed a new genetic tool to do the same thing in just recruited macrophages.   Cindy St. Hilaire:        What was the most challenging aspect of this study? There's a lot of moving parts. I'm sure probably the data analysis alone is challenging, but what would you say is the most challenging?   Kory Lavine:               I think you alluded to this early on, but the most challenging thing is collecting the right tissues to analyze, and that's not a small feat or a small effort here. All the technologies are a lot of fun, and everything works so well today compared to many years ago when we trained, so it's an exciting time to do science. The most challenging and time-consuming component was assembling a group of tissues that we could do single-cell sequencing on between our group and our colleagues at Duke, and then creating validation cohorts that we did across several different institutions, including our own as well as Stanford. That team effort in building that team is the most important, challenging, and honestly, enjoyable part of this.   Chieh-Yu Lin:             I cannot agree more what Kory just said. I think that that's the challenging and the fun part, and that we're very fortunate to really have a great team to tackle this questions in multiple from multiple institute. I just want to add one more thing that, particularly for me as a cardiopathologist, one of the hardest things is I've known how to look or diagnose sarcoidosis for years, but seeing the data emerging that is so complicated and then beyond my reliable eyes in understanding, it's kind of mentally very challenging but very fun to really open and broaden the vision. It's not just how it looks like just giant cells in macrophages.   Cindy St. Hilaire:        What do you think about in terms of diagnostics or even potential therapies? How do you think this data that you have now can be leveraged towards those objectives, whether it's screening for new cell types that are really key to this granuloma formation versus therapeutically targeting them?   Kory Lavine:              This study opens new doors, and right now, diagnosis of sarcoids islimited by trying to biopsy, which, in the heart, is limited by sample bias. You certainly can biopsy the wrong area because you don't know whether a granuloma is in the area or not. We do do some cardiac and other imaging studies like FDG-PET scans, which are helpful but are not perfect, and each of them has their individual limitations. One of the beauties of our study is it identifies new markers of macrophage populations that live within the granuloma, many of which are unique to this disease.   That suggests that there's maybe an opportunity to develop imaging tracers that can identify those populations more specifically than our current PET imaging studies do, which rely simply on glucose uptake. It also opens up the possibility that we may able to take blood samples and identify some of these cell types within the blood, and have more simple testing for our patients. I think in terms of therapy, you alluded to it earlier, these concepts about mTOR signaling, that could be a new therapeutic avenue that needs to be rigorously explored in preclinical models. We're lucky already to have very good mTOR inhibitors available in clinical practice today.   Cindy St. Hilaire:        Obviously, opening new doors is amazing because it's more information, but often a good study leads to even more questions to be asked. What question, or maybe what questions, are you guys going to go after next?   Chieh-Yu Lin:             Well, that list is very long, and then that's actually the exciting thing about doing this research. There's no bad questions, in some sense. All the way from diagnosis, management, monitoring, therapeutic, how we predict where the patient can respond, that's the whole clinical side. Even the basic science side, we still haven't really answered the question, although our data suggests where that multinucleated giant cells coming from. It's very eye catching. How do they form, even though our data suggests it's from the recruited macrophages. But that's still a long way from the recruited macrophage,  monocyte to that gigantic bag of nuclei in the very fluffy cytoplasm.   And then, how the granuloma, as we discussed earlier in this discussion, really initially from a relatively normal background myocardium to form this disease process. There are just so many questions that we can ask. There are, of course, several fronts that we would like to focus on. Kory already nicely listed some of them. First and foremost is actually to establish animal model to enable us to do more details in mechanistic studies, because human tissue, as good as it is, it's kind of like a snapshot, just one time point, and it really limits our ability to test our hypothesis. Animal model, certainly, is one of the major directions that we are going forward, but also the other side, like more clinical science also to develop novel noninvasive methodologies to diagnose and to hopefully monitor this patient population in a better way.       Cindy St. Hilaire:        Well, it's beautiful work. I was actually reading this paper this weekend at a brunch place just next door to my house, and the guy sitting next to me happened to see over my shoulder the title and said that his father had passed away from it. This is hopefully going to help lots of people in the future, and really help to make the models that we need to ask, "What's happening in this disease?" Thank you so much for taking the time to speak with me, and congratulations on what seems to be a landmark study in understanding what's going on in this disease.   Chieh-Yu Lin:             Thank you so much. It's a pleasure.   Cindy St. Hilaire:        That's it for our highlights from the September 30th and October 14th issues of Circulation Research. Thank you so much for listening. Please check out the Circ Res Facebook page, and follow us on Twitter and Instagram with the handle @CircRes, and hashtag Discover Circ Res. Thank you so much to our guests, Dr Kory Lavine and Dr Chieh-Yu Lin from Washington University St. Louis. This podcast is produced by Ashara Retniyaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy texts for highlighted articles was provided by Ruth Williams. I'm your host, Dr Cynthia St. Hilaire, and this is Discover Circ Res, your on-the-go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association, 2022. The opinions expressed by speakers in this podcast are their own, and not necessarily those of the editors of the American Heart Association. For more information, please visit ahajournals.org.  

This month on Episode 40 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the September 2 and September 16 issues of the journal. This episode also features an interview with Dr Jun Yoshioka, and Dr Yoshinobu Nakayama, from the City University of New York, about their study, Interaction of ARRDC-4 with GLUT1 Mediates Metabolic Stress in the Ischemic Heart.   Article highlights:   Jin, et al. Gut Dysbiosis Promotes Preeclampsia   Mengozzi, et al. SIRT1 in Human Microvascular Dysfunction   Hu, et al. Racial Differences in Metabolomic Profiles and CHD   Garcia-Gonzales, et al. IRF7 Mediates Autoinflammation in Absence of ADAR1   Cindy St. Hilaire:        Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh. And today I'm going to be highlighting some articles from September 2nd, and September 16th issues of CircRes. And I'm also going to have a conversation with Dr Jun Yoshioka, and Dr Yoshinobu Nakayama, from the City University of New York, about their study, Interaction of ARRDC-4 with GLUT1 Mediates Metabolic Stress in the Ischemic Heart. But, before I get to the interview, I'm going to highlight a few articles.                                       The first article is from our September 2nd issue, and it's titled, Gut Dysbiosis Promotes Preeclampsia by Regulating Macrophages, and Trophoblasts. The first author is Jiajia Jin, and the corresponding author is Qunye Zhang from the Chinese National Health Commission.                                       Preeclampsia is a late-stage pregnancy complication that can be fatal to the mother, and the baby. It's characterized by high blood pressure, and protein in the urine. The cause is unknown, but evidence suggests the involvement of inflammation, and impaired placental blood supply. Because gut dysbiosis can influence blood pressure, and inflammation has been observed in preeclamptic patients, Jin and colleagues examined this link more closely. They found that women with preeclampsia had altered gut microbiome. Specifically, a reduction in a species of bacteria that produced short-chain fatty acids, and lower short-chain fatty acid levels in their feces, in their serum, and in their placentas. And preeclamptic women had lower short-chain fatty acid levels in their feces, in their serum, and in their placentas compared with women without preeclampsia.                                       They found that fecal transfers from the preeclampsia women to rats with a form of the condition exacerbated the animals' preeclampsia symptoms, while fecal transfers from control humans alleviated the symptoms. Furthermore, giving rats an oral dose of short-chain fatty acids or short-chain fatty acid producing bacteria decreased the animals' blood pressure, reduced placental inflammation, and improved placental function. This work suggests that short-chain fatty acids, and gut microbiomes could be a diagnostic marker for preeclampsia. And microbial manipulations may even alleviate the condition.                                       The second article I want to share is also from our September 2nd issue, and it's titled, Targeting SIRT1 Rescues Age and Obesity-Induced Microvascular Dysfunction in Ex Vivo Human Vessels. And this study was led by Alessandro Mengozzi from University of Pisa.                                       With age, the endothelial lining of blood vessels can lose its ability to control vasodilation, causing the vessel to narrow and reduce blood flow. This decline in endothelial function has been associated with age related decrease in the levels of the enzyme, SIRT1. And artificially elevating SIRT1 in old mice improves animals' endothelial function. Obesity, which accelerates endothelial dysfunction, is also linked to low SIRT1 levels.                                       In light of these SIRT1 findings, Mengozzi, and colleagues examined whether increasing the enzyme's activity could improve the function of human blood vessels. The team collected subcutaneous microvessels from 27 young, and 28 old donors. And both age groups included obese, and non-obese individuals. SIRT1 levels in the tissue were, as expected, negatively correlated with age and obesity, and positively correlated with baseline endothelium dependent vasodilatory function. Importantly, incubating tissue samples from older, and obese individuals with a SIRT1 agonist, restored the vessel's vasodilatory functions. This restoration involved a SIRT1 induced boost to mitochondrial function, suggesting that maintaining SIRT1 or its metabolic effect might be a strategy for preserving vascular health in aging, and in obesity.                                       The third article I want to share is from our September 16th issue. And this one is titled, Differences in Metabolomic Profiles Between Black And White Women and Risk of Coronary Heart Disease. The first author is Jie Hu, and the corresponding author is Kathryn Rexrode, and they're from Brigham and Women's Hospital, and Harvard University.                                       In the US, coronary heart disease, and coronary heart disease-related morbidity, and mortality is more prevalent among black women than white women. While racial differences in coronary heart disease risk factors, and socioeconomic status have been blamed, this group argues that these differences alone cannot fully explain the disparity. Metabolomic variation, independent of race, has been linked to coronary heart disease risk. Furthermore, because a person's metabolome is influenced by genetics, diet, lifestyle, environment and more, the authors say that it reflects accumulation of many cultural, and biological factors that may differ by race.                                       This group posited that if racial metabolomic differences are found to exist, then they might partially account for differences in coronary heart disease risk. This study utilized plasma samples from nearly 2000 black women, and more than 4500 white women from several different cohorts. The team identified a racial difference metabolomic pattern, or RDMP, consisting of 52 metabolites that were significantly different between black, and white women. This RDMP was strongly linked to coronary heart disease risk, independent of race, and known coronary heart disease risk factors. Thus, in addition to socioeconomic factors, such as access to healthcare, this study shows that racial metabolomic differences may underlie the coronary heart disease risk disparity.                                       The last article I want to share is also from our September 16th issue, and it is titled, ADAR1 Prevents Autoinflammatory Processes in The Heart Mediated by IRF7. The first author is Claudia Garcia-Gonzalez, and the corresponding author is Thomas Braun, and they are from Max Planck University.                                       It's essential for a cell to distinguish their own RNA from the RNA of an invading virus to avoid triggering immune responses inappropriately. To that end, each cell makes modifications, and edits its own RNA to mark it as self. One type of edit made to certain RNAs is the conversion of adenosines to inosines. And this is carried out by adenosine deaminase acting on RNA1 or ADAR1 protein. Complete loss of this enzyme causes strong innate immune auto reactivity, and is lethal to mice before birth. Interestingly, the effects of ADAR1 loss in specific tissues is thought to vary. And the effect in heart cells in particular has not been examined.                                     This study, which focused on the heart, discovered that mice lacking ADAR1 activity specifically in cardiomyocytes, exhibit autoinflammatory myocarditis that led to cardiomyopathy. However, the immune reaction was not as potent as in other cells lacking ADAR1. Cardiomyocytes did not exhibit the sort of upsurge in inflammatory cytokines, and apoptotic factors seen in other cells lacking ADAR1. And the animals themselves did not succumb to heart failure until 30 weeks of age. The author suggests that this milder reaction may ensure the heart resists apoptosis, and inflammatory damage because, unlike some other organs, it cannot readily replace cells.   Cindy St. Hilaire:        Today I have with me, Dr Jun Yoshioka, and Dr Yoshinobu Nakayama, and they're from City University of New York. And today we're going to talk about their paper, Interaction of ARRDC4 With GLUT1 Mediates Metabolic Stress in The Ischemic Heart. And this is in our September 2nd issue of Circulation Research. So, thank you both so much for joining me today.   Jun Yoshioka:             Thank you for having us. We are very excited to be here.   Cindy St. Hilaire:        It's a great publication, and also had some really great pictures in it. So, I'm really excited to discuss it. So, this paper really kind of focuses on ischemia, and the remodeling in the heart that happens after an ischemic event. And for anyone who's not familiar, ischemia is a condition where blood flow, and thus oxygen, is restricted to a particular part of the body. And in the heart, this restriction often occurs after myocardial infarctions, also called heart attacks. And so, cardiomyocytes, they require a lot of energy for contraction, and kind of their basic functions. And in response to this lack of oxygen, cardiomyocytes switch their energy production substrate. And so, I'm wondering if before we start talking about your paper, you can just talk about the metabolic switch that happens in a cardiac myocyte in the healthy state versus in the ischemic state.   Jun Yoshioka:             Sure. As you just said, that the heart never stops beating throughout the life. And it's one of the most energy demanding organs in the body. So, under normal conditions, cardiac ATP is mainly derived from fatty acid oxidation, and glucose metabolism contributes a little bit less in adult cardiomyocytes. However, under stress conditions such as ischemia, glucose uptake will become more critical when oxidative metabolism is interrupted by a lack of oxygen. That is because glycolysis is a primary anaerobic source of energy. We believe this metabolic adaptation is essential to preserve high energy phosphates and protect cardiomyocytes from lethal injuries. The concept of shifting the energy type of stress preference toward glucose, as you just said, has been actually long proposed as an effective therapy against MI. For example, GIK glucose insulin petition is classic.                                       Now, let me explain how glucose uptake is regulated. Glucose uptake is facilitated by multiple isophones of glucose transporters in cardiomyocytes. Mainly group one and group four, and the minor, with a minor contribution of more recently characterized STLT1. In this study, we were particularly interested in group one because group one is a basal glucose transporter.                                       Dr Ronglih Liao, and Dr Rong Tian's groups reported nearly two decades ago that the cardiac over-expression of group one prevents development of heart failure, and ischemic damage in mice. Since they are remarkable discoveries, the precise mechanism has not yet been investigated enough, at least to me. Especially how acute ischemic stress regulates group one function in cardiomyocytes. We felt that this mechanism is important because there is a potential to identify new strategies around group one, to reduce myocardiac ischemic damage. That is why we started this project hoping to review a new mechanism by which a protein family, called alpha-arrestins, controls cardiac metabolism under both normal, and diseased conditions.   Cindy St. Hilaire:        That is a perfect segue for my next question, actually, which is, you were focusing on this arrestin-fold protein, arrestin domain-containing protein four or ARRDC4. So, what is this family of proteins? What are arrestin-fold proteins? And before your study, what was known about a ARCCD4, and its relationship to metabolism, and I guess specifically cardiomyocyte metabolism?   Jun Yoshioka:             So, the arrestin mediated regulation of steroid signaling is actually common in cardiomyocytes. Especially beta, not the alpha, beta-arrestins have been well characterized as an adapter protein for beta-adrenergic receptors. Beta-arrestins combine to activate beta-adrenergic receptors on the plasma membrane, promote their endosomal recycling, and cause desensitization of beta-adrenergic signaling. Over the past decade, however, this family, the arrestin family, has been extended to include a new class of alpha-arrestins. But unlike beta-arrestins, the physiological functions of alpha-arrestins remain largely unclear based in mammalian cells. Humans, and mice have six members of alpha-arrestins including Txnip, thioredoxin interacting protein called Txnip, and five others named alpha domain-containing protein ARRDC1 2, 3, 4 and 5. Among them Txnip is the best studied alpha-arrestin. And Txnip is pretty much the only one shown to play a role in cardiac physiology.                                       Txnip was initially thought to connect alternative stress and metabolism. However, it is now known that the Txnip serves as an adapter protein for the endocytosis of group one, and group four to mediate acute suppression of glucose influx to cells. In fact, our group has previously shown that the Txnip knockout mice have an enhanced glucose uptake into the peripheral tissues, as well as into the heart. Now, in this study, our leading player is ARRDC4. The arrestin-domains of ARRDC4 have 42% amino acid sequence similarities to Txnip. This means that the structurally speaking ARRDC4 is a brother to Txnip. So, usually the functions of arrestins are expected to be related to their conserved arrestin-domains. So, we were wondering whether two brothers, Txnip, and ARRDC4, may share the same ability to inhibit the glucose transport. That was a starting point where we initiated this project.   Cindy St. Hilaire:        That's great. And so, this link between ARRDC4, and the cardiac expression of gluten one and gluten four, I guess, mostly gluten one related to your paper, that really wasn't known. You went about this question kind of based on protein homology. Is that correct?   Jun Yoshioka:             That is right.   Cindy St. Hilaire:        And so, ARRDC4 can modulate glucose levels in the cell by binding, and if I understand it right, kind of helping that internalization process of glute one. Which makes sense. You know, when you have glucose come into the cell, you don't want too much. So, the kind of endogenous mechanism is to shut it off, and this ARRDC4 helps do that. But you also found that this adapter protein impacts cellular stress, and the cellular stress response. So, I was wondering if you could share a little bit more about that because I thought that was quite interesting. It's not just the metabolic impact of regulating glucose. There's also this cellular stress response.   Jun Yoshioka:             Right? So, Txnip is known to induce oxidative stress. But about the ARRDC4, we found that ARRDC4 actually does not induce oxidative stress. Instead, we found that it reproducibly causes ER, stress rather than oxidative stress. So, let Yoshinobu talk about the ER stress part. Yoshinobu, can you talk about how you found the ER stress story?   Yoshinobu Nakayama: So, then let's talk about the, yeah, ER stress caused by ARRDC4. The ER stress caused by ARRDC4, year one was the biggest challenge in this study, because it's a little bit difficult to how we found a link of the glucose metabolism to the effect of the ARRDC4, only our stress. And at the other point of the project, we noticed that a ARRDC4 causes ER stress reproducibly, but we did not know how. So, both group one, and ARRDC4 are membrane proteins mainly localized near the plasma membrane. Then how does ARRDC4 regulate the biological process inside in the plasma radical? So, we then hypothesize that ARRDC4 induces intercellular glucose depravation by blocking cellular glucose uptake, and then interferes with protein glycosylation, thereby disturbing the ER apparatus. That makes sense because inhibition of group one trafficking by ARRDC4 was involved in the unfolded protein response in ischemic cardiomyocytes.   Cindy St. Hilaire:        So how difficult was that to figure out? How long did that take you?   Yoshinobu Nakayama: How long? Yeah. Is this the question?   Cindy St. Hilaire:        It's always a hard question.   Yoshinobu Nakayama: I think it's not several weeks. Maybe the monthly, months project. Yeah.   Cindy St. Hilaire:        Okay. It's always fun when, you know, you're focusing on one angle, and then all of a sudden you realize, oh, there's this whole other thing going on. So, I thought it was a really elegant tie-in between the metabolism, but also just the cellular stress levels. It was really nice.                                       So, you created a full body knockout of ARRDC4 in the mouse, and you did all the proper kind of phenotyping. And at baseline everything's normal, except there's a little bit of changes in the blood glucose levels. But I also noticed when you looked at the expression of ARRDC4 in different tissues, it was very high in the lungs, and also in the intestines. And so, I know your study didn't focus on those tissues, but I was wondering if you could possibly speculate what ARRDC4 is doing in those tissues? Is it something similar? Do those cells under stress have any particular metabolic switching that's similar?   Jun Yoshioka:             Well, actually we don't have any complete answer for that question, because like you said, we didn't focus on lung, and other tissues. But I could say that actually the brother of ARRDC4, Txnip, is also highly expressed in lung, and bronchus, and in those organs. So, it's interesting because, which means that, the molecule is very oxygen sensitive, I will say. Both brothers. But that's all we know for now. But that's a very great point. And then we are excited to, you know.   Cindy St. Hilaire.        Yeah.   Jun Yoshioka:             Move on to the other tissues.   Cindy St. Hilaire:        I was thinking about it just because I've actually recently reviewed some papers on pulmonary hypertension. So, when I saw that expression, that was the first thing I thought of was, oh, they should put these mice in a sugen/hypoxia model, and see what happens.   Jun Yoshioka:             Right?   Cindy St. Hilaire:        So, there's an idea for you, Yoshinobu. A K-99 grant or something. And also, because it's a full body knockout, even when you're looking at the heart, obviously the cardiomyocytes are really the most metabolically active cell, but cardiac fibroblasts are also a major component of the heart tissue. And so, do you know, is the, I guess, effects or the protectiveness of the ARRDC4 knockout heart, is it mostly because of the role in the cardiomyocytes or is there a role for it also in the fibroblast?   Yoshinobu Nakayama: Yeah, that's a very great question. Yeah. So, although we use the systemic knockout mice in the study, we believe that the beneficial effect of ARRDC4 deficiency is cardiac, autonomous. But this is because cardioprotection was demonstrated in the isolated heart experiments. But, you know, root is still uniformly expressing all cell types within the heart.                                       To address this, we have tested the specific effects of ARRDC4 on cardiac fibroblasts, and inflammatory cells. ARRDC4 knockout hearts had a twofold increase in myocardial glucose uptake over wild-type hearts during insulin-free perfusion. However, an increase in glucose uptake in isolated cardiac fibroblast or inflammatory cells was relatively mild, with about 1.2 fold increase over wild-type cells.                                       Thus we conclude that cardiomyocytes are the measure contributed to the cardiac metabolic shift. And then the mechanism within cardiomyocytes should play the major role in cardioprotection.   Jun Yoshioka:             I might, at one point, because, you know, the fibroblasts, they don't need to beat, right?   Cindy St. Hilaire:        Right.   Jun Yoshioka:             The inflammasome cells. They don't need to beat neither. So, they don't need that much energy. So, the cardiomyocytes energy metabolism is very important. So, that's why this mechanism is kind of more important in cardiomyocytes than other cell types.   Cindy St. Hilaire:        Yeah. And I think, you know, your phenotyping of the mice at baseline show that there's really no effect in a cell that's not under stress. So, it's really, really nice finding. Yeah.                                       This article, I should say, is featured on the cover of the September 2nd Circulation Research issue. And it's got this really nice 3D modeling of the binding of ARRDC4 to glute one. And I was reading the paper, and the methods said, you use some AI for that. So, I'm sure other people have heard, too, AI in protein modeling is important. But AI in art, right? There's that new DALL-E 2 program. So how are you able to do this? How did that work?   Jun Yoshioka:             So, our study used is called AlphaFold, which applies the artificial intelligence-based deep learning method. AlphaFold, nowadays, everybody really is interested in AlphaFold. AlphaFold uses structural, and genetic data to come up with a model of what the protein of interest should look like. So, that is also how we got the protein structure, ARRDC4. We think that the ability of AlphaFold to precisely predict the protein structure from amino acid sequence would be a huge benefit to life sciences, including of course, cardiovascular science research, because of high cost, and technical difficulties in experimental methods.                                       It's very useful if you can computationally predict the complex from individual structures of ARRDC4. And group one, which is actually structure of group one, is available in a protein data bank. But ARRDC4, it was not available. That's why we used AlphaFold.                                       And then we use the docking algorithm called Hdoc. So, based on these AI analysis, we could successfully identify specific residues in a C terminal arrestin domain as an international interface, that regulates group one function. So, we believe this AI method will pretty much accelerate efforts to understand the protein, protein interactions. And we believe that will enable more advanced drug discovery, for example, in very near future.   Cindy St. Hilaire:        Yeah, it's really great. I started thinking about it in terms of some of the things I'm studying. So yeah, it was really nice. Jun Yoshioka:             Try next time.   Cindy St. Hilaire:        Yeah, I will, I will. Actually, I went to the website, and was playing with it before I got on the call with you. So, how do you think your findings can be leveraged towards informing clinical decision making or even developing therapeutics?   Jun Yoshioka:             So, let me talk about what needs to be done. There are more things we must do.   Cindy St. Hilaire:        Always. Yeah.   Jun Yoshioka:             One of the most clinically relevant questions is whether ARRDC4 inhibition actually can mitigate development of post MI heart failure, and reduce mortality in the chronic phase, not the acute phase. Because in this paper we just did the seven day post MI, which is kind of like acute to subacute phase. But you never know what's going to happen in the chronic phase, right? And that is actually not so simple to answer because there are so many issues that you should consider. For example, Dr E. Dale Abel's lab has reported previously that cardiomyacites, specific group one, knockout in mice does not really accelerate the transition from compensated hypotrophy to heart failure. Also, the same group has shown that the overexpression group one does not actually prevent LV dysfunction in the mouse model of pressure overload. So, it is possible that ARRDC knockout can be, do much, or even harmful to LV remodeling in a chronic phase because chronic phase, it's not, it's getting hypoxy conditions, right?   Cindy St. Hilaire:        Yeah. So, it really might be something, I guess, personalized medicine is not the phrase I'm looking for. But I guess temporarily modulated, it would be something maybe we can figure out in an acute phase versus.   Jun Yoshioka:             Chronic phase.   Cindy St. Hilaire:        Yeah. Yeah.   Jun Yoshioka:             This makes sense. Because, you know, high capacity of ATP synthesis, by oxidating metabolism, could be important for chronic heart failure. So, it's selecting substrates. Energy substrates is no longer, you know, that issue. So, I'm not sure I'm answering your question, but this is the point that we consider to move on to the next.   Cindy St. Hilaire:        Well, that's great. And I think that was my next question, really. What is next? Are you really going to try to pinpoint where you could possibly target?   Jun Yoshioka:             Right. So, the first point we have to figure out about chronic phase, and another point we are interested in, is what's going on at the level of mitochondria. Does ARRDC4 knockout hearts have a different activity of electron transport chain or glycolytic enzymes within mitochondria?   Cindy St. Hilaire:        Or even mitochondrial fission infusion because it's, you know, it's a machinery.   Jun Yoshioka:             Yeah. And how about the other essential pathways in glucose metabolism such as mTOR, AMPK and HEF1, and so on. So, all these must be determined to help understand the more precise role of ARRDC4 in cardiac metabolism, we believe. Cindy St. Hilaire:        It's a wonderful study, and now we have even more questions to ask using your great model. Congratulations again.   Yoshinobu Nakayama: Thank you so much.   Cindy St. Hilaire:        Dr Yoshioka, and Dr Nakayama.   Jun Yoshioka:             Thank you.   Cindy St. Hilaire:        A wonderful paper, and congrats on getting the cover, and thank you so much for joining me today.   Jun Yoshioka:             Thanks well so much for having us.   Yoshinobu Nakayama: Thank you.   Cindy St. Hilaire:        That's it for the highlights from our September 2nd, and our September 16th issues of Circulation Research. Thank you so much for listening. Please check out our CircRes Facebook page, and follow us on Twitter, and Instagram with the handle @circres, and hashtag discovercircres. Thank you to our guests, Dr Jun Yoshioka, and Dr Yoshinobu Nakayama.                                     This podcast is produced by Ishara Ratnayaka, edited by Melissa Stonerm, and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on the go source for the most exciting discoveries in basic cardiovascular research.                                       This program is copyright of the American Heart Association 2022. The opinions expressed by speakers in this podcast are their own, and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahajournals.org.

This month on Episode 39 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the August 5th and 19th issues of the journal. This episode also features an interview with Dr Annet Kirabo and Dr Ashley Pitzer from Vanderbilt University on their article, Dendritic Cell ENaC-Dependent Inflammasome Activation Contributes to Salt-Sensitive Hypertension.   Article highlights:   Jain, et al. Role of UPR in Platelets   Orlich et al: SRF Function in Mural Cells of the CNS   Xue et al: Gut Microbial IPA Inhibits Atherosclerosis   Wang et al: Endothelial ETS1 on Heart Development   Cindy St. Hilaire:        Hi, welcome to Discover CircRes, the podcast of the American Heart Association's journal Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to be highlighting articles from our August 5th and August 19th issues of Circulation Research. I'm also going to have a chat with Dr Annet Kirabo and Dr Ashley Pitzer from Vanderbilt University about their study, Dendritic Cell ENaC-Dependent Inflammasome Activation Contributes to Salt-Sensitive Hypertension.   But before I get to the interview, I first want to share an article from our August 5th issue, and that article is titled, Unfolded Protein Response Differentially Modulates the Platelet Phenotype. The first author of this study is Kanika Jain and the corresponding author is John Hwa from Yale University. Self-stress can lead to protein misfolding, and the accumulation of misfolded proteins can lead to a reduction in protein translation and may alter gene transcription, a process collectively known as the unfolded protein response, or UPR. UPR is well documented in nucleated cells; however, it has not been studied in platelets, which are anuclear, but do have a rapid response to cellular stress. In this study, they investigated the UPR in anucleate platelets and explore its role, if any, in platelet physiology and function.   They found that treating human and mouse platelets with various stressors caused aggregations of misfolded proteins and induction of UPR-specific factors. Oxidative stress, for example, induced the UPR kinase PERK, while an endoplasmic reticulum stressor induced the transcription of the UPR factor XBP1. The team went on to study the UPR in platelets from people with type II diabetes, which is a population in which platelet mediated thrombosis is a major complication. They showed that protein aggregation and upregulation of the XBP1 pathway in diabetic patient platelets correlated with disease severity. Furthermore, treating the diabetic patient platelets with a chemical chaperone that helps to correct protein misfolding reduced protein aggregations and prevented the cells prothrombotic activation. This work confirms that even without transcription, platelets display stress-induced UPR, and that targeting this response may be a way to reduce thrombotic risk in diabetic patients.   Cindy St. Hilaire:        The second article I want to share with you is from our August 5th issue and is titled, Mural Cell SRF Controls Pericyte Migration, Vessel Patterning and Blood Flow, and it was led by Michael Orlich from Uppsala University in Sweden. Blood vessels are lined with endothelial cells and surrounded by mural cells. Vascular smooth muscle cells are the mural cells in the case of veins and arteries, and pericytes are the mural cells in the case of capillaries. In the capillaries, pericytes maintain blood-brain and blood-retina barrier function and can mediate vascular tone, similar to smooth muscle cells. While these pericytes and smooth muscle cells are related, they have distinct roles and characteristics.   To learn more about the similarities and the differences between pericytes and smooth muscle cells, this group examined how each would be affected by the absence of SRF in the other. SRF is a transcription factor, essential for nonvascular or visceral smooth muscle cell function. In visceral smooth muscle cells, SRF drives expression of smooth muscle actin and other smooth muscle genes. Using mice engineered to lack SRF in mural cells, they show that SRF drives smooth muscle gene expression in these pericytes and smooth muscle cells, and its loss from smooth muscle cells causes atrial venous malformations and diminishes vascular tone. In pericytes, loss of SRF impaired cell migration in angiogenic sprouting. In a mouse model of retinopathy, activation of SRF drove pathological growth of pericytes. This work not only highlights the various functions of SRF in mural cell biology, but it also suggests that it has a role in pathological capillary patterning.   Cindy St. Hilaire:        The third article I want to share is from our August 19th issue of Circulation Research and is titled, Gut Microbially Produced Indole-3-Propionic Acid Inhibits Atherosclerosis by Promoting Reverse Cholesterol Transport and its Deficiency Is Causally Related to Atherosclerotic Cardiovascular Disease. The first authors are Hongliang Xue and Xu Chen, and the corresponding author is Wenhua Ling from Sun Yat-Sen University in Guangzhou, China. Recent studies provide evidence that disorders in the gut microbiota and gut microbiome derived metabolites affect the development of atherosclerosis. However, which and how specific gut microbial metabolites contribute to the progression of atherosclerosis and the clinical relevance of these alterations remain unclear. Gut microbiome derived metabolites, such as short-chain fatty acids and trimethylamine N-oxide, or TMAO, have been found to correlate with atherosclerotic disease severity.   This study has now found that serum levels of indole-3-propionic acid, or IPA, are lower in atherosclerosis patients than controls. The team performed unbiased metagenomic and metabolomic analyses on fecal and serum samples from 30 coronary artery disease patients and found that, compared with controls, patients with atherosclerosis had lower gut bacterial diversity, depletion of species that commonly produce IPA and lower levels of IPA in their blood. Examination of a second larger cohort of atherosclerosis patients confirmed this IPA disease correlation. The team also showed serum IPA was reduced in a mouse model of atherosclerosis, and that supplementing such mice with dietary IPA could slow disease progression. Analysis of the macrophages from these mice showed that IPA increased cholesterol efflux, and the team went on to elucidate the molecular steps involved. The results of this study not only unraveled the details of IPA's influence on atherosclerosis, but suggest boosting levels of this metabolite could slow atherosclerotic disease progression.   Cindy St. Hilaire:        The last article I want to share is also from our August 19th issue, and it's titled, Endothelial Loss of ETS1 Impairs Coronary Vascular Development and Leads to Ventricular Non-Compaction. The first author is Lu Wang and the corresponding author is Paul Grossfeld, and they are at UCSD. Congenital heart defects, or CHDs, are present in nearly 1% of the human population. In some cases, the heart defects result from a genetic error, which can give researchers clues to its etiology. Jacobson syndrome is a complex condition caused by deletions from one end of chromosome 11, and the occurrence of a congenital heart defect in this syndrome has been associated with the loss of the gene ETS1. ETS1 is an angiogenesis promoting transcription factor, but how ETS1 functions in heart development was not known.   Wang and colleagues now show that both global or endothelial-specific loss of ETS1 in mice caused differences in embryonic heart development that ultimately led to a muscular wall defect known as ventricular non-compaction. The mice also had defective coronary vasculogenesis associated with decreased abundance of endothelial cells in the ventricular myocardium. RNA sequencing of ventricular tissue revealed that, compared with controls, mice lacking ETS1 had reduced expression of several important angiogenesis genes and upregulation of extracellular matrix factors, which together contributed to the muscular and vascular defects.   Cindy St. Hilaire:        Today I have with me, Dr Annet Kirabo and Dr Ashley Pitzer, both from Vanderbilt University, and we're going to talk about their paper, Dendritic Cell ENaC-Dependent Inflammasome Activation Contributes to Salt-Sensitive Hypertension. This article is in our August 5th issue of Circulation Research. Thank you both so much for joining me today.   Annet Kirabo:             Yeah, thank you so much for having us.   Ashley Pitzer:              Yeah, thank you for having us.   Cindy St. Hilaire:        Yeah, it's a great paper. I think we're all familiar with hypertension and this idea that too much salt is bad for our cardiovascular system. When I was a kid, my grandparents had those salt replacements on their kitchen table, Mrs. Dash and whatever. But, like you said in the start of your paper, the exact mechanism by which salt intake increases blood pressure and also increases cardiovascular risk, it's not really well understood, and you guys are focusing on the contribution of immune responses in this process or in this pathogenesis. Before we dig into the details of your paper, I was wondering if you could give us a little bit of background about what's known regarding the role of inflammation in this salt-sensitive hypertension pathogenesis.   Annet Kirabo:             Yeah. It's difficult to know where begin to from, but the role of inflammation in cardiovascular disease have been known for many, many decades. Right now, Dr David Harrison showed more than 10 years ago that T cells contribute to hypertension, but the mechanisms were not known. Back when I was a post doc in David Harrison's lab, we discovered a new mechanism, how immune cells are activated in inflammation and hypertension, whereby we found that there is increased oxidative stress in antigen-presenting cells. This leads to formation of oxidative products known as arachidonic acid or lipid products known as isolevuglandin, or IsoLGs. These IsoLGs are highly, highly reactive and they adapt to lysines on proteins. This is a covalent binding, which leads to permanent alteration of proteins, and so these proteins act as neoantigens that are presented as self-antigens to T cells, leading to an autoimmune-like state in hypertension.   Annet Kirabo:             We found that these antigen-presenting cells are activated and they start producing a lot of cytokines that paralyze T cells to IL-17 producing T cells that contribute to hypertension. And so, when I started my lab back in 2016, we discovered that excess dietary salt profoundly activates this pathway, and we found for the first time that these antigen-presenting cells, they express ENaC, the epithelial sodium channel, and sodium goes into these antigen-presenting cells and activates the NADPH oxidase, which is an enzyme which produces this reactive oxygen species, leading to this IsoLG formation, which I've talked about, and leading to inflammation.   So, three years ago when Ashley joined my lab, she had extensively studied the inflammasome in her PhD program, and she suggested why don't we look at the role of the inflammasome in this pathway and how IsoLG may contribute to this. In her paper that we are discussing right now, she found that in a dependent manner, sodium enters the cell and activates this pathway, and the NLRP3 inflammasome is involved in this process.   Cindy St. Hilaire:        That's such a wonderful story that fits together so many pieces. One of the things you talk about, which I guess I didn't even appreciate myself is, there are certain individuals out there who are more salt-sensitive than others.   Annet Kirabo:             Yeah.   Cindy St. Hilaire:        What is that difference? Do we know the root cause of that? And then also, how many individuals are we talking about are salt-sensitive?   Annet Kirabo:             Salt-sensitive blood pressure, it is a variable trait and it's normally distributed in the population, but it happens more in some individuals than others. It happens even in 25% of people without any hypertension. These people go to that doctor, that doctor thinks they're normal, they don't have any hypertension, but these people can be at a risk of sudden heart attack or cardiovascular risk or even a stroke, simply because when they eat a salty meal, their blood pressure will go up.   Cindy St. Hilaire:        Yeah, that's one of my questions. How much salt are we talking about here? And not only how much in a meal, but a sustained amount? How bad is a miso soup a day?   Annet Kirabo:             Yes. The American Heart Association and the World Health Organization have recommendations. American Heart Association recommends one spoon per day. We have refused to adapt to this recommendation, but that is the recommendation that they have recommended per day to eat. But this is difficult because most of the salt, as you know, is already in our food through processing in our processed foods and we don't have any control over how much salt we have, and there's also a lot of adding of salt at a table.   Cindy St. Hilaire:        Ashley, your background was more the inflammasome. What were your thoughts entering into this project? Did you have much of a hypertension background?   Ashley Pitzer:              No. My graduate thesis focused mainly on endothelial dysfunction and cardiovascular disease, and so it was a pretty easy segue. But it was just with Annet, so excited about the project and showing me all the data and this robust IL-1 beta production that she was seeing after these immune cells being exposed to high salt, I, with my inflammasome background, was immediately like, this could be playing a role. And so it was, like I said, a pretty easy transition and, as is in the paper, we're doing human studies. All of my research back in grad school was very basic research, so it was very exciting to see how our research was being translated with people having this condition and potentially finding mechanisms where we can target this to help actual people.   Cindy St. Hilaire:        I think a lot of us who are not in the hypertension field, and maybe this was you before you joined Annet's lab, we really only kind of think of the kidneys and the blood vessels when we think about hypertension, but studies like this are changing that. And I think a lot of Annet's earlier work, as well as the work of others, have shown a role for this epithelial sodium channel as an important player in this salt-induced hypertension. New to me, it's not just found in the kidney, which I totally did not appreciate that. And it's this channel sensing the salt that can trigger this IL-1 beta production that does a whole bunch of other things.   Cindy St. Hilaire:        What are those other things? What are those cells that are affected and where is this happening? Obviously it's not just kidney cells, but is it only in the kidney or are these systemic cells? What do we think is happening?   Ashley Pitzer:              That's the question, is, where is this happening? There's been studies at Vanderbilt by Jens Titze and his lab showing, where are these immune cells sensing the salt? And so they've shown that sodium accumulates in the skin, a huge argument is for they're sensing the sodium in the kidney because that's where a lot of it is being processed. But these immune cells travel through the whole body, so they're seeing it where there are the highest amounts of sodium concentration, and so I would argue it's in the kidney.   Annet Kirabo:             Indeed, because we're now collaborating with Tina Kon, and we have recently published with her a paper in the International Journal of Science, where we have done sodium MRI and we find this accumulation of sodium in the kidney even much more than in the skin. And we know that the kidney is where sodium is highly concentrated. So the working hypothesis in the lab is that these immune cells can be activated wherever they are, in the lymph nodes or not, in other tissues, but they can travel to the kidney.   We find that in high salt, if you feed high salt to the mouse, the endothelium in the kidney becomes dysfunctional and it expresses molecules, chemoattractants, that attract these immune cells in the kidney. We think that the high salt accumulation in the kidney can activate these, and then these immune cells are activated and they produce cytokines. Dr Steve Crowley showed that they can produce IL-1 beta, which induces activation of sodium channels that can be induced. We have also actually found that even IL-17 can be produced by these immune cells in the kidney and they can activate sodium channels in the kidney, leading retention of sodium and water and hypertension.   Cindy St. Hilaire:        Very cool. You used a lot of mice in this paper. Can you tell us, I just want to know a little bit about the models you chose to use, but also how similar is hypertension in mouse and humans? Obviously for atherosclerosis, we have to do lots of things to get them to form a plaque. Is hypertension similar in a mouse and do mice also show this salt-sensitive phenotype?   Annet Kirabo:             That is an extremely important point. If you read our paper, we use a slightly different approach. Most people do benchside to bed approach. We did the opposite. We did a bed to benchside approach.   Cindy St. Hilaire:        Always smart.   Annet Kirabo:             Yeah. We first started humans, and then with some references, we went to the mice, because I think when it comes to salt-sensitive blood pressure, mice are different from humans. In fact, if we look in the lab, we find that female mice are protected from salt-sensitive blood pressure, but we find that in the humans, it's the opposite. Females are more prone to salt-sensitive hypertension. Those are studies that we are doing right now. We haven't published. But we know that it can be different.   The model we use most of the time in the lab, the C57 mice, are resistant to salt-sensitive hypertension. These C57 mice would rather die before they raise their blood pressure in response to salt. We can induce salt-sensitivity in these mice like in the paper that we are discussing. When we induce the endothelial dysfunction using L-NAME and we wash it out, then these mice, when you give them, subsequently, salt, suggests that they become salt-sensitive. But we also have a salt-sensitive mouse model that we use, the 129/SV mouse. So we use several models to kind of prove the same thing over and over again with the findings that we found in humans.   Cindy St. Hilaire:        And you used a technique, which I'm a little bit familiar with, but I'd like to hear, A, about it from you, but also your experience in using it, and that is CITE-seq. So, how does that work?   Ashley Pitzer:              That was with our human study where we actually had patients come in, who were hypertensive, took them off medication for 2 weeks. They come in, we get baseline samples, we give them a salt load on one day, and then the next day we completely salt deplete them.   Cindy St. Hilaire:        How much is a salt load? Like a Big Mac? What's a salt load?   Ashley Pitzer:              Yeah, it's pretty much just like eating Lays chips all day. It's a lot of salt. It's a very salty meal.   Annet Kirabo:             And then in addition, we also infuse saline too.   Cindy St. Hilaire:        Oh, wow.   Annet Kirabo:             Because these people, when they come into the hospital, some them have already eating high salt. This approach is to just maximize the whole system so that then when we sort deplete everybody, it's at the same level and it's just to unify the whole process. But sorry, Ashley, you go ahead.   Ashley Pitzer:              With the CITE-seq, we're able to take different patients on different days. So we take samples each day, and we can give each sample a barcode, basically. Give them a barcode, we can pool them all together, process them, and we can sequence their RNA, we can probe for a certain amount of protein expression as well. So then when we analyze, we can look at protein expression, so you get the translation and the transcription for each person on each day, and then you're able to compare. And so you get this huge picture and it's a lot of data.   Cindy St. Hilaire:        How long did it take you to sort through?   Ashley Pitzer:              Well, we have a statistician who does all of that, because my wheelhouse is here and it is on a different planet. So we have somebody who helps us with that who does an unbiased approach. And then once he does an analysis, gives us back what are the things that are changing the most, and one of those was IL-1 beta.   Annet Kirabo:             As you can see, our list is huge, this is a massive input of so many collaborators. We have computational people on there that help us with this. I can't even begin to learn these techniques, but with all this collaboration and the resources at Vanderbilt, these things are possible. And so, this is a really powerful approach where you can combine protein expression and you get the specific cells that express the genes and you couple the channel type to the gene expression.   Annet Kirabo:             We actually found that not all monocytes are the same. There's a specific class that of monocytes, A small class of monocytes that is so angry, and the inflammasome is activated and producing this IL-1 beta, and that is enough to contribute to this phenotype of salt-sensitive hypertension, which dynamically changed according to blood pressure, suggesting that this is a targetable salt-sensitive blood pressure, even in normotensive people, is a targetable trait. And because these monocytes are in blood, can we get a blood sample and routinely diagnose salt-sensitive blood pressure so that doctors are aware and they can appropriately advise patients.   Cindy St. Hilaire:        This was samples obviously taken from a blood draw, right? So they're circulating.   Annet Kirabo:             It was a blood draw, yes.   Cindy St. Hilaire:        What do you think about these immune cells, perhaps, native in the kidney? Do you think the small population of angry cells, like you said, is escaping from the kidney environment? What do you think?   Annet Kirabo:             When I was a post-doc in David Harrison's lab, we found that the most angry dendritic cells that contribute to this inflammation and hypertension are monocyte-derived. So that's why in the human study we focused on monocytes, because there are so many subtypes of dendritic cells, plasmacytoid dendritic, classical dendritic cells. We have studied all of these subtypes, and we have focused on monocyte-derived dendritic cells because they're the ones that seem to be contributing to this phenotype the most.                                     Cindy St. Hilaire:        You guys focused in on the NLRP3 inflammasome, which, obviously it's a really critical component broadly for the innate immune system. Do you think that this is going to be a targetable approach that can be leveraged for hypertension? Or do you think it's too broad? What do you think about that as a therapeutic potential?   Ashley Pitzer:              Even when you look in our paper, and we use a knockout model, where we use a completely global knockout model, put them on high salt, and we give them back only dendritic cells that are from wild-type mice, so they have that NLRP3, that have been exposed to high salt. We were able to increase blood pressure, but I also did, in mice, where I gave them an IL-1 beta neutralizing antibody, similar to canakinumab, which is the CANTOS trial, and there's not much of a difference. There is, but it's minor. It's very minor.   Ashley Pitzer:              So, to be able to target in specific cell types in humans one thing, it's very difficult, and maybe one day we can get there. But I think it at least gives us a better idea of what is the full picture, what's the big mechanism going on with immune cells? In part of our human study, we are looking at something to try and be able to identify who is salt sensitive. So if anything, we're able to sit here and potentially have a way of identifying salt-sensitive patients, where, right now, all we can do is have them come in like we do and do a 3-day study, and not everybody can do that.   Annet Kirabo:             To add onto that, perhaps you know, we are talking about precision medicine. This is an era of precision medicine where you need to really tailor treatments if we can get there, and I think this is one way. CANTOS trial. They had no way of knowing who is salt-sensitive and who is not, it was a global approach, and the lack of differences in blood pressure might be explained that this IL-1 beta pathway is targetable in a specific population whose blood pressure is probably driven by inflammation. There are so many, many mechanisms that drive hypertension, and so perhaps we need to focus this on salt-sensitive people, and maybe we can really use this approach to target. Plus, this is ENaC-dependent.   As you know, amiloride has lost favor in the clinic as a treatment of hypertension, because in the majority, it's not effective. But studies have shown that in Black men, for example, who had been categorized salt-resistant, when they give them amiloride, their blood pressure went down, and yet it's not effective in the majority of the people.   So, can we bring back, can we take another look at amiloride. As our studies indicate that blockade of ENaC is anti-inflammatory and it's also antioxidant agent, can we at least bring back amiloride and look at it again and we focus it for specific populations of people that may be more prone to salt-sensitive hypertension?   Here we have so many targets for potential precision treatment of salt-sensitive potential in this paper. You can target SGK1, which we know is possible, we listed a number of clinical trials that they have used NLRP3 inflammasome inhibitors, you can use amiloride for these people, and you can also potentially scavenge IsoLGs.     Cindy St. Hilaire:        What was the most challenging aspect of this study? There's a lot of moving parts, so what was the biggest challenge? And then, also, what was the most surprising part or the most pleasantly surprising part?   Ashley Pitzer:              You have to think, most of this was going on right when the pandemic hit. And right before that, we had started our human recruitment for the human study. And so that put a little bit of a time damper on it.   Ashley Pitzer:              Other than that, it was just, we were finding one thing, developing a new experiment, doing it again, doing it again. And honestly, what was the most surprising and rewarding was just seeing the same thing in, because we took just PBMCs from normotensive patients, treated them with high salt, and saw the changes that we did with the inflammasome. And to see that exactly again in an in vivo model of giving patients high salt and seeing the same thing, it was very rewarding and confirmed that, okay, we're on the right path. Seeing the same thing over and over and over again, it kind of reaffirms that you had a good idea.   Annet Kirabo:             I might add, one of the most challenging was, initially, the computational. Oh, part of the pandemic I was, the pandemic hit, I had a baby during the pandemic, and it was my time to leave my home, and then all these things were going on. We had a clinical trial where patients had to come in. Vanderbilt was so super supportive ,even checking for COVID-19. Our patients could not have COVID-19. We needed to check them.   Cindy St. Hilaire:        Yeah.   Annet Kirabo:             They also had to check for COVID-19. And so during that time, I realized, wait, I need learn computation analysis. I realized I cannot learn, and then reached out to collaborators that helped. That was extremely challenging. And then the other challenging thing that we faced later during the pandemic is vaccinations. In our criteria, these people cannot be vaccinated for reasons. We've studied inflammation, hypertension, and so vaccination was confounding. And even COVID-19 is even more for confounding. So we had this exclusion criteria where we could not recruit anyone.   Annet Kirabo:             Everybody was having COVID, everybody was being vaccinated, and everybody was in that exclusion criteria, so it was difficult to get people. We have had some slow down, but right now it's beginning to build up.   Cindy St. Hilaire:        So, what's next? What's the next question?   Annet Kirabo:             We have so many.   Cindy St. Hilaire:        That means it was a great study. If you have more, that means it was a great study.   Annet Kirabo:             Yeah. This study and us, it kind of warms. The inside seat just opened up, we have primary data in the genetic regulation of ENaC, we have primary data where we found. We are trying to figure out the specific ENaC channel in these antigen-presenting cells. We don't know. We found that ENaC delta, for example, it's not found in a kidney or you talked about a kidney contribution versus immune cells. ENaC delta is not found in the kidney, but we have primary data that show that ENaC delta is the most correlated with cardiovascular risk, is the most correlated with kidney disease and all forms of hypertension. So now we're like, ENaC delta expressed in the immune cells, not in the kidney, it is the one that is most involved in cardiovascular disease, so how are we going to tell the world that.   Cindy St. Hilaire:        Yeah, very cool.   Annet Kirabo:             Those cells, not necessarily the kidney. The kidney plays a part because the cells are going there, but it's very, very exciting. Plus a number of other lines that we are investigating.   Cindy St. Hilaire:        It's great. Well, congratulations, again, on this publication, on just getting all this done with what sounds like extremely difficult patient recruitment. So, Dr Kirabo and Dr Pitzer, thank you so much for joining me today and I'm looking forward to these next studies on maybe ENaC delta.   Annet Kirabo:             Thank you. Thank you so much.   Ashley Pitzer:              Thank you for having us.   Cindy St. Hilaire:        That's it for the highlights from the August 5th and August 19th issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and hashtag Discover CircRes. Thank you to our guests, Dr Annet Kirabo and Dr Ashley Pitzer.   This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on the go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2022. Opinions expressed by speakers in this podcast are their own, and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.  

This month on Episode 38 of Discover CircRes, host Cynthia St. Hilaire highlights original research articles featured in the Jue 24th, July 8th and July 22nd issues of the journal. This episode also features an interview with the 2022 BCBS Outstanding Early Career Investigator Award finalists, Dr Hisayuki Hashimoto, Dr Matthew DeBerge and Dr Anja Karlstadt.   Article highlights:   Nguyen, et al. miR-223 in Atherosclerosis.   Choi, et al. Mechanism for Piezo1-Mediated Lymphatic Sprouting   Kamtchum-Tatuene, et al.  Plasma Interleukin-6 and High-Risk Carotid Plaques   Li, et al. 3-MST Modulates BCAA Catabolism in HFrEF   Cindy St. Hilaire:        Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, from the Vascular Medicine Institute at the University of Pittsburgh. And today I'm going to be highlighting articles from our June 24th, July 8th and July 22nd issues of Circulation Research. I'm also going to have a chat with the finalists for the 2022 BCBS Outstanding Early Career Investigator Award, Dr Hisayuki Hashimoto, Dr Matthew DeBerge and Dr Anja Karlstadt.   Cindy St. Hilaire:        The first article I want to share is from our June 24th issue and is titled, miR-223 Exerts Translational Control of Proatherogenic Genes in Macrophages. The first authors are My-Anh Nguyen and Huy-Dung Hoang, and the corresponding author is Katey Rayner and they're from the University of Ottawa. A combination of cholesterol accumulation in the blood vessels and subsequent chronic inflammation that's derived from this accumulation drive the progression of atherosclerosis. Unfortunately, current standard medications tackle just one of these factors, the cholesterol. And this might explain why many patients on such drugs still have vascular plaques. In considering treatments that work on both aspects of the disease, meaning lipid accumulation and inflammation, this group investigated the micro RNA 223 or miR-223, which is a small regulatory RNA that has been shown to suppress expression of genes involved in both cholesterol uptake and inflammatory pathways in both liver and immune cells.   Cindy St. Hilaire:        The team showed that mouse macrophages deficient in miR-223, exhibited increased expression of pro-inflammatory cytokines and reduced cholesterol efflux compared with control cells. Overexpression of miR-223 had the opposite effects. Furthermore, atherosclerosis prone mice, whose hematopoietic cells lacked miR-223, had worse atherosclerosis with larger plaques and higher levels of pro-inflammatory cytokines than to control animals with normal levels of miR-223. These findings highlight miR-223's dual prompt, antiatherogenic action, which could be leveraged for future therapies.   Cindy St. Hilaire:        The second article I want to share is from our July 8th issue of Circulation Research and is titled, Piezo1-Regulated Mechanotransduction Controls Flow-Activated Lymph Expansion. The first author is Dongwon Choi and the corresponding author is Young-Kwon Hong, and they're from UCLA.   As well as being super highways for immune cells, lymph vessels are drainage channels that help maintain fluid homeostasis in the tissues. This network of branching tubes grows as fluids begin to flow in the developing embryo. This fluid flow induces calcium influx into the lymphatic endothelial cells, which in turn promotes proliferation and migration of these cells, leading to the sprouting of lymph tubules. But how do LECs, the lymphatic endothelial cells, detect fluid flow in the first place? Piezo1 is a flow and mechanosensing protein known for its role in blood vessel development and certain mutations in Piezo1 cause abnormal lymphatic growth in humans.   Cindy St. Hilaire:        This script found that Piezo1 is expressed in the embryonic mouse LECs and that the suppression of Piezo1 inhibits both flow activated calcium entry via the channel ORAI1, as well as downstream target gene activation. Overexpression of Piezo1, by contrast, induced the target genes. The team went on to show that mice lacking either Piezo1 or ORAI1 had lymphatic sprouting defects and that pharmacological activation of Piezo1 in mice enhanced lymphogenesis and prevented edema after tail surgery. Together, the results confirmed Piezo1's role in flow dependent lymphatic growth and suggest it might be a target for treating lymphedema.   Cindy St. Hilaire:        The third article I want to share is also from our July 8th issue and is titled, Interleukin-6 Predicts Carotid Plaque Severity, Vulnerability and Progression. The first and corresponding author of this study is Joseph Kamtchum-Tatuene from University of Alberta.   Excessive plasma cholesterol and systemic inflammation are contributing factors in atherosclerosis. While traditional remedies have been aimed at lowering patient's lipid levels, drugs that tackle inflammation are now under investigation, including those that suppress Interleukin-6, which is an inflammatory cytokine implicated in the disease. Focusing on carotid artery disease, this group conducted a prospective study to determine whether IL-6 levels correlated with disease severity. 4,334 individuals were enrolled in the cardiovascular health study cohort. They had their blood drawn and ultrasounds taken at the start of the study and five years later. This group found IL-6 was robustly correlated with and predicted plaque severity independent of other cardiovascular risk factors. This study also determined that an IL-6 blood plasma level of 2.0 picograms/mls, identified individuals with the highest likelihood of plaque, vulnerability and progression. This threshold value could be used to select patients who might benefit from novel IL-6 lowering medications.   Cindy St. Hilaire:        The last article I want to share is from our July 22nd issue of Circulation Research and is titled, Mitochondrial H2S Regulates BCAA Catabolism in Heart Failure. The first author is Zhen Li, and the corresponding author is David Lefer from Louisiana State University. Hydrogen sulfide, or H2S, is a compound that exerts mitochondrial specific actions that include the preservation of oxidative phosphorylation, mitochondrial biogenesis and ATP synthesis, as well as inhibiting cell death. 3-mercaptopyruvate sulfurtransferase, or 3-MST, is a mitochondrial H2S producing enzyme, whose functions in cardiovascular disease are not fully understood.   Cindy St. Hilaire:        This group investigated the global effects of 3-MST deficiency in the setting of pressure overload induced heart failure. They found that 3-MST was significantly reduced in the myocardium of patients with heart failure, compared with non failing controls. 3-MST knockout mice exhibited increased accumulation of branch chain amino acids in the myocardium, which was associated with reduced myocardial respiration and ATP synthesis, exacerbated cardiac and vascular dysfunction, and worsened exercise performance, following transverse aortic constriction. Restoring myocardial branched-chain amino acid catabolism, or administration of a potent H2S donor, ameliorated the detrimental effects of 3-MST deficiency and heart failure with reduced injection fraction. These data suggest that 3-MST derived mitochondrial H2S, may play a regulatory role in branch chain amino acid catabolism, and mediate critical cardiovascular protection in heart failure.   Cindy St. Hilaire:        Today, I'm really excited to have our guests, who are the finalists for the BCVS Outstanding Early Career Investigator Awards. Welcome everyone.   Hisayuki Hashimoto:   Thank you.   Anja Karlstaedt:          Hi.   Hisayuki Hashimoto:   Hi.   Matthew DeBerge:      Hello. Thank you.   Cindy St. Hilaire:        So the finalists who are with me today are Dr Hisayuki Hashimoto from Keio University School of Medicine in Tokyo, Japan, Dr Matthew Deberge from Northwestern University in Chicago and Dr Anja Karlstaedt from Cedar Sinai Medical Center in LA. Thank you again. Congratulations. And I'm really excited to talk about your science.   Hisayuki Hashimoto:   Thank you. Yes. Thanks, first of all for this opportunity to join this really exciting group and to talk about myself and ourselves. I am Hisayuki Hashimoto, I'm from Tokyo, Japan. I actually learned my English... I went to an American school in a country called Zaire in Africa and also Paris, France because my father was a diplomat and I learned English there. After coming back to Japan, I went to medical school. During my first year of rotation, I was really interested in cardiology, so I decided to take a specialized course for cardiology. Then I got interested in basic science, so I took a PhD course, and that's what brought me to this cardiology cardiovascular research field.   Matthew DeBerge:      So I'm currently a research assistant professor at Northwestern University. I'm actually from the Chicagoland area, so I'm really excited to welcome you all to my hometown for the BCVS meeting.   Cindy St. Hilaire:        Oh, that's right. And AHA is also there too this year. So you'll see a lot of everybody.   Matthew DeBerge:      I guess I get the home field advantage, so to speak. So, I grew up here, I did my undergrad here, and then went out in the east coast, Dartmouth College in New Hampshire for my PhD training. And actually, I was a viral immunologist by training, so I did T cells. When I was looking for a postdoctoral position, I was looking for a little bit of something different and came across Dr Edward Thorpe's lab at Northwestern university, where the interest and the focus is macrophages in tissue repair after MI. So, got into the macrophages in the heart and have really enjoyed the studies here and have arisen as a research assistant professor now within the Thorpe lab. Now we're looking to transition my own independent trajectory. Kind of now looking beyond just the heart and focusing how cardiovascular disease affects other organs, including the brain. That's kind of where I'm starting to go now. Next is looking at the cardiovascular crosstalk with brain and how this influences neuroinflammation.   Anja Karlstaedt:          I am like Hisayuki, I'm also a medical doctor. I did my medical training and my PhD in Berlin at the Charité University Medicine in Berlin, which is a medical faculty from Humboldt University and Freie University. II got really interested in mathematical modeling of complex biological systems. And so I started doing my PhD around cardiac metabolism and that was a purely core and computationally based PhD. And while I was doing this, I got really hooked into metabolism. I wanted to do my own experiments to further advance the model, but also to study more in crosstalk cardiac metabolism. I joined Dr Heinrich Taegteyer lab at the University of Texas in the Texas Medical Center, and stayed there for a couple of years. And while I was discovering some of the very first interactions between leukemia cells and the heart, I decided I cannot stop. I cannot go back just after a year. I need to continue this project and need to get funding. And so after an AHA fellowship and NIHK99, I am now here at Cedars Sinai, an assistant professor in cardiology and also with a cross appointment at the cancer center and basically living the dream of doing translational research and working in cardio-oncology.   Cindy St. Hilaire:        Great. So, Dr Hashimoto, the title of your submission is, Cardiac Reprogramming Inducer ZNF281 is Indispensable for Heart Development by Interacting with Key Cardiac Transcriptional Factors. This is obviously focused on reprogramming, but why do we care about cardiac reprogramming and what exactly did you find about this inducer ZNF281?   Hisayuki Hashimoto:   Thank you for the question. So, I mean, as I said, I'm a cardiologist and I was always interested in working heart regeneration. At first, I was working with pluripotent stem cells derived cardiomyocyte, but then I changed my field during my postdoc into directly programming by making cardiomyocyte-like cells from fiberblast. But after working in that field, I kind of found that it was a very interesting field that we do artificially make a cardiomyocyte-like cell. But when I dissected the enhanced landscape, epigenetic analysis showed that there are very strong commonalities between cardiac reprogramming and heart development. So I thought that, hey, maybe we can use this as a tool to discover new networks of heart development. And the strength is that cardiac reprogramming in vitro assay hardly opens in vivo assay, so it's really time consuming. But using dark programming, we can save a lot of time and money to study the cardiac transitional networks. And we found this DNF281 from an unbiased screen, out of 1000 human open reading frames. And we found that this gene was a very strong cardiac reprogramming inducer, but there was no study reporting about any functioning heart development. We decided to study this gene in heart development, and we found out that it is an essential gene in heart development and we were kind of able to discover a new network in heart development.   Cindy St. Hilaire:        And you actually used, I think it was three different CRE drivers? Was that correct to study?   Hisayuki Hashimoto:   Ah, yes. Yeah.   Cindy St. Hilaire:        How did you pick those different drivers and what, I guess, cell population or progenitor cell population did those drivers target?   Hisayuki Hashimoto:   So I decided to use a mesodermal Cre-driver, which is a Mesp1Cre and a cardiac precursor Cre-driver, which is the Nkx2-5 Cre and the cardiomyocyte Cre, which is the Myh6-Cre. So three differentiation stages during heart development, and we found out that actually, DNF281 is an essential factor during mesodermal to cardiac precursor differentiation state. We're still trying to dig into the molecular mechanism, but at that stage, if the DNF281 is not there, we are not able to make up the heart.   Cindy St. Hilaire:        That is so interesting. Did you look at any of the strains that survived anyway? Did you look at any phenotypes that might present in adulthood? Is there anything where the various strains might have survived, but then there's a kind of longer-term disease implicating phenotype that's observed.   Hisayuki Hashimoto:   Well, thank you for the question. Actually, the mesodermal Cre-driver knocking out the DNF281 in that stage is embryonic lethal, and it does make different congenital heart disease. And they cannot survive until after embryonic day 14.5. The later stage Nkx2-5 Cre and Myh6-Cre, interestingly, they do survive after birth. And then in adult stage, I did also look into the tissues, but the heart is functioning normally. I haven't stressed them, but they develop and they're alive after one year. It looks like there's really no like phenotype at like the homeostatic status.   Cindy St. Hilaire:        Interesting. So it's kind of like, once they get over that developmental hump, they're okay.   Hisayuki Hashimoto:   Exactly. That might also give us an answer. What kind of network is important for cardiac reprogramming?   Cindy St. Hilaire:        So what are you going to do next?   Hisayuki Hashimoto:   Thank you. I'm actually trying to dig into the transitional network of what kind of cardiac transitional network the ZNF281 is interacting with, so that maybe I can find a new answer to any etiology of congenital heart disease, because even from a single gene, different mutation, different variants arise different phenotypes in congenital heart disease. Maybe if I find a new interaction with any key cardiac transitional factors, maybe I could find a new etiology of congenital heart disease phenotype.   Cindy St. Hilaire:        That would be wonderful. Well, best of luck with that. Congratulations on an excellent study. Hisayuki Hashimoto:   Thank you.   Cindy St. Hilaire:        Dr DeBerge, your study was titled, Unbiased Discovery of Allograft Inflammatory Factor-1 as a New and Critical Immuno Metabolic Regulatory Node During Cardiac Injury. Congrats on this very cool study. You were really kind of focused on macrophages in myocardial infarction. And macrophages, they're a Jeckel Hyde kind of cell, right? They're good. They're bad. They can be both, almost at the same time, sometimes it seems like. So why were you interested in macrophages particularly in myocardial infarction, and what did you discover about this allograft inflammatory factor-1, or AIF1 protein?   Matthew DeBerge:      Thank you. That's the great question. You really kind of alluded to why we're interested in macrophages in the heart after tissue repair. I mean, they really are the central mediators at both pro-inflammatory and anti-inflammatory responses after myocardial infarction. Decades of research before this have shown that inflammation has increased acutely after MI and has also increased in heart failure patients, which really has led to the development of clinical efforts to target inflammatory mediators after MI. Now, unfortunately, the results to target inflammation after MI, thus far, have been modest or disappointing, I guess, at worst, in the respect that broadly targeting macrophage function, again, hasn't achieved results. Again, because these cells have both pro and anti-inflammatory functions and targeting specific mediators has been somewhat effective, but really hasn't achieved the results we want to see.   Matthew DeBerge:      I think what we've learned is that the key, I guess, the targeting macrophage after MI, is really to target their specific function. And this led us to sort of pursue novel proteins that are mediating macrophage factor function after MI. To accomplish this, we similarly performed an unbiased screen collecting peri-infarct tissue from a patient that was undergoing heart transplantation for end stage heart failure and had suffered an MI years previously. And this led to the discovery of allograft inflammatory factor-1, or AIF1, specifically within cardiac macrophages compared to other cardiac cell clusters from our specimen. And following up with this with post-mortem specimens after acute MI to show that AIF1 was specifically increased in macrophages after MI and then subsequently then testing causality with both murine model of permanent inclusion MI, as well as in vitro studies using bone marrow drive macrophages to dig deeper mechanistically, we found that AIF1 was crucial in regulating inflammatory programing macrophages, which ultimately culminated in worse in cardiac repair after MI.   Cindy St. Hilaire:        That's really interesting. And I love how you start with the human and then figure out what the heck it's doing in the human. And one of the things you ended up doing in the mouse was knocking out this protein AIF1, specifically in macrophage cells or cells that make the macrophage lineage. But is this factor in other cells? I was reading, it can be intracellular, it can be secreted. Are there perhaps other things that are also going on outside of the macrophage?   Matthew DeBerge:      It's a great question. First, I guess in terms of specificity, within the hematopoietic compartment, previous studies, as well as publicly available databases, have shown that AIF1 is really predominantly expressed within macrophages. We were able to leverage bone marrow chimera mice to isolate this defect to the deficiency to macrophages. But you do bring up a great point that other studies have shown that AIF1 may be expressed in other radio-resistant cell populations. I mean, such as cardiomyocytes or other treatable cells within the heart. We can't completely rule out a role for AIF1 and other cell populations. I can tell you that we did do the whole body knockout complementary to our bone marrow hematopoetic deficient knockouts, and saw that deficiency of AIF1 within the whole animal, recapitulate the effects we saw within the AIF1 deficiency within hematopoietic department.   Matthew DeBerge:      It was encouraging to us that, again, the overall role of AIF1 is pro-inflammatory after MI.   Cindy St. Hilaire:        I mean, I know it's early days, but is there a hint of any translational potential of these findings or of this protein?   Matthew DeBerge:      Yeah, I think so. To answer your question, we were fortunate enough to be able to partner with Ionis that develops these anti-sensible nucleotides so that we could specifically target AIF1 after the acute phase during MI. We saw that utilizing these anti-sensible nucleotides to deplete AIF1, again, within the whole mouse, that we were able to reduce inflammation, reduce in heart size and preserve stock function. I think there really is, hopefully a therapeutic opportunity here. And again, with it being, perhaps macrophage specific is, even much more important as we think about targeting the specific function of these cells within the heart.   Cindy St. Hilaire:        Very cool stuff. Dr Karlstaedt, the title of your submission is, ATP Dependent Citrate Lyase Drives Metabolic Remodeling in the Heart During Cancer. So this I found was really interesting because you were talking about, the two major killers in the world, right? Cardiovascular disease and cancer, and you're just going to tackle both of them, which I love. So obviously this is built on a lot of prior observations about the effects of cancer on cardiac metabolic remodeling. Can you maybe just tell us a little bit about what is that link that was there and what was known before you started?   Anja Karlstaedt:          Yeah. Happy to take that question. I think it's a very important one and I'm not sure if I will have a comprehensive answer to this, because like I mentioned at the beginning, cardio-oncology is a very new field. And the reason why we are starting to be more aware of cancer patients and their specific cardiovascular problems is because the cancer field has done such a great job of developing all these new therapeutics. And we have far more options of treating patients with various different types of cancers in particular, also leukemias, but also solid tumors. And what has that led to is an understanding that patients survive the tumors, but then 10, 20 years later, are dying of cardiovascular diseases. Those are particular cardiomyopathies and congestive heart failure patients. What we are trying, or what my lab is trying to do, is understanding what is driving this remodeling. And is there a way that we can develop therapies that can basically, at the beginning of the therapy, protect the heart so that this remodeling does not happen, or it is not as severe.   Anja Karlstaedt:          Also, identifying patients that are at risk, because not every tumor is created equally and tumors are very heterogeneous, even within the same group. To get to your question, what we found is, in collaboration actually with a group at Baylor College of Medicine, Peggy Goodell's group, who is primarily working on myeloid malignancies, is that certain types of leukemias are associated with cardiomyopathies. And so when they were focusing on the understanding drivers of leukemia, they noticed that the hearts of these animals in their murine models are enlarged on and actually developing cardiomyopathies. And I joined this project just very early on during my postdoc, which was very fortunate and I feel very lucky of having met them. What my lab is now studying here at Cedars is how basically those physiological stress and mutations coming from the tumors are leading to metabolic dysregulation in the heart and then eventually disease.   Anja Karlstaedt:          And we really think that metabolism is at the center of those disease progressions and also, because it's at the center, it should be part of the solution. We can use it as a way to identify patients that are at risk, but also potentially develop new therapies. And what was really striking for us is that when we knock down ACLY that in a willdtype heart where the mouse doesn't have any tumor disease, ACLY actually is critically important for energy substrate metabolism, which seems counterintuitive, because it's far away from the mitochondria, it's not part of directly ADP provision. It's not part of the Kreb cycle. But what we found is that when we knock it out using a CRISPR-Cas9 model, it leads to cardiomyopathy and critically disrupts energy substrate metabolism. And that is not necessarily the case when the mouse has leukemia or has a colorectal cancer, which upregulated in the beginning, this enzyme expression. And so we have now developed models that show us that this could be potentially also therapeutic target to disrupt the adverse remodeling by the tumor.   Cindy St. Hilaire:        That is so interesting. So one of the things I was thinking about too is we know that, I mean, your study is showing that, the tumor itself is causing cardiac remodeling, but we also know therapies, right? Radiation, chemotherapy, probably some immune modulatory compounds. Those probably do similar, maybe not exactly similar, but they also cause, adverse cardiac remodeling. Do you have any insights as to what is same and what is different between tumor driven and therapy driven adverse remodeling?   Anja Karlstaedt:          So we do not know a lot yet. It's still an open question about all the different types of chemotherapeutics, how they are leading to cardio toxicities. But what we know, at least from the classic anti-cyclic treatments, is right now at the core, the knowledge is that this is primarily disrupting cardiac mitochondrial function. And through that again, impairing energy provision and the interaction, again, with the immune system is fairly unknown, but we know through studies from Kathryn Moore and some very interesting work by Rimson is that myocardial infarction itself can lead to an increase in risk for tumor progression. And what they have shown as independent of each other, is that the activation of the immune system in itself can lead to an acceleration of both diseases, both the cardiac remodeling, and then also the tumor disease. We don't fully understand which drivers are involved, but we do know that a lot of the cardiomyopathies on cardiotoxicities that are chemotherapeutically driven, all have also metabolic component.   Cindy St. Hilaire:        Nice. Thank you. When I prepare for these interviews, I obviously read the abstracts for the papers, but I found myself also Googling other things after I read each of your abstracts. It was a rabbit hole of science, which was really exciting.                               I now want to transition to kind of a career angle. You all are obviously quite successful, scientifically, at the bench, right? But now you are pivoting to a kind of completely opposite slash new job, right? That of, independent researcher. I would love to hear from each of you, if there was any interesting challenge that you kind of overcame that you grew from, or if there was any bit of advice that you wish you knew ahead of time or anything like that, that some of our trainee listeners and actually frankly, faculty who can pass that information onto their trainees, can benefit from.   Anja Karlstaedt:          I think the biggest challenge for me in transitioning was actually the pandemic. Because I don't know how it was for Hisa and Matt, but trying to establish a lab, but also applying for faculty position during a major global pandemic, is challenging is not quite something that I expected that would happen. And so I think saying that and looking more conceptually and philosophically at this as, you can prepare as much as you want, but then when life just kicks in and things happen, they do happen. And I think the best is to prepare as much as you can. And then simply go with the flow. Sometimes one of my mentors, Dave Nikon, mentioned that to me when I was applying for faculty positions, it's sometimes good to just go with the flow. And as a metabolism person, I absolutely agree. And there are some things that you can do as a junior investigator.   Anja Karlstaedt:          We need to have a good network. So just very important to have good mentors. I was blessed with have those mentors, Peggy Goodell's one of them, Heinrich Taegtmeyer was another. And now with this study that we are publishing, Jim Martin and Dave Nikon were incredible. Without them, this study wouldn't have been possible and I would not be here at Cedars.   Anja Karlstaedt:          You need to reach out to other people because those mentors have the experience. They have been through some of this before. Even if they have never had a major event, like COVID-19 in their life before, because none of us had before, they had other experiences and you can rely on them and they set you then up for overcoming these challenges. And the other thing I would say, is put yourself out there, go and talk to as many people as possible or set conferences, present a poster, not only talks. Don't be disappointed if you don't get a talk, posters are really great to build this network and find other people that you probably wouldn't have encountered and apply for funding. Just again, put yourself out there and try to get the funding for your research. Even if it's small foundations, it builds up over time and it is a good practice to then write those more competitive grants.     Cindy St. Hilaire:        Dr Hashimoto, would you like to go next?   Hisayuki Hashimoto:   Just my advice is that, could be like a culture of difference, but in east Asia, like in Japan, we were taught to, do not disturb people, don't interrupt people and help people. But I realized that I wasn't really good at asking for help. After I am still not like fully independent, but I do have my own group and I have to do grant writing. I still work at the bench and then have to teach grad students, doing everything myself. I just realized it's just impossible. I didn't have time. I need like 48 hours a day. Otherwise, you won't finish it. I just realized that I wasn't really good at asking for help. So my advice would be, don't hesitate to ask for help. It's not a shame. You can't do everything by just yourself. I think, even from the postdoc, even from grad school, I think, ask for help and then get used to that. And then of course, help others. And that is the way I think to probably not get overwhelmed and not stress yourself. Science should be something fun. And if you don't ask for help and if you don't help someone, I think you are losing the chance of getting some fun part from the science.   Cindy St. Hilaire:        That's great advice. I really like that, especially because I find at least, I started my lab seven years ago now. And I remember the first couple months/year, it was extremely hard to let go, right? Like I taught my new people how to do the primary cell culture we needed, but I was terrified of them doing it wrong or wasting money or making too many mistakes. But you realize, you got to learn to trust people. Like you said, you got to learn to ask for help. And sometimes that help is letting them do it. And you doing, you're being paid now to write grants and papers. That's a big brain, you're not paid to do the smaller things. That's really great advice. I like that. Thank you. Dr DeBerge, how about you?   Matthew DeBerge:      So I guess towards a bit of life advice, I think two obvious things is one, be kind, science is hard enough as it is. So I think we should try to lift each other up and not knock each other down. And along those lines as the others have alluded to as well, one of the mantras we sort of adapted on the lab, is a rising tide raises all ships, this idea that we can work together to elevate each other's science and really, again, collaborate.   Towards the career side of things I'll just touch on, because I guess one thing I'll add, there's more than one path, I guess, to achieving your goals. I've been fortunate enough to have an NIH post-doctoral fellowship and had an AHA career development award, but I'm not a K99 recipient. Oftentimes, I think this is the golden ticket to getting the faculty job, so I'm trying to, I guess, buck trend, I just submitted an RO1. So fingers crossed that leads to some opportunity.   Even beyond academia, I'm not certain how much everyone here is involved in science Twitter, it's really become a thing over the last couple years, but I think, kind of the elephant in the room is that academia, it's really hard on the trainees nowadays to have a living wage, to go through this. I mean, I'm really excited to see my, fellow finalists here are starting their own groups and stuff, but for many, that's not the reality for many, it's just not financially feasible. So I think, kind of keeping in mind that there's many, many alternative careers, whether it's industry, whether it's consulting, science writing, etcetera, going back to what Dr Hash says, find what you love and really pursue that with passion.   Cindy St. Hilaire:        I think it's something only, I don't know, five to 10% of people go into or rather stay in academia. And that means, 90 to 95% of our trainees, we need to prepare them for other opportunities, which I think is exciting, because it means it can expand our network for those of us in academia.   Anja Karlstaedt:          I think right now it's even worse because it's about 2% of old postdocs that are actually staying and becoming independent researchers, independent or tenure track or research track. And I think I second, as what Matt said, because I play cello. I do music as a hobby and people always ask me if I'm a musician. And at the beginning I felt like, no, of course not. I'm not like Yoyo Ma. I'm just playing, it's a hobby. And then I, that got me thinking. I was like, no, of course you are because there's so many different types. And what we need to understand is that scientists, like you are always a scientist. It doesn't matter if you are working at Pfizer or if you are working at a small undergrad institution and you're teaching those next generation scientists, you are still scientist and we all need those different types of scientists because otherwise, if everybody is just a soloist, you are never going to listen to symphony. You need those different people and what we need to normalize beyond having those different career paths, is also that people are staying in academia and becoming those really incredible resources for the institutions and labs, quite frankly, of being able to retain those technologies and techniques within an institution. And I think that's something to also look forward to, that even if you're not the PI necessarily, you're the one who is driving those projects. And I hope to pass this on at some point also to my trainees that they can be a scientist, even if they're not running a lab and they become an Institute director and that's also critically important.   Cindy St. Hilaire:        There's lots of ways to do science. Thank you all so much for joining me today. Either waking up at 5:00 AM or staying up past midnight, I think it is now in Japan or close to it. So Matt and I kind of made it out okay. It's like 8:00 or 9:00 AM.   Matthew DeBerge:      Thank you.   Hisayuki Hashimoto:   My apologies for this time zone difference.   Cindy St. Hilaire:        I'm very glad to make it work. Congratulations to all of you, your presentations. I forget which day of the week they are on at BCVS, but we are looking forward to the oral presentations of these and congratulations to all of you. You are amazing scientists and I know I'm really looking forward to seeing your future work so best of luck.   Matthew DeBerge:      Thank you.   Hisayuki Hashimoto:   Thank you.   Anja Karlstaedt:          Thank you so much.   Cindy St. Hilaire:        That's it for the highlights from the June 24th, July 8th and July 22nd issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle at CircRes and hashtag Discover CircRes. Thank you to our guests. The BCVS Outstanding Early Career Investigator Award Finalists, Dr Hisayuki Hashimoto, Dr Matthew DeBerge and Dr Anja Karlstaedt. This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire. And this is Discover CircRes, you're on the go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association, 2022. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information visit ahajournals.org.  

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This month on Episode 36 of Discover CircRes, host Cynthia St. Hilaire highlights original research articles featured in the April 29 and May 13 issues of Circulation Research. This episode also features a conversation with Dr Patricia Nguyen and Jessica D'Addabbo from Stanford University about their study, Human Coronary Plaque T-cells are Clonal and Cross-React to Virus and Self.   Article highlights:   Zanoli, et al. COVID-19 and Vascular Aging   Wang, et al. JP2NT Gene Therapy in a Mouse Heart Failure Mode   Harraz, et al. Piezo1 Is a Mechanosensor in CNS Capillaries   Zhao, et al. BAT sEVs in Exercise Cardioprotection   Cindy St. Hilaire:        Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cyndy St. Hilaire, from the Vascular Medicine Institute at the University of Pittsburgh. And today, I'll be highlighting the articles from our April 29th and May 13th issues of Circulation Research. I also will speak with Dr Patricia Nguyen and Jessica D'Addabbo from Stanford University about their study, Human Coronary Plaque T-cells are Clonal and Cross-React to Virus and Self.   Cindy St. Hilaire:        The first article I want to share is titled Vascular Dysfunction of COVID 19 Is Partially Reverted in the Long-Term. The first author is Agostino Gaudio and the corresponding author is Luca Zanoli. And they're from the University of Catania. Cardiovascular complications, such as endothelial dysfunction, arterial stiffness, thrombosis and heart disease are common in COVID 19. But how quickly such issues resolve, once the acute phase of the illness has passed, remains unclear. To find out, this group examined aortic and brachial pulse wave velocity, and other measures of arterial stiffness in 90 people who, several months earlier, had been hospitalized with COVID 19. These measurements were compared with data from 180 controls, matched for age, sex, ethnicity and body mass index, whose arterial stiffness had been assessed prior to the pandemic. 41 of the COVID patients were also examined 27 weeks later to assess any changes in arterial stiffness over time. Together, the data showed arterial stiffness was higher in COVID patients than in controls. And though it improved over time, it tended to remain higher than normal for almost a year after COVID.   Cindy St. Hilaire:        This finding could suggest residual structural damage to the arterial walls or possibly, persistent low-grade inflammation in COVID patients. Either way, since arterial stiffness is a predictor of cardiovascular health, its potential longterm effects in COVID patients deserves further longitudinal studies.   Cindy St. Hilaire:        The second article I want to share is titled Gene Therapy with the N-Terminus of Junctophilin-2 Improves Heart Failure in Mice. The first author is Jinxi Wang and the corresponding author is Long-Sheng Song from the University of Iowa. Junctophilin-2 is a protein with a split personality. Normally, it forms part of the heart's excitation contraction coupling machinery. But when the heart is stressed, JP2 literally splits in two, and sends its N-terminal domain, JP2NT, to the nucleus, where it suppresses transcription of genes involved in fibrosis, hypertrophy, inflammation and other heart failure related processes. However, if this stress is severe or sustained, the protective action of JP2NT is insufficient to halt the progressive failure. This group asked. "What if this N-terminal domain could be ramped up using gene therapy to aid a failing mouse heart?"   Cindy St. Hilaire:        To answer this question, they injected adenoviral vectors encoding JP2NT into mice either before or soon after transaortic constriction, or TAC, tack, which is a method of experimentally inducing heart failure. They found, in both cases, that the injected animals fared better than the controls. Animals injected before TAC showed less severe cardiac remodeling than control mice, while those treated soon after TAC exhibited slower loss of heart function with reduced ventricle dilation and fibrosis. These data suggest that supplementing JP2NT, via gene therapy or other means, could be a promising strategy for treating heart failure. And this data provides a basis for future translational studies.   Cindy St. Hilaire:        The third article I want to share is titled Piezo1 Is a Mechanosensor Channel in Central Nervous System Capillaries. The first and corresponding author is Osama Harraz from the University of Vermont. Neurovascular coupling is the process whereby transient activation of neurons leads to an upsurge in local blood flow to accommodate the increased metabolic needs of the cell. It's known that agents released from active neurons trigger changes in local capillaries that prompt vasodilation, but how these hemodynamic changes are sensed and controlled is not entirely clear. This group suspected that the mechanosensory protein Piezo1, a calcium channel that regulates dilation and constriction of other blood vessels, may be involved. But whether Piezo1 is even found in the microcirculation of the CNS was unknown. This group shows that Piezo1 is present in cortical capillaries of the brain and the retina of the mouse, and that it responds to changes in blood pressure and flow.   Cindy St. Hilaire:        Ex vivo preparations of mouse retina showed that experimentally induced changes in hemodynamics caused calcium transients and related currents within capillary endothelial cells, and that these were dependent on the presence of Piezo1. While it is not entirely clear how Piezo1 influences cerebral blood flow, its pressure induced activation of CNS capillary endothelial cells suggest a critical role in neurovascular coupling.   Cindy St. Hilaire:        The last article I want to share is titled Small Extracellular Vesicles from Brown Adipose Tissue Mediate Exercise Cardioprotection. The first authors are Hang Zhao and Xiyao Chen. And the corresponding authors are Fuyang Zhang and Ling Tao from the Fourth Military Medical University. Regular aerobic exercise is good for the heart and it increases the body's proportion of brown adipose tissue relative to white adipose tissue. This link has led to the idea that brown fat, possibly via its endocrinal activity, might somehow contribute to exercise related cardioprotection. Zhao and colleagues now show that, indeed, brown fat produces extracellular vesicles that are key to preserving heart health. While mice subjected to four weeks of aerobic exercise were better protected against subsequent heart injury than their sedentary counterparts, blocking the production of EVs prior to exercise significantly impaired this protection. Furthermore, injection of brown fat derived EVs into the hearts of mice lessened the impact of subsequent cardiac injury.   Cindy St. Hilaire:        The team went on to identify micro RNAs within the vesicles responsible for this protection, showing that the micro RNAs suppressed an apoptosis pathway in cardiomyocytes. In identifying mechanisms and molecules involved in exercise related cardio protection, the work will inform the development of exercise mimicking treatments for people at risk of heart disease or who are intolerant to exercise.   Cindy St. Hilaire:        Lastly, I want to bring up that the April 29th issue of Circulation Research also contains a short Review Series on pulmonary hypertension, with articles on: The Latest in Animal Models of Pulmonary Hypertension and Right Ventricular Failure, by Olivier Boucherat; Harnessing Big Data to Advance Treatment and Understanding of Pulmonary Hypertension, by Christopher Rhodes and colleagues; New Mutations and Pathogenesis of Pulmonary Hypertension: Progress and Puzzles in Disease Pathogenesis, by Christophe Guignabert and colleagues; Group 3 Pulmonary Hypertension From Bench to Bedside, by Corey Ventetuolo and colleagues; and Novel Approaches to Imaging the Pulmonary Vasculature and Right Heart, by Sudarshan Rajagopal and colleagues; and Understanding the Pathobiology of Pulmonary Hypertension Due to Left Heart Disease, by Jessica Huston and colleagues.   Cindy St. Hilaire:        Today, Dr Patricia Nguyen and Jessica D'Addabbo, from Sanford University, are with me to discuss their study, Human Coronary Plaque T-cells are Clonal and Cross-React to Virus and Self. And this article is in our May 13th issue of Circulation Research. So, Trisha and Jessica, thank you so much for joining me today.   Jessica D'Addabbo:    Thank you for having us.   Patricia Nguyen:         Yes. Thank you for inviting us to your podcast. We're very excited to be here.   Cindy St. Hilaire:        Yeah. And I know there's lots of authors involved in this study, so unfortunately we can't have everyone join us, but I appreciate you all taking the time.   Patricia Nguyen:         This is like a humongous effort by many people in the group, including Roshni Roy Chowdhury, and Xianxi Huang, as well as Charles Chan and Mark Davis. So, we thank you.   Cindy St. Hilaire:        So atherosclerosis, it stems from lipid deposition in the vascular wall. And that lipid deposition causes a whole bunch of things to happen that lead to a chronic inflammatory state. And there's many cells that can be inflammatory. And this study, your study, is really focusing on the role of T-cells in the atherosclerotic plaque. So, before we get into the nitty gritty details of your study, can you share with us, what is it that a T-cell does normally and what is it doing in a plaque? Or rather, let me rephrase that as, what did we know a T-cell was doing in a plaque before your study?   Patricia Nguyen:         So, T-cells, as you know, are members of the adaptive immune system. They are the master regulators of the entire immune system, secreting cytokines and other proteins to attract immune cells to a diseased portion of the body, for example. T-cells have been characterized in plaque previously, mainly with immunohisto chemistry. And their characterization has also been recently performed using single cell technologies. Those studies have been restricted to mainly mirroring studies, studies in mice in their aortic walls, in addition to human carotid arteries. So, it is well known that T cells are found in plaque and a lot of attention has been given to the macrophage subset as the innate immune D. But let's not forget the T-cell because they're actually composed about... 50% in the plaque are T-cells.   Patricia Nguyen:         And we were particularly interested in the T-cell population because we have a strong collaboration with Dr Mark Davis, who's actually the pioneer of T-cell biology and was the first to describe the T-cell receptor alpha beta receptor in his lab in the 1970s. So, he has developed many techniques to interrogate T-cell biology. And our collaboration with him has allowed us and enabled us to perform many of these single cell technologies. In addition, his colleague, Dr Chen, also was pivotal in helping us with the interrogation and understanding of the T-cells in plaque.   Cindy St. Hilaire:        And I think one of the really neat strengths of your study is that you used human coronary artery plaques. So, could you walk us through? What was that like? I collect a lot of human tissue in my lab. I get a lot of aortic valves from the clinic. And it's a lot of logistics. And a lot of times, we're just fixing them, but you are not just fixing them. So, can you walk us through? What was that experimental process from the patient to the Petri dish? And also, could you tell us a little bit about your patient population that you sampled from?   Jessica D'Addabbo:    So, these were coronary arteries that we got from patients receiving a heart transplant. So, they were getting a heart transplant for various reasons, and we would receive their old heart, and someone would help us dissect out the coronary arteries from these. And then, we would process each of these coronary arteries separately. And this happened at whatever hour the hearts came out of the patient.   Jessica D'Addabbo:    So sometime, I was coming in at 3:00 AM with Dr Nguyen and we would be working on these hearts then, because we wanted the samples to be as fresh as possible. So, we would get the arteries. We would digest out the tissue. And then, we would have certain staining profiles that we wanted to look at so that we could put the cells on fax to be able to sort the cells, and then do all the downstream sequencing from there.   Cindy St. Hilaire:        So, in terms of, I don't know, the time when you get that phone call that a heart's coming in to actually getting those single cells that you can either send a fax or send a sequencing, how long did that take, on a good day? Let's talk only about good days.   Jessica D'Addabbo:    Yeah. A lot of factors went into that, sometimes depending on availability of things. But usually, we were ready with all of the materials in advance. So, I'd say it could be anywhere from six to 12 hours, it would take, to get everything sorted. Then, everything after that would happen. But that was just that critical period of making sure we got the cells fresh.   Patricia Nguyen:         So we have to credit the CT surgeons at Stanford for setting up the program or the structure, infrastructure, that enables us to obtain this precious tissue. That is Jack Boyd and Joseph Woo of CT surgery. So, they have enabled human research on hearts by making these tissues available. Because as you know, a transplant... They can say the transplant's happening at 12:00 AM, but it actually doesn't happen until 4:00 AM. And I think it's very difficult for a lab to make that happen all the time. And I think having their support in this paper was critical. And this has allowed us, enabled us, to interrogate kind of the spectrum of disease, especially focusing on T-cells, which are... They make a portion of the plaque, but the plaque itself has not like a million cells that are immune. A lot of them are not immune. So, enabling us to get the tissue in a timely fashion where they're not out of the body for more than 30 minutes enables us to interrogate these small populations of cells.   Cindy St. Hilaire:        That's actually the perfect segue to my next question, which is, how many cells in a plaque were you able to investigate with the single cell analysis? And what was the percentage again of the T-cells in those plaques or in... I guess you looked at different phases of plaque. So, what was that spectrum for the percentage of T-cells?   Patricia Nguyen:         So, for 10X, for example, you need a minimum of 10,000 captured cells. You could do less, but the utility of the 10X is maximized with 10,000. So, many times before the ability to multiplex these tissues, we were doing like capturing 5,000 for example. And the number of cells follows kind of the disease progression, in the sense that as a disease is more severe, you have more immune cells, in general. And it kind of decreases as it becomes more fibrotic and scarred, like calcified. So, it was a bit challenging to get very early just lipid-only cells. And a lot of those, we captured like 3000 or something like that. And efficiency is like 80% perhaps. So, you kind of capture…   Cindy St. Hilaire:        And also, how many excised hearts are going to have early athero? So, it's...   Patricia Nguyen:         Well, there are... nonischemics will have...   Cindy St. Hilaire:        Oh, okay. Okay.   Patricia Nguyen:         So, the range was nonischemic to ischemic.   Cindy St. Hilaire:        Oh great.   Patricia Nguyen:         So, about a portion... I would say one third of the total heart transplants were ischemic. And a lot of them were non ischemic. But as you know, the nonischemic can mix with ischemia. And so, they could have mild to moderate disease in the other arteries, for example, but not severe like 70%/90% obstruction.   Cindy St. Hilaire:        Wow. That's so great. That's amazing. Amazing sample size you have. So T-cell, it's kind of an umbrella term, right? There's many different types of T-cells. And when you start to get in the nitty gritty, they really do have distinct functions. So, what types of T-cells did you see and did you focus on in this study?   Jessica D'Addabbo:    So, the two main types of T-cells are CD4 positive T-cells and CD8 positive T-cells. And we looked at both of these T-cells from patients. We usually sorted multiple plates from each. And then, with 10X, we captured both. But our major finding was actually that the CD8 positive t-cell population was more clonally expanded than the CD4 population, which led us to believe that these cells were more important in the coronary artery disease progression and in the study that we were doing because for a cell to be clonally expanded, it means it was previously exposed to an antigen. And so, if we're finding these T-cells that are clonally expanded in our plaques, then we're hypothesizing that they were likely exposed to some sort of antigen, and then expanded, and then settled into the plaque.   Cindy St. Hilaire:        And when you're saying expansion, are you talking about them being exposed to the antigen in the plaque and expanding there? Or do you think they're being triggered in the periphery and then honing in as a more clonal population?   Patricia Nguyen:         So, that's a great question. And unfortunately, I don't have the answer to that. So basically-   Jessica D'Addabbo:    Next paper, next paper.   Patricia Nguyen:         Exactly. So, we... Interesting to expand on Jessica's answer. Predominantly what was found, as you said, was memory T-cells, so memory T-cells expressing specific markers, so memory versus naive. And these were effector T-cells. And memory meaning they were previously expanded by antigen engagement, and just happened to be in the plaque for whatever reason. We do not know why T-cells specifically are attracted to the plaque, but they are obviously there. And they're in a memory state, if you will. And some of them did display activation markers, which suggested that they clonally expanded to an antigen. What that antigen is, is the topic of another paper. But certainly, it is important to understand that these patients that we recruit, because they were transplant patients, they're not actively infected, right? That is a exclusionary criteria for transplants, right?   Patricia Nguyen:         So, that means these T-cells were there for unclear reasons. Why they're there is unclear. Whether they are your resident T-cells also is unclear, because the definition of resident T-cell still remains controversial. And you actually have to do lineage tracking studies to find out, "Okay, where... Did they come from the bone marrow? Did they come from the periphery? How did they get there?" Versus, "Okay. They were already there and they just expanded, for whatever reason, inside the plaque."   Cindy St. Hilaire:        So, your title... It was a great title, with this provocative statement, "T-cells are clonal and cross react to virus and self." So, tell us a little bit more about this react to virus and self bit. What did your data show?   Jessica D'Addabbo:    So, because of the way we sequenced the T-cell receptor, we were able to have paired alpha and beta chains. And because we knew the HLA type of the patients, we were able to put the sequences that we got out after we sequenced these through an algorithm called GLIPH, which allows us to look at the CDR3 region of the T cell receptor, which is the epitope binding region. And there are certain peptide. They're about anywhere from three to four amino acids long. These are mapped to certain binding specificities to known peptides. And so, basically, we were able to look at which epitopes were most common in our plaques. And we found that after comparing these to other epitopes, that these were actually more binding to virus. Patricia Nguyen:         So let me add to what Jessica stated, and kind of emphasize the value of the data set, if you will. So, this is, I believe, the first study that provides the complete TCR repertoire of coronary plaque, and actually any plaque that I know of, which is special because we know that there is specificity of TCR binding. It's more complicated than the antibody that binds directly from B cells to the antigen, because the T-cells bind processed antigen. So, the antigens are processed by antigen presenting cells like Dendritic cells and macrophages. And they have a specific HLA MHC class that they need to present to. And they need both arms, the antigen epitope and the MHC, to activate the T-cell. So unfortunately, it's not very direct to find the antigen that is actually activating the T-cell because we're only given a piece of it. Right?            Patricia Nguyen:         But we have provided a comprehensive map of all the TCRs that we find in the plaque. And these TCRs have a sequence, an immuno acid sequence. And luckily, in the literature, there is a database of all TCR specificities. Okay. So, armed with our TCR repertoire, we can then match our TCR repertoire with an existing database of known TCR specificities. Surprisingly, the matching TCRs are specific to virus, like flu, EBV and CMB. And also, because this was done in the era of COVID, we thought it would be important to look at the coronavirus database. We did find that there were matches to the coronavirus database. Even though our finding is not specific to SARS, it does lend to some potential mechanistic link there as well.   So, because this is all computational, it is important to validate. So, the importance of validation requires us to put the TCR alpha beta chain into a Jurkat cell, which is a T-cell line that does not have alpha beta chains on it, and then expose it to what we think is the cognate antigen epitote, with the corresponding HLA MHC APC. Because you don't have all those pieces, it will not work. Yes. So importantly, we did find that what we predicted to have the specificity of a flu peptide had specificity to a flu peptide.   Patricia Nguyen:         So then, the important question was, "Okay, these patients aren't infected, right? Why are these things here? Is there a potential cross reactivity with self peptides?"   Patricia Nguyen:         So luckily, our collaborator, Dr Charles Chan, was able to connect us with another computational algorithm that he was familiar with, whereby we were able to take the peptide sequences from the flu and match them with peptide sequencing from proteins that are self and ubiquitous. And we demonstrated, again, these T-cells were activated in vitro. That is why we concluded that there's a potential cross reactivity between self and virus that can potentially lead to thrombosis associated with viral infections. Of course, this all needs to be proved in vivo.   Cindy St. Hilaire:        Sure, sure.   Patricia Nguyen:         It's that first step for other things.   Cindy St. Hilaire:        The other big immune cell that we know is in atherosclerotic plaques and that's macrophages. And they can help to present antigens and things like that. And they also help to chew up the necrotic bits. And so, do you think that this T-cell component is an earlier, maybe disease driving, process or an adaptive process that goes awry as a secondary event? Patricia Nguyen:         So, I'm a fan of the T-cell. So... I'm with team T cell. I would like to think that it is playing an active role in pathology in this case and not a reactive role, in the sense of just being there. I think that the T-cell is actively communicating with other cells within the plaque, and promoting pro fibrotic and pro inflammatory reactions, depending on the T-cell. So, a subset of this paper was looking at kind of the interactions between the T-cell and other cells within the plaque, like macrophages and smooth muscle cells. And as we know, T-cells are activated and they produce cytokines. Those cytokines then communicate to other cells. And we found that, computationally, when you look at the transcriptome, there is a pro-inflammatory signature of the T-cell that resides in the more complex stage. And then, there's an anti-inflammatory signature that kind of resides in the transition between lipid and fibro atheroma, if you will.   Cindy St. Hilaire:        So, do you know, or is it known, how dynamic these populations are? Obviously, the hearts that you got, the samples you got, didn't have active infections. But do you know perhaps even how long ago they happened, or even how soon after there might be an infection or an antigen presented that you could get this expansion? And could that be a real driver of rupture or thrombosis?   Patricia Nguyen:         So, in theory, you would suppose that T-cells expanding and dividing and producing more and more cytokines would then lead to more macrophages coming, more of their production of proteinases that destroy the plaque. Right? So yes, in theory, yes. I think it's very difficult to kind of map the progression of T cell clonality in the current model that we have, because we're just collecting tissues. However, in the future, as organoids become more in science and kind of a primary tissue, where we can... For example, Mark Davis is making organoids with spleen, and also introducing skin to that.   Patricia Nguyen:         And certainly, we could think of an organoid involving the vasculature with immune cells introduced. And so, I think, in the next phase, project 2.0, we can investigate what... like over time, if you could model atherosclerosis and the immune system contribution, T-cells as well as macrophages and other immune cells, you can then kind of map how it happens in humans. Because obviously, mice are different. We know that mice... Actually, the models of transgenic mice do not rupture. It's very hard to make them rupture. Right?   Cindy St. Hilaire:        Well, if you stop feeding them high fat diet, the plaque goes away.   Patricia Nguyen:         For sure, for sure. So I think.. I mean, Mark Davis is a huge proponent of human based research, like research on human tissue. And as a physician scientist, obviously I'm more inclined to do human based research. And Jessica's going to be a physician someday soon. And I'm sure she's more inclined to do human based research. And certainly, the mouse model and in vitro models are great because you can manipulate them. But ultimately, we are trying to cure human diseases.   Cindy St. Hilaire:        Mice are not little humans. That's what we say in my lab. I similarly do a lot of human based stuff and it's amazing how great mice are for certain things, but still how much is not there when we need to really fully recapitulate a disease model.   So, my last question is kind of regarding this autoimmune angle of your findings. And that is, women tend to have more autoimmune diseases than men, but due to the fact that you are getting heart transplants, you've got a whole lot more men in your study than women. I think it was like 31 men to four women. But, I mean, what can you do? It's the nature of heart transplants. But I'm wondering, did you happen to notice...Maybe the sample size perhaps is too small, but were there any differences in the populations of these cells between women and men? And do you think there could be any differences regarding this more prevalence of autoimmune like reactions in women?   Patricia Nguyen:         So, that's an interesting question, but you hit it on the nose when you said "Your sample is defined mainly by men." And in addition, the samples that were women tend to have less disease. And they tend to be nonischemic in etiology. So, I think that kind of restricts our analysis. And perhaps, I guess, future studies could model using female tissues, for example, instead of only male. But the limitation of all human studies is sample availability. And perhaps, human organoid research can be less limited by that. And certainly, mouse research has become more evenly distributed of male and female mice.   Cindy St. Hilaire:        Yeah. Suffice it to say, human research is hard, but you managed to do an amazing and really important study. It was really elegant and well done. Congratulations on what is an epic amount of time. 12-hour experiments are no joke, and really beautiful data. So, thank you so much for joining me today, Dr Nguyen and Miss almost Dr D'Addabbo. Congrats and I'm really looking forward to seeing your future work.   Jessica D'Addabbo:    Thank you so much.   Patricia Nguyen:         Thanks so much.   Jessica D'Addabbo:    Thank you for having us. This is wonderful.   Cindy St. Hilaire:        That's it for the highlights from the April 29th and May 13th issues of Circulation Research. Thank you so much for listening. Please check out the Circ Res Facebook page and follow us on Twitter and Instagram with the handle @Circres and #Discover CircRes. Thank you to our guests: Dr Patricia Nguyen, and soon to be Doctor, Jessica D'Addabbo, from Stanford University.   This podcast was produced by Ishara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Copy text for the highlighted articles was provided by Ruth Williams. I'm your host, Dr Cindy St. Haler. And this is Discover CircRes, you're on the go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2022. The opinions expressed by the speakers of this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, visit aha journals.org.  

This Fresh Take interview featured Ruth Williams-Brinkley, Regional President of Kaiser Permanente (Mid-Atlantic states). Ruth joined Francesca Ioffreda (VP, Inclusive Growth & Talent Initiatives) to discuss supporting inclusive growth initiatives, Kaiser's role in the community, and the importance of mental health.Hosted by Francesca Ioffreda.  Produced by Jenna Klym, Ramir Cena, , Nina Sharma, and Christian Rodriguez. Edited by Christian Rodriguez. and Ramir Cena Learn from leaders doing the work across the Capital Region and beyond. These conversations will showcase innovation, as well as history and culture across our region, to bridge the gap between how we got here and where we are going.About our guest:Ruth Williams-Brinkley is president of Kaiser Foundation Health Plan of the Mid-Atlantic States, Inc. In this role, Williams-Brinkley oversees all of Kaiser Permanente's care delivery and health plan operations in Washington, D.C., and suburban Maryland, Baltimore, and Northern Virginia. The Mid-Atlantic States Region operates 36 medical office buildings and has 825,288 members.Williams-Brinkley has more than 40 years of executive experience in health care. She joined Kaiser Permanente in November 2017, serving as president of Kaiser Foundation Health Plan and Hospitals of the Northwest. She oversaw all of Kaiser Permanente's care delivery and health plan operations in Oregon and markets in Vancouver and Longview/Kelso, Washington.Prior to that, she served as CEO of KentuckyOne Health, Kentucky's largest integrated health system. KentuckyOne was a division of CommonSpirit Health, one of the nation's largest nonprofit health systems.Before joining KentuckyOne, Williams-Brinkley served as president and CEO of Carondelet Health Network in Tucson, Arizona, and as president and CEO of Memorial Healthcare System in Chattanooga, Tennessee.Williams-Brinkley serves on the boards of Travere Therapeutics, DePaul University, University of Phoenix, and the Clinical Center Research Hospital Board of the National Institutes of Health. She has been recognized by Modern Healthcare as one of the 100 Most Influential Leaders in Healthcare, one of the Top 25 Women in Healthcare, and one of the Top 25 Minority Executives in Healthcare, as well as one of Becker's most admired CEOs in health care.She holds a bachelor's degree and Master of Science degree from De Paul University, and an honorary doctoral degree from Spaulding University in Louisville, Kentucky.

Ils sont originaires de pays différents, ne partagent pas la même culture ou la même religion, n'ont pas la même couleur de peau, mais ont fait le choix de vivre ensemble.  En France, les mariages mixtes représentaient 14% des unions célébrées en 2015. Si les chiffres progressent, les obstacles rencontrés demeurent, entre différences et pressions sociales et familiales. De l'autre côté de l'Atlantique, aux États-Unis, les mariages interraciaux sont autorisés depuis 1967. Les couples noirs-blancs sont mis en avant comme celui de la nouvelle vice-présidente Kamala Harris et son mari Doug Emhoff, mais ils restent en réalité ultra-minoritaires. En Afrique du Sud, aussi, près de 30 ans après la fin de l'Apartheid, les mariages mixtes sont encore mal acceptés. Quelles sont les difficultés rencontrées par ces couples ? Comment faire évoluer le regard de la société ?   Cette émission est une rediffusion du mardi 1er juin 2021. Avec : Cécile Coquet-Mokoko, professeure de Civilisation américaine à l'Université de Versailles-Saint-Quentin. Autrice de Love Under the Skin Interracial Marriages in the American South and France (non traduit).  Peggy Caperet, psychologue à Paris, spécialisée en psychologie interculturelle. Le témoignage d'Annaëlle. En couple avec son compagnon comorien, ils tentent de se marier depuis 2019, mais sont confrontés aux difficultés juridiques.  Et une chronique de Kpenahi Traoré, journaliste à RFI. Elle revient sur une histoire d'amour entre un homme noir et une femme blanche qui a pris une tournure politique, celle de Seretse Khama et Ruth Williams.

Ils sont originaires de pays différents, ne partagent pas la même culture ou la même religion, n'ont pas la même couleur de peau, mais ont fait le choix de vivre ensemble.  En France, les mariages mixtes représentaient 14% des unions célébrées en 2015. Si les chiffres progressent, les obstacles rencontrés demeurent, entre différences et pressions sociales et familiales. De l'autre côté de l'Atlantique, aux États-Unis, les mariages interraciaux sont autorisés depuis 1967. Les couples noirs-blancs sont mis en avant comme celui de la nouvelle vice-présidente Kamala Harris et son mari Doug Emhoff, mais ils restent en réalité ultra-minoritaires. En Afrique du Sud, aussi, près de 30 ans après la fin de l'Apartheid, les mariages mixtes sont encore mal acceptés. Quelles sont les difficultés rencontrées par ces couples ? Comment faire évoluer le regard de la société ?   Cette émission est une rediffusion du mardi 1er juin 2021. Avec : Cécile Coquet-Mokoko, professeure de Civilisation américaine à l'Université de Versailles-Saint-Quentin. Autrice de Love Under the Skin Interracial Marriages in the American South and France (non traduit).  Peggy Caperet, psychologue à Paris, spécialisée en psychologie interculturelle. Le témoignage d'Annaëlle. En couple avec son compagnon comorien, ils tentent de se marier depuis 2019, mais sont confrontés aux difficultés juridiques.  Et une chronique de Kpenahi Traoré, journaliste à RFI. Elle revient sur une histoire d'amour entre un homme noir et une femme blanche qui a pris une tournure politique, celle de Seretse Khama et Ruth Williams.

Has everyone heard of this obscure musical small-fry named Joni Mitchell?

This month on Episode 35 of Discover CircRes, host Cynthia St. Hilaire highlights two original research articles featured in the April 1 issue of Circulation Research, as well as highlights from the Stroke and Neurocognitive Impairment Compendium in the April 15th issue.  This episode also features a conversation with Dr Shubing Chen and Dr Yuling Han from Weill Cornell Medical College to discuss their study, SARS-CoV-2 Infection Induces Ferroptosis of Sinoatrial Node Pacemaker Cells.   Article highlights:   Pabel, et al. Effects of Atrial Fibrillation on the Ventricle   Pattarabanjird, et al. P62-Mediated B1b Cell Atheroprotection   Iadecola, et al. Introduction to the Compendium on Stroke and Neurocognitive Impairment   Cindy St. Hilaire:        Hi and welcome to Discover CircRes, the podcast of the American Heart Association's Journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, from the Vascular Medicine Institute at the University of Pittsburgh. And today I'm going to be highlighting articles from our April issues of Circulation Research.                                     I'll also speak with Dr Shubing Chen and Dr Yuling Han from Weill Cornell Medical College, and they're with me to discuss their study, SARS-CoV-2 infection induces ferroptosis of Sinoatrial node pacemaker cells.   Cindy St. Hilaire:        The first article I want to share is titled, Effects of Atrial Fibrillation on the Human Ventricle. The first author is Steffen Pabel and the corresponding author is Samuel Sossalla and they're from Regensburg University. Atrial fibrillation, or AFib, is the most common form of heart arrhythmia. Patients with AFib may experience shortness of breath, dizziness and weakness. And they're also at risk for more life-threatening complications, such as clot-induced stroke and heart failure. Focusing on heart failure, this study investigated how disruptions to rhythm in the atria might lead to changes in the ventricular myocardium. The team studied ventricular muscle tissue from 24 patients with AFib and 31 without AFib. While the levels of fibrosis were equivalent in ventricular myocytes from both the AFib and the non AFib patients, other cellular features were distinct. For example, patients with AFib had reduced systolic calcium release, prolonged action potential duration and increased oxidative stress, compared with the non AFib patient controls. These differences were largely recapitulated in ventricular myocytes derived from human induced pluripotent stem cells that had been electrically stimulated to either mimic AFib or normal sinus rhythm. The results indicate that AFib affects the ventricles just as well as the atria and might therefore be best studied and treated with the whole heart in mind.   Cindy St. Hilaire:        The second article I want to share is titled B-1b Cells Possess Unique bHLH-Driven P62-Dependent Self-Renewal and Atheroprotection. The first author is Tanyaporn Pattarabanjird and the corresponding author is Colleen McNamara, from the University of Virginia.   Atherosclerosis is a complex and dynamic chronic inflammatory condition. However, not all immune cells exacerbate this disease. Some immune cells are actively dampening the inflammation. B-1 cells are such cells that do this, and they produce IgM antibodies that bind cholesterol, preventing its uptake into macrophages and therefore limiting macrophage driven inflammatory responses. Increased number of B1 cells, therefore, might be atheroprotective. In mice, deletion of the transcription factor ID3 leads to a boost in B-1 cell IgM production.   Cindy St. Hilaire:        In this work the authors investigated the molecular mechanism underlying this effect and found that upon deletion of ID3 in mice B-1b cells, the level of P62 protein was increased. B-1b cell proliferation was found to be dependent on P62 and over expression of P62 in mouse B-1b cells increased cell numbers, raised plasma IgM levels and importantly, ameliorated diet-induced atherosclerosis in animals. The team went on to show that people with an ID3 mutation had an unusually high level of serum IgM and B-1b cell P62. This suggests that results from mice may hold true for humans, and if so, could inform the development of immunomodulatory treatments for atherosclerosis.   Cindy St. Hilaire:        So the April 15th issue of Circulation Research is our Stroke And Neurocognitive Impairment Compendium. The last Circulation Research Compendium on Stroke was published about five years ago. In this year Dr Costantino Iadecola, Dr Mark Fisher and Dr Ralph Sacco focused this update on advances made over the past five years, with a focus on topics that were not addressed in the previous compendium, that best reflect the leading edge of basic in clinical science related to cerebral vascular diseases. Seemant Chaturvedi, Brian Mac Grory and colleagues provide an overview of preventative strategies according to stroke mechanism, including stroke of unknown cause. And the challenges of stroke prevention with antithrombotic therapy and subjects with increased hemorrhage risk are also considered.   Cindy St. Hilaire:        Stéphanie Debette and Hugh Markus provide an account of the most recent developments in the genetics of cerebrovascular diseases. The gut microbiota is another factor that has recently been linked to stroke risk and Pedram Honarpisheh, Louise McCullough and colleagues provide a comprehensive overview of the microbiology and the microbiota, and the influence that stroke risk factors exert on its composition and homeostatic relationship with mucosal surfaces. Karin Hochrainer and Wei Yang provide a systematic review of the large amount of data and stroke proteomic from animal models and human patients. Matthias Endres and colleagues cover the dramatic effect that innate and adaptive immunity exert on stroke risk and on acute brain damage and post stroke sequelae, such as post-stroke cognitive impairment and depression.                                     Cindy St. Hilaire:        Manuela De Michele, Alexander Merkler and colleagues discuss the cerebral vascular diseases that have emerged as a frequent manifestation of the maladaptive immune response to severe SARS-CoV-2 infection. Jessica Magid-Bernstein and Lauren Sansing review the current concepts on epidemiology, risk factors in etiology, clinical features, as well as the medical and surgical interventions for cerebral hemorrhage. Yunyun Xiong and Marc Fisher cover the progress that has been achieved in the treatment of acute ischemic stroke and Natalie Rost and Martin Dichgans and colleagues address the long term impact of stroke on cognitive function, which is becoming a significant healthcare challenge in the world's aging population.   Cindy St. Hilaire:        So today I have Dr Shubing Chen and Yuling Han from Weill Cornell Medical College. And they're with me to discuss their study SARS-CoV-2 infection induces ferroptosis of Sinoatrial node pacemaker cells. And this article is in our April 1st issue of Circulation Research. So thank you both for joining me today.   Shubing Chen:             Thank you. It's really nice to join the program, and it's really a great honor.   Cindy St. Hilaire:        It's a really great article. I'm so excited to talk about. So there's a lot of research happening regarding SARS-CoV-2 virus and the patients who are infected and have COVID-19. And this paper is focusing on the impact of viral infection on the heart and specifically on the sinoatrial node, which is the primary cardiac pacemaker that keeps our hearts beating. So I was wondering if you could tell us what led you to focus on this particular aspect of COVID-19 symptoms, and also how early in the pandemic did you start this?   Shubing Chen:             Yeah, so we started working on SARS-CoV-2 through back to early 2020 when very unfortunately, New York City was a pandemic center and we had a lot of patients in the hospital unit, and also postdoc students working very hard in the lab. So that's the time we start working on SARS-CoV-2. And I was trained as a stem cell biologist. And what we're really interest is to set up a platform to basically understand which type of cells can be infected by SARS-CoV-2 and if they can, how they respond to SARS-CoV-2 infection. Not only for SARS-CoV-2, we sent it as like a viral infection platform, but SARS-CoV-2 is one of the virus we study now. And it's kind of very surprising. We have a pretty broad platform. We have a lung organoid, we have colon organoids, we have pancreas, we have cardiomyocytes, pacemaker cells. And as expected, we see lung can be infected like colon and because patient had GI tract, liver can be infected, but very surprisingly we see very high cardiomyocytes infection as well as pacemakers.                                       So as we'll know that still big controversy in the field, whether we can detect SARS-CoV-2 like viral protein or viral RA in the heart, in particular, cardiomyocytes. But I think now everyone agree that the cardiomyocytes really can be very well infected actually. Because it's very difficult to get the pacemaker tissue and the sinoatrial tissue from the COVID patient. So we collaborate with Dr Ben Andora's lab at NYU to get this hamster model. So we basically take SA tissue from hamster and then other colleagues basically did the section imaging, and we confirm that the hC4 polymerase cells can be infected by SARS-CoV-2. And at that time we start to learn a more clinical studies they report the COVID patient, they develop arrhythmia, or some other problem, not only with cardiomyocyte, as well as the conduction system. So at that time, that's the time that we say maybe we should do something on the pacemaker and focus on that. So that's how the project was developed.   Cindy St. Hilaire:        That is so interesting. And so I know humans infected, like you just said with SARS-CoV-2, they can develop arrhythmias. What's that timeframe? Is there a common timeframe that this happens? Does it normally happen very close to the infection or only in later stage? What's that window of when these arrhythmias are happening?   Shubing Chen:             At least based on the clinical study we show right now, actually the patient can develop acute arrhythmia. So it can be very soon after they developed symptom for COVID.   Cindy St. Hilaire:        Wow. That's amazing. So you mentioned this, your study utilized a hamster model, which you actually don't see a lot of. Most studies use a lot of rats or most studies I'm familiar with, especially in Circulation Research, they use more rats or more mouse models. So what advantages does that hamster model have and why were you interested in using it?   Shubing Chen:             Yeah, that's actually really specific for SARS-CoV-2. As SARS-CoV-2 mainly use ACE2 as a key entry factor to enter the cells. Of course, there's additional receptor, like neutrophils is one. Like all this enzyme involved, but human and mouse ACE2, they have very different structure. So the SARS-CoV-2 virus combine with human ACE2 very well but not mouse ACE2. So from the beginning, the rat and mouse was not used as a very good model to study SARS-CoV-2 infection. Of course there are other models, like knockin human ACE2 in the mouse and also like ACE2 transgenic mice. That's how different mouse model use. But hamster you don't need any modification, but they are very promising to SARS-CoV-2 infection. And so that's a reason we decide to use that as an animal model to basically run in parallel with our human stem cell model.   Cindy St. Hilaire:        We joke in my lab, mice are not little humans, but it's really true in a lot of cases, they're beautiful models in so many ways, but then when they don't work, they really don't work.   Shubing Chen:             Yeah. Before COVID every time when we try to talk about our human stem cell, derived cells, organoids as a disease model. People always ask, why do you want to work on human organoids? Right? It's that we have all these beautiful animal models like as you mentioned, mouse or rats, that's very broadly used. And we have to find different reasons. And now when we start working on SARS-CoV-2, which is very clear example, that mouse are not identical to human. Yeah.   Cindy St. Hilaire:        Yeah. That's great. I love finding additional models to use that are the best one for the question. So in order to investigate, I guess kind of the mechanism of how this was happening in the SAN cells, the sinoatrial node cells, you had to develop a new differentiation protocol that took the human embryonic stem cells, I think it was the H9 line you used, and essentially differentiate that cell line into a sinoatrial node-like cell. So I was wondering if you could tell us a little bit about A) how did you figure out that protocol and B) how does it work?   Shubing Chen:             So it's actually a long story to cell line.   Cindy St. Hilaire:        We can condense it. Let's get-   Shubing Chen:             At least based on the clinical study we show right now, actually the patient can. Let's condense it. But it's as you can imagine, we did not develop this cell line only for this particular project. Actually, we start working on this cell line back to maybe six, seven years ago. The first postdoc we have who basically knockin the mCherry, Myh6. Which basically label the atrial cardiomyocytes. And another postdoc, Zanir, he basically put a GFP in the SARS2 locus. So now we have this duel reporter line we can visualize the SA nodal cells. And we really spend a lot of time on that because we think that unfortunately in our hand, there is not really no good antibody for SARS2. We think it's very, very important that you can see these cells. So after developing these lines and because my lab run a lot of chemical screening, where we run Zanir, we run several chemical screening to develop the protocol.                                       And Jialing Zhu, another postdoc in the lab, also pick up the project to further develop the protocol. And there is several years' work. We do have this good protocol to make pretty efficiently to make the cells. And it's not only our work. I want to say that. For example, Dr Sean Wu from Stanford, they did this beautiful study on the single cell RNC mouse conduction system and Dr Gordon Keller and many other labs also basically published protocol in the field. We are very excited about this duel reporter line. I think they gave us a lot of new opportunity and we are very happy to share this line. Yeah. So if anyone in the field are interested in that, just contact us.   Cindy St. Hilaire:        Yeah. Anyone listening. That's great. So were you surprised to find the entry factors that SARS-CoV-2 uses to get into a cell, were you surprised to find them on these sinoatrial node cells? And I guess in the context of comparing these particular cells to other cells in the heart, are those entry factors higher in the sinoatrial node cells?   Shubing Chen:             So it can be either surprised or not surprised let's say this way. So because one, we see the cardiomyocytes that can be infected, we were kind of surprised. And then we find actually several type of cells in the heart can be infected, like endothelial cells. I will say that the ACE2 expression of like ACE2 aminophenol in pacemaker cell, it's not significantly higher than cardiomyocytes. So we are not really saying, or seeing that SA nodal cells are more permissive to SARS-CoV-2 infections compared to cardiomyocytes, even in the petri dish, but they can be infected.   Cindy St. Hilaire:        So you found SARS-CoV-2 infection in these sinoatrial nodal cells induces a process called ferroptosis. So Yuling, I was wondering if you could tell us what is ferroptosis and what is it doing in these pacemaker cells?   Yuling Han:                 For the ferroptosis, they was surprised so far that its by the RA sequencing of the SARS-CoV-2 infection make our cells. And the first process is mainly caused by the-   Shubing Chen:             Error in iron.   Yuling Han:                 Yes. So more intake of the iron error and induced the RA's pathway and caused the cell deaths. So by our RA sequencing, we found the key factor involved in ferroptosis pathway is the GPS score was checked after the SARS-CoV-2 infection. So we focused on the ferroptosis pathway and found other key factors or checked after the infection makes in the pacemaker cells.   Cindy St. Hilaire:        What is the ferroptosis doing that disrupts the SNA cells?   Shubing Chen:             Ferroptosis is a type of cell death mechanism. So eventually it will cause cell death. And we think something that is really surprising, but we think it's very interesting, is we only see ferroptosis in the SARS-CoV-2 infected general atrial cells. So SA cells, we actually, as Yuling mentioned, when we develop this platform, we see different type of cell can be affected. And we are very curious what happened. So we see that we run a sequence on each individual cells we can see infection and along, we can see cell death like apoptosis in cardiomyocytes. We see apoptosis and only in SA nodal cells, we actually see the ferroptosis pathway as we come up.   Cindy St. Hilaire:        Why do you think that is in that cell type versus in another? Do you have any ideas about why?   Shubing Chen:             No, we don't have any idea yet to be honest, but we are working on that. But at least I think that it gave us some clue that we really need to use different type of whole cells to study the whole cell response. Because traditionally when we study viral infection and when we see lung, we always say, oh, the cell died. It's fairly simple. But now if we really study the details and we think it's maybe over simplified way to think about how cells can respond to viral infection, not only to SARS-CoV-2 infection. So it gives us the motivation, very strong motivation to now really study how different host tissues response to viral infection.   Cindy St. Hilaire:        I thought that was really interesting, not all cell death is the same.   Shubing Chen:             Yeah. And another thing is kind of a little bit surprising is we actually did a very careful comparison between the SA nodal cells and the cardiomyocyte. We only see ferroptosis come up as SA nodal cell, but not cardiomyocyte. Again, we don't understand why as maybe some host factor that is specific, we're working on that.   Cindy St. Hilaire:        So in addition to working out this mechanism of what is going wrong when these cells are infected with the virus, you also used this embryonic stem cell like tool for a drug screen. So can you walk us through that process in terms of what you did to do that? Did you focus in on one specific type of drugs or was it just kind of an unbiased screen?   Yuling Han:                 For the sinoatrial pacemaker cells, we focus on the antiviral drugs screening. And we also did several other projects, like lot of night or some neuron cells. For the [they did drug screening to find some drugs to inhibit the SARS-CoV-2 entry. And for the dominic neuron, we found SARS-CoV-2 infection can cause neuro cells synapses. So we focus on the synapses associated drug screening, but for the pacemaker cells, they only did the antiviral drug screen.   Cindy St. Hilaire:        And you came up with two drugs that you wrote about in the paper, deferoxamine and imatinib. So what are the mechanisms of action of those drugs? Are they targeting the same thing or are they targeting slightly different things?   Yuling Han:                 For the imatinib, we also found this drug inhibit SARS-CoV-2 entry and we did several other screenings, like the lung organoids and neuro cells. We also found this drugs. And the six drug, the mechanism is kept and the spec protein of SARS-CoV-2. And this was found by several other groups and published some paper this year. And we found this in 2020 maybe. And we published this paper before and we found this mechanism. And for another drug, we checked the RA sequencing data of SARS-CoV-2 affect the peacemaker cells. And we did several run of RA sequencing. And we compared the key factors, involved in SARS-CoV-2 entry. Several key factors like CTSL and like TMPS2 and among several run of RA sequencing. We only found the drug can decrease the expression of CTSL. So we also did PTR immunostaining, and then we found the drug decrease the expression level of CTSL.   Shubing Chen:             Yeah. So actually the other drug, it's also an antiferroptosis drug. So we did the mechanism study and it's very nice to see, we also identify the drug from an unbiased chemical screen. And for the chemical screening, we actually have a pretty large platform and we have around 1200 FDA approved drugs. We have like a 2000 anatrofin amino acid that signal pathway regulators for most of the SARS-CoV-2 screening, as you did mention, we have multiple screening platform. We focus on FDA approved drug. So it's more like for the drug repurposing and for other screening we also write larger skills.   Cindy St. Hilaire:        So we got a mechanism, we got a super specific cell type and we now have some drugs. So what are the translational implications of these findings? And I guess I'm thinking about that in terms of the time course of when a patient gets infected, has symptoms, has arrhythmia, like where could you possibly target this ferroptosis pathway? Meaning if someone already is exhibiting AFib as a result of the infection, is that actually too late? Or can you start to treat it to reverse it or prevent it from getting worse? Like what do you see as a therapeutic potential for using these drugs?   Shubing Chen:             That's a very good question. I will say this way, I think when we identify all these drugs, it's very, very exciting. But for antiviral drug development perspective, we definitely want a drug that show broader spectrum. So for COVID patient, of course we want to protect their heart, but we also want to protect their lungs.   Cindy St. Hilaire:        Exactly. Protect everything.   Shubing Chen:             Exactly. Exactly. So for the real drug that can clinical use, I think the lack of broad spectrum antiviral drug, I think that will be the way to go for drug development and for the cardioprotective respective. So if the patient do have very severe cardio symptom, particularly like arrhythmia symptom, I think that can be considered. But I don't want to really say this is the drug to treat the COVID patient. I don't think that's a way to go, particularly for ferroptosis is a cell type. This is a phenotype, very specific for the pacemaker. And I think for us, as a basic scientist, is very, very important that we understand the biology and we can identify these normal chemical tools that we can manipulate the system that can facilitate the future drug development.   Cindy St. Hilaire:        So do you think your findings and I mean findings at multiple levels, that a viral infection can induce apoptosis in one cell, but ferroptosis in another cell, but also the findings of viral infection in general, sufficient enough to drives sinoatrial node cell dysfunction. Do you think this is specific to SARS-CoV-2 and corona viruses or do you think this is something that is more broad with other viruses that maybe we just haven't recognized possibly because we don't have the tools yet?   Shubing Chen:            That's a great question. I will say some other type of virus can also infect heart, at least cardiomyocyte, like a Coxsackie virus, regular virus three. And there's actually a lot of study on the viral infection on the cardiomyocytes. And for us, the most exciting part is we really have now in serious, limited starting materials to get these pacemaker cells. Like I SA nodal cells. So we can use this as a platform to study how other virus infect, how the viral infection in general cause cell dysfunction. Because in the study we also do the calcium blocks assay, we can monitor their beating and then we can do RN-seq to monitor their transcription changes. Because this we have this still reporting system, we can purify cells, we can even run larger scale, like epigenetic level, how they change. So that's a very useful tool to study how cell responds to viral infection. I'm very excited about that.   Cindy St. Hilaire:        That's great. Well, Dr Chen and Dr Han, thank you so much for joining me today. Congratulations on a beautiful story. And I look forward to hearing more out all these different organoid and cell models you have.   Shubing Chen:            Cindy, thank you. Thank you for so much for having us.   Cindy St. Hilaire:        That's it for the highlights from the April issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Dr Shubing Chen and Dr Yuling Han. This podcast was produced by Ishara Rantikac edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles was provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, you're on the go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association, 2022. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American heart Association. For more our information visit ahajournals.org.  

This month on Episode 34 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the March 4 and March 18th issues of Circulation Research. This episode also features a conversation with Dr Mireille Ouimet and Sabrina Robichaud from the University of Ottawa Heart Institute to discuss their study, Autophagy is Differentially Regulated in Leukocyte and Non-Leukocyte Foam Cells During Atherosclerosis.   Article highlights:   Pauza, et al. GLP1R in CB Suppress Chemoreflex-Mediated SNA   Lim, et al. IL11 in Marfan Syndrome   Hohl, et al. Renal Denervation Prevents Atrial Remodeling in CKD   Liu, et al. Smooth Muscle Cell YAP Promotes Arterial Stiffness   Cindy St. Hilaire:        Hi and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to be highlighting articles from our March issues of Circulation Research. I'm also going to speak with Dr Mireille Ouimet and Sabrina Robichaud from the University of Ottawa Heart Institute, and they're with me to discuss their study, Autophagy is Differentially Regulated in Leukocyte and Non-Leukocyte Foam Cells During Atherosclerosis.   The first article I want to share is titled GLP1R Attenuates Sympathetic Response to High Glucose via Carotid Body Inhibition. The first author is Audrys Pauza, and the corresponding authors are Julian Paton and David Murphy at the University of Bristol.   Cindy St. Hilaire:        Hypertension and diabetes are risk factors for cardiovascular disease. And yet, for many patients with these two conditions, lowering blood pressure and blood sugar is insufficient for eliminating the risk. The carotid body is a cluster of sensory cells in the carotid artery, and it regulates sympathetic nerve activity. Because hypertension and diabetes are linked to increased sympathetic nerve activation, this group investigated the role of the carotid body in these disease states. They performed a transcriptome analysis of crowded body tissue, from rats with and without spontaneous hypertension. And they found among many differentially-expressed genes that the transcript encoding glucagon-like peptide-1 receptor or GLP1R, was considerably less abundant in hypertensive animals.   Cindy St. Hilaire:        This was of particular interest because the gut hormone GLP-1 promotes insulin secretion and tends to be suppressed in Type 2 diabetes. Moreover, GLP1R agonists are already used as diabetic treatments. This group showed that treating rat carotid body with GLP1R agonist suppresses sympathetic nerve activation and arterial blood pressure, suggesting that these drugs may provide benefits in more than one way. Perhaps the carotid body could be a novel target for lowering cardiovascular disease risk in metabolic syndrome.   Cindy St. Hilaire:        The second article I want to share is titled Inhibition of IL11 Signaling Reduces Aortic Pathology in Murine Marfan syndrome. The first author is Wei-Wen Lim, and the corresponding author is Stuart Cook and they're from the National Heart Center in Singapore. People with the genetic connective tissue disorder Marfan syndrome, are typically tall and thin with long limbs and are prone to skeletal, eye and cardiovascular problems, including a life-threatening weakening of the aorta. While Marfan syndrome patients commonly take blood pressure-lowering treatments to minimize risk of aortic aneurysm and dissection, there's currently no cure for Marfan syndrome or targeted therapy.   Cindy St. Hilaire:        The cytokine IL11 is strongly induced in vascular smooth muscle cells upon treatment with the growth factor TGF-beta, which is over activated in Marfan syndrome patients. And TGF-beta is also considered a key feature of the syndrome's molecular pathology. This study found that IL11 is strongly upregulated in the aortas of Marfan syndrome model mouse, and that genetically eliminating IL11 in these animals protected them against aortic dilation, fibrosis, inflammation, elastin degradation and loss of smooth muscle cells. Treating Marfan syndrome mice with anti-IL11 neutralizing antibodies exhibited the same beneficial effects. These results suggest that perhaps inhibiting IL11's activity could be a novel approach for protecting the aortas of Marfan syndrome patients.   Cindy St. Hilaire:        The next article I want to mention is titled Renal Denervation Prevents Atrial Arrhythmogenic Substrate Development in Chronic Kidney Disease. The first authors are, Mathias Hohl, Simina-Ramona Selejan and Jan Wintrich, and the corresponding authors also Mathias Hohl, and they're from Saarland University. People with chronic kidney disease have a two to three fold higher risk than the general population of developing atrial fibrillation, which is a common form of arrhythmia that can be life-threatening. Chronic kidney disease is associated with activation of the sympathetic nervous system, which can be damaging to the heart. Thus, this group examined myocardial tissues from atrial fibrillation patients with and without chronic kidney disease to see how they differ. They found that atrial fibrosis was more pronounced in patients with both conditions than in patients with atrial fibrillation alone, suggesting that chronic kidney disease perhaps exacerbates or even drives arterial remodeling.   Cindy St. Hilaire:        Sure enough, induction of chronic kidney disease in rats led to greater atrial fibrosis and incidence of atrial fibrillation than seen in the control animals. Renal denervation is a treatment in which the sympathetic nerves are ablated, and it's a medical procedure that's used for treating uncontrolled hypertension, and it has also been shown in animals to reduce atrial fibrillation. Performing renal denervation in the rats with chronic kidney disease reduced atrial fibrosis and atrial fibrillation susceptibility. This study not only shows that chronic kidney disease induces atrial fibrosis and in turn atrial fibrillation, but also suggests that renal denervation may be used in chronic kidney disease patients to break this pathological link and prevent potentially deadly arrhythmias.   Cindy St. Hilaire:        The last article I want to highlight is titled YAP Targets the TGFβ Pathway to Mediate High-Fat/High-Sucrose Diet-Induced Arterial Stiffness. First author is Yanan Liu and the corresponding author is Ding Ai from Tianjin Medical University. Metabolic syndrome is characterized as a collection of conditions that increase the risk of cardiovascular diseases, such as obesity, hypertension and diabetes. Among the tissue pathologies associated with metabolic syndrome is arterial stiffness, which itself is a predictor of cardiovascular disease incidence and mortality. To specifically investigate how arterial stiffness develops in metabolic syndrome, this group fed mice a high-fat, high-sugar diet, which is known to induce metabolic syndrome and concomitant arterial stiffness.   Cindy St. Hilaire:        After two weeks on the diet, the animals' aorta has exhibited significant upregulation of TGF-beta signaling, which is a pathway known for its role in tissue fibrosis, and the aorta has also exhibited increased levels of yes-associated protein, or YAP, which has previously been implicated in vascular remodeling, collagen deposition and inflammation. YAP gain and loss of function experiments in transgenic mice revealed that while knockdown of protein in the animals' smooth muscle cells attenuated arterial stiffness, increased expression exacerbated the condition.   Cindy St. Hilaire:        The team went on to show that YAP interacted with and prevented the activation of PPM-1 B, which is a phosphatase that normally inhibits TGF-beta signaling and thus fibrosis. Together the results suggest that targeting the YAP, PPM-1 B pathway, could be a strategy for reducing arterial stiffness and associated cardiovascular disease risk in metabolic syndrome.   Cindy St. Hilaire:        Today, Sabrina Robichaud and Dr Mireille Ouimet from University of Ottawa Heart Institute are with me to discuss their study Autophagy is Differentially Regulated in Leukocyte and Non-Leukocyte Foam Cells During Atherosclerosis, which is in our March 18 issue of Circulation Research. So thank you both for joining me today.   Sabrina Robichaud:    Thank you so much for having us. It's a pleasure.   Mireille Ouimet:         Thank you for having us.   Cindy St. Hilaire:        Yeah, and congrats on the study. So we know that LDL particles contain cholesterol and fats, and these are the initiating factors in atherosclerosis. And it's also really now appreciated that inflammation in the vessel wall is a secondary consequence to this lipid accumulation. Macrophages are an immune cell that, in the context of the plaque, gobble up this cholesterol to the point that they become laden with lipids and exhibit this foamy appearance, which we now call foam cells. And these foam cells can exhibit atheroprotective properties, one of them called reverse cholesterol transport, and that's really one of the focuses of your paper. So before we dig into what your paper is all about, could you give us a little bit of background about what reverse cholesterol transport is in the context of the atherosclerotic plaque? And maybe introduce how it links to this cellular recycling program, autophagy, which is also a big feature of your study.   Mireille Ouimet:         Yes, so the reverse cholesterol transport pathway is a pathway that's very highly anti-atherogenic. It's linked to HDL function and the HDL protective effects, in that HDL can serve as a cholesterol acceptor for any excess cholesterol from arterial cells or other cells of the body and return this excess cholesterol to the liver for excretion into the feces. There is also trans-intestinal cholesterol efflux that can help eliminate any excess bodily cholesterol. Mireille Ouimet:         So reverse cholesterol transport is a way that we can eliminate excess cholesterol from foam cells in the vascular wall, and that's why we're really interested in the process. But the rate-limiting step of cholesterol efflux out of foam cells in plaques is actually, they have to be mobilized in the form of free cholesterol to be pumped out of the cells through the action of the ATP-binding cassette transporters. And so the rate-limiting step of the process is the hydrolysis of the cholesterol esters and the lipid droplets, because that's where the excess cholesterol is stored in foam cells.   Mireille Ouimet:         And so for years, people investigated the actions of cytosol like lipases in mobilizing free cholesterol from lipid droplets, although the identity of those lipases are not well-known and in macrophage themselves, but our recent work showed a role for autophagy in the catabolism of lipid droplets. And in fact, in macrophage foam cells, 50% of lipid droplet hydrolysis is attributable to autophagy while the other half is mediated by neutral lipases, which makes it really important to investigate the mechanisms of autophagy-mediated lipid droplet catabolism.   Cindy St. Hilaire:        That is so interesting. I guess I didn't realize it was that significant a component in that kind of rate-limiting step. That's so cool. So really, a lot of the cholesterol efflux studies, and maybe this is just limited to my knowledge of a lot of these cholesterol efflux studies, but to my knowledge, it's been really focused on the foam cell itself, the macrophage foam cell. However, there's been a lot of recent work that has now implicated vascular smooth muscle cells in this process. So could you share some of the research specific to smooth muscle cells and smooth muscle-derived foam cells that led you to want to investigate the contributions of smooth muscle cell-derived foam cells in cholesterol efflux?   Mireille Ouimet:         Yeah, so you're right in the sense that macrophages have always been the culprit foam cells in the atherosclerotic plaques but pioneering work from several groups, including Edward Fisher and Gordon Francis, they've shown that the smooth muscle cells can actually acquire a macrophage-like phenotype becoming lipid-loaded and foamy. And there's been work specifically looking at the ABC transporters, and their ability to efflux cholesterol from these vascular smooth muscle cell-derived foam cells, because as they trans-differentiate into macrophage-like cells, they acquire the expression of ABCA1, but this is to a lower extent, as compared to their macrophage counterparts.   Mireille Ouimet:         And the efflux is defective because there's an impairment in liposomal cholesterol processing of the lipoproteins that's really important to activate a like cell, and the expression of the ABC transporters, so vascular smooth muscle cell-derived foam cells are very poor effluxes.   Sabrina Robichaud:    There's very few studies that look at the vascular smooth muscle cell foam cells, and the very few that did look at it mostly focused on the ABCA1 transporters, and did show that they were poor effluxes. And as we all know, ABC1 is not the only cholesterol transporters that can transport cholesterol out of cells, there's also ABCG1 which is also one of our major findings in our paper.   Cindy St. Hilaire:        Can you tell us a little bit about the models you chose in the study and why you picked them? And also maybe a step back in terms of, what are the pros and cons of using mouse models in atherosclerotic studies?   Sabrina Robichaud:    So we chose to use the GFP-LC3 reporter mouse model because it allows us to track in lifestyle the movement of LC3, which is the main component of the autophagosome which is involved in pathology. So by using this reporter model, we could infer whether or not the cells had high autophagy or low autophagy. And to induce atherosclerosis in these mice, instead of backcrossing them to either an LDLR knockout or an ApoE knockout, we chose to do the adeno-associated virus that encode the gain of function PCSK9 instead to kind of minimize the time for breeding. It did have the effect that we needed in terms of raising plasma cholesterol to induce the atherosclerosis. So that was one of the models that we used in our paper.   Mireille Ouimet:          There's not very many good mouse models to study autophagy flux in vivo and GFP-LC3 is kind of the main one currently. We're working on developing some other tools to track lipophagy in vivo, but these things take time to put in place. So in the future, we hope to have some better tools to track lipophagy in real-time in vivo.   Cindy St. Hilaire:        How difficult is it to measure autophagy flux in vivo? I know there's certain part like LC3 or P62, a lot of people use a western blot and it's like, oh, it's high, it must be active, but it's a flux. So it's a little bit more... There's more subtleties to that, dynamic than that. So how difficult is it to really measure this flux in in vivo tissues?   Mireille Ouimet:         Yes, so now there are more recent mouse models that have been developed more recently to replace kind of the GFP-LC3 is the Rosella LC3. So it has both a red and a green tag, and so two LC3, so when autophagosomes are fused to lysosomes and are degraded, then there's preferential quenching of the GFP first, and then you have the red appearance that predominates so we know that then it's kind of like it a live flux measurements. Because we use the GFP-LC3 mouse, Sabrina treated her cells ex vivo. When we dissected out the aortic arches, digested the cells then we divided those into two components and added bafilomycin so that we can inhibit lysosome acidification to see the changes in the flux. And that's really to get the differences in untreated versus bafilomycin-treated.   Mireille Ouimet:         When we inhibit the lysosome, then we're sure that it is a functional flux or not. But it's kind of an indirect way of measuring it, and it reads very complex when we're talking about P62 and LC3 degradation with or without lysosome inhibition, but you really need that lysosomal inhibition, to show that if you block the degradation of the autophagosomes that fuse in with a lysosome, then you get an increase in the LC3 and the P62, and that's when you know that the flux is you intact.   Mireille Ouimet:         Because you could get an increase in LC3, that's just related to a defect in the breakdown of the autophagosome. But in our study, we've used phosphorylated ATG16L1, which is a now better marker of active autophagy. And I would recommend researchers to begin to use that rather than the combination of P62 and LC3 together with or without a lysosome inhibitors such as- Cindy St. Hilaire:        Oh, interesting. So let's repeat that, phosphorylated ATG-   Mireille Ouimet:         16L1, yes. So there's been an antibody that was developed by a colleague at the University of Ottawa, Dr Ryan Russell, and it's commercially available through cell signaling now, and it really has been a great tool to track active autophagy.   Cindy St. Hilaire:        That's great. I remember my lab was looking at that at one point, and I was trying to explain the flux as... I don't know if people are going to remember this, but there's this amazing, I Love Lucy skit, where her and Ethel are working on a chocolate factory conveyor belt, and it picks up speed. And because she can't get it all done quick, she starts stuffing them in her mouth. And it's like, if you just took a snapshot of that, you would not know whether it's going too fast, or not functioning properly. And so I equate the flux experiments to that. Which are probably aging myself a lot on so.   Cindy St. Hilaire:        All right, so sticking to kind of the autophagy angle, what were the differences you found in autophagy in early and late atherosclerotic plaques? Because I know you looked at those two time points, but also, importantly, between the macrophage foam cells and the smooth muscle cell-derived foam cells?   Sabrina Robichaud:    So surprisingly, there weren't that big of a difference between each time point when we were looking at the individual cell type by themselves. Surprisingly, we did find that the macrophages did have a functional autophagy flux, even at the later stages of atherosclerosis, which was kind of interesting in itself. But when we looked at the vascular smooth muscle cell foam cells, though, that was a whole other story, and we found that these were actually defective at a very early stage and stayed defective up until the very late stage of atherosclerosis.   Cindy St. Hilaire:        And what is the very early stage like? What's that definition with the smooth muscle cell?   Sabrina Robichaud:    So we did a six-week time points in terms of our atherosclerosis study, and then a 25-week time point. So there are far apart, which shows like the very early, early stage and what would be considered the most effective autophagy at that point with the necrotic core and everything. So surprisingly, the two phenotype were quite similar at early and both late stages for both cell types, but were functional in the macrophages but dysfunctional in the smooth muscle cells.   Cindy St. Hilaire:        So you mentioned at one point in the discussion that you observed inconsistent lipid loading of the smooth muscle cells, and you mentioned that a lipase, which is excreted from the foam cells can then be internalized by, I assume kind of neighboring or in the vicinity, smooth muscle cells. And so the question I had it's kind of one of those chicken-and-egg question, and it's, is the smooth muscle cell-derived foam cell an independent process? Does it happen alone or de novo as a function of a smooth muscle-mediated process? Or is it really dependent first on this macrophage foam cell providing this lipid that is efflux that is then internalized by a smooth muscle cell that kind of goes on to become a foam cells. It's kind of a question of like the continuum of an atherosclerotic plaque and what do you think is happening, either based on your data or just kind of a hunch?   Mireille Ouimet:         That's an excellent question. And there's no doubt that macrophages really drive the initiating events of atherosclerosis. So I don't think that without the macrophage there would ever be a vascular smooth muscle cell, or there would be minimal vascular smooth muscle cell-derived foam cells. Definitely the inconsistencies that we observed in our study, were if we added like aggregated LDL on its own to a primary mouse vascular smooth muscle cell, we would get poor lipid loading and a very low percentage of those cells that would become foamy, relative to treating them with cyclodextrin complex cholesterol, for instance.   Mireille Ouimet:         So free cholesterol, that's cell permeable, will go into the vascular smooth muscle cell, no problem, and generate the foaminess and then allow that cell to acquire the macrophage-like phenotype. But aggregated LDL on its own in our hands, just gave very poor loading. And when we treated the vascular smooth muscle cells with aggregated LDL along with macrophage-derived condition media, we got some improvements, but it was still kind of inconsistent. But then we thought if we treat the vascular smooth muscle cells with aggregated LDL in the presence of conditioned media from macrophage foam cells that were preloaded with the aggregated LDL, would that promote their foaminess to a greater extent? And it did.   Mireille Ouimet:         So, there have been studies from Gordon Francis's lab that showed that adding recombinant lysosomal acid lipase to vascular smooth muscle cells that contained aggregated LDL, promoted the lysosomal hydrolysis of the aggregated LDL and to generate the foamy macrophages and allow the lysosomal processing. So we know that that vascular smooth muscle cells take up lysosomal acid lipase, and we know that macrophages undergo lysosome exocytosis and they can secrete lysosome acid lipase and acidify the extracellular milieu.   Mireille Ouimet:         So work from Fred Maxfield group has shown the presence of these cell surface connected compartments that are acidified, containing macrophage-derived lysosomal acid lipase, that even hydrolyze extra cellularly-aggregated LDL for macrophages. So we're not sure whether there's probably a local production of free cholesterol in the plaque by macrophages, this free cholesterol could be taken up by the vascular smooth muscle cell. And also the vascular smooth muscle cells do express some scavenger receptors, whether the expression of these scavenger receptors like LRP or CD36 even goes up when they've taken up a little bit of the free cholesterol. And then that allows the aggregated LDL to come in and then there would be some lysosomal acid lipase secreted by the macrophage foam cells that would promote the lysosomal processing of this aggregated LDL. All of those are very complex questions that will require some addressing in vivo models.     Cindy St. Hilaire:        You also mentioned in the paper that studies... There's a handful of them now. Studies have shown that between 30% and 70% of the cells that are staining positively for macrophage markers, meaning they're foam cells, are of the smooth muscle cell lineage. And so I believe people have seen that in mouse plaques with lineage tracing, but they've also used newer techniques to really see this also in human atherosclerotic plaques. So we know it's not just from a mouse, we know that smooth muscle cells can turn into a macrophage-like foam cell, and it's 30% to 70%, which is a huge range. Cindy St. Hilaire:        So do we know the factors that dictate whether a specific plaque is going to have more or less smooth muscle cell derived foam cells? And I guess more important to what you found in your paper is, how important would it be to know whether a plaque is on the 30% end or on the 70% end in terms of therapeutic strategies?   Sabrina Robichaud:    Yeah, most of these studies, the range can be attributed to the different time points at which these studies have been collected early on will be a little bit more macrophage understanding would be at a later time point. Now of course in terms of therapeutics, as we saw in our paper, metformin actually will positively increase cholesterol efflux in the vascular smooth muscle cell foam cells, but not in the macrophages. So obviously, being able to know at which point there's a majority of macrophages versus vascular smooth muscle cells, definitely going to determine which therapeutic we're going to be able to use.   Sabrina Robichaud:    Ideally, we would be able to find a therapeutic that would work in both foam cell, but from what we've seen, the mechanistic behind the autophagy dysfunction between both cell types are so different, that I'm not entirely sure that that would be possible, we would need some sort of combination therapy. But again, we need to be a little bit more targeted depending on the percentage of the foam cells that are comprising the plaque at that particular moment in time.   Cindy St. Hilaire:        Yeah, so you mentioned there's a function of time there. If you look earlier, there's more macrophage, if you look later, the percent of smooth muscle cell-derived foam cell increases. Is there a point in a very advanced atherosclerotic plaque where it's just mostly smooth muscle cells? Or do those macrophage foam cells stay, and it's just the increasing number of smooth muscle cell-derived foam cells? Do we know?   Mireille Ouimet:         This is an excellent question, and I was going to bring up the topic of clonal expansion of the vascular smooth muscle cells. So it's a very heterogeneous population and understanding that might be some of the differences that we see in different studies. It could be the model has one type of a smooth muscle cell that's expanding more than another, what are the factors that govern that? Does one clone take over at the later stages versus the earlier stages? We don't know.   Mireille Ouimet:         But we were surprised in our studies to see that the macrophages that are present at least on the lumen of the plaques were very active in autophagy. They had the highest staining for the phospho-ATG16L1 in that late stage. So we're not sure if it's newly-recruited macrophages that come in, that are more active and in autophagy, and then have good lysosomal capacity that keeps degrading the lipid present in the plaque and tries to ingest it, but also as a consequence keeps releasing some of the degraded cholesterol into the milieu where the smooth muscle cells that are proliferating are internalizing it and becoming more foamy. So these are really great open questions that need to be addressed in the field.   Cindy St. Hilaire:        So drug-eluting stents are coated with rapamycin or the various chemical compositions that are derived from rapamycin. And rapamycin itself induces autophagy. So while the thought behind using this coating on stents was to prevent smooth muscle cell proliferation, and thus restenosis or ingrowing of the stent, your study suggests that this could also help to promote autophagy in the cells underlying the stent. So has anyone gone in and looked at plaques that have been stented and either failed or not, and investigated the foam cell content or markers for autophagy activity?   Mireille Ouimet:         Not to my knowledge, and this has been something we've definitely... We think that this is what's happening. Some of the protective effects of these drug-eluting stents that have everolimus or sirolimus or the rapamycin or rapamycin analogs, we do believe that some of their protective effect can be attributed to autophagy activation, but this remains to be demonstrated. We think that autophagy activation locally would promote reverse cholesterol transport and would be one of the processes that prevents restenosis because we can promote the efflux of cholesterol out.   Cindy St. Hilaire:        Great. So I guess stemming from my question on the stents, what are the other translational implications of the findings of your study? And what would you like to see come out of this?   Mireille Ouimet:         So one of the things is, as Sabrina mentioned, would be to target both foam cell populations because it seems as though the vascular smooth muscle cell foam cells are very much defective in their autophagy capacity, and they're very poor effluxes, but we could potentially restore autophagy in the cell population to promote reverse cholesterol transport.   And looking at prevention of atherosclerosis is a bit different than looking at regression, because regression is at a later stage where the plaques are more advanced. And if they're mostly vascular smooth muscle cell-derived, maybe then those drugs that we're considering that protect against the development of atherosclerosis are effective on the macrophage themselves early on, but might not be mimicking what we would see in the clinic where the patients that present are older.   Cindy St. Hilaire:        Yeah, it's kind of really reminiscent of like the CANTOS trial and like, where do we want to target the therapy? It's going to be very different if it's an early smaller plaque, versus a late-stage possibly pro close to rupturing type of plaque. Well, Sabrina Robichaud and Dr Ouimet, thank you so much for joining me today. Congratulations again on a wonderful study, and I'm really looking forward to hearing more about this from your group.   Sabrina Robichaud:    Thank you.   Mireille Ouimet:         Thank you very much. And we also want to thank all the co-authors on the study, specifically also Adil Rasheed, who is co-first author on the work and Katey Rayner's group for all the support and involvement in this study.   Cindy St. Hilaire:        That's it for the highlights from the March issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @circres and #DiscoverCircRes. Thank you to our guests, Sabrina Robichaud and Dr Mireille Ouimet Sabrina. This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles was provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire and this is Discover CircRes, you're on-the-go source for the most up-to-date and exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2022, The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.

This month on Episode 33 of Discover CircRes, host Cynthia St. Hilaire highlights two original research articles featured in the February 4 issue of Circulation Research. In addition, she previews Circulation Research's Compendium on Women and Cardiovascular Health, featured in the February 18th issue. This episode also features a conversation with Dr Alastair Poole and Dr Laura Corbin from the University of Bristol and Dr Stephen White from the Manchester Metropolitan University about their study, Epigenetic Regulation of F2RL3 Associates with Myocardial Infarction and Platelet Function.   Article highlights:   Samargandy, et al. Blood Pressure Trajectories and Menopause   Gilchrist, et al. Research Goes Red Registry   Cindy St. Hilaire:        Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's Journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh and today I'm going to be highlighting articles from our February issues of Circulation Research. I'm also going to speak with Dr Alastair Poole and Dr Laura Corbin from the University of Bristol and Dr Stephen White from the Manchester Metropolitan University about their study, Epigenetic Regulation of F2RL3 Associates with Myocardial Infarction and Platelet Function.   Cindy St. Hilaire:        The first article I want to share is titled Trajectories of Blood Pressure in Midlife Women: Does Menopause Matter? The first author is Saad Samargandy, and the corresponding author is Samar El Khoudary from the University of Pittsburgh. Blood pressure increases with age, but after midlife, the rate of increase for women generally exceeds that for men. This observation has led to debate over whether menopause might influence the blood pressure trajectory.   Cindy St. Hilaire:        To find out, this group examined data on over 3300 women of diverse ethnicity enrolled in the Study of Women's Health Across the Nation, or SWAN study. The women began the study between 42 and 52 years old, and they had 17 follow-up visits at roughly one-year intervals. At these visits, blood pressure, hormone levels, weight and other health parameters were measured.   Cindy St. Hilaire:        Analysis of the data revealed women fell largely into three blood pressure trajectory groups. Those with low blood pressure before menopause and accelerated blood pressure after menopause, those with a linear increase linked to age, and those with high blood pressure before and a slower ascent afterwards. White, Chinese and Japanese women were more likely to be in the low to accelerated group, as were those with early menopause, while Latino and Black women were more likely to have high blood pressure in general. Together, the results indicate that for many women, menopause itself does not accelerate age-related blood pressure increase, and that women of menopausal age should be advised of this risk and have their blood pressure monitored regularly.   Cindy St. Hilaire:        The second article I want to highlight is titled Research Goes Red: Early Experience With a Participant-Centric Registry. The first author is Susan Gilchrist and the corresponding author is Jennifer Hall from the American Heart Association. Cardiovascular disease is a leading cause of death for men and women alike, but there are particular factors such as pregnancy and menopause that may specifically influence the genesis, presentation and management of the condition in women.   Cindy St. Hilaire:        With that in mind, for the past two decades, the AHA's Go Red for Women campaign has been raising awareness of and driving research into women's cardiovascular health issues. The latest Go Red initiative, an online platform called Research Goes Red, was launched in 2019 with the aim of empowering women to contribute to health research by, among other things, taking part in health surveys. In the last two years, the platform has garnered 15,000 registered individuals between the ages of 30 and 60. It has deployed six targeted health surveys and prompted two AHA-funded research studies based on participant responses: one on perimenopausal weight gain, and one on the use of social media to engage young women in cardiovascular disease awareness. While Research Goes Red has successfully amassed middle aged participants, the authors say that future goals should include increasing the number and the diversity of the registrants and encouraging researchers to use the registry not just for data, but for identifying potential trial participants.   Cindy St. Hilaire:        I want to now mention the 15 articles that are featured in our Compendium on Women and Cardiovascular Disease that is featured in our February 18th issue of Circulation Research. And this also happens to correspond with February being the American Heart Month. So Susan Cheng and colleagues present A Scientific Imperative As Seen Through a Sharpened Lens: Sex, Gender and the Cardiovascular Condition. Genetic, molecular and cellular determinants of sex-specific cardiovascular traits is discussed by Teemu Niiranen and colleagues. Bonnie Ky et al. describe sex-specific cardiovascular risks of cancer and its therapies. Sex differences and similarities in valvular heart disease is presented by Francis Delling and colleagues. Cecile Lahiri and colleagues wrote about the cardiovascular implications of immune disorders in women. Joshua Smith and colleagues discuss sex differences in cardiac rehabilitation outcomes.   Cindy St. Hilaire:        Pregnancy and reproductive risk factors of cardiovascular disease in women is reviewed by Michael Honigberg and colleagues. The impact of sex and gender on stroke is presented by Kathryn Rexrode and colleagues. Ersilia DeFilippis and colleagues cover heart failure subtypes and cardiomyopathies in women. Demilade Adedinsewo and colleagues wrote about cardiovascular disease screening in women, leveraging artificial intelligence, and digital tools. Sexual dimorphism in cardiovascular biomarkers, clinical research implications, is discussed by Jennifer Ho and colleagues. Connie Hess et al. review sex differences in peripheral artery disease. Janet Wei and colleagues provide an update on coronary arterial function and disease in women with non-obstructive coronary arteries. Sex differences in myocardial and vascular aging is presented by Hongwei Ji and colleagues. And lastly, arrhythmias in female patients, incidence, presentation and management, is reviewed by Andrea Russo and colleagues.   Cindy St. Hilaire:        Today I have with me Drs  Alastair Poole and Laura Corbin from the University of Bristol and Dr Stephen White from the Manchester Metropolitan University. And they're here with me to discuss their study, Epigenetic Regulation of F2RL3 Associates with Myocardial Infarction and Platelet Function. And this article is in our February 4th issue of Circ Res. Well, Drs Corbin, Poole and White, thank you so much for joining me today.   Laura Corbin:             Thank you very much.   Stephen White:            Thank you.   Alastair Poole:            Thanks.   Cindy St. Hilaire:        So this is a really neat study. It's bringing in a couple different fields. It's investigating what I'm calling a Venn diagram of these intersecting topics all related to cardiovascular disease: cigarette smoking, epigenetic modification and platelet activation. So can you maybe give us a little bit of background on the status of the field and how these three topics intersected at the start of your study?   Laura Corbin:             So yeah, our working hypothesis was based on existing literature and it was really to look at whether smoking-induced epigenetic DNA hypermethylation of F2RL3 could increase risk of myocardial infarction and whether the route to that could be through platelet function. So there's quite a lot of literature going back probably to around about 2015 that shown that there are changes to the methylome in response to smoking. And DNA methylation is a way of cells controlling gene expression, but without having to actually make changes in the DNA sequence itself. So this could be a really important way that we know that smoking increases the risk of a number of cardiovascular diseases, but we don't really know how that happens. And one way that that could happen is through changes to methylation.   Cindy St. Hilaire:        What is known about how cigarette smoke impacts the status of DNA methylation? How has that switched or changed? Maybe when someone is actively smoking, when someone quits, is it dynamic? What is known about that relationship?   Laura Corbin:            Okay. So yeah, going back to about 2015, there was a number of studies that looked at methylation across the whole genome. So in a hypothesis-free untargeted manner, developments and technology meant that we could look at many, many sites across the genome at the same time. And so studies were done to look at changes that were associated with smoking. And what was found was those changes, actually quite a lot of changes across the genome in a number of different genes, but not really anything much beyond that. So F2RL3 was one of the first sites to be identified as being associated, methylation at that site associated with smoking. And it was replicated in several studies.   Laura Corbin:            And it was also showing that there was a dose-response relationship. So the more a person smoked, the less methylated that site appeared to be. And then there's been some work done already, but we also did it in our paper to show that those methylation marks actually hang around for quite a long time once somebody quits smoking. But also that there's a lot of variation within an individual, so even if you smoke, it doesn't necessarily mean that you'll definitely have low methylation, there's still variation. So there's other factors that are involved in that.   Cindy St. Hilaire:        So you were looking at a specific population of patients, can you tell us a little bit about that group of patients you were looking at? And you mentioned the variability in the amount of smoke they were exposed to, do you know that information? And I guess one of the base questions I had is I'm in Pittsburgh, which back in the '80s and earlier was a steel mill town that had a lot of pollution. And so I'm wondering if you're able to clearly separate out a cigarette smoker from maybe someone who is a light cigarette smoker, but lives in a more polluted area?   Laura Corbin:             Okay. So there's two parts of the study that were looking at this in a human context, so in a whole person context. One of those was using data from the Copenhagen City Heart Study, and that's the one where we looked at the relationship between smoking and methylation and then between methylation and myocardial infarction. So that study is great because it's been tracking people over time and so we're able to use the samples that were collected before they had their cardiovascular event and look at methylation at that point. So we know that the event occurs after that point, which is important. And so we were able to verify in that population that we did see an association between smoking and methylation. We were able to show that it was a dose-dependent relationship. So if we look at something like in that dataset, we had things like the intensity of which people smoke, so pack years is one of the things that we looked at. And it did appear to correspond in an approximately linear fashion.   Laura Corbin:             So we don't really know, I don't think, at this point, what impact other environmental exposures would have on the methylome and how that would interact with the cigarette smoking. That's actually a really interesting point that we'll probably come onto later about whilst we were looking here at the smoking effect on this methylation site, in the second part of the work, we were able to show that even in non-smokers, there's variability in methylation at this site, and that can still have impacts on the biology downstream. So yeah, it's an interesting point.   Stephen White:            Just to maybe just jump in, there's very good amounts of literature now that show quite a good correlation between changes in air quality and cardiovascular events. So smogs, wildfires and so on, clearly correlate with an increase in cardiovascular events. But actually the opposite's also been observed in the more recent COVID lockdowns, where reduction in air pollution also mirrored a reduction in the number of cardiovascular events. So I think you raise a really interesting point about is it cigarette smoke alone or does air quality in general play an effect? And clearly it does play an effect, although we didn't correlate that within this current dataset.   Cindy St. Hilaire:        Your study looked at DNA methylation patterns at cytosine, phosphate, guanidine or CPG sites in the genome. Can you tell us a little bit more about what these islands are and how they change throughout maybe different cells in the body, but also maybe in the same cell, but throughout the course of life or the course of, in this case, cigarette exposure?   Stephen White:            So if we just want to focus in on our study, what we showed was that exposure to cigarette smoke changes endothelial cell methylation. It also changes megakaryocyte methylation patterns in the same way. And I think one of the surprising things was that only 48 hours of exposure to cigarette smoke significantly changes the methylation pattern of the F2RL3 locus. So it's quite a dynamic event, but it does show that these can be quite rapidly regulated. And Laura's really nice work shows that the methylation on cessation of smoking, that pattern does actually go down, but it's a 20-year process. So it looks like it can be rapidly induced, but may actually remain as a methylation mark for a considerable length of time.   Stephen White:            And I think one of the things we did in our study was actually to triangulate not only the observational data and the association data in patients, but actually start to look at a mechanism of how that might actually relate to changes in gene expression. So we showed that this particular CPG site is right next to a binding site for a transcription factor, and transcription factors are the cell's way of regulating how much of a particular gene is expressed. And we show that changes in methylation changed the binding of this transcription factor and therefore change the amount of this particular gene that was made.   Cindy St. Hilaire:        Yeah. Actually, I want to start to talk about that locus you were interested in. So what was known about the F3RL2 locus? How big is it, but also what genes are there and what did you start to investigate with your in vitro modeling?   Stephen White:            So I think when we started, we had the observation that a change in methylation at the F2RL3 locus was associated with the risk of cardiovascular events. And then it was a detective expedition into the gene using various in silico analyses that identified the methylation site that we are interested in, or most interested in, is right next to a transcription factor binding site.   Stephen White:            So we then went on to show that binding of that transcription factor is sensitive to methylation, that if we would just excise that piece of DNA, we can show that that has the ability to regulate F2RL3 expression or the expression of a reporter gene. And then if you knocked out the transcription factor binding site, you lose that regulation. So it was a series of detective work and experimental steps that allowed us to put a mechanism behind the observation that changes in methylation might truly affect the level of gene expression of the F2RL3, otherwise known as PAR4 to platelet biologists. So get that in there.   Alastair Poole:            I first came across it when another member of our team actually mentioned it to me over a casual conversation actually a few years ago that F2RL3 gene was regulated in this way. To me as a platelet biologist, F2RL3 didn't mean a lot, but when I was told then it was the gene that encodes PAR4, it meant everything. And so platelet biologists, we talk about PAR4, which is of course the protein product of the F2RL3 gene. And PAR4 is one of several really key receptors on a platelet surface that responds to, in this case, to changes in thrombin generation, thrombin activity, which is of course the major effectively end product of the coagulation cascade.   Alastair Poole:            So it's what couples coagulation and platelet biology together, thrombin. And there are two major receptors on platelets that operate in response to changes in thrombin and that's PAR1 and PAR4. And they're both very important genes, but yeah, really interestingly, you have this rather selective effect on PAR4 and the paper actually shows it is indeed a selective effect on PAR4 as opposed to PAR1 in terms of epigenetic regulation of its responsiveness to PAR4 activation.   Cindy St. Hilaire:        So I want to tap back onto something that Laura had mentioned briefly, and that is talking about your platelet assays where you isolated platelets from a specific subset of the patients. And I believe it was figure three, and you looked at patients who in adolescence had exhibited differences in the methylation pattern at the site in the F3RL2 locus. What do we know about that innate or early-age change? And then I would love to hear more about this actual experiment, how you looked at the patients earlier versus current and what the thinking was behind that.   Laura Corbin:            So yeah, this part of the work was done in a birth cohort study called the Avon Longitudinal Study of Parents and Children, which is based at the University of Bristol. And this is a really great study, a great resource that we have, and in fact, it's open to all researchers so anyone could use it, where mothers were recruited during pregnancy, which was in around 1991 to 1992. And then those children that were born from those pregnancies have then been followed up ever since.   Laura Corbin:             So that was the data that we were able to use for this part of the study. And what we wanted to do was to look at how this could work functionally, so look at the platelet function, but we really wanted at this point to step away from the smoking. Because obviously if you're going to look at platelet function in smokers versus non-smokers, it's incredibly difficult then to say that that's coming through a specific pathway, because we know that smoking induces lots and lots of changes in methylation, in proteins, all sorts of things going on. So we couldn't see a way of doing that part of the experiment with a comparison of smokers versus non-smokers. But what we know is that there's natural variation in methylation across all sites, including F2RL3.   Laura Corbin:             So we had historic data from earlier time points, so two earlier time points from when the children were under 20. And we looked at those measures for F2RL3 and then just simply ranked people according to whether they had high or low methylation, and then used those two ranks together to then work out who had a consistently high versus consistently low level. And then we invited participants back into the clinic to have samples taken from those up and lower ends of the distribution. At that point, we were just really hoping that that methylation pattern would continue because this was then, I think they aged about 24 by the time we did this work, so it was some time after. And we restricted our selection just to people who were non-smokers, so never smokers based on the information they provided, but also asking them when they came in for that clinic just to verify that they were non-smokers.   Laura Corbin:             And then we had a look at the methylation again. This time we looked across four sites in the region, which are the sites presented in the paper. And luckily for us, there was still that mean difference between the high group and the low group. But what we were able to then do is to compare people with high and low methylation, but without all of the trouble of isolating that pathway in amongst all the other smoking effects. And also not just the smoking effects, but the other confounding factors that come with smoking. So we know that smoking is correlated with a lot of other lifestyle factors. So if you ever do a smoking versus non-smoking comparison, it's really hard to work out exactly which bits are coming from smoking and which pathways it might be going down. So this was the idea behind this part of the study was to just really zoom in on F2RL3 methylation in the absence of all of the other noise in the other experimental designs.   Laura Corbin:             So yeah, the natural variation we see in the non-smoking healthy participants in this part of the study is actually quite a lot less than we see when we look at smokers compared to non-smokers, but it was still enough to then go on and look at the platelet function. And then the differences we saw in the platelet's responses, there is nothing pathological there. It was just very subtle changes in the response when stimulated in the lab.   Alastair Poole:            The only other thing I could add would be that platelets are very complicated cells. Every cell of the body is very complicated. Platelets are certainly very complicated. PAR4, F2RL3, is just one of very many components of the platelet that modulate its activity. So platelets are controlled by multiple forces sort of thing, at which F2RL3 and PAR4 is just one of them. So biology is very good at compensating for one level going up in one part of a pathway and going down compensatory wise in another part of a pathway. There isn't necessarily a direct relationship between one pathway enhancement and an overall effect because of the compensation.   Cindy St. Hilaire:        Why would it be easy?   Alastair Poole:            Yeah. Yeah. It's just very complex, the biology. So yeah, I completely get what you're saying, Laura, that we obviously don't want to frighten people that maybe they've got a propensity to enhance thrombosis based upon a single gene methylation difference because it will be much more complex than that.   Cindy St. Hilaire:        Yeah. I think that's one of the beautiful things about your study is with the luck of having this sample population, you were able to ask these really precise questions that... You can't just start a study now and ask that sort of question. So it was really elegant in that sense.   Cindy St. Hilaire:        Do we know the mechanism of how cigarette smoke induces these methylation changes, or maybe even the specific components of the smoke? And I guess I'm thinking that in terms of vaping that's becoming more and more popular, obviously the company selling those products want to advertise them as safer, but it comes down to is it all of the mixture of the cigarette smoke or is it one component that we know impacts the methyltransferases and demethyltransferases in this process?   Alastair Poole:            Those are two follow-on routes of our study that I have to say that we discussed previously amongst ourselves and identified those as definitely very important follow-up areas. So do e-cigs have similar effects and that's a study that definitely needs to be done. We have done a little bit of work to try to investigate that initially, but I think that's a very important follow-on study. But yeah, you're also right that one of the key things that we want to understand and is, the missing piece in a way, is how is methylation at a molecular mechanistic level altered by smoking? Steve, I don't know whether you have any further details to add to that.   Stephen White:            I think one of the key molecular pathways seems to be the antioxidant response. And so that's largely controlled by another transcription factor called NRF2. And so if you think about smoking or poor air quality, all of those things do combine through this particular pathway that senses free radical damage, free radical stress. So as Alastair said, it's an area we are going to carry on to look at and it's a big area of my own lab's investigations. But oxygen stress is probably the mediating factor, but the actual nuts and bolts about how the demethylase is targeted to this particular locus is still an area of active investigation.   Cindy St. Hilaire:        All right, well, I will be on the lookout for those future studies because it's a really interesting topic, just the whole interplay of all of this. Are there any translational implications for these findings? Do you think potentially we could screen patients, say, to see their methylation status? I don't know if megakaryocytes are easy to isolate, but is it in a circulating cell, would this possibly be able to be turned into a screening tool or a diagnostic tool to predict thrombic events in patients?   Alastair Poole:            It is possible. I think it would not be possible to isolate megakaryocytes very easily. There are a small number in the peripheral circulation, but the majority are not in the peripheral circulation. But we and others have used other blood cells as proxy measures. So actually, the gene methylation changes that we identified here come from other leukocytes, white blood cells, and those effectively are a cell that are exposed to smoke in the same way, or the smoke products in the same way. So we'd use a proxy cell for that.   Alastair Poole:            Yes, I suppose it is possible. As you say, there's a natural variation in methylation status of that gene and there's, layered on top of that, a smoking induced. And I suppose that it would be an interesting further investigation to understand whether, effectively, your natural methylation status of that gene happened to give you an enhanced risk of a cardiovascular event. The work we've done seems to suggest that that may well be the case and therefore you could imagine possibly a personalized medicine approach that might include understanding the methylation status of F2RL3 as part of that.   Cindy St. Hilaire:        Well, it was a beautiful study. I love these studies that bring in lots of different fields or specialties to ask interesting questions. So Dr Corbin and Dr Poole from the University of Bristol and Dr White from Manchester Metropolitan University, thank you so much joining me today.   Stephen White:            Thank you. Our pleasure.   Alastair Poole:            Thank you.   Laura Corbin:             I'd also just like to acknowledge all of our co-authors as it really was a big team effort, especially the guys who are not represented on the call today, which is the folk from the Copenhagen City Heart Study, and also to all of the participants of that study and the Children of the '90s Study that contributed to the work. Thanks very much.   Cindy St. Hilaire:        That's it for the highlights from our February issues of Circulation Research. Thank you so much for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and the hashtag #DiscoverCircRes. Thank you to our guests, Drs  Alastair Poole, Laura Corbin and Stephen White.   Cindy St. Hilaire:        This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discoverer CircRes, your on-the-go source for the most exciting discoveries and basic cardiovascular research.   Cindy St. Hilaire:        This program is copyright of the American Heart Association 2022. The opinions expressed by speakers of this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahajournals.org.    

This month on Episode 32 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the January 7 and January 21 issues of Circulation Research. This episode also features a conversation with Ms Natalie Harris and Dr Kathleen Caron from the University of North Carolina Chapel Hill about their study, VE-Cadherin Is Required for Cardiac Lymphatic Maintenance and Signaling.   Article highlights:   Carlson, et al. AKAP18δ Controls CaMKIIδ Activity   Gan, et al. sEV and Adipocyte ER Stress Following MI/R   Khan, et al. Long-term Risk Prediction of Heart Failure   Awan, et al. Wnt5a Is Essential for Cholesterol Homeostasis   Cindy St. Hilaire:        Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh. Today, I'm going to be highlighting the articles from our January issues of Circulation Research. I'm also going to speak with Ms Natalie Harris and Dr Kathleen Caron from the University of North Carolina Chapel Hill about their study, VE-Cadherin Is Required for Cardiac Lymphatic Maintenance and Signaling.   Cindy St. Hilaire:        The first article I want to share is titled AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and Ryanodine Receptors. The first and corresponding author for this article is Cathrine Carlson, and the study was conducted at University of Ohio. In cardiac muscle cells, calcium is continuously released and taken up by the sarcoplasmic reticulum to drive alternating contractions and relaxations. The kinase, CaMKII, regulates this calcium signaling via phosphorylation of the sarcoplasmic reticulum proteins ryanodine receptors also called RYR.   Cindy St. Hilaire:         These receptors promote calcium release, and phospholamban promotes calcium uptake via the transporter SERCA, but how CaMKII localizes to and associates with these sarcoplasmic reticulum factors was unclear. Because AKAP18 delta enables phosphorylation of phospholamban and calcium uptake into the sarcoplasmic reticulum, this group suspected it might be involved. The team's immuno precipitation and functional experiments in rodent cardiomyocytes show that AKAP18 delta associates with CaMKII and phospholamban SERCA2 as well as with CaMKII and ryanodine receptors, and that these interactions are linked to CaMKII activity.   Cindy St. Hilaire:         The team identified two separate CaMKII binding domains within the AKAP18 delta protein, one that inhibits the kinase and one that actuates it, suggesting they may somehow serve to fine tune CaMKII activity. While such regulatory details remain to be resolved, the isolated domains may be utilized as tools for studying calcium handling in cardiomyocytes, and for developing therapeutic CaMKII regulating reagents for treating arrhythmia.   Cindy St. Hilaire:         The second article I want to share is titled Ischemic Heart-Derived Small Extracellular Vesicles Impair Adipocyte Function. The first author is Lu Gan, and the corresponding authors are Yajing Wang and Yu Cao from Thomas Jefferson University. While diabetes and obesity increase a person's risk of myocardial infarction, suffering a myocardial infarction itself can lead to metabolic dysfunction. One of the main regulators of systemic metabolic homeostasis is the body's adipose tissue, but whether and how an injured heart communicates with adipocytes was unclear.   Cindy St. Hilaire:         The infarcted heart is known to release microRNA containing extracellular vesicles, also called EVs, and so this group hypothesized that these EVs might constitute a heart-to-fat communication system. They isolated circulating EVs before and after myocardial infarction in mice, and incubated these vesicles with cultured adipocytes. After 24 hours, differences in adipocyte gene and protein expression were apparent. Notably, a key cardioprotective metabolic factor called adiponectin was downregulated in cells treated with the extracellular vesicles from myocardial infarcted mice, while genes involved in endoplasmic reticulum stress were increased.   Cindy St. Hilaire:         Analysis of the myocardial infarction extracellular vesicle content showed an increased abundance of specific microRNAs, and the team went on to show that inhibiting production of these microRNAs or the EVs themselves, prevented adipocyte ER stress and adiponectin production in mice after myocardial infarction. Together, these data hints that such microRNA inhibition may be a clinical strategy that can be used to prevent infarction-associated metabolic dysfunction in humans.   Cindy St. Hilaire:         The next article I want to share is titled Development and Validation of A Long-Term Incident Heart Failure Risk Model. The first and corresponding author of this study is Sadiya Khan from Northwestern University. Heart failure contributes to approximately 1.2 million hospitalizations, and 300,000 deaths in the U.S. annually. Heart failure also has an estimated healthcare cost of over $10 billion. With both the incident rates and costs expected to rise in the future, a method for predicting an individual's heart failure risk would enable preventative interventions such as diet and blood pressure treatments to be initiated early, thus prolonging the number of healthy years.   Cindy St. Hilaire:         To develop such a prediction tool, this group studied decades of health data from over 24,000 individuals that was collected as part of five separate, long-running national heart, lung and blood institute studies. The individuals included in the model for development were at baseline aged between 20 and 59 years old, and had no cardiovascular disease diagnosis at that time. Analysis of their body mass indices, blood pressures, total cholesterol levels, high density lipoprotein levels, smoking statuses, diabetes diagnoses, and other cardiovascular health data over several decades enabled the team to develop an equation for predicting an individual's likelihood of developing heart failure in the next 30 years. The hope is such personalized risk assessments will help to guide patient-doctor discussions regarding cardiovascular health, lifestyle choices and medical interventions.   Cindy St. Hilaire:        The last article I want to share is titled Wnt5a Promotes Lysosomal Cholesterol Egress and Protects Against Atherosclerosis. The first authors are Sarah Awan and Magalie Lambert, and the corresponding author is Philippe Boucher from the University of Strasbourg. The Wnt family of signaling proteins drives many developmental processes, such as cell fate determination, proliferation and migration. Recently, Wnt signaling has been implicated in lipid homeostasis. Mutations that impair Wnt signaling have been shown to cause hyperlipidemia in mice, and in humans, decreased Wnt signaling activity inversely correlates with atherosclerosis severity.   Cindy St. Hilaire:        Because the protein Wnt5a in particular has been shown to inhibit cholesterol accumulation in cells, this group investigated the role of Wnt5a protein in mice and human cells. Mice whose vascular smooth muscle cells lacked Wnt5a developed more severe atherosclerosis compared to control animals, and human smooth muscle cells lacking Wnt5a accumulated far greater amounts of cholesterol in the lysosomes than did cells with normal levels of Wnt5a. The group then showed that Wnt5a normally associates with lysosomes, where it promotes the catabolism of lysosomal cholesterol via activating lysosomal lipase, and promoting cholesterol egress via the endoplasmic reticulum. In revealing how cholesterol efflux is trafficked by Wnt5a, these findings may help to inform future cholesterol regulating therapies.   Cindy St. Hilaire:         Today, Natalie Harris and Dr Kathleen Caron from the University of North Carolina Chapel Hill are here with me to discuss their study, VE-Cadherin Is Required for Cardiac Lymphatic Maintenance and Signaling, which is featured in our January 7th issue of Circulation Research. Thank you both for joining me today.   Kathleen Caron:          Thanks, Cindy, for having us. We're really honored and excited to talk with you.   Cindy St. Hilaire:         I'm excited too, because I think this is my first lymphatic paper I'm talking about. That's where I'm going to start my questions. Your study is investigating cardiac lymphatics. But like I said, I haven't talked a lot about lymphatics here, so I was wondering if you could at least give a little bit of background about what the role is of the lymphatic system, especially because I feel like it's the unappreciated member of the circulation, and also give us a little bit of background on what cardiac lymphatics are.   Kathleen Caron:          That's a really great question. We sometimes talk about lymphatic vessels as the third vascular system or the understudied vascular system. I'm hoping that that's not the case so much anymore, because the lymphatic field has really boomed in the past 15 years or so. I think where we are right now in the field is in early days, we and others had discovered key signaling molecules, and transcription factors, and growth factors that are important and specific to the lymphatic vasculature as compared to blood endothelial cells. Through those unique tools, now, the field has fast forwarded where we're starting to look into organ-specific functions of lymphatics.   Kathleen Caron:          We're appreciating that perhaps a little unlike the blood vascular system, which has one main function of delivering blood, lymphatics actually have very different functions depending on the organ that they're in. Some of the more common ones that you'll read about in textbooks in about a paragraph in a medical textbook are that lymphatics are important for immune cell trafficking through the lymph nodes, so they're the major route of trafficking for immune cells and for their maturation. Lymphatics are also important for draining interstitial fluid, and maintaining the homeostasis of tissue fluid balance.   Kathleen Caron:          A third really big one, which is sometimes underappreciated, is that lymphatics are the key vessels within the intestine that absorb lipid, and so all of our dietary lipids are absorbed through lymphatic vessels as opposed to the blood vasculature. Those three hallmark functions of lymphatics are the cornerstone of what they do throughout our body. But when you start to look into different organs and recognizing the different extrinsic and intrinsic forces that govern the function of these endothelial cells and different organs, you start to realize that they're even more complex, and that brings us to the heart.   Kathleen Caron:          The heart just has this beautiful network of lymphatic vessels that begin in the subendocardial space, and then project out and cover the subepicardial surface of the heart. And because the heart is always pumping, and because lymphatic vessels don't have an intrinsic mechanism for the flow of fluid through them, they rely on the movement of the tissue that they're in to help propel the fluid. So, this really raises the question of how are lymphatics functioning physically within a myocardium that is pumping with a very strong extrinsic force, and what is the function of those vessels if the heart is a very dense, thick organ that is not necessarily prone to edema necessarily as maybe our peripheral tissue and our skin is? Kathleen Caron:          We've been studying this for many years now, and we've had several studies exploring genetic factors that are important for the growth and development of cardiac lymphatics. That's the focus of this paper today. They're quite unique and very different vessels.   Cindy St. Hilaire:         Reading your paper, I definitely learned a lot about lymphatics in general. One of the things I was thinking about, obviously, you're looking at VE-Cadherin, which is an endothelial cell marker. When I think of VE-Cadherin, and when I think of endothelial cells, my mind goes primarily to those that are in arteries and veins. In those conduits, their role is to really keep a tight seal to keep things out. But in the lymphatic system, it's very different, so how exactly different are the endothelial cells in the lymphatic tissue, and are they different, say, in the cardiac lymphatics versus, like you said, the mesenteric lymphatic?   Kathleen Caron:          Lymphatics are very different than the blood vasculature. First of all, the lymphatic vasculature has key differences in terms of its architecture and structure. The lymphatic endothelial cells themselves, as they exist in vessels, don't put down a basement membrane, and in general, the dermal capillaries or the initial collector lymphatics that are the ones that are taking in fluid also don't have smooth muscle cells surrounding them like our typical vasculature does. All of this is guided and precedented by the differences in gene expression patterns of these very specialized endothelial cells.   Kathleen Caron:          They also have very different cell-cell junctions. So when we think of a blood endothelial cell, we typically think of these tight junctions that bring them together, but the lymphatic endothelial cells have oak leaf shaped overlapping junctions. They're really beautiful to see on an EM, and they're very different than the blood vasculature, because, Cindy, as you mentioned, the function is very different. You're supposed to let things leak out, and big things too, right, like immune cells and large proteins.   Cindy St. Hilaire:         One of the neat things that really made your study possible is this really nice PROX1 inducible CRE that you crossed with the flox-cadherin5 gene. I was wondering a little bit about that protein. Is that one of these, I guess, markers that allows lymphatic EC to be a lymphatic EC, and how specific is that protein for those specific ECs?   Natalie Harris:            The PROX1 CRE that we use is based off of the PROX1 transcription factor, which we consider to be one of the master transcription factors of lymphatics. In fact, that was one of the very first lymphatic specific transcription factors that help maintain the lymphatic identity. So in this case, PROX1 turns on from blood endothelial cells, because many lymphatics are of venous origin, so actually, PROX1 turning on is a hallmark of them becoming a lymphatic endothelial cell.   Natalie Harris:            Those are really great CRE specifically to look at lymphatics in this case, and it actually is a perfect model system because VE-Cadherin itself is only expressed in lymphatics and blood vessels, and then we have PROX1 as our free driver. Therefore, it will only be lymphatic, so it's a very specific lymphatic knockout of VE-Cadherin.   Cindy St. Hilaire:         That's so wonderful when we discover things that are so specific like that. So using this really nice model that's also Tamoxifen inducible, you then have control to look at things temporally. One of the neat things that you did was you looked at this in terms of an embryonic level knockout, but then another one postnatally, and then another one, it was an adult mouse, which not a lot of people do that intricate, temporal spacing of things. So I was wondering if you could just share with us what you were thinking behind doing that, and then really importantly, what those different models actually taught you about the cardiac lymphatics?   Kathleen Caron:          That's a great question, Cindy. It would take me 20 minutes to answer. It really represents work by all of the co-authors. Really, it's the first effort to look at the different stages. That's because the growth and development of lymphatics, particularly within the myocardium, differs a lot during embryogenesis, and then the vessels themselves are quiescent in an adult animal. Then of course, we were interested in seeing what might happen in an injured myocardium, and that was also part of the study.   Kathleen Caron:          We felt that it was important to address the changing and dynamic role of this protein in a developing lymphatic, because it's growing and forming these nascent vessels, and then as it's starting to remodel an early life, and then in adulthood when it's in a quiescence state. That was the rationale for looking at this. It was also... Sometimes, science just takes you where it takes you, and it was a co-author of ours, and collaborator of ours, who had noted a phenotype in the hearts of these animals that he generated and suggested that maybe it would be a good idea to look early in development. Then as one thing leads to another, you start looking later in development and so on and so forth, so the science just kind of…   Cindy St. Hilaire:         Sometimes tells you where to go on its own.   Kathleen Caron:          Exactly. It was a long project.   Natalie Harris:            Part of the reason too is that the cardiac lymphatics have been shown to have a little bit of a different development and maintenance and pruning cycle than some of the other lymphatics. Some other lymphatics are totally fully formed in embryonic development, but the cardiac lymphatics have been shown to develop through birth and a little bit postnatally as well. That makes them a little bit unique in the sense that their maturation is very prolonged, so that's part of the reason as well we wanted to look both in embryonic development as well as that postnatal period.   Cindy St. Hilaire:         That's so interesting. There are a lot of little nuggets that my antennas would perk up as I read your paper, really neat observations. One of them was that I think it was the postnatal and the adults. There was lymphatic endothelial cells in the cardiac tissue were disrupted. They were discontinuous and fragmented, yet there was no cardiac edema. I thought that was interesting because normally, you'd think about any of these mice with lymphatic issues. You think of edema. You think of swelling, and yet it wasn't happening in the heart. What do you think that means either about the lymphatic system in the heart or in lymphatics as a whole?   Kathleen Caron:          That's a really great question, and one that we think about all the time. I think it goes back to the first question or the first comment about the really remarkable differences in the functions of lymphatics and different tissues, right? And within the myocardium, because it is continuously moving and pumping with great force, the extrinsic forces within that tissue will help to mitigate the formation of edema. This is not to say that you can't get myocardial edema, and we've actually developed surgical models in our lab to form myocardial edema in mice.   Kathleen Caron:          It is a very common clinical condition in humans as well, but the lymphatics themselves being fully invested within this myocardium probably are being regulated differently in their function in draining fluid than, for example, the lymphatics that you might have in the skin or in your thigh or in other organs in your body. The fact that there wasn't edema, even though you had leaky vessels, didn't alarm us too much because we knew and sensed that with this constant pressure and pumping of the myocardium, that in itself helps to keep the tissue fluid balanced.   Natalie Harris:            That might be another reason why we're not seeing such extremes in edema, and then going back to what Kathrine said, again, because lymphatics have multiple functions, perhaps it's more in the immune cell realm or even other functions we haven't uncovered yet.   Cindy St. Hilaire:         One of the other neat observations you had was that you were doing a myocardial infarction model on the adult animals, and you noticed that the infarct size and the fibrosis was indeed larger in the knockouts, but the cardiac function wasn't exactly affected. What does this mean, and were you surprised by this?   Kathleen Caron:          Yeah, we were surprised. We absolutely were surprised, and we think that's actually one of the key big reveals for the field. To balance this, to counterbalance the absence of a phenotype, that was really remarkable to us, and I hope to many others as well, is that other studies including work from our lab and Paul Riley's lab and Eva Brackinham's lab have very convincingly shown in multiple different ways that if you stimulate lymphangiogenesis after injury, if you have a model, either genetic or induced, where there are more lymphatics for whatever reason, that's a beneficial thing. That's a great thing, and having more lymphatics is positive and beneficial to improving heart repair, and mitigating heart injury, and helping in the context of myocardial infarction.   Kathleen Caron:          Of course, it was really surprising that now we have a mouse model where we essentially have little to no lymphatics with very little to no function, and yet the ejection fractional shorting of the heart was doing just fine. I think that was a big moment and a big discovery for us, but very convincing. Then I think it leads us to really asking while more might be better, what really could be the critical function of the lymphatics in an injured myocardium? As Natalie just mentioned previously, it might be related to immune cell trafficking. Paul Riley's group has made some really seminal discoveries in that regard.   Natalie Harris:            It's just very interesting, because it's really against everything that you would expect from, again, all the previous studies. It just goes to show again that the lymphatics are so heterogeneous in their organ level function that that's really worth exploring more, because maybe if you can figure out strategies to selectively target certain beds, you can really do a treat on the disease by disease, organ by organ basis. That makes the lymphatics just really cool in my opinion, because they are so different, but it's all the same system, so it's just a very interesting organ, in my opinion.   Kathleen Caron:          I should also say serendipitously or right about a few months ago... Shout out to Mark Kahn's lab at University of Pennsylvania. They had a recent paper, I believe, in JCI that had a similar finding to ours. It's always gratifying when another lab says, "Oh, wow, really?" Their study was very different than ours and on a different series of signaling molecules, but similarly, they ablated or reduced cardiac lymphatics through different mechanisms, and then had an injury model. Also, were rather surprised to see that it didn't have this negative effect.   Cindy St. Hilaire:        It's so neat. The whole observations that you saw with these knockouts was a paper in itself, but the next half of the paper, you dig into the mechanism, which is also interesting. Can you share a little bit about the links that you found between VE-Cadherin and the VEGF receptor signaling, and is your mechanism you think specific to all lymphatic ECs or even all ECs, or is it specific just to the cardiac lymphatic ECs?   Kathleen Caron:          Yes, the mechanism, I find one of the funnest parts of this paper, because I think it really synergizes a lot of the key signaling molecules within our field. Also, I think it bridges together a G-protein-coupled-receptor signaling pathway that my lab has been interested in for decades now, and that is a pathway with the VEGFR3 signaling pathway. I think that's been a big open question in the field. How do these two critical requisite signaling paradigms for lymphatics converge together to maintain lymphatic function development?   Kathleen Caron:          I think we've really made some really great inroads in the study, and VE-Cadherin is central to that because it forms a structural scaffold to keep a GPCR signaling pathway in register with the receptor tyrosine kinase signaling pathway, and basically allow for the transactivation of these two really powerful pathways. The mechanism really is gratifying to be able to finally pull how these molecules all interface together and regulate one another.   Natalie Harris:            It's very interesting in the fact that VE-Cadherin, it's not necessarily like a lymphatic-specific molecule, but a lot of work in terms of VE-Cad has been more in studying mechanosensing and mechanotransductions. That's where a lot of little nuggets about maybe our mechanism has occurred that we know from really just on protein level studies that VE-Cadherin does interact with VEGFR2 and VEGFR3 by the transmembrane domain interaction. That was clue number one, and then clue number two is that we know that a lot of different mechanical signals that might affect VEGFR3 happened in the presence of VE-Cad.   Natalie Harris:            So in a sense, this particular paper is just piecing together a lot of these nuggets of information, and it all makes sense. One thing that you were saying in terms of maybe specific to the heart, going back to some of the earlier studies on these papers on these mice, that we found very vessel-bed-specific effects. One of the vessel beds that is really impacted is the lacteals and the mesentery, so the gut lymphatics. We do know that these lymphatic beds are very sensitive to VEGFC. In fact, they require constant VEGFC signaling. So if you're not having VEGFR3 stable at the membrane to receive these signals, it makes sense if you would have really extreme effects. That might be, again, some of the case in the heart as well. We do know after a cardiac injury, we do see an increase in things like adrenomedullin, and an increase in VEGFC has been shown to increase lymphangiogenesis, so perhaps also the heart, the gut lymphatics also has a special requirement for VEFGR3 signaling.   Cindy St. Hilaire:         So in terms of, I guess, the future of this line of research and maybe thinking about translation, what do you see as maybe a role for this in terms of developing therapeutic strategies or even preventative measures, I guess, specifically in the cardiac lymphatic area?   Natalie Harris:            Like we mentioned earlier, there's been a lot of studies in mice that have looked at increasing lymphangiogenesis post-injury, so it would be interesting to see more when those hit the clinical end, and if you're seeing similar effects. Then the other thing that's interesting about lymphatics, you can think of them as both a target and also as a drug delivery route. There's a huge, huge field totally dedicated to using the lymphatics to deliver drugs like nanoparticles. That's very big in the cancer realm, and pretty much for any kind of drug delivery, if you can imagine using that as a super highway to deliver drugs as well.   Natalie Harris:            That could be a potential avenue in terms of the heart as well, getting a more specific administration of cardiovascular drugs to the heart. So whether or not we're thinking of them as being modulated by disease, we can also use them to modulate the disease itself by delivering drugs as well, so it's interesting. You can think of the lymphatics as a therapeutic target and as a therapeutic administrator. That's going to be really interesting to see where the field goes.   Cindy St. Hilaire:         I like that, a new super highway to deliver drugs. Thank you so much, soon to be Dr Harris and Dr Caron from UNC Chapel Hill. This was a wonderful conversation and a beautiful paper. Congratulations on all the hard work. Kathleen Caron:          Well, thanks so much, Cindy, and to the whole Circ Research team. We really appreciate your advocacy for our work and giving us this wonderful opportunity.   Natalie Harris:            Thank you so much.   Cindy St. Hilaire:         That's it for the highlights from our January issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page, and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Natalie Harris and Dr Kathleen Caron. This podcast was produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles was provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on-the-go source for the most up-to-date and exciting discoveries in basic cardiovascular research.   Cindy St. Hilaire:         This program is copyright of the American Heart Association, 2022. The opinions expressed by speakers on this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.  

MONROE COUNTY CIVIL RIGHTS HISTORY MR. H B WILLIAMS & NANNY RUTH WILLIAMS Traveling back in time to visit Harper Lee's hometown, the setting of her 1960 masterpiece and ... with her husband, Homer Beecher Williams—“H.B.” to his friends—and anyone else they invited. ... Monroeville styles itself “the Literary Capital of Alabama. ... That it appeared, like a revelation, at the very moment the civil rights .. Conversation about the NAACP With Mr H.B.Williams Monroeville, Al.Homer Beecher Williams—“H.B A long time Civil RIGHT Activitiest. Community Teacher. Special Guest Gwendolyn P. Kyles ALABAMA  CIVIL RIGHTS HISTORY INTRO  INTERVIEW MR. H B WILLIAMS & NANNY RUTH WILLIAMS FREDDIE C.HOWARD WITH AUNDRETTA WILLIAMS INTRO H. B. WILLIAMS CIVIL RIGHTS  ACTIVITIST The following documents contain excerpts, quotes, and news articles about racism, social, economic and environmental justice, criminal justice and prison reform, community policing and Equality in America. https://www.blogtalkradio.com/howcee-productions-gospel/2020/02/04/civil-right-activist-mr-hb-williams-mrs-nanny-ruth-williams-w-host-howcee https://www.blogtalkradio.com/howcee-productions-gospel/2015/07/20/sunday-night-conversation-about-the-naacp-with-mr-hbwilliams-monroeville-al https://www.blogtalkradio.com/howcee-productions-gospel/2015/07/20/sunday-night-conversation-about-the-naacp-with-mr-hbwilliams-monroeville-al  

This month on Episode 31 of Discover CircRes, host Cynthia St. Hilaire highlights two original research articles featured in the December 3 issue of Circulation Research. This episode also features a conversation with Drs Xavier Revelo, and Jop van Berlo from the University of Minnesota about their study, Cardiac Resident Macrophages Prevent Fibrosis and Stimulate Angiogenesis.   Article highlights:   Tong, et al. Alternative Mitophagy Protects Obesity Hearts Soetkamp, et al. Myofilament Phosphorylation in CDC Treated HFpEF   Cindy St. Hilaire:        Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hillaire from the Vascular Medicine Institute at the University of Pittsburgh. And today I'll be highlighting two articles presented in our December 3rd issue of CircRes, and I'll also speak with doctors, Xavier Revelo, and Jop van Berlo from University of Minnesota about their study, Cardiac Resident Macrophages Prevent Fibrosis and Stimulate Angiogenesis.   Cindy St. Hilaire:        The first article I want to share is titled, Alternative Mitophagy Protects the Heart Against Obesity-Associated Cardiomyopathy. The first doctor is Ming Ming Tong, and the corresponding author is Jun Sadoshima from Rutgers University. People with obesity or diabetes have an increased risk of developing cardiomyopathy, a condition which can eventually lead to heart failure. One of the major pathological features of obesity-related cardiomyopathy at the cellular level is a decrease in mitochondrial function. This decrease in mitochondrial function is likely due to a decrease in the canonical mitophagy pathway, which is a process by which dysfunctional mitochondria are degraded. However, a new process termed alternative mitophagy was recently discovered. When mice were fed a high fat diet for 24 weeks after only eight weeks, canonical mitophagy ceased. However alternative mitophagy steadily increased over the 24 weeks. Alternative mitophagy is regulated via the protein ULK1 and Rab9. The team went on to show that suppressing alternative mitophagy by knocking out ULK1, or expressing a loss of function, Rab9 mutant exacerbated the high fat diet induced cardiac dysfunction. Over expression of Rab9 in mouse hearts increased the alternative mitophagy pathway and protected the animals from cardiac dysfunction. These results suggest that pharmacological boosting of this ULK1 Rab9 mediated alternative mitophagy pathway might be a treatment strategy for preventing obesity related cardiomyopathy.   Cindy St. Hilaire:        The second article I want to share is titled Myofilament Phosphorylation in Stem Cell Treated Diastolic Heart Failure. The first doctor is Daniel Soetkamp and the corresponding author is Jenny Van Eyk from Cedar Sinai Medical Center. Weakness, fatigue and troubled breathing are among the symptoms experienced by someone suffering from heart failure with preserved ejection fraction, which is frequently called HFpEF. The pathology of the condition includes hypertrophy, fibrosis and stiffening of the heart and hyperphosphorylation of the cell sarcomere proteins. Because this hyperphosphorylation is a key contributor to HFpEF pathology, and because cardio sphere derived stem cells or CDCs have shown promise as a potential HFpEF treatment, this group investigated whether CDC treatment reduces phosphorylation levels of the sarcomere proteins in the heart. They found that administering CDCs to rats with HFpEF decreased the associated protein hyperphosphorylation, compared with that seen in untreated animals. Bioinformatic analysis revealed that protein kinase C or PKC is a prime suspect behind the phosphorylation. The authors suggest that CDCs alleviate HFpEF in part by reversing PKC-induced phosphorylation, and that PKC inhibition may be a desirable alternative treatment strategy, especially as it avoids regulatory issues associated with cell-based therapies.   Cindy St. Hilaire:        Today, I have Dr Xavier Revelo and Dr Jop van Berlo from the University of Minnesota and they're with me to discuss their study, Cardiac Resident Macrophages Prevent Fibrosis and Stimulates Angiogenesis. And this article is in our December 3rd issue of Circ Res. So, thank you both so much for joining me today.   Jop van Berlo:             Thanks for having us.   Cindy St. Hilaire:        Absolutely. So, your study is investigating the contribution of resident and monocyte drug of macrophages in cardiac remodeling, specifically in hypertrophy remodeling. So can you just introduce the topic of cardiac hypertrophy in humans, why that's not great to have, and then maybe tell us a little bit about what is known or what was known about the role of inflammatory cells in that hypertrophic remodeling.   Jop van Berlo:             Yeah, so absolutely. Cardiac hypertrophy is not a disease in and of itself in humans, but it is often a consequence of pathologies that can happen in patients, such as high blood pressure, hypertension or aortic valve stenosis, or if you've had a myocardial infarction the remaining myocardial may also become hypertrophic. We know that cardiac hypertrophy has downsides to it. People can develop sudden cardiac death when they have hypertrophic heart disease. We notice from population studies like the Framingham Heart Study and other studies, but it also increases the chance of developing heart failure later on. So even though cardiac hypertrophy by itself is not a disease, it is contributing to the cardiac pathology that can develop in patients and that can contribute to the development of heart failure.   Cindy St. Hilaire:        Great. So, what's the base level of knowledge of what is known regarding inflammatory cells in cardiac hypertrophy or cardiac hypertrophic remodeling?   Xavier Revelo:             So previous forecast focused on the role of infiltrating cells, specifically monocyte-derived macrophages, and generally these cells are pro-inflammatory and they aggravate the progression of heart failure. With Jop, we focus on, and what we think is exciting is the role of cardiac resident macrophages. And so, in our experiments, we decided to look at what's the role of these cardiac meta macrophages during pressure overload.   Cindy St. Hilaire:        That's a perfect segue to my next question, which is you obviously modeled this in mice, you used mice as your model and the method that you used to induce this hypertrophy is a technique called Transverse Aortic Constriction. So how does that actually work in a mouse and are there certain pros and cons to using that as a model for cardiac hypertrophy and, does it really recapitulate well, what happens in humans?   Jop van Berlo:             So, you're absolutely right that we use model systems to mimic what happens in humans and every model system has pros and cons to it. What we're trying to do here is to induce essentially acute cardiac pressure overload in a mouse model by inducing a constriction of the transverse aorta, right between the anomia and the left carotid artery. And we do this by ligating a needle on top of the transverse aorta that is of a specific size. And then we pull the needle out of the ligation and that immediately induces constriction. This is known to induce cardiac hypertrophy, and there are thousands of papers about this model as a model to induce cardiac hypertrophy. I think Howard Rockman was the first to publish this as a model of cardiac hypertrophy. Over the past decades, most of the research has focused on how cardiomyocytes within the heart respond to their stress and how they become hypertrophic. And I think what is new about our study is that instead of really focusing on the cardiomyocyte, we are focusing more on the non-cardiomyocyte compartments early after this stress is induced on the heart.   Cindy St. Hilaire:        That was one of the things I liked about this paper. We read about TAC a lot, the transaortic constriction model, but a lot of it is looking at either just the fibrotic cells or the scarring or the cardiomyocytes. So, this was, I thought a really nice unique take. So, one of the things I'm wondering is what are the functional differences between the systemic macrophages and these resident macrophages? I guess, resident to the cardiac tissue. And how does one tell the difference between these cells in the mice, but also in the humans? What is the human equivalent of those cells?   Xavier Revelo:             So, these cells, they rest in the macrophages in the cardiac tissue. One of the key differences from circulatory cells is the origin of the cells. In the heart, these cells self-renew, and they are from embryonic origins, as opposed to circulatory immune cells that come from the bone marrow. In terms of similarities between mice and humans, there are some markers that we can use to specifically study the cardiac resident macrophages. And these markers fortunately seem to be consistent between people and mice, which is advantages.   Cindy St. Hilaire:        That is good. That's always nice when it works out that way. So, you, you actually answered my next question, which was, are these residents macrophages a) able to self-replicate or are they from their own source? And so, regarding that developmental origin, how far apart are these lineages of the circulating monocytes versus the resident or the cardiac resident? How similar and how different, and how far back on the tree do they diverge? If we know it?   Xavier Revelo:             It's a complicated question.   Jop van Berlo:             And it's an active area of study right now, not just by us, but also by many other groups.   Xavier Revelo:             So, what we know is that regardless of origin, the cells are myeloid cells. So, they're the same lineage within the big family of immune cells. Having said that, the function of the cells is dictated by the origin, as well as the issue of residency. I forgot a second part of your question. Cindy St. Hilaire:        I'm wondering how much they diverge functionally from the circulating monocytes?   Xavier Revelo:             They do. It seems like the tissue factors and the residency dictates the function of the cells in general. This is a general comment. Resident cells seem to have a protective role. Sometimes they help with the repair and healing as opposed to infiltrating cells that come into the tissue and they cause inflammation, generally they aggravate disease progression.   Jop van Berlo:             But what I also find fascinating about these resident macrophages is they are not only found in the heart, but they're also found in all organs, and they all come from developmental origins. But if you compare the macrophages between these different organs, they resemble the organ itself more than macrophages between organs and that's based on recent work where people have compared resident macrophages from different organs. And I think that's just fascinating how this develops in the heart, but also in other organs as a way to protect specific organs from potentially dangerous signals.   Cindy St. Hilaire:        Yeah, that's so interesting. So, it's almost like their niche, their new residential home, is really informing their function. So, there's some kind of back and forth between that environment and the cell itself.   Jop van Berlo:             That's what we presume, but I don't think we truly understand how the niche is important in dictating the function of these resident macrophages. And I think we need to do a lot more research into how the niche of tissue resident macrophages has formed and how that then dictates the differentiation of these resident macrophages to give rise to certain functionalities.   Cindy St. Hilaire:        Maybe you can summarize in a couple short sentences or so what, what your key findings were.   Jop van Berlo:             The main findings of our study is that very early after the induction of acute cardiac pressure overload, there is a high level of inflammation happening in the heart. And this allows the replication of resident macrophages and our study showed that these resident macrophages are really important for a protective mechanism within the heart to allow the heart to deal with this increased pressure in a heart. And what they do is they stimulate the formation of new block vessels, also known as angiogenesis and furthermore, they inhibit the formation of scar tissue or fibrosis, and we used different ways to substantiate these conclusions.   Xavier Revelo:             We studied cardiac-resident macrophages as one population. But one thing we learned in this study is that these macrophages are highly diverse. And so, using our techniques, we discover that within cardiac macrophages, we have 11 different subsets. And so, our future studies will be aiming at understanding the precise role of these different subsets that we think have different roles in pressure overload.   Cindy St. Hilaire:        One of the things I was thinking about is these 11 subsets, do they represent kind of end stage fully differentiated resident macrophages, meaning 11 different types, or are they kind of representing maybe the different stages that get to the one end type? Do we have a sense of what's going on?   Xavier Revelo:             I don't think it's completely understood my take on that is that these different subsets they can represent different activation states or functional subsets that we don't really understand why is that we have this diversity?   Jop van Berlo:             I think one of the aspects that we as a field need to work on is to better understand that complexity of immune cells that reside within an organ and associate that complexity to specific functionalities. And right now, the field is mostly lacking in technologies that allow us to do this. For example, we cannot culture these resident macrophages right now. We don't know the proper culturing conditions that allow us to test functional differences between subsets of macrophages. We don't have very good genetic tools to dissect these specific subsets of macrophages. And I think those are important areas that the field and us of course need to work on in the coming years.   Cindy St. Hilaire:        Every layer of discovery, just brings like 10 more layers complexity, or 11 more co-layers of complexity in this case.   Jop van Berlo:             Which is why we all love science!   Cindy St. Hilaire:        Exactly, exactly. It's a drug that, that keeps on giving. So, one of your experiments, I forget which number, I think figure five or six or something like that, but in wild type mice, you went on to use an anti CD115 antibody. And because that treatment others, as well as yourselves has shown depletes the resident macrophages. And, and one thing I thought was really interesting. I just want to hear how you unpack it. And that is in the wild type mice that were treated with the anti CD115 antibody. You found that the depletion of the resident macrophages exacerbated the adverse remodeling and it increased fibrosis, it decreased angiogenesis, but when you did the same thing in a CCR2 knockout mouse in that mouse, they don't have the circulating macrophages, but they also don't have the resident macrophages. They were protected from the increased fibrosis, but there was no change in the angiogenesis. And I was just wondering if you could unpack these results for me and kind of talk about the competing roles of the resident and the non-resident macrophage in this pathogenesis.   Jop van Berlo              So I think you highlight a really important experiment that we performed that try to dissect the protective versus damaging effects of different subsets of macrophages within the heart. We know that if you delete the receptor CCR2, that circulating monocytes cannot extravasate and enter the tissue in response to the cytokine CCL2 that is produced by the myocardium. So, using the CCR2 knockout, we essentially blocked the invasion of circulating monocytes into the myocardium to become monocyte-derived macrophages. And we knew from the literature that, especially the monocyte-derived macrophages, were pro fibrotic. So, we wanted to discern the effects on fibrosis between resident macrophages and monocyte derived macrophages. So, we were happy to indeed see that when we blocked extravasation of circulating monocytes and blocked them to become macrophages, that we indeed reduce the amount of fibrosis that we observed within the heart.                                       I think the difficulty here that we observed that we don't have a very good explanation for right now are the effects on angiogenesis. And I think what this highlights is that there are many, many more complexities than just the resident and recruited macrophages on the development of angiogenesis because when we block tissue resident macrophages, are we actually depleting tissue resident macrophages? We didn't completely block the development of angiogenesis. We merely inhibited this by a little bit. And so, I think there are many more actions happening within the heart in response to stress than just the immune cells. And I think it highlights how complex a living organ really is. And we always try to do reductionist experiments to try to understand the functioning of specific aspects of that organ, but it's much more complex than just one cell type doing one thing and another cell type doing another thing.   Xavier Revelo:             One potential explanation to this complexity is the fact that when we deplete resident macrophages, the monocyte-derived macrophages, the infiltrating macrophages, they can replenish those resident macrophages. And so, whether there's a difference between the original resident macrophages compared to the replacing macrophages is unknown. And so, all these complexities can perhaps explain that different phenotypes that we observed in terms of angiogenesis.   Cindy St. Hilaire:        What do your findings suggest about potential therapies or you even potential therapeutic targets? Is it possible in a human to be able to target one or the other macrophage population? I know a lot of your experiments, because it's an experiment, you're targeting the depletion of macrophages before to see the effects, but are we able to possibly activate or stimulate their production, post MI for example?   Xavier Revelo:             Yeah, absolutely. So, thinking about cardiomyocyte independent interventions that can enhance the preparation process of any stressed heart, we could see potential in manipulating resident macrophages, specifically enhancing the functions of these resident macrophages that will help us heal and prevent fibrosis and enhance angiogenesis. So, we think that future studies need to look at what factors can be manipulated to enhance the function and survival of these resident macrophages.   Jop van Berlo:             One important aspect of our study that we don't highlight is that after this large increase in tissue resident macrophages, that we observed within the first week after cardiac pressure overload, these cells actually disappear. And right now, we don't really know the signals that are important for mediating that disappearing of cells. And we don't know this whether maintenance of these signals could improve longer beneficial effects of tissue resident macrophages.   Cindy St. Hilaire:        Interesting. I guess we know some questions you're going to start to ask in the future.   Jop van Berlo:             Absolutely. There's always more questions to answer in science.   Cindy St. Hilaire:        Well, great. Well, Dr Revelo, Dr van Berlo. Thank you so much for joining me today. Congrats on a wonderful paper and we look forward to these future studies.   Jop van Berlo:             Thank you.   Xavier Revelo:             Thank you.   Cindy St. Hilaire:        That's it for the highlights from the December 3rd issue of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle at @CircRes and #DiscovererCircRes. Thank you to our guests, Dr Xavier Revelo and Jop van Berlo. This podcast is produced by Ishara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hillaire. And this is Discover CircRes, your-on-the-go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association, 2021. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or the American Heart Association for more information visit aha journals.org.  

This month on Episode 30 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the October 29 and November 12 issues of Circulation Research. This episode also features a conversation with Dr Elisa Klein from the University of Maryland about her study, Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity.   Article highlights:   Subramani, et al. CMA of eNOS in Ischemia-Reperfusion Liu, et al. Macrophage MST1 Regulates Cardiac Repair Van Beusecum, et al. GAS6/Axl Signaling in Hypertension Pati, et al. Exosomes Promote Efferocytosis and Cardiac Repair   Cindy St. Hilaire:        Hi and welcome to Discover CircRes, the podcast of the American Heart Association's Journal Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh and today I'll be highlighting articles presented in our October 29th and November 12th issues of Circulation Research. I also will speak with Dr Elisa Klein from the University of Maryland about her study, Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity.   Cindy St. Hilaire:        The first article I want to share is titled, Chaperone-Mediated Autophagy of eNOS in Myocardial Ischemia Reperfusion Injury. The first author is Jaganathan Subramani and the corresponding author is Kumuda Das from Texas Tech University Health Sciences Center. Reestablishing blood flow to ischemic heart muscle after myocardial infarction is critical for restoring muscle function but the return of flow itself can cause damage, a so-called reperfusion injury. The generation of reactive oxygen species or ROS and loss of nitric oxide or NO both contribute to reperfusion injury.                                       Reperfusion injury is exacerbated when the NO producing enzyme, endothelial nitric oxide synthase or eNOS, produces damaging super oxide anions instead of NO. This switch in eNOS function is caused by glutathionylation of the enzyme, termed SG-eNOS. But how long this modification lasts and how it is fixed is unclear. This group used an in vitro model of ischemia reperfusion where human endothelial cells are exposed to several hours of hypoxia followed by reoxygenation. In this model, they found the level of SG-eNOS steadily increases for 16 hours and then sharply decreases. By blocking several different cellular degradation pathways, they discovered that this decrease in S-G eNOS was due to chaperone mediated autophagy with the chaperone protein, HSC70, being responsible for SG-eNOS destruction. Importantly, this team went on to show that pharmacological D-glutathionylation of eNOS in mice promoted NO production and reduced reperfusion injury, suggesting this approach may be of clinical benefit after myocardial infarction.   Cindy St. Hilaire:        The second article I want to share is titled Macrophage MST1/2 Disruption Impairs Post-Infarction Cardiac Repair via LTB4. The first author is Mingming Liu and the corresponding author is Ding Ai and they're from Tianjin Medical University. Myocardial infarction injures the heart muscle. These cells are unable to regenerate and instead a non-contractile scar forms and that fibrotic scar can lead to heart failure.                                     Cardiomyocytes specific inhibition of the kinase MST1 can prevent infarction induced death of the cells and preserve the heart function, suggesting that it may have clinical utility. However, MST1 also has anti-inflammatory properties in macrophages. So inhibition of MST1 in macrophages may delay inflammation resolution after MI and impair proper healing. Thus, targeting this enzyme for therapy is not a straightforward process. This study examined mice lacking MST1 in macrophages and found that after myocardial infarction, the inflammatory mediator leukotriene B4 was upregulated in macrophages and the animal's heart function was reduced compared to that of wild type controls. Blocking the action of leukotriene B4 in mice reduced infarction injuries in the hearts of MST1-lacking animals and enhanced repair in the injured hearts of wild type animals given an MST1 inhibitor. The results suggest that if MST1 inhibition is used as a future post infarction regenerative therapy, then leukotriene B4 blockade may prevent its inflammatory side effects.   Cindy St. Hilaire:        The next article I want to share is titled Growth Arrest Specific-6 and Axl Coordinate Inflammation and Hypertension. The first author is Justin Beusecum and the corresponding author is David Harrison and they're from Vanderbilt University. Inflammation contributes to hypertension pathology but the links of this relationship are unclear. It's thought one trigger of inflammation may be the hypertension-induced mechanical stretch of vascular endothelial cells. Mechanical stretch causes endothelial cells to release factors that convert circulating monocytes into inflammatory cells. And one such factor is the recently identified Axl and Siglec-6 positive dendritic cells, also called AS DCs.                                       AS DCs produce a large amount of inflammatory cytokines but little is known about the role of AS DCs or their cytokines in hypertension. This group found elevated levels of AS DCs in hypertensive people compared to normal tensive individuals. Mechanical stretch of human endothelial cells promoted the release of GAS6, which is an activator of the AS DC cell surface kinase, Axl. This stretch induced GAS6 release also promoted conversion of co-cultured monocytes to AS DCs. Inhibition of GAS6 or Axl in the co-cultured system prevented conversion of monocytes to AS DCs. This team went on to show that hypertensive humans and mice have elevated levels of plasma GAS6 and that blocking Axl activity in mice attenuated experimentally induced hypertension and the associated inflammation. This work highlights a new signaling pathway, driving hypertension associated inflammation and identifies possible targets to treat it.   Cindy St. Hilaire:        The last article I want to share is titled Novel Mechanisms of Exosome- Mediated Phagocytosis of Dead Cells in Injured Heart. The first author is Mallikarjun Patil and Sherin Saheera and the corresponding author is Prasanna Krishnamurthy from the University of Alabama, Birmingham. After myocardial infarction inflammation must quickly be attenuated to avoid excessive scarring and loss of muscle function. Macrophage mediated efferocytosis of dead cells is a critical part of this so-called inflammation resolution process. And resolution depends in part on the protein. MFGE8. MFGE8 helps macrophages engage with eat me signals on the dead cells and loss of macrophage MFGE8 delays inflammation resolution in mice. Because stem cell-derived exosomes promote cardiac repair after infarction and are anti-inflammatory and express MFGE8, this group hypothesized that perhaps part of a stem-cell derived exosomes proresolven activity may be due to boosting macrophage efferocytosis.                                     They showed that stem cell derived exosomes did indeed boost efferocytosis of apoptotic cardiomyocytes in vitro and in vivo. An in vitro experiments showed that if exosomes lacked MFGE8 then efferocytosis by macrophages was reduced. Furthermore, after myocardial infarction in mice, treatment with MFGE8 deficient exosomes did not reduce infarct size and did not improve heart function compared to control exosomes. These results suggest MFGE8 is important for the cardioprotective effects of stem cell-derived exosomes. And that this protein may be of interest for boosting efferocytosis after myocardial infarction and in other pathologies where inflammation is not readily resolved.   Cindy St. Hilaire         So today, Dr Elisa Klein from the Department of Biomedical Engineering at the University of Maryland is with me to discuss her study Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity and this article is in our November 12th issue of Circulation Research. So Dr Klein, thank you so much for joining me today.   Elisa Klein:                 Thank you for having me.   Cindy St. Hilaire:        Yeah. So broadly your study is investigating how blood flow patterns specifically, kind of, laminar and oscillatory flow, how those blood flow patterns impact protein modifications and activity. So before we, kind of, get to the details of the paper, I was wondering if you could just introduce for us the concept of blood flow patterns, how they change in the body naturally but then how they might influence or contribute to disease pathogenesis in the vessels?   Elisa Klein:                 Sure. So obviously we have blood flow through all of our vessels and since we are complex human beings, we have complex vascular beds that turn and that split or bifurcate. And so every place we get one of these bifurcations or a turn in a vessel, the blood flow can't quite make that turn or split perfectly. So you get a little area where the flow is a oscillatory or what we call disturbed. There's lots of different kinds of disturbed flow. And the reason why that's important is because you tend to develop atherosclerotic plaques at locations where the blood flow is disturbed. So in my lab, we look a lot at what it is about that disturbed flow that makes the endothelial cells there dysfunctional and that leads to the atherosclerotic plaque development.   Cindy St. Hilaire:        That is so interesting. So I can picture how this is happening in a mouse at the bifurcation of different arteries but how are you able to model this in vitro? Can you describe the setup and then also how that setup can mirror the physiological parameters?   Elisa Klein:                 Sure. So we have a couple of different systems we can use to model this and they all have their advantages and disadvantages, right? So a few years ago we made a system that's a parallel plate flow chamber. So you basically have your cells that you see that on a microscope slide and you use a gasket that's a given shape and that either drives the flow… Usually it drives the flow straight across the cells. So that's a nice laminar steady flow. And we see that the cells align and they produce nitric oxide in that type of flow which are measures that they are responding to the flow in vitro. So, a few years ago we made a device that actually makes the flow zigzag as it goes across the endothelial cells. And that creates these little pockets of disturbed flow and we did that in our parallel plate flow chamber.                                       And that parallel plate flow chamber is really good for visualizing the cells. So you can stick it on a microscope. You can see what's happening, we can label for specific markers but it's not good for doing the things that we did in this Circ Research paper, where we want it to measure metabolism, because you need a lot more cells to measure metabolism and we needed a better media to cell ratio, so less media and more cells. So for this one, we designed and built a cone-and-plate device. So what it is, it's a cone and you spin that cone on top of a dish of endothelial cells and that cone produces flow. So it's going around in a circle. And if we just make it go around in a circle, it'll produce a steady laminar flow but if we oscillated it, so basically we kind of turn it back and forth, it'll make this oscillating disturbed flow. And then we have our dish of cells.                                     We do this in a 60-millimeter dish and then we have a small amount of media in there and a lot of cells. And we can culture the cells in there for a while.                                     Cindy St. Hilaire:        That is so neat. And so I'm assuming that then your cone system is very tuneable. You could either speed it up, slow it down or change that oscillatory rate with different, I guess, shifts of it? Elisa Klein:                 Yeah, that's exactly right. So we can do all those things. It's programmable with a motor and so we can run whatever type of flow we want. Cindy St. Hilaire:        That's great. So before your study, what was known regarding this link between hemodynamics and endothelial cell dysfunction and also endothelial cell metabolism? Because I feel like that's a really interesting space that a lot of people look at, kind of, metabolism and EC dysfunction or they just look at shear stress and EC dysfunction and you're, kind of, combining the three. So what was kind of the knowledge gap that you were hoping to investigate? Elisa Klein:                 Yeah, so we're really interested in macrovascular endothelial cell dysfunction. So this pro atherosclerotic phenotype that you can get in endothelial cells. And most of the work on endothelial cell metabolism had actually been done in the context of angiogenesis. So how much energy and how do cells get their energy to make new blood vessels? And that's more of a microvascular thing. So there was a study that came out before ours, actually, before we started this study, that was looking at how steady laminar flow could decrease endothelial cell glycolysis. And so that was after 72 hours of flow and they showed some gene expression changes at that time. Our study is shorter than that and we were still able to see a decrease in glycolysis in our cells in laminar flow. Before we started this study, no one had really looked at disturbed flow. So in the meantime, there are a few other papers that came out showing that the cells don't decrease glycolysis when they're in disturbed flow but not so much connecting them back to this function of making nitric oxide. Cindy St. Hilaire:        So we were kind of dancing to the topic of O linked N acetylglucosamine or how do you say it? Elisa Klein:                 GlcNAC. Cindy St. Hilaire:        GlcNAC? O- GlcNAC. So, O- GlcNAC is a sugar drive modification and I think it's added to Syrian and three Indian residues and proteins. Elisa Klein:                 Yup, that's right. Cindy St. Hilaire:        Okay, good. And that modification, it does help dictate a protein's function. And you were investigating the role of this moiety on endothelial nitric oxide synthase or eNOS and so what exactly does this GlcNAC do for eNOS' function and under what conditions or disease states is this modification operative? Elisa Klein:                 Yeah. So there's some really important studies from a little bit ago that showed that eNOS gets GlcNAcylated in animals with diabetes, right? So if you have constantly high sugar levels, you get this modification of eNOS. The thought was that eNOS gets GlcNAcylated at the same site where it gets phosphorylated. But a more recent study came out and said, well, maybe that's not the case but it definitely gets GlcNAcylated somewhere where it affects this phosphorylation site. So it may be near it and prevent the folding or prevent the phosphorylation site availability. So if the eNOS gets GlcNAcylated, the thought is that it can't get phosphorylated and therefore it can't make nitric oxide. Cindy St. Hilaire:        And so an interesting thing about this GlcNAcylation, which is probably the hardest thing I've ever said on this podcast, is that it's integrated with lots of different things. Obviously you need glycolysis and the substrates from the breakdown of sugars to make that substrate but also the enzymes that make that substrate are required. And so what's known about that balance in endothelial cells? Is there much known regarding the metabolic rate of the cells and this N-Glcynation? Elisa Klein:                 Yeah. So endothelial cells are thought to be highly glycolytic in terms of how they use glucose but they definitely take up glutamine to fuel the tricarboxylic acid or TCA cycle. And another paper came out a few years ago showing that quiescent and endothelial cells metabolize a lot of fatty acids. So they're fueling their energy needs that way. So there wasn't a lot known about GlcNAcylation in endothelial cells.                                     A lot of this work has been done in cancer cells, which are also highly glycolytic but their metabolism actually seems like it's maybe more diverse than people have thought for a long time. So the weird thing about GlcNAcylation, which if you're used to working with phosphorylation there's a thousand different enzymes that can phosphorolate right. But with GlcNAcylation there's one enzyme that's known to put the GlcNAC on and one enzyme that's known to take it off. And so they're global, right? So in our studies, if we say, okay, we're going to knock down that enzyme, you're effecting every single protein in the cell that's GlcNAcylated. And obviously ourselves in particular, we're not a big fan of that. Especially once you put them in flow, they were, like, nope, we're not going to make it. Cindy St. Hilaire:        Well, and that's a perfect segue to my next question because your results show that this flow really did not alter the expression of these enzymes that either add or subtract to the moiety. And rather it was the Hexosamine Biosynthetic Pathway that was decreased itself. So can you maybe give us a quick primer on what that is exactly and how that pathway feeds into the glycosylation... I think you wrote in the paper of over 4,000 proteins? So how would that fit in and why eNOS then? Elisa Klein:                 Yeah, so the Hexosamine Biosynthetic Pathway is one of these branch pathways that comes off glycolysis and there are these numbers sometimes there are these pathways out there and people say for the HBP in particular, 2% to 5% of the glucose that's going down through glycolysis gets shunted off into the HBP. We've done a lot of looking to try and figure out exactly where that 2% to 5%- Cindy St. Hilaire:        Yeah, what exact percentage? Elisa Klein:                 Yeah, but some percentage of it comes down and we really thought there were going to be changes in these enzymes that do the GlcNacylation, we thought there might be changes in the localization of the proteins and it's possible that those things do occur. We just couldn't detect them in our cells. And in the end, what we showed was the main thing was that when you have cells and steady laminar flow, you just decreased glycolysis. And therefore, that 2% to 5% goes down. So you seem to make less of this UDP- GlcNAC, which is the substrate that gets put on to eNOS in this case. The really strange thing that we could not explain despite a lot of work and obviously we don't get to put all of our experiments that didn't work in the paper- Cindy St. Hilaire:        The blood, sweat and tears gets left out. So- Elisa Klein:                 Exactly. So we tried really hard to figure out why it was eNOS specifically, right? Because in steady laminar flow, you see a lot of these like GlcNAcylated  proteins and a lot of them didn't change but eNOS changed hugely, essentially this GlcNAcylation just went away for the cells and steady laminar flow. So we couldn't quite answer that. We're still working on that part of the question and looking at some of the other proteins that maybe get GlcNAcylated more in this case and trying to figure out what they are. Cindy St. Hilaire:        I thought one of the cool results in your paper was one of the last ones. It was the one in healthy mice. In that you looked at healthy mice, just normal C57 black 6 mice that were 10 weeks old. So they just, kind of, reached maturity but you looked at their kind of these bifurcations and you looked at the inner aortic arch where there is more disturbed flow and you saw, similar to your in vitro studies, that there was this higher level of O-GlcNAcylation compared to the outer arch in the descending order. So my question is, these are healthy mice that are relatively young, they're not even full adults yet. That takes a couple more months. And so what are your thoughts about the role of this O-GlcNAcylation specifically on eNOS in driving atherogenesis. Where do you think this is happening in the disease process? It appears if it's in these wild type mice, it's already happening early. So where do you think this is most operative in the disease pathogenesis? Elisa Klein:                 I mean, I think it's very early, the effects of disturbed flow on endothelial cells. I can't imagine that there's a time when it's not having an effect on the cells. So I teach college students and I tell them all the time you think you're invincible now but these choices you're making today are going to affect your cardiovascular future in 50 years, which is very hard to accept. So I think it's very early in the process and I think it's only made worse by the things that we eat, in particular, that changed our blood sugar and our blood fatty acids and things like that. And our lab is looking into this more to try and see how when you change your blood metabolites then how does that then also affect this GlcNAcylation and the endothelial cell metabolism and then how does that affect endothelial cell function? Cindy St. Hilaire:        Yeah. And it's funny, it's really making me think of those, kinds of, extreme diets like keto diets and things like that where you're just like depleting sugar. And obviously there's lots of controversy in that field, but if you just think about the sugar aspect what is that doing to those EC cells? Why do you think endothelial cells have this response? Meaning why do you think it is that they've adapted to induce a metabolic shift in response to disturbed flow? Because, obviously it's not going to be perfect laminar flow everywhere. So what do you think it is that provides some sort of advantage in the shift? Elisa Klein:                 That's a really good question. I haven't thought about the advantage that it might provide. There are a lot of things that are going on in this area of disturbed flow. So there is the shear stress, the differential shear stress that the cells are experiencing. There's also transport issues, right? So if you have this area of disturbed flow, you have blood and the contents of the blood, including the white blood cells and the red blood cells, everything else that's, kind of, sitting around in that area and not getting washed downstream as quickly. So it is possible that maintaining glycolysis provides energy for repair or for protecting the endothelial cell from some sort of inflammatory insult or something like that, that's happening in the area of disturbed flow. And I feel like I just read something recently, it was in a different genre but... if they stopped the increased glycolysis or stop the metabolic shifts, it actually was worse.                                     Right? So I also believe that we treat humans for a single metabolic change, right? So if you have diabetes, I'm going to give you this drug and if you have high triglycerides, I'm going to give you this drug. But it's possible that if you have this metabolic abnormality, your body shifts the rest of your metabolism to protect the cells because of that metabolic abnormality. And so part of what we do as engineers is try and build computational models or we can take into account some of this complexity. So that's a really interesting question and my guess is that there are some protective aspects of this maintenance of high glycolysis and disturbed flow. Cindy St. Hilaire:        Yeah, maybe it would be perfectly fine until we get athero and then it all goes awry. So in terms of... obviously it's early days and I know you're a bioengineer but in terms of translational potential, what do you think your findings suggest about future potential therapies or future targets for which we can use to develop therapies? Is modulating this O-GlcNAcylation itself, a viable option? Elisa Klein:                 I don't think that modulating it is a super viable option, right? Because as I said, when we tried to change those enzymes ourselves did not enjoy going through flow or anything else. So it's very hard to change it overall. What I think is these things that are coming out about how metabolism may shift for endothelial cells when they're activated versus when they're quiescent, right? So when laminar flow or cells are quiescent, they decrease glycolysis, they increase fatty acid oxidation. Those things are important to take into consideration when you are treating a person who has a metabolic disorder. So that's the biggest translational piece that I think is, how do we give therapies that modify the metabolism of a cell holistically instead of trying to hit one pathway in particular.                                     We have done some studies where we tried to give endothelial cells something to inhibit a specific metabolic pathway and you see the cell shifts its entire metabolism to account for that. So we're starting to look at some of these other drugs like statins or metformin that do change endothelial cell metabolism, possibly even the SGLT2 inhibitors and trying to see not just how they change glycolysis but how they change metabolism as a whole and how that then affects endothelial cell function. Cindy St. Hilaire:        So what are you going to do next on this project? Elisa Klein:                 So on this project, so we have some stuff in the works like I said on statins and how statins work together. And one of our big goals is to sort of build a comprehensive metabolic model of the endothelial cell. So this study really focused on glucose but there are other things that endothelial cells metabolize, glutamine, and fatty acids, and trying to look at some of those and then seeing how changes in the glycolytic pathway may affect some of those other pathways. We also have some really nice mass spec data part of which is in this paper but part of which is going to go into our next work, which is looking at how laminar flow impacts some of the other side branch pathways that are in metabolism and coming off of glycolysis as well as the TCA cycle, right? So we don't think of endothelial cells as being big mitochondrial energy producers but they do use their mitochondria. And so we think it's really interesting and part of our goal of building an endothelial cell model and then hopefully a model of the complexity of the whole vascular wall. Cindy St. Hilaire:        Wow. That would be amazing. Well, Dr Elisa Klein from the University of Maryland, thank you so much for joining me today. This is an amazing study and I'm looking forward to seeing hopefully more of your future work. Elisa Klein:                 Thank you so much. It was a pleasure. Cindy St. Hilaire:        That's it for the highlights the from October 29th and November 12th issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes or #DiscoverCircRes. Thank you to our guest, Dr Elisa Klein. This podcast is produced by Asahara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy texts for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on-the-go source for the most exciting discoveries and basic cardiovascular research. This program is copyright of the American Heart Association, 2021. The opinions expressed by speakers on this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, visit AHAjournals.org.

This month on Episode 29 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the September 17th and October 1st issues of Circulation Research. This episode also features conversations with BCVS Outstanding Early Career Investigator Award finalists, Dr Jiangbin Wu from the University of Rochester, Dr Chen Gao from UCLA, and Dr Chris Toepfer from Oxford University.   Article highlights:   Raftrey, et al. Dach1 Extends Arteries and Is Cardioprotective   Zhang, et al. Blood Inflammatory Exosomes and Stroke Outcome   Joyce, et al. Cardiovascular Health and Epigenetic Age   Liu, et al. Wls Suppresses Fibrosis in Heart Regeneration   Cindy St. Hilaire:        Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh. And today, I'll be highlighting articles presented in our September 17th and October 1st issues of Circulation Research. I also am going to speak with the BCVS Outstanding Early Career Investigator Award finalists, Dr Jiangbin Wu from the University of Rochester, Dr Chen Gao from UCLA, and Dr Chris Toepfer from Oxford University. Cindy St. Hilaire:        The first article I want to share is titled, Dach1 Extends Artery Networks and Protects Against Cardiac Injury. The first author is Brian Raftrey, and the corresponding author is Kristy Red-Horse from Stanford University. Coronary artery disease occurs when blood vessels supplying the heart develop atherosclerotic plaques that limit blood flow, which prevents oxygen and nutrients from reaching the cardiac tissue and often leads to a heart attack or cardiac arrest. The suggested strategy for treating coronary artery disease is to promote the growth of new blood vessels to compensate for the dysfunctional ones. Several factors are known to control coronary blood vessel development, including the transcription factor, DACH1. In mice lacking DACH1, embryonic coronary artery development is stunted. But whether increasing DACH1 protein levels boosts heart vessel development, and whether this would work in mirroring coronary arteries, were unanswered questions. Cindy St. Hilaire:        This group engineered inducible gain-of-function DACH1 mice and found that DACH1 over expression in the embryo boosted coronary artery development. The team then used the same model to induce DACH1 in adult mice for six weeks. While there was no apparent differences in the artery growth between the animals and the controls under normal conditions, after myocardial infarction, the mice over expressing DACH1 had better recovery and survival with increased artery growth and heart function. The results paved the way for studying the mechanisms of DACH1-mediated protection, and how they might be leveraged as potential coronary artery disease treatments. Cindy St. Hilaire:        The second article I want to share is titled Circulating Pro-Inflammatory Exosomes Worsen Stroke Outcomes in Aging. The first author is Hongxia Zhang, and the corresponding author is Kunlin Jin from University of North Texas Health Science Center. Aging is associated with declining tissue function and an assortment of health issues. But in rodents at least, certain factors, including the plasma of youthful animals and the exosomes of stem cells, can have rejuvenating effects on old animals. Exosomes are small membrane-bound particles containing cellular contents that circulate in the blood after they're released from cells. This group has shown that as rats age, the animals' serum exosomes accumulate pro-inflammatory mediators, such as C3a and C3b. Cindy St. Hilaire:        When these aged rats were subjected to stroke, and then injected with serum exosomes isolated from either old or young rats, those receiving youthful exosomes fared much better in terms of infarct size and sensory motor deficits, while those receiving aged exosomes fared worse. The team went on to show that injected exosomes accumulate at the site of stroke injury, but those from old donors caused more neuronal damage, as seen by reduced synaptic function. Preventing C3a activity on microglia reversed the effects of the old exosomes and improved stroke outcome, suggesting that such modulation of inflammatory molecules might be a treatment strategy for stroke. Cindy St. Hilaire:        The next article I want to share is titled Epigenetic Age Acceleration Reflects Long-Term Cardiovascular Health. The first author is Brian Joyce, and the corresponding author is Donald Lloyd-Jones. And they're from Northwestern University. DNA methylation is an epigenetic modification that regulates gene transcription. Studies of young and old individuals have shown that at certain locations in the genome, methylation status is highly correlated with age. These methylation patterns are also linked to measures of cardiovascular health, including blood pressure, cholesterol level and body mass index. This suggests that if a person has particularly good or particularly poor cardiovascular health, their DNA may appear younger or older than the individual's actual age. Cindy St. Hilaire:        This group tested the hypothesis that people with poor cardiovascular health exhibit methylation changes more commonly found in elderly individuals than those with good cardiovascular health. And if so, DNA methylation patterns might be useful for predicting future cardiovascular risk. Cindy St. Hilaire:        The team examined DNA methylation of over a thousand individuals enrolled in a prospective heart health cohort, testing them around age 40 and then again at around age 45. Changes in methylation status were then compared to individuals' cardiovascular health scores over a longer period. Sure enough, faster epigenetic changes did correlate with poor cardiovascular health later in life. Data from the second cohort of individuals supported the initial findings. This study indicates that DNA methylation status may be an early biomarker that signals cardiovascular issues, and may therefore allow for prompt implementation of treatment and prevention strategies. Cindy St. Hilaire:        The last article I want to share is titled, Yap Promotes Noncanonical Wnt Signaling from Cardiomyocytes for Heart Regeneration. The first author is Shijie Liu, and the corresponding author is James Martin. And they're from Baylor College of Medicine. After a heart attack, cardiomyocytes are destroyed and replaced with a fibrotic scar that interferes with the contractile function of the heart. While adult mouse and human hearts are similar in this regard, the hearts of newborn mice possess greater regenerative capacity, and this regeneration capacity persists for approximately one week. The transcription factor YAP is known to regulate regenerative processes in neonatal hearts of mice. And its deletion eliminates regeneration, and its over-activation in adult cardiomyocytes reduces fibrosis. Cindy St. Hilaire:        These experiments suggest cardiomyocytes transmit signals to cardiac fibroblasts. Wntless protein regulates the release of Wnt signaling molecules and also is a target of YAP. Mice that lack Wntless in their cardiomyocytes appear to have normal heart development and function. However, their neonatal regenerative capacity was impaired. In the weeks after heart injury, the mice that lack Wntless had reduced heart function, increased scar size and increased numbers of activated cardiac fibroblasts compared with that seen in controls. The study indicates that Wntless is critical to the regeneration of cardiac tissue, and may perhaps be leveraged to minimize scarring after heart attacks. Cindy St. Hilaire:        I'm really excited to have with me today the three finalists of the BCVS Outstanding Early Career Investigator Award. The first person I'm going to be speaking with is Jiangbin Wu, who is a research assistant professor at the Aab Cardiovascular Research Institute at the University of Rochester. Thank you so much for joining me today. Jiangbin Wu:               Thank you. Cindy St. Hilaire:        And congratulations, actually. I know this is a highly competitive award that gets a lot of applications, so congrats on becoming a finalist. Before we get to your abstract, which is related to mitochondria and calcium influx in cardiomyocytes, I was wondering if you could share a bit about yourself. Maybe what your research path was, and what brought you to study cardiomyocytes and the mitochondria that are within them? Jiangbin Wu:               Yeah. Right now, I'm an assitant professor at Cardiovascular Research Institute of University of Rochester. Previous, I was actually studying in the cancer field and also some kind of mitochondria work in some cancer cells. Although when I came to the University of Rochester and I switched to cardiovascular and then we are working on a kind of microRNA[at the initial. The way we screen for these is just by doing the RNA-Seq is target the microRNA. and then we start to study the function of these genes, and found that it's a mitochondria calcium channel regulator. Cindy St. Hilaire:        The title of your abstract is FAM210A Maintains Cardiac Mitochondrial Homeostasis Through Regulating LETM1-Dependent Calcium Efflux. So before we unpack what all those words in the abstract title mean, could you tell me how you ended up focusing on FAM210A? What does this protein do, and why'd you focus on it? Jiangbin Wu:               Yeah. As I mentioned that we just gathered this protein actually is by some kind of chance as a microRNA target. And this protein full name is family with similarity 210 A, actually is a family of proteins. This is just one of them. And the way discover is localized in mitochondria in the membrane. And also, there is some other people's report is in mitochondria. And we want to sort out its function inside the mitochondria and in the cardiac background. So we do some kind of omics or mass spec to get its interlocking interacting proteins. And then we found LETM1. It's a calcium channel inside the mitochondria in the membrane. So we figured out is, this FAM210 protein regulate LETM1 function in calcium, pump calcium is part of the mitochondria matrix. And I think this is a very important, because calcium overload is always happening in the very heart of the cardiomyocytes. Cindy St. Hilaire:        That's a perfect segue, because my next question was really what is the gap in knowledge that your study was trying to address? Were you really focused on just the function of this one protein, or what was the greater goal of this study? Jiangbin Wu:               Actually, the function this protein is the initial step. Our final aim is to use this protein, to over expression this protein in the heart failure patient or in some kind of heart failure models to do the, sort of do the work in some heart failure patients. Cindy St. Hilaire:        Maybe a gene therapy approach, or if there's a pharmacological way to up regulate this protein? Jiangbin Wu:               Yeah, because we've proposed that the self expression of this proteins will reduce the calcium overloading cardiomyocytes, which is a major cause for the cardiomyocytes death in heart failure process. So over expression will reduce this kind of process. And then it will make the cardiomyocytes survival in the failure heart. Cindy St. Hilaire:        That is interesting. I mean, obviously you were using a mouse knockout model, so you know what's driving the expression down in that case. But in humans, what do we know about the regulation of this protein? Is anything known, or any known causes that cause its reduction in expression? Jiangbin Wu:               Actually, we do. Its expression in heart failure is slightly increased in heart failure. So we feel it's a kind of some kind of compensating effect to try to save the heart from failing. Cindy St. Hilaire:        Interesting. It's just not turned on early enough, in that case then. Jiangbin Wu:               Yeah. And for the regulating protein for this one, I think we find microRNA can suppress its expression, but not too many other influences on these regulator proteins. Cindy St. Hilaire:        That is so interesting. So what's next? What are you going to do next on this project? Jiangbin Wu:               Yeah. I think currently, we are just at the start to do some kind of therapeutic effect that use to these proteins. I think we will do more deep in the therapeutic effects for over expression of these genes in... Currently, we are working on mouse models. Maybe in different heart failure models to prove that it's very benefiting to the heart failure patients. Cindy St. Hilaire:        Wonderful. Well, congratulations on an excellent study. Really looking forward to your presentation, which is coming up shortly, and really looking forward to your future research in this field. Jiangbin Wu:               Okay, thank you. Cindy St. Hilaire:        So I also have with me, Dr Chris Toepfer, who's another finalist for the BCVBS outstanding early career investigator award. He's a principal investigator from the University of Oxford, and his abstract is titled, Defining Diverse Disease Pathway Mechanisms Across Thick And Thin Filament, Hypertrophic Cardiomyopathy Variance. So congratulations, Chris, and thank you for joining me today. Chris Toepfer:             Thank you very much. It's great to be here. Cindy St. Hilaire:        Before we start to discuss your abstract, I was wondering if you could just share a little bit about yourself. Maybe your career path, and how you came to study hypertrophic cardiomyopathy? Chris Toepfer:             Yeah, sure. I guess this story gets longer and longer every time somebody asks it,right, in your career? Cindy St. Hilaire:        That's a good thing. Chris Toepfer:             Yeah. I started out as an undergraduate in London, and actually during the second year of my undergraduate degree, I fell into a lab kind of out of interest. It was starting to study cardiac muscle mechanics. And that was the lab of Professor Michael Ferenczy. And ended up, after I finished my undergraduate degree, I joined him for a PhD. I had a PhD program that also took me overseas to the NIH to work with Dr James Sellers, who was a muscle motor protein biochemist. And we really, I sort of really fell in love, with the idea of studying disease of multiple levels, and understanding how the heart would function from the basic molecule up to the entire organ and looking at different systems in between. Chris Toepfer:             And that's what led me to then, so my postdoctoral position to seek out a completely different direction in some ways, but something that could also extend how we could look at the heart. And that's where I moved to Boston to work with Christine and Jonathan Seidman. I'm looking at more of the genetic basis then of hypertrophic cardiomyopathy rather than just, sort of more diffusely the mechanisms underlying cardiac muscle contraction. And then two years ago, I moved back to the UK to Oxford to sets up my own group, which has been fun during the pandemic as you can imagine. Cindy St. Hilaire:        It's hard enough starting up a lab under normal times. I can't imagine doing it during a pandemic. Chris Toepfer:             And we are now completely focused on stem cell models and CRISPR CAS engineering, and trying to understand hypertrophic cardiomyopathy in a dish. Cindy St. Hilaire:        That's wonderful. And actually I looked at your CV. We actually overlapped a little bit. I was doing my postdoc at NIH in the NHLBI while you were there for your graduate school. So I too fell in love with kind of the starting with the human as the model path of research. So maybe you can  kind of fill in all the listeners in who aren't cardiomyopathy experts. So what is, I guess, in a nutshell, hypertrophic cardiomyopathy, and what gap in knowledge was your study specifically addressing? Chris Toepfer:             So in general, about one in 500 people have hypertrophic cardiomyopathy. And for those that are genetically linked, a lot of them are in the key contractile proteins of the heart, the drive muscle contraction. And what you often see in those people is they have thickened hearts. And what happens is actually the heart begins to be too hard, and it actually relaxes very poorly in between beats. Chris Toepfer:             So what we are really trying to understand in this disease and with this abstract was how are different forms of hypertrophic cardiomyopathy created? Because it can be a couple of different forms. There are different proteins involved that have very vastly different functional mechanisms within the cell. So would this, we went away, we generated some stem cell models where we could then differentiate into cardiomyocytes. Model the disease in a dish. And we made kind of a group of good methods to go and look at what was happening inside the cells. And then we could screen drugs against what's happening inside those cells, so that was kind of the idea of what we were looking at, at the time. And what's fallen out of all of that is a drug now called Melacamptin that's starting to get to the clinic, which addresses some of these underlying mechanisms we were beginning to study. So that's what I'll talk about a bit later on in our session today. Cindy St. Hilaire:        It's great. One of the things you focused on in the abstract is comparing these thick and thin filament variants. What are the implications of those, I guess, in the human disease state, but also in how you could design or use your stem cells as a model, and were any of the results that you found surprising? Chris Toepfer:             So I think what was the really key finding that we saw was that the thick filament variants seemed to be switching myosin, which is a molecular motor that drives cardiac muscle contraction very much to arm”ON”. And my sort of analogy to that is they're all very sort of bodybuilder like. Myosin switched on, ready to go to work causing way too much contraction. And the compound that we were using at the time Myocamptin, we could turn those off and resolve the disease. Whereas with the thin filament variants, they were operating through a completely different mechanism. And when we tried to treat them with the same compound, they wouldn't always salvage disease. So though the face of it, they look the same in the dish, in that they contracted too much, relaxed very poorly. You're clearly doing it via complete different mechanism. And that's what we're starting to dig into now. And that's what we'll be talking about. Cindy St. Hilaire:        Yeah. And that's actually kind of the question I was going to finish up with you. What are the, I guess translational implications? No, yes. You're using this drug. Is that only good for thick filament-like variants? And are you going to be able to screen patients to tell which variant they have, and therefore if this or that drug might be useful? Chris Toepfer:             So we're in a real golden age now for genomics where I guess patients can come into the clinic and they can be sequenced and you could maybe tell them now what might be the underlying cause of their disease. I am not a clinician, but what we, as a basic scientist can say is, well, we can go away and try and understand whether this variant you may have in your genome is causative of disease. And if it is what mechanism that may fall under, what may be causing them to have this phenotype? Chris Toepfer:             And I think what we can do is we can try and then bin the subpopulations of variants, and try and find novel drugs or novel pathways that we could try and find drugs for to treat the disease, and to differentiate them from each other. So I think it's too early to say whether Mylocamptin will be able to sort this for everybody, I guess we will find out in the next years. But I think already we can start thinking about, well, what would be the next step after this? We can bring precision medicine even further. And that's, I think the goal where we're heading towards. Cindy St. Hilaire:        Well, that's wonderful, and this is a wonderful abstract. I'm really looking forward to seeing the full study and your presentation later on. And thank you so much for joining. Chris Toepfer:             No. Yeah. Thank you for having me. I'm really looking forward to it later on. Cindy St. Hilaire:        Great. Dr Chen Gaol is the third finalist for the BCBS Outstanding Early Career Investigator Award. She's an assistant researcher at UCLA, and her abstract is titled, Functional Impact of RBFox1C in Cardiac, Pathological Remodeling through Targeted MRNA Stability Regulation. So congratulations, and thank you so much for joining me today. Chen Gal:                    Absolutely, thank you for having me. Cindy St. Hilaire:        Before we jump into your abstract, could you share with us a little bit about your career path, and how you came to study the role of RNA binding proteins, I guess specifically in pathological cardiac remodeling? Chen Gal:                    Yes, I think my research over the years has been into the very basic questions, which is I'm interested in looking at how the RNA is being regulated. For example, how the RNA is being spliced, is being ideated, and how the RNA is being degraded if it's ever been translated into protein. And the second half of my research is of course, physiological driven, because I'm interested in different type of cardiac disease, starting from the traditional heart attack to the now more emerging medical need, which is the cardiometabolic disease. So I was trained as a molecular biologist. I started in molecular biology Institute at UCLA. My PhD supervisor is Dr Yibin Wang, who first introduced me to understand there is actually a whole new world of R regulation at a post-transcription level. Chen Gal:                    So at that time we basically utilized the R sequencing. Just look for the easiest to heart, and try to understand how these RNA are differentially spliced in the heart. And I was so interested in understanding more about a cardiology. So I decided, even if I move out to my postdoc research I still want to continue working in the heart, although at a totally different angle. And that is when I started to really try to understand different aspects of RNA regulation. So now I am starting to be a junior faculty, establishing my own lab. And I really wanted to understand more how different steps of our metabolism is regulated. Cindy St. Hilaire:        Really timely research. And I really like how you are doing a great job combining extremely basic biochemical processes with advanced disease states. An extra, that's why this abstract made it as a finalist. So congrats on that. So your study was focused on the RNA binding protein, RB Fox one, which has several isoforms. And so can you tell us which isoform you were looking at, and why you were interested in that particular isoform? Chen Gal:                    Yes, actually I've studied about ISO form of RPFox1. It itself, is actually subject to alternative splicing, while generating one nuclear, and another simosolic isoform. Where I was a PhD student, I was very simple minded, just trying to screen for the R binding protein that actually is expressed in the diseased heart. So RBFox1 is at least at a transcriptional level, the only one that we identify to be to decreased in the fatal heart. The nuclear function, the nucelo ISO form of RPFox1 is mainly regulating alternative splicing. But it is when I was studying this nuclear function of the RBFox1, I identified there is actually another isoform where she is in the set ourselves based on the different of c terminal domains of the RFox1. So I was just wondering, apparently you shouldn't be regulating and splicing anymore. I just move on to another layer of RA regulation. And then what I found most interesting is these RBFox1 is regulating the R stability, which is something that we'll talking about later today. Cindy St. Hilaire:        That's great. So to do this study, you actually created a new knockout mouse model where you specifically deleted this one C isoform. What was kind of the baseline and maybe the disease state phenotypes that you saw in that mouse? Chen Gal:                    The result and phenotype so far is very striking. We utilize the CAS nine CRISPR technology simply because for, we were lucky the settle the Fox warehouse, one extra axon. So that does allow us to coach the lox P side, just blanking in that particular AXA. And in theory we could across it with different CRE, and to generate either cardiac or different tissue, specifically knock out. Even at a baseline we see a decreased cardiac function when we inactivate this isoform in the adult heart. And when we look at the gene expression profile is, I call mind-blowing type of experience, because turns out this gene not only is regulating some of the inflammatory genes, but also is helping involve protein translation and delivery metabolism, which I hope in the future will set us on the path to really understand the role of this RP Fox1. Not only into HFpEF, but also in the cardiometabolic disorder. Cindy St. Hilaire:        Yeah, that's great. It's so rewarding when you do this one really big kind of risky experiment, and it turns into not just one interesting path to study, but multiple. One of the things that you mentioned in the abstract is clip seek. I was wondering if you could tell us a little bit about this technology, and how you used it in your study? Chen Gal:                    Yeah. I think one of the rewarding parts for me focusing on the R metabolism is really driving different accounting and sequencing tools, and utilize that in the heart. So cardiomyocyte has been traditionally viewed now to be very easy to work with type of model comparing helo cells, right? And I think in the field, we are still so short of knowledge, what type of the cutting-edge tools that we can use in the heart. My research involved clip seek, which is to use UV crosslinking the RNA with the R binding protein. So that will allow us to understand which are the RNA targets that are directly interacting with the RNA binding protein. I'm also using great seek, which is to find dynamically label the recency size to RNA. And that will allow us to look forward to RA degradation profile at a global level in the baseline or under disease. So I thought those are really cool technologies, and that's something that makes me excited about my work on a daily basis. Cindy St. Hilaire:        Yeah, that's wonderful. So what's next? What are you going to do after this initial study? What's the next question you're going to go after? Chen Gal:                    Yeah, like I mentioned, I'm interested in, honestly, different type of heart disease, not just the stress induced heart failure, but also the recent years, I started to branch out a little bit to understand more of the biology of HFpEF. For example, how the R binding protein that we are studying right now is playing a role in the development of HFpEF. Or we actually understand very little about them, the micromechanism for HFpEF development, right. What are the RNA splicing profile in the cardio metabolic disorder on account? We also find differential regulation of R stability in the HfPEF compared to the HFpEF compared to the HFrEF. So I thought those are really interesting questions that I would like to pursue in the future. Cindy St. Hilaire:        That's great and best of luck in those future studies. Chen Gal:                    Thank you. Cindy St. Hilaire:        Before we leave, I was wondering if you could share with us any advice that you would give to a trainee, maybe something that you wish you knew ahead of time in this kind of early career stage. Chen Gal:                    I consider myself a really, really lucky person. And if I have one word to give to the younger people, younger than me, is to find great mentors for your career. And luckily our field has a lot of good mentors who are ready to help us every single step of our career. For example, my PhD supervisor, Dr Wang. And I have met a lot of good mentors inside and outside of UCLA. I'm pretty sure this is the same thing for Chris, who is trained by Dr Seidman, and everybody know how great a mentor she is. So I think having a great mentor will help you every step of your career development to making sure you're always on the right track. And that, that is also something that you will do when we have our own lab, because we want to be great mentors for our trainees as well. Cindy St. Hilaire:        I know. That's something I strive for too, is to emulate my amazing mentors that I've had. What do you think is a good quality for a good mentor? Like what's one of the, I guess key features that you look for in someone that you would like to be your mentor? Chen Gal:                    For me, I think my mentors are all cheerleaders. They never try to push me to move out one career path versus the other. They are good listeners, and they are also my role models. Cindy St. Hilaire:        That's wonderful. Chris, what's a piece of advice that you would like to share with trainees that your former self wish you knew of? Chris Toepfer:             I think it's very important to echo the message of a good mentorship, and a good lab environment that allows you to flourish and really helps you to grow yourself to the future. And also helps you understand the bits of you that you could actually grow as well, a little bit better. So you become a more rounded scientist. I think something that's really important or something that I've always found very infectious is to find mentorship and mentors that are also incredibly enthusiastic about you as an individual, as well as the science. I think that that can really drive you. And I think that's also an important thing to have in yourself, to have, to find that question for yourself that really drives you and you can be really enthusiastic about. Cindy St. Hilaire:        I totally agree. Well, thank you again for joining me today. Congratulations on being a finalist, and I wish everyone the best of luck in their presentations later on at BCBS. Chen Gal:                    Thank you so much. Jiangbin Wu:               Thank you. Chris Toepfer:             Thank you very much. Cindy St. Hilaire:        That's it for the highlights from the September 17th and October 1st issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page, and follow us on Twitter and Instagram with the handle  @CircRes and #Discover CircRes. Thank you to our guests, BCBS Outstanding Early Career Investigator Award Finalists, Dr Jaobing Wu, Dr Chen Gal, and Dr Chris Toepfer. And a special congratulations to Dr Toepfer who won this year's competition. This podcast is produced by Asahara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of circulation research. Some of the copy texts for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire. And this is Discover CircRes, you're on the go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American heart association, 2021. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American heart association. For more information, please visit AHAjournals.org  

Im Rahmen des Tag der Stiftung sprechen wir mit Ruth Williams, Generalsekretärin des Verbandes für gemeinnütziges Stiften in Österreich, über alles, was wir immer schon einmal über Stiftungen wissen wollten.

This month on Episode 28 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the August 20th and September 3rd issues of Circulation Research. This episode also features an in-depth conversation with Dr Scott Cameron from the Cleveland Clinic and Dr Milka Koupenova from the University of Massachusetts Medical Center about their study, SARS-CoV-2 Initiates Programmed Cell Death in Platelets.   Article highlights:   Gupta, et al. Electronic Cigarettes and Oxidized Lipids   Bartosova, et al. Glucose Derivative Induced Vasculopathy in CKD   Atmanli, et al. DMD Correction Attenuates Cardiac Abnormalities   Ma, et al. Length Dependent Activation in Porcine Myocardium   Cindy St. Hilaire:        Hi, and welcome to Discover CircRes, the podcast for the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I will be highlighting articles presented in our August 20th and September 3rd issues of Circulation Research. I also will speak with Dr Scott Cameron from the Cleveland Clinic and Dr Milka Koupenova from the University of Massachusetts Medical Center about their study, SARS-CoV-2 Initiates Programmed Cell Death in Platelets. Cindy St. Hilaire:        The first article I want to share is titled Electronic and Tobacco Cigarettes Alter Polyunsaturated Fatty Acids and Oxidative Biomarkers. The first author is Rajat Gupta and the corresponding author is Jesus Araujo from UCLA. E-cigarettes have surged in popularity in the last decade and while many people switching from traditional cigarettes to smokeless ones view the latter as a safe alternative to smoking tobacco, emerging data shows that E-cigarettes cause adverse effects such as oxidative stress, inflammation and endothelial dysfunction in users. The aerosols produced during vaping contain similar levels of reactive oxygen species, also called ROS, as the vapors of tobacco smoke. However, data on the extent to which E-cigarettes, E-cigarette ROS, influences cardiovascular health is lacking. Cindy St. Hilaire:        To address this, this group recruited 32 chronic users of E-cigarettes, 29 chronic tobacco smokers, and 45 individuals that used neither and they measured their plasma levels of oxidative biomarkers. The team found both similarities and differences between the E-cigarettes and the tobacco users. For example, both smoking groups had increased plasma antioxidant capacity and decreased levels of oxidized linoleic acid compared with the levels seen in non-users, while arachidonic acid levels were raised in tobacco smokers and reduced in E-cigarette users. Overall, however, the biomarker levels were deemed to be intermediate for E-cigarette users between the non-users and the tobacco users. This study suggests that while E-cigarettes carry a lower health risk than tobacco, they are by no means safe.     Cindy St. Hilaire:        The second article I want to share is titled Glucose Derivative Induced Vasculopathy in Children on Chronic Peritoneal Dialysis. The first author is Maria Bartosova and the corresponding author is Claus Schmitt and they're from the University of Heidelberg. Diabetes, high blood pressure and obesity are risk factors for both cardiovascular disease and chronic kidney disease. Worse still, loss of kidney function and even dialysis itself are thought to exacerbate cardiovascular issues. In the case of dialysis, it's thought that high levels of glucose degradation products, or GDPs, in the dialysis fluids can promote the addition of sugar moieties to vascular proteins and lipids causing vascular damage. To investigate this theory, Bartosova and colleagues studied vascular tissue from children with chronic kidney disease receiving dialysis fluids with either high levels or low levels of glucose degradation products and compared these to tissues from children not on dialysis at all. Cindy St. Hilaire:        Proteome and transcriptome analysis of the vessel tissues revealed that compared with patients or no to low GDP fluids, patients receiving high GDP fluids had higher levels of damaging glycation, increased transcription of genes involved in cell death, and decreased transcription of genes involved in cell survival and cytoskeletal reorganization. In line with these findings, vessels from high GDP patients displayed considerable evidence of damage, such as markers of apoptosis, skeletal disintegration and thickened intimas. The results confirmed GDPs can cause vasculopathy and suggest low GDP fluids should be used for dialysis patients. Cindy St. Hilaire:        The next article I want to share is titled Cardiac Myoediting Attenuates Cardiac Abnormalities in Human and Mouse Models of Duchenne Muscular Dystrophy. The first author is Ayhan Atmanli and the corresponding author is Eric Olson from UT Southwestern. Duchenne Muscular Dystrophy, or DMD, affects one in 5,000 baby boys and is caused by mutations in gene for dystrophin, an architectural protein essential for muscle cell integrity. Patients display profound muscle degeneration and weakness, with respiratory and heart muscle dysfunction being a major cause for death. With the recent improvements in respiratory medicine that extend the lives of patients, this group now focused on heart dysfunction and specifically, whether gene editing could mitigate it. The team created induced pluripotent stem cells, or iPSCs, from Duchenne Muscular Dystrophy patient and his healthy brother and showed that gene editing from the DMD cells enabled their development into normal-looking cardiomyocytes with normal contractile function and calcium handling, equivalent to that seen in healthy control cells. The unedited DMD cells, by contrast, did not develop normally. For great clinical relevance, the team edited DMD cells after cardiomyocyte differentiation showing that this reduced their propensity for arrhythmia, compared with that of unedited cells. Cindy St. Hilaire:        Lastly, the team provided evidence to suggest gene editing may improve heart abnormalities in mice with the same mutation. All together the results are proof of principle and support of the development of gene editing therapy as treatment for DMD. Cindy St. Hilaire:        The last article I want to share is titled The Super-Relaxed State and Length Dependent Activation in Porcine Myocardium. The first authors are Weikang Ma and Marcus Henze and the corresponding author is Thomas Irving and they're from the Illinois Institute of Technology. Myofilament length-dependent activation or LDA is the fundamental mechanism coupling the force of the heart's contraction to it's proceeding diastolic volume. In other words, LDA ensures that the more the heart fills, the stronger it contracts. Studies of rodent hearts have given insights into LDA mechanics. However, how it operates in large mammalian hearts is unknown. Using structural and biochemical analysis of pig myocardial fibers, this group found that compared with small stretches of the fibers which were equivalent to small diastolic volumes, long stretches induced greater ATP turnover and greater numbers of cross bridges between myosin and actin filaments which are critical contractile machinery proteins. Cindy St. Hilaire:        Myosin motors can be found in three stages, engaged with actin, unengaged in a disordered, relaxed state but ready to engage, or super-relaxed state where they are essentially switched off. The team showed that as muscle stretch increased, the amount of super-relaxed myosin motors diminished with more myosin motors becoming engaged to enable a stronger contraction. When the fibers were treated with a myosin motor inhibitor, these stretch effects were impaired. In revealing the mechanisms of myofilament length-dependent activation, this study provides a platform for studying cardiomyopathies in which this system goes awry. Cindy St. Hilaire:        So today, Dr Scott Cameron from the Cleveland Clinic and corresponding author of the paper, Dr Milka Koupenova from the University of Massachusetts Medical Center, are both with me to discuss their study, SARS-CoV-2 Initiates Programmed Cell Death in Platelets. And this article is in our September 3rd issue of Circ Research and for full disclosure, the editor of Circ Res, Dr Jane Freedman is also an author on this manuscript. And for full double disclosure, I know Dr Koupenova quite well as we were both graduate students together back in the Ravid Lab at Boston University. However, the full Editorial Board selects these articles, not just me alone and this one is timely, novel, and an amazing story. So thank you both for joining me today. Milka Koupenova:       Thank you for having us. Scott Cameron:           Privileged to be here. Cindy St. Hilaire:        So before we jump into the story that is your paper, can you give us a little bit of background about platelets? I know for years, I guess certainly before Katya's lab, I just thought of platelets as little nucleus-free particles that clot. But we know they are so much more than that. So why are they so important? And how do they function to do more than just stop a bleed? Milka Koupenova:       So this is a great question, Cindy, and I am happy that you alluded exactly to the anucleated nature of platelets. So platelets are cell fragments. They're precursors in the bone marrow, the megakaryocyte. They are the second most abundant blood component after the red blood cells. And traditionally, platelets have been known, as what you pointed out, as these little units that change their conformation once there is some form of a problem with either the vascular, which we have a cut, they come together, they form this clot, and bleeding is prevented. But as we have learned perhaps in the past 20 years that platelets have a profound immune role during various immune processes and infections for different kind of microbes. And particularly relevant to this paper is that we understand that platelets have clearly a role responding to the viruses and activating the immune system. Cindy St. Hilaire:        Yeah, and that was actually my next question. You and Jane are the world-leading experts on platelets and viral responses. So what was known about that interaction, I guess before we started looking at SARS-CoV-2, what was known about that platelet virus or even type of virus interaction? Milka Koupenova:       So SARS-CoV-2 is a RNA virus--respiratory virus that we actually thought similarly to influenza that it mostly stays in the lower respiratory tract where it becomes problematic. However, from our work with influenza, when we saw that in certain patients you actually can detect the virus in platelet. In the beginning of the pandemic, we hypothesized that perhaps, in some people, the virus crosses over into the circulation. And based on our previous studies with influenza, we wanted to see if that indeed is the case. Hence we initiated a study here at UMass with the department head who is also on the paper, Dr Finberg, who is a leading expert in influenza and novel virus and we collected platelets from people to see if we can detect it. And so in the beginning, we were not able to detect SARS-CoV-2 in platelets. So we collected platelets from 17 patients and by qPCR with the primers that the CDC has, for whatever reason I couldn't detect anything. And I was really frustrated because previous reports have shown that about 25%, in some people even 35% of the study population, SARS can be detected. So very interesting observations. Milka Koupenova:       I could see it by immunofluorescence but I couldn't detect the RNA. And the story goes, that I attended a seminar on SARS-CoV-2 and the person was actually referencing a company that started from University of Pitt where you are. Cindy St. Hilaire:        Oh, very nice. Milka Koupenova:       And they do specific, it's called amplicon ARTIC v3 sequencing so they enrich for the SARS-CoV-2 RNA and screen by sequencing. And when we did that, we were able to detect it in all patients. So I freaked out and I said, "Oh my gosh, something is wrong." Milka Koupenova:       And so I sent plasma, and I sent controls, and actually RNA from the virus and you can see beautifully that it's only in platelets. Four of the 17 people actually had RNA in the plasma, but what you can observe in all these people is that the virus is fragmented, meaning it's not infectious. And in a way what this tells us, it suggests that platelets are super important in the removing it from the circulation and they probably serve as a dead-end for the virus because you cannot find virus coming out of platelets and the RNA is chopped off. So what I would say, is that platelets are these amazing little units that serve as removal of the viral RNA for these particular viruses, respiratory viruses that are RNA viruses. Cindy St. Hilaire:        I think that is so interesting. So essentially, they're almost like little composters that are chewing it up and preventing it from spreading in the organism. Milka Koupenova:       Yes, and as a result there is a response. Cindy St. Hilaire:        Scott, probably the most common thing that people know with SARS is that loss of smell, or taste, and things like that, but really that doesn't send anybody to the hospital. So really what are the symptoms of COVID-19 patients that tie in with platelets specifically? I feel like that's a lot of things that we maybe in the public, or on Twitter, and things didn't hear as much about. So really what are those big symptoms linking COVID and platelets and what are the implications of platelet death in the pathogenesis of COVID? Scott Cameron:           So certainly I think several investigators are in the world of now showing that platelets are hyperactivated, Robbie Campbell and Matt Rondina put a really nice paper in Blood last year showing that platelets are hyperactive and there are other investigators who found something similar. And so the question is, what are the symptoms of hyperactive platelets in the SARS-CoV-2 patient? So what most of them would find is shortness of breath or dyspnea, and when they present to the emergency department, and certainly we saw this, the oxygen saturation which should be in the mid to high 90s on room air on an average person, was quite often low. It was in the 80s or 70s, sometimes even the 60s. Scott Cameron:           And the real surprising thing was those are patients that would normally immediately be on a ventilator, but yet they could still be talking to you. And so if you have a platelet that's activated in a hyperthrombotic condition, like SARS-CoV-2, COVID-19, and then that forms a blood clot, you have a situation where the amount of oxygen the patients taking in and the amount of oxygen you're measuring in the artery is quite discrepant and we call that the alveolar arterial or oxygen gradient. So if you've got lots of platelet plugs through the microvasculature, it's going to take up some space the oxygen should be using for diffusing in. And so that would be manifested as shortness of breath and that's certainly one of the biggest tip-offs that a patient might have a blood clot, particularly in the lung. Cindy St. Hilaire:        Some of these symptoms of COVID-19 are really worse in patients with comorbidities, diabetes, obesity and heart failure. Are platelets central to kind of the pathogenesis of those disease or the symptoms of those diseases? I guess the root of my question is, why do the comorbidities of diabetes, obesity, and heart failure make COVID worse? Is it something about those disease states themselves or is there a role for platelet? Scott Cameron:           That's a brilliant question, no one's ever asked that before. And as Dr Koupenova said, I'm a little bit biased too because I firmly believe that in different disease states, the disease educates the platelets so you've got a different platelets phenotype. So focusing on diabetes, we know the platelet phenotype is different in diabetic patients. We know that platelet reactivity seems to be higher through the P2Y12 receptor.  In terms of obesity, it is true, we know that, and this has been published also, and we know that the platelet phenotype is hyperactive in a patient with obesity and so that tells me that, that's a comorbidity that might affect platelet function and also vice versa for that case. And then in terms of why is it affecting males more prominently and more severely than females, well one of the beefs, I guess, that I had is that we treat diseases in women the same as we do in men assuming that the platelet phenotype in disease must be the same, but that's absolutely not true. And that's actually a theme that we have in our lab right now, we know that the behavior of platelets, and how platelets are educated in diseases is not all the same in women as in men and I think it's a huge disservice that we really had to have a pandemic that would make that quite clear to us. Cindy St. Hilaire:        You kind of hit onto something that's really, I think it's now becoming more recognized certainly in the cardiovascular field and that is so many studies are really only on male mice, or only younger or older men, and we are missing not only a huge patient population, but probably some really interesting biology that is distinct. Milka Koupenova:       So expanding on that, we know that in platelets, the toll-like receptors, and we've looked at the expression of all 10 in a study that we published in ATVB in 2015, actually, significantly if you look at Farmingham Heart Study data and the expression of these toll-like receptors they are increased in women versus men. And also, an interesting observation that never got published, once upon a time when I was doing studies with TLR7 mice is that if you inject TLR7 agonists, male mice would have a higher level of reduced platelet count than female mice at the same time points, right? And at that time it wasn't published. Definitely there are differences, but I also want to extrapolate a little bit on what was said at the beginning. We have to understand that when it comes to these comorbidities, everything affects a unit that doesn't have a nucleus, right? And diabetes and obesity have the so called profound, chronic inflammation of cytokines, such as IL6, that keep circulating. These things have effect on platelets. So we have two responses, we have the environment that affects platelets and we have the direct response of the virus that affects platelets. And that cumulative response truly can exhaust them and once they become exhausted, once they release their contents, as we show in this paper, then you're compromising their function and you will be compromising taking out the virus from one side and from the other side you're going to be compromising the environment because all of the content that comes out from a unit that already has free form proteins, it exhibits a true insult on what's being surrounded. So these clots that form in the lung or the platelets that circulate they no longer can be resolved properly. Cindy St. Hilaire:        Yeah. Milka Koupenova:       It's a balance. Cindy St. Hilaire:        Yeah, so really it's like destroying the platelet not only are you destroying the vacuum that has to suck up those particles, you're then just dumping a whole bunch of pro-inflammatory things on all of the endothelial cell vasculature that those platelets are nearby. Cindy St. Hilaire:         Actually that was one thing that I thought you spent a decent portion of the discussion on, and that is the method by which the blood is collected really impacts the outputs you observe in quote unquote platelets. Can you talk about the importance of that because I think that's one thing, certainly as a PhD who's just like, "Oh, yeah. I'm just going to collect blood from my mice and do this thing," how critical is that point in the experiment, in the blood collection? Milka Koupenova:       So I am very adamant when it comes to platelets for the blood to be drawn in citrate. And I have to say that a lot of the studies that you would see in the literature are done using EDTA blood or serum. They all have their importance. I'm not going to dismiss it, but if you want to truly measure what's inside in plasma, versus what's inside in platelets, or what's inside in any cell for that matter, you got to go for citrate. You have to be very careful not to shake the blood. You have to be very careful not to cool down the blood. So the nurses probably hated me because often I would be like, "You can't do this. You can't put it on ice. You can't warm it up to above certain degrees. Everything has to be controlled and done correctly." Milka Koupenova:       And so I had done in the past studies in which I would take plasma from the same patient in EDTA, in citrate and then isolate the RNA, have my tech isolate the RNA, and we send it to a fragment analyzer, and you can see how much more RNA you will get in the EDTA plasma. I'm not even talking about serum. Milka Koupenova:       Serum is a very different thing, then you're definitely going to get platelet content in it, in the serum, right? So it's important to distinguish that perhaps when you're getting EDTA plasma you are looking at a content that could have been inside in platelet and I can't stress enough that when it comes to these particular studies, citrate, dextrose, phosphate is your place to go and be. Cindy St. Hilaire:        So in terms of translational potential, what do your findings suggest about future therapies or targets to investigate as therapy? And is modulating platelets a potential for combating viral infections or mitigating their severity? Milka Koupenova:       Well, Scott and I actually talk a lot about that. Scott Cameron:           That's right. Milka Koupenova:       I personally would say, control the inflammation, never let it go to platelet. Let me back up a little bit, if you have to, you have to, right? But your go to method should be inflammation, if you don't get to the point that you need to control platelets then you're in a better place because it becomes very fickle. From everything that you hear me say, you push it to one side and the balance is destroyed. You deactivate platelets or inhibit platelets well, are they now not able to pick up the virus and then you're now having the virus circulating somewhere. Now, if you don't treat platelets that's also not good. So you're in the very fickle situation if you get to the point that you need to control the activation of platelets and there are trials currently that are trying to look at those things. Scott, I'm going to refer this a little bit more to you because you have done some interesting things with that particular point. Scott Cameron:           No, it's a great question, Milka, and I think that as platelet biologists, nobody more than I wanted it to be true that platelets would be the ultimate target. I mean, clearly patients with SARS-CoV-2 have thrombosis, clearly platelets are activated, so should we inactivate them? That was the whole point of the RECOVERY trial and one of the benefits I'll tell you before I sort of go into that is, working in a large organization like the Cleveland Clinic and we have access to data and lots of it extremely quickly, and so because of that I of course could see how many patients were coming into our hospital with thrombotic events. And I could see what the independent predictors of thrombotic events was and it wasn't the platelet count, sometimes platelet count was low, sometimes it's high in the SARS-CoV-2 patient. And if you took those individuals that were on aspirin, comparing them to those that are not in a propensity match study,  one of the things that we find is that aspirin doesn't seem to affect or improve mortality or the number of blood clots in the patient with SARS-CoV-2. Scott Cameron:           We compared that to all non-steroidal anti-inflammatory medications that patients may have been taking also in a propensity match study just in case it was the mechanism action of the drug, rather than the drug itself, and we found that NSAIDs not only did not protect patients, but they were not necessarily harmful either, which was one of the things that came out at the start of the pandemic. Among, I'll add, the absence of evidence based medicine and a lot of cases where naturally people, including clinicians, were scared and so they were going off label and they were trying a lot of different medications with really not a shred of randomized controlled data. Scott Cameron:           But now that we're 18 months into it, the first and biggest study that came back was the RECOVERY trial, which we were all waiting on, where patients were given aspirin and short term mortality was examined over an observational period of one month. And just like we found in a propensity match study, which is as close as you'll get to a clinical trial in a retrospective manner, the prospect of RECOVERY trial actually showed the curves were almost super imposeable, those that got aspirin versus those that didn't. So I think low dose aspirin clearly is not going to be enough for those patients, but I'll also add that over the observational period of one month they also didn't see a higher incidence of death in those patients. And I think Milka's point is really well taken that you have to remember that as well being an entity of thrombosis, platelets are immunological entities and so you've got to really consider should we be inhibiting them and if you are inhibiting them, I think the time point at which you should inhibit them is what we should examine, not just an all or nothing, inhibited or not. Milka Koupenova:       It's just in our linear brains we prefer to think of it as one straight, linear pathway, but it isn't, and I think platelets are actually a great example of how many pathways are feeding into one tiny fragment and that particular blood cell is inducing this profound response during these infections. Cindy St. Hilaire:        I think most people have heard that angiotensin-converting enzyme 2, also called ACE2 is the receptor of SARS-CoV-2. The virus itself uses it to bind and become internalized into the cell, but there's been some discussion or even some discrepancy of data as to whether platelets truly express ACE2 and if that is the means for the virus to enter the platelets. So can you share with us what is the current state of knowledge about that? Scott Cameron:           Yeah, just as a segue of some of the things that Milka said, I think the preparation of your sample is part of the answer. If you draw in the incorrect tube, if you the tube is not completely filled, and the ratio of citrates to whole blood isn't correct you're going to have discrepant results. If you biomechanically activate the platelets by drawing through a short needle, in a small-bore needle for example, that's going to activate the platelets. If you cool them, it's going to activate them. But then also, depending on how you decide to separate them, we always washed platelets in my lab, we wash them two or sometimes three times, and I can tell you if you use flow cytometer we get one white blood cell for every 12,000 platelets. Scott Cameron:           And some investigators might go one step further and they'll a CD45 depletion set, which is certainly important if you're studying RNA. But one of the issues, as you well know, a CD45 is also on the surface of platelets, so if you start with a low expressing protein and you CD45 deplete them, you are actually going to get a decrease in your platelet yields. I've seen it, I think Milka's seen it, various other investigators have, and you might find yourself at the threshold of what your antibody can detect. It's also variably expressed. If you look at even healthy individuals, some of them have almost none. So if you look at 10 individuals, you might actually find none, but then if you look at another 10, the amount of expression that we see is kind of all over the place. It's not like other receptors where one tends to express a certain amount and that's the way it is in health. ACE2 doesn't seem to be that way for whatever reason. Milka Koupenova:       We were able to detect in some of the people by qPCR, but what was interesting is that from the three primers that I used there was never the same person who we were able to detect all three primers with for that receptor. That tells you that maybe they are changes of one base that is not enough for the primer to detect it, right? That becomes another possibility of not being able to detect. Milka Koupenova:       And so I go to confocal microscopy where I use 100 lens and tons of hours in the microscope room, and Scott is completely right, it's really hard to see it particularly in healthy people. And it starts to pick a little bit more in people with cardiovascular disease or people with COVID that are old. So it's a bit complicated, but the important thing here is, besides the fact that we are detecting ACE2 and we're detecting proteins and I use controls, biological controls to prove that this is the case and it's not just an antibody problem, is that the virus will get picked up by platelets even if you don't have ACE2. That is the take home message from this paper is that the platelet has evolved various mechanisms by which is utilizes getting it inside. It is that important for this virus. This type of virus is not recirculating. In this case, what we observed is that the virus is attached to microparticles that are of platelet origin for that matter. Cindy St. Hilaire:        So really what you're saying, what I'm hearing is the platelet is the superhero of the body. Milka Koupenova:       Definitely. Absolutely. No bias, absolutely. Cindy St. Hilaire:        Unbiasedly, it is a superhero. Well, Dr Cameron and Dr Koupenova, thank you so much not only for this amazing discussion, but for really an elegant, elegant paper that is really bringing to light the complex interaction between SARS-CoV-2 and platelets. So thank you so much for joining me and keep publishing amazing stories like this. Milka Koupenova:       Thank you for having us. Scott Cameron:           Thank you, an honor to be here. Thanks again. Cindy St. Hilaire:        That's it for the highlights from August 20th and September 3rd issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Dr Scott Cameron and Dr Milka Koupenova. This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cynthia St. Hilaire, and this is Discover CircRes, your on-the-go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association, 2021. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahajournals.org.  

This month on Episode 27 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the July 23rd and August 6th issues of Circulation Research. This episode also features an in-depth conversation with Drs Ana Gomez and John Pierre Benitah, from INSERM and the Paris-Saclay University, about their study, Impaired Binding to Junctophilin 2 and Nanostructural Alterations in CPVT Mutation.   Article highlights:   Glasenap, et al. Imaging Inflammation and Fibrosis in Heart Failure   Shi, et al. Cardiomyocyte Pyroptosis Aggravates MI/R Injury   Koenis, et al. SPM Temper Phagocyte Responses in COVID-19   Zhang, et al. Common Origin of Heart and Extraembryonic Lineages   Cynthia St. Hilaire:     Hi, and welcome to Discover CircRes, the podcast to the American Heart Association's journal, Circulation Research. I'm your host, Dr Cynthia St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'll be highlighting articles presented in our July 23rd and August 6th issues of Circulation Research. I also will speak with Drs Ana Gomez and John Pierre Benitah, from Inserm and the Paris-Saclay University, about their study, Impaired Binding to Junctophilin 2 and Nano-structural Alterations in CPVT Mutation. Cynthia St. Hilaire:     The first article I want to share comes from the July 23rd issue of Circ Res, and it's titled Molecular Imaging and Inflammation and Fibrosis in Pressure Overload Heart Failure. The first author is Aylina Glasenapp and the corresponding author is James Thackeray, and they're from Hanover Medical School in Germany. After a heart attack, inflammation and fibrosis of the heart alter cardiac contraction and can lead to its failure. Currently, for ischemic heart failure, doctors use imaging techniques such as positron emission tomography, and cardiac magnetic resonance imaging, to measure the inflammation and fibrosis to provide a prognosis. Cynthia St. Hilaire:     However, whether these imaging techniques are useful for non-ischemic heart failure was unknown. To find out, this group performed transverse aortic constriction on mice, which is a commonly used method to model non-ischemic heart failure, and then they analyzed the animal's hearts with positron emission tomography to assess the inflammation and cardiac magnetic resonance imaging to quantify scar tissue. Compared with Sham-operated animals, those that underwent TAC exhibited increased heart inflammation for at least three weeks and significant fibrosis for at least six weeks. The degree of scarring and inflammation was inversely correlated with heart function. The team also found that reversal of TAC led to reduced inflammation and fibrosis over time. Together, the results confirm that these imaging modalities are valuable for monitoring fibrosis and inflammation in non-ischemic heart failure, and they could potentially be useful for assessing the effectiveness of interventions. Cynthia St. Hilaire:     The second article I want to share is titled GSDMD Mediated Cardiomyocyte Pyroptosis Promotes Myocardial Ischemia Reperfusion Injury. The first author is Huairui Shi and the corresponding author is Junbo Ge, and they're from Fudan University in China. After myocardial infarction, restoring blood flow is essential to saving muscle function. However, restoration of flow itself causes damage by inducing inflammation and cell death. This study found that the cell death aspect of a reperfusion injury occurs via a process called pyroptosis, which is a controlled form of necrosis that is due to excessive inflammation. Cynthia St. Hilaire:     The team developed an in vitro model of reperfusion injury, where cultured cardiomyocytes are starved and then resupplied with oxygen. Using this model, they found that cells exhibited features of pyroptosis, including the release of inflammatory factors, increased production of the pyroptotic factor gasdermin D and cell death. Cardiomyocytes lacking gasdermin D did not display signs of pyroptosis under these same conditions. The team went on to show that gasdermin D was significantly increased in the hearts of mice following ischemia reperfusion. And compared with control animals, mice whose cardiomyocytes were engineered to lack gasdermin D, suffered less necrosis and smaller reperfusion injuries in their hearts. Together, these findings provide insights into the mechanisms that should be targeted to minimize pyroptosis and subsequent ischemia reperfusion injury, following myocardial infarctions. Cynthia St. Hilaire:     The next article I want to share is titled Disruptive Resolution Mechanisms Favor Altered Phagocyte Responses in COVID-19. The first authors are Duco Steven Koenis, Issa Beegun and Charlotte Camille Jouvene, and the corresponding author is Jesmond Dalli. And they're from Queen Mary University of London. Inflammation is essential in the early stages of battling and invading pathogen, but at the same time, inflammation can become damaging to the host if it is not resolved in a timely manner. Prolonged and unresolved inflammation is responsible for the hospitalizations and deaths of many COVID-19 patients. An excess of circulating pro-inflammatory cytokines is one of the key features of severe COVID-19. And now, Koenis and colleagues show that certain pro-resolving factors are out of balance in these severe patients. Cynthia St. Hilaire:     Blood samples from patients with mild COVID-19 showed an increase in specialized pro-resolving lipid mediators. However, blood from patients with severe COVID-19 had lower levels of these pro-resolving lipid factors. Expression of specialized pro-resolving lipid mediator receptors on phagocytes was also higher in patients with mild disease than those with severe COVID-19. And, in line with this, the proportion of activated pro-inflammatory phagocytes was higher in patients with severe disease. Cynthia St. Hilaire:     When patients were treated with the steroid dexamethasone, they subsequently inhibited the increased levels of the specialized pro-resolving lipid mediators in the blood. Together, these results reveal specialized pro-resolving lipid mediators are dysregulated in severe cases of COVID-19, and the findings suggest increasing these pro-resolving lipid mediators could promote resolution of out-of-control inflammation. Cynthia St. Hilaire:     The last article I want to share is titled Unveiling Complexity and Multi Potentiality of Early Heart Fields. The first authors are Qinqguan Zhang and Daniel Carlin, and the corresponding authors are Sylvia Evans, Joshua Bloomekatz, and Neil Chi, and they're from UC, San Diego. The developing heart is thought to originate from two populations of cells; the first and the second heart fields. And these are first identifiable at stages E 7.5 in the mouse, or on day 15 in the human embryo. Genes controlling the development of these fields have been linked to congenital heart defects, but interestingly, congenital heart defects are also sometimes linked to placental abnormalities. However, the mechanisms underlying this link have been unclear. Now this study has gone on to discover an unexpected link between the first heart field and extra embryonic tissues, which give rise to the yolk sack and the placenta. Cynthia St. Hilaire:     Through lineage tracing experiments and single cell transcriptomics, the team discovered that the first heart field consists of two sources of mesoderm progenitor cells, one source that is embryonic in nature and the other source arises from the interface between the extra embryonic and the embryonic tissue of the early gastrula. This latter population of progenitor cells, which is defined by the expression of the transcription factor hand one, gives rise to extra embryonic mesoderm cells in addition to the two Hartfield cell populations. The discovery of this shared source of mesodermal progenitors not only blurs the lines between the embryo and its supporting tissue but may also explain the link between placental abnormalities and congenital heart defects. Cynthia St. Hilaire:     Today I have with me Drs Ana Gomez and Jean-Pierre Benitah, and they're from Inserm and the Paris-Saclay University. And today we'll discuss their study Impaired Binding of Junctophilin 2 and Nano-structural Alterations in CPVT Mutation. And this article is in our July 23rd issue of Circulation Research. So thank you both very much for joining me today. Jean-Pierre Benitah:    Thank you. Ana Gomez:                Thank you. Cynthia St. Hilaire:     You're in Paris, so we're trying to match it so we're all meeting our normal workday on a Friday. So I very much appreciate you taking the time to meet with me. So this study is investigating a rare disease called Catecholaminergic Polymorphic Centricular Tachycardia, or CPVT. So can you describe to us what is CPVT and how does this disease present in patients? Ana Gomez:                Okay, so CPVT stands for Catecholaminergic Polymorphic Centricular Tachycardia. So it is a genetic disease that appears mainly in childhood and youth with sudden death. So the patients don't have any remarkable problem, either in the electrocardiogram or arteries, or in the cardiac structure by echocardiography, and they seem healthy. But when they have stress, it can be emotional or it can be physical, so during exercise, it presents with syncope or sudden cardiac arrest. So the problem is that, many of the times, the first symptom is the death of a child playing soccer or doing exercise and then the only treatment that they, so far, it's beta blockers, to avoid this stress, and also flecainide and propafenol. But these treatments are still not completely efficacious, or sometimes the people need to get implant defibrillator. It's a big cost and it's also stressful because if the patient feels that they have to recharge, that supposes stress, and this stress is bad for them. Cynthia St. Hilaire:     Right, so it's like if they feel a flutter, it makes them more stressful, which can exacerbate. That is terrifying. And so the goal, I guess, regarding gaps in knowledge that are leading to your investigation, what was known about this disease before you started your study? And where did you leap off from that? Jean-Pierre Benitah:    Up to now, what we know about the disease is an alteration of the calcium homeostasis in cardiac myocyte. That could induce trivial activity, and then arrhythmia and cardiac sudden death. So mainly the mutation related to an intracellular calcium channel called Ryanodine receptor. So it's up to 60% of the patient with this mutation, but also you have a mutation related also to proteins that are in-buried in the control of the Ryanodine receptor activity, priadine, calmodulin. Cynthia St. Hilaire:     Yeah, that was actually going to be my next question. So I know this cardiac Ryanodine receptor 2, or RYR2, it's obviously the channel component that helps to release that calcium signal, but it's part of a larger complex. I believe it's called the Calcium Release Unit. Can you talk about what is in that unit in terms of proteins and then where those other genetic mutations fit into that? Ana Gomez:                Yeah, so the Calcium Release Unit is formed by a cluster of Ryanodine receptors. So in the reticular cardiomyocytes, these are mostly in the junction of sarcoplasmic reticulum that is very close to the sarcoplasmic reticulum membrane inside the cardiomyocyte, inside the cell. So the channel is internal. But it's very close to the sarcolemma in the T-tubule invaginations where the L-type calcium channels are located. So this is... The channels are very important to activate contraction, so it's heartbeat. The calcium entry through the attached calcium channel on the surface makes some calcium get into these very restricted spaces, like 20 nanometers, and in this space this calcium activates the Ryanodine receptor. So the Ryanodine receptor is activated by calcium and these release much more calcium than is needed for the contraction. So the problem of the CPVT is that the channels may release calcium during diastole, so when calcium should be low because they had to relax. Ana Gomez:                For your new question, which proteins? So the main proteins are the Ryanodine receptor. But Ryanodine receptors are a very big macro complex. They are the biggest channels that are known and they have a big cytoplasmic portion with proteins that can bind to them, and most of them just keep the channel quiet. So this may be calmodulin, FKPB 12.6, or 12, sorcin. And then there are also some other proteins that scaffold kinases, like PKA and CaM kinase. And also they have some proteins that moderate the channel from the luminal side. So, calsequestrin, triadin and junctin. And this agents to fill in that we will speak later. It's important because it binds to the L-type calcium channel and to the ryanodine receptor. So it's important to keep the dyad structure. It's not only a structural role. Cynthia St. Hilaire:     Yeah, that is so interesting. So your study focused on a very specific mutation. It's the RYR2 arginine in the 420 spot to glutamine mutation. So I guess my first question is based on the patient population, how common is this specific mutation? How common is that? Ana Gomez:                Yeah. So in fact, I'm going to say that it's very common, because normally CPVT is one mutation, one family. Cynthia St. Hilaire:     I see. Ana Gomez:                Even if they are located in hotspots, but these particular mutations, we were approached by a cardiologist working in Spain who had this family with a child that died at the age 14, playing soccer game. And so Dr Zorio in Valencia, she found this RyR2 420Q mutation. And at this time this was the first mutation in this site. I mean, not really in the site, there was already RyR2 420W that was already, so it was the same spot, but different. Cynthia St. Hilaire:     That was my next follow up question to that. My PhD was biochemistry, so this brought back having to memorize the amino acid structure. So arginine is large and positively charged to glutamine is neutral. So what were the experiments that you designed to help determine the functional causes of this mutation? You know, in addition to just, okay, obviously there's a charge change, so there's probably a structural or a binding change, but how did you determine the functional consequences of this mutation? Ana Gomez:                The structure, as you say, this has been shown. In fact, they was the first family, but then also in this region, there was another family and in Israel also there is another family. So there are three, but the structural limitations that these arginine is neutral. It has been shown by a laboratory, who works in Vancouver, in a structural and the end terminal has like three logs and these are 420. It's important to hold a chloride that in the middle and, and to hold the position. So, but this is not the functional, the functional is what we were going to analyze. So the first thing that we did is to analyze calcium sparks because calcium sparks is the functional, let's say elementary event, of calcium release to RyR2 receptors. So we start analyzing calcium sparks in the cells and we found strange things, like very long calcium sparks that was not so clear in other CPVT models, even one that we studied earlier. And so then we started to continue to know why we have longer calcium sparks and different kind of analysis. So we also collaborate with some other laboratories to do the ultrastructure of the dyad by electromicroscopy. Ana Gomez:                And then we found that the sarcoplasmic reticulum, junctional sarcoplasmic reticulum, was enlarged. So we thought, well, maybe the channel, the calcium spark is longer because locally they delayed depletion. So we did another kind of experiment changing the volume of the SR and it was not so concluded so we found that it may contribute to longer calcium sparks, but it doesn't explain for it. So then we start with to analyze different proteins candidates, also the phosphorylation of course. And then we didn't find in most of these proteins, like FKVP. Cynthia St. Hilaire:     Kind of a standard go-tos. None of them were involved. Yeah. Ana Gomez:                Yeah. And then, because there is this ultrastructural alteration, we thought of junctophilin and that is how we found that junctophilin binding was impaired. Cynthia St. Hilaire:     That's a perfect segue. You're hitting all of my next questions. So can you tell us a little bit about, what did you find regarding junctophilin and the RyR2 channel? Jean-Pierre Benitah:    So mainly, junctophilin act to us the good structural design between the ryanodine receptor and the trigger L-type calcium channel. And people say that junctophilin binds to both proteins to keep them close to each other. So mainly what we found is that we don't have activation of the expression of junctophilin, but it seems that with this mutation the junctophilin is less in contact with ryanodine receptor. But it's not the case for the L-type calcium channel. It seems that coimmunoprecipitation experiments that we've done show that junctophilin stayed still with the L-type calcium channel, but have a lower affinity to the ryanodine receptor when you have this mutation. What was really important is that we saw that not only in the mouse model where we induce this mutation, but also in cardiomyocytes derived from induced pluripotent stem cells from patients that have this mutation. Cynthia St. Hilaire:     I think that's one of the great strengths of your study. You know, I like how you took a multi-faceted approach, you know, using these IPS cells from the patients and also created a knock in model. Previous studies had used more global or whole exon deletions. So how is your knock-in able to identify additional information that built upon those former studies? Ana Gomez:                Maybe this is not an exact answer to your question, but what I think is that the strength of our study or one of the strengths of our study is that we have the patients with electrocardiograms working, we have the cells from the patients. So we have...Our IPS cell is from one of the persons that have been patient, and the control line is from his brother. So we have the two brothers. They are still living, and we have the mice and everything is in the same point mutation. So in this thing, because there is a lot of, let's say, critics to the IPS cells studies because they are not mature and they don't look like an adult cardiomyocyte. And I think that besides CPVT, we can also show that of course cardiomyocytes derive from IPS cells. They are not adult, but they are still a good model because we recapitulate the same thing. Ana Gomez:                So we can mix the human context to really have what happened in patients, because that is the important thing, but we also need to manipulate the in vivo animals and there are some things that we cannot do. We cannot get adult cardiomyocytes from patients, so for that, we have the mice and we can also analyze from in vivo to the molecular level. So I think that it's a big strong point from our study that you take compared to others, that they are only in mice or only in IPS, cannot do this correlation. Then, each mutation, we think that it may, or at least each region of the mutation, may have different mechanisms. So if we find these longer calcium sparks in these R420Q mutation, it doesn't mean that because we also have other studies in C-terminal mutation, and we don't find longer calcium sparks, we just find more. So this is not because of the design of the study, but because the mechanism of the mutation is different. Cynthia St. Hilaire:     In terms of translational potential, what do your findings suggest about either the ability to screen patients potentially for the development of CPVT or actually more importantly, you know, therapies to help treat these patients when they're identified? Jean-Pierre Benitah:    Yeah. It's one of the big problems with the CPVT, especially since when you look at the different mutations, those are different mutations that have been reported on the ryanodine receptor located on different hotspots on the ryanodine receptor. And it's seems that each hotspot could have a different type of mechanism behind that. So, for example, we show, there you see, you know, different mutations in collaboration with CPVT or 420Q mutation. So the mechanism was related to an alliteration of the sensitivity of the ryanodine receptor to the calcium. So the group of branching show that in other mutations, in other spots, hot spots, it was related in fact, to a modification of this. Also the sensitivity of calcium of the ryanodine receptor calcium, but from the luminal side. Ana Gomez:                Regarding your first question was diagnosis. I think that after our work, we may also include junctophilin, because so far there has not been any link to junctophilin for sensitivity. So when a patient has CPVT, they start screening for mutations in the ryanodine receptor, since it was found that this child was involved and then in other proteins. So I think now if they don't find in a patient, because there are still like 40% of CPVT patients that the mutation has not been found. Ana Gomez:                For therapeutic side maybe find a molecule that stimulates the binding of junctophilin to ryanodine receptor, but also maybe some smaller molecule that may interact between the N-terminal and the core solenoid because we found that in the interim molecular structure, they show tighter association between the N-terminal and the core solenoid. So maybe it's more of a tide or something that can be in between too. I mean, I don't know, but it's first line there. Cynthia St. Hilaire:     Potential, but still far off. That's wonderful. So are some of these mechanisms, I assume, they would also be relevant in non-genetic forms of tachycardia? Is that the case? Could some of your findings also perhaps be applied to the tachycardia related to heart failure or other types of disease states? Ana Gomez:                I think it's actually, for example, junctophilin binding to ryanodine receptor in heart failure. It has not been yet studied, but we want to do it. It's something because as you say heart failure, it's a very common disease. So it's also very relevant to the public health. This is something that we need to know. Jean-Pierre Benitah:    One of the things that happens in heart failure is that it seems also that you are a dissociation between the calcium channels and the ryanodine receptor because you have less tissue formation. So perhaps this is difficult to try to figure out whether it would be the same, but perhaps this activation between the communication between the two channels is one of the main points that we have in CPVT and in heart failure related to tachycardia. Ana Gomez:                Yeah. In fact, many years ago we showed that. We showed that in heart failure there is a defect in calcium channel and ryanodine receptor. So in this study it was only functional. We didn't do the structure, but of course it is something that we have to keep in mind, continue investigating. Cynthia St. Hilaire:     Yeah. Well that sounds like a great future project. Well, I want to thank you so much for joining me today and helping to discuss your paper. I love it when we take rare diseases and figure out the mechanism with hopefully applying it to more common disease states. That's what I do in my lab with vascular calcification, and so thank you so much for joining me and for this great publication. And we look forward to your future work that is hopefully in Circ Res. Jean-Pierre Benitah:    Thank you for the invitation. Ana Gomez:                Yeah, thank you very much for your time. Cynthia St. Hilaire:     That's it for the highlights from the July 23rd and August 6th issues of Circulation Research. Thank you for listening. Cynthia St. Hilaire:     Please check out the Circ Res Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, doctors Ana Gomez and John-Pierre Benitah. Cynthia St. Hilaire:     This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles was provided by Ruth Williams. I'm your host, Dr Cynthia St. Hilaire, and this is Discover CircRes, your on-the-go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association, 2021. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.  

Meet Ruth Williams-Brinkley:Ruth Williams-Brinkley is Regional President, Kaiser Permanente, Mid-Atlantic. In this role, Ruth oversees all of Kaiser Permanente's care delivery and health plan operations in Washington, D.C., and suburban Maryland, Baltimore, and Northern Virginia. Previously, Ruth served as CEO for multiple health systems, including Kentucky's largest integrated health system, KentuckyOne. She began her career as a nurse, earning a MS in Nursing Administration from DePaul University.Key Insights:Ruth's experience spans from direct patient care, to operations management across four states and districts. She is well-versed in Kaiser Permanente's unique patient care and payment model, and oversees the building of healthy ecosystems around the patient.Filling Gaps with Technology. Ruth discusses the platform developed by Unite Us and how it addresses social-determinants of health. (32:27)Leading People. Healthcare is full of promising technology, but at its core, patient care and leadership are about people. (10:10)Problem Solving. Ruth explains how her background influenced her career and developed her ability to problem-solve. (34:49)Mentorship. Mentors can provide powerful, early-career advice. And once you are established, you can return the favor to others. (37:58)

Join host Dr. Rupa Wong as she interviews Dr. Ruth Williams, past President of the American Academy of Ophthalmology and practicing physician for over 30 years.  They discuss recent statements made in the medical community that women physicians all want to prioritize family over work, leading to a doctor shortage. Work-life balance (or imbalance), the need to overexplain ourselves and whether working part time is feasible are addressed.

In this special edition of Her Story, we continue our leadership series by asking a few of our guests whether they consider themselves accidental or intentional leaders. Join the conversation for stories about the social determinants of health, problem-solving, empowerment, and more.

When the King of Bechuanaland fell in love with a white Londoner in 1946, everyone was... surprisingly cool with it! But due to the influence of Apartheid, he wasn't allowed to reclaim his throne. Find out how their marriage totally redrew the map of Africa and led to the new, independent nation of Botswana. Learn more about your ad-choices at https://www.iheartpodcastnetwork.com

 A Conversation  with Civil Rights Activitist Homer Beecher Williams “H.B.” to his friends &  wife Nannie Ruth Williams Nannie Ruth Williams her father a  sharecropper as well as her grandfather ., talks about her role as family protector vs wife/supporter for Civil Rights Activitist Homer Beecher Williams “H.B.”  H.B., Williams  then a school principal life changing/rights  began when he was offered the job of assistant principal, and when he refused, pointing out that it was a demotion, he was fired. He spent years fighting for his reinstatement. Durning this battle about for justic about  ten years, was his awaking to real life "Civil Rights" on a personal scale set in motion by the Monroe County Board of Education.  Real life injustic at the hands of segregated school system where black  educators role of leadership were set in stone in class rooms, but not as administrators.  Who is "Popy Chat" Monroe County First Black Talk Radio host during the civil right battles? @ Monroeville AL. WMFC 99.3Firebrand named Ezra Cunningham, who had taken part in the Selma march. Using a magnifier device, she is a reader, mainly of history, but also of crime novels. ... H.B. Williamssighed when any mention of the book came up. ..... and a firebrand named Ezra Cunningham, who had taken part in the Selma march. All this ...

This week we welcome Amma Asante, director of the acclaimed 'Belle' and this week's 'A United Kingdom' on to the Curzon Film Podcast. The true story of Ruth Williams, a typist from London, and Seretse Khama, the prince of Bechuanaland, who fell in love in the late 1940's. Asante's film uses a sweeping romance as a gateway for a poignant and powerful film about race and politics; that feels as current now as it did then. On the podcast this week are Sam Howlett, Harry Chapman and Jake Cunningham.Produced and edited by Jake CunninghamStudio services by CSRFM.comMusic supplied by incompetech.com Hosted on Acast. See acast.com/privacy for more information.

David Oyelowo and Rosamund Pike discuss A United Kingdom, a new film which tells the true story of Seretse Khama, the future King of Bechuanaland, and Ruth Williams, a clerk from South London. When they married in 1948 they not only faced fierce opposition from both of their families but from the British and South African governments. It had been claimed that the lost sketchbook from Van Gogh's time in Arles, France, has been discovered. However, in a statement released this afternoon, the Van Gogh Museum in Amsterdam has said they are of the opinion that these sketches 'could not be attributed to Vincent van Gogh'. We talk to the museum and the expert behind the 'discovery'.Cape Town City Ballet, the oldest ballet company in South Africa, has been resident at Cape Town University for eight decades. It's now caught in the long-running student protests for decolonisation of the curriculum. With the university deciding not to renew the company's lease, Gerard Samuel, Director of the School of Dance at Cape Town University and a Cape Town City Ballet board member, discusses the troupe's uncertain future.And 60 years after Ray Charles made his eponymous album, the music critic Kevin Le Gendre re-evaluates the moment that an artist who played rhythm & blues, the music from which rock & roll was born, was about to change the music world.Presenter: Samira Ahmed Producer: Ella-mai Robey.

Tyler and Mitch are talking with Ruth Williams, the leader of a group known as the Heart Mothers based in Laramie, Wyoming. Ruth supports the mission to rescue and heal girls in Cambodia lead by Somaly Mam Foundation. In this episode Ruth gives her introduction into the dark world of sex trafficking, talks about her organization and what their mission is and how her family and faith have lead her to continue fighting this fight for over a decade.