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This month on Episode 26 of Discover CircRes, host Cindy St. Hilaire highlights four original research articles featured in the June 25th and July 9th issues of Circulation Research. This episode also features an in-depth conversation with Dr Hirofumi Watanabe, Dr Ariel Gomez, and Dr Maria Luisa Sequeira-Lopez from the University of Virginia about their study, The Renin Cell Baroreceptor, A Nuclear Mechanotransducer Central for Homeostasis.   Article highlights:   Mesirca, et al. Electrical Remodeling of the AV Node in Athletes   Yang, et al. Macrophage-Mediated Inflammation in COVID-19 Heart   Örd, et al. Functional Fine-Mapping of CAD/MI GWAS Variants   Akhter, et al. EC-S1PR1 Activity Directs Vascular 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 the articles presented in our June 25th and July 9th issues of Circulation Research. I'm also going to speak with Dr Hirofumi Watanabe, Dr Ariel Gomez and Dr Maria Luisa Sequeira-Lopez from the University of Virginia about their study, The Renin Cell Baroreceptor, A Nuclear Mechanotransducer Central for Homeostasis. Cindy St. Hilaire:        The first article I want to share comes from the June 25th issue of Circ Res and is titled Intrinsic Electrical Remodeling Underlies Atrial Ventricular Block in Athletes. The first authors are Pietro Mesirca, Shu Nakao, Sarah Dalgas Nissen, and the corresponding author is Alicia D'Souza. And they're from the University of Manchester in the UK. Cindy St. Hilaire:        Endurance training has cardiovascular benefits, but when taken to extremes, it can elicit heart problems such as atrial ventricular block or AV block. AV block is the impaired conduction through the AV node. In fact, some endurance athletes require pacemakers later in life due to AV block. One hypothesis for this conundrum is that the problem stems from disruptions in the autonomic nervous system. This study shows that in fact, the intrinsic electrophysiology of the heart is to blame. They used trained race horses, as well as mice, subjected to endurance swimming as models for human endurance athletes. Electrocardiograms on the animals showed that just like human athletes, the race horses and the swim-trained mice exhibited signs of AV node dysfunction that is not seen in sedentary controls. Cindy St. Hilaire:        Because the dysfunction also persisted when the autonomic nervous system was blocked, the team examined molecular changes within the heart itself. They found that ion channels, HCN4 and Cav1.2, were less abundant in the AV nodes of trained animals than those of the controls. The team went on to identify two microRNAs regulating HCN4 and Cav1.2 production and showed that suppression of these microRNAs restored normal heart electrophysiology in the mice. If the result holds true for humans, this could pave the way for novel treatments for AV block. Cindy St. Hilaire:        The second article I want to share is titled An Immuno-Cardiac Model for Macrophage-Mediated Inflammation in COVID-19 Hearts. The first authors are Liuliu Yang, Yuling Han, Fabrice Jafre, Benjamin Nilson-Payant and Yaron Bram. And the corresponding author is Shuibing Chen. And they're from Cornell University Medical Center. Cindy St. Hilaire:        COVID-19 is primarily a respiratory disease, but cardiac complications are common and appear to be linked with worsening outcomes. Post-mortem examinations of COVID-19 patients' hearts have revealed abnormally high numbers of macrophages, suggesting that these cells have a role in the heart pathology. To investigate this possibility, this group co-cultured macrophages and cardiomyocytes, both which were derived from human induced pluripotent stem cells and infected the cultures with SARS-CoV-2 virus. Upon infection, both cell types increased their rates of apoptosis. However, the number of cardiomyocytes succumbing to the cell death process was far higher than that of macrophages. When cardiomyocytes were infected with the virus in the absence of macrophages, their rate of apoptosis dropped. Cindy St. Hilaire:        The team showed that macrophages produced large amounts of the inflammatory cytokines, IL-6 and TNF, in response to the virus and that trading the cardiomyocytes directly with the cytokines could similarly induce apoptosis. Blocking IL-6 and TNF alpha signaling prevented the macrophage-driven cardiomyocyte death. The team then identified two FDA approved drugs, ranolazine and tofacitinib, that prevented the virus-induced cardiomyocyte death in vitro and suggest that these drugs now be investigated in larger animal models. Cindy St. Hilaire:        The next article I want to share is titled Single-Cell Epigenomics and Functional Fine-Mapping of Atherosclerosis GWAS Loci. The first author is Tiit Ord, and the corresponding author is Minna Kaikkonen, from the University of Eastern Finland. Cindy St. Hilaire:        Genome-wide association studies, or GWAS studies, have identified hundreds of genetic loci associated with coronary artery disease and myocardial infarction. And many of these genes likely play a role in atherosclerotic development. However, most of these loci are located in non-coding intergenic regions of the genome. Thus, their functional effects on atherosclerosis development are not clear. Non-coding regions of the genome may contain gene regulatory elements, including cell type specific enhancers. And because such enhancer elements often have open chromatin structures, this team profiled the chromatin accessibility of single cells in human atherosclerotic plaques. Cindy St. Hilaire:        They found that many cell type-specific assessable regions overlapped with both transcription factor binding motifs, as well as GWAS-identified coronary artery disease loci. Using an algorithm called Cicero, the team was able to predict likely genes under the control of these accessible intergenic regions. They found that in more than 30 cases, they were able to confirm these intergenic regions control gene expression in in vitro assays. This work highlights the power of chromatin accessibility mapping for homing in on GWAS loci with transcriptional effects, and for identifying the likely genes they regulate. Cindy St. Hilaire:        The last article I want to share is titled Programming to S1PR1+ Endothelial Cells Promote Restoration of Vascular Integrity. The first author is Mohammed Zahid Akhter, and the corresponding author is Dolly Mehta, and they're from the University of Illinois College of Medicine. Cindy St. Hilaire:        Endothelial cells line the lumen of our blood vessels, forming a barrier that regulates the transport of nutrients, fluids and circulating cells to and from tissues. The lipid signaling molecule, sphingosine-1-phosphate, or S1P, and its receptor, S1PR1, promote endothelial barrier integrity. But how S1P and S1PR1 signaling might restore barrier function to inflammation-induced leaky vessels is unclear. Cindy St. Hilaire:        Using mice with fluorescently tagged S1PR1, this group showed that when mice are given a dose of the bacterial endotoxin, LPS, which induces lung inflammation, there's a dramatic boost in the proportion of growing lung endothelial cells. This boost in S1PR1+ endothelial cells is due to their increase in proliferation. Cindy St. Hilaire:        The authors go on to show that this proliferation is accompanied by increased production of the transcription factors involved in S1P synthesis and secretion. When they transplanted S1PR1+ cells into mice whose endothelial cells lacked the receptor, they could rescue the leaky blood vessels. By detailing the cells and molecular players responsible for vessel recovery after inflammation, this work may inform repair boosting therapies for chronic inflammatory conditions. Cindy St. Hilaire:        So today with me, I have Dr Hirofumi Watanabe, Dr Ariel Gomez and Dr Maria Luisa Sequeira-Lopez, from the University of Virginia. And they are all with me to discuss their study, The Renin Cell Baroreceptor, a Nuclear Mechanotransducer Central for Homeostasis. And this article is in our July 9th issue of Circulation Research. So thank you all for joining me today. I think we're spanning 13 time zones, so I appreciate you all making the effort. Maria Luisa Sequeira-Lopez:  It's our pleasure. Thank you. Ariel Gomez:               Thank you. Hirofumi Watanabe:   Thank you. Cindy St. Hilaire:        I won't lie, the Renin-Angiotensin-Aldosterone System is quite complex, so we're not going to try to break it all down here, but it is essential for the regulation of fluid balance and blood pressure in the body. Without it, things go quite awry. And your study is focusing on the kidney cell that produces renin in response to the minute changes in the blood pressure and the composition and the volume of the extracellular fluid in the body. So I'm wondering if, before we jump into the study, if you can give us a bit of background about these renin-producing cells and what is known about the renal pressure sensing system? Maria Luisa Sequeira-Lopez:  So in the adult mammalian kidney, renin cells are located at the tip of the afferent arterioles at the entrance to the glomeruli. So that's why they are called juxtaglomerular cells. They synthesize and release renin. This is then, as you mentioned, the rate-limiting enzyme for the renin-angiotensin system that controls blood pressure and fluid-electrolyte homeostasis. However, during early embryonic development, as demonstrated many years ago, renin cells are widely distributed along the renal arterial tree and inside the glomerulus and the interstitium. And with maturation they differentiated to vascular smooth muscle cells and they end up being located in the juxtaglomerular area. Maria Luisa Sequeira-Lopez:  But in response to a homeostatic challenge, such as hypertension, dehydration, hemorrhage, there is an increase in the number of renin-expressing cells along the renal arterial tree, resembling the embryonic counter. And this occurs mostly by re-expression of renin from vascular smooth muscle cells that descended from originally renin-expressing cells. And when the challenge passes, then they stop expressing renin and become vascular smooth muscle cells again. So renin cells are extremely plastic and they can switch back and forth from an endocrine to a contractile phenotype.   Cindy St. Hilaire:        I'm really glad you mentioned the vascular smooth muscle cell angle because I actually have a question about that later on. But before I get to that question, one of the things that I love reading in studies is when a current paper references much older work that often has a really intricate or insightful observation. And in your paper you cited, I believe it was in 1957, was the first real hypothesis that there is an existence of this pressure sensing mechanism in the kidney, what we're calling this baroreceptor. Yet, that was a long time ago and the identity has really been elusive. So I was wondering why has it just been so difficult to really pin down this baroreceptor and how this pressure and fluid sensing works in these cells? Ariel Gomez:               So it was elusive, as you said. The reason is the researchers didn't have the tools to actually study it. It really requires an evolution, conceptual evolution, and scientific evolution, as well as technical development. And so we were fortunate over time, over the years. We developed ways to mark the cells endogenously with the appropriate fluorescent markers, genetically engineer, then develop models that allowed to drop the blood pressure in a consistent manner, and so forth. And we could follow the lineage of these cells and study them as they move back and forth from their phenotypes. So I think it was a matter of even Dr Tovian, who is the person that you mentioned, Lou Tovian, who I actually met a long time ago. So he even postulated that maybe it was a stretch mechanism, and that's one of the great contributions of Hirofumi who figure out how to stretch the cells using different ways of doing that. Cindy St. Hilaire:        So in your quest to identify this baroreceptor, you use several murine models. A surgical tool, but also several genetic tools. And I was wondering if you could share a little bit about that initial surgical model, that aortic constriction and maybe the pros and cons about that method? Hirofumi Watanabe:   And so we established surgical model of in mice. We created inductation between the roots of the right and left renal arteries. By the surgery, and our right kidney receives high pathogen pressure, and the left kidney receives low pathogen pressure. And this surgery model resulted in a marked difference in the expression of renin in each kidney. And by RT2 PCR and in situ hybridization, renin was decreased in the right kidneys and increased in the left kidneys. Cindy St. Hilaire:        Excellent. So it's a really powerful model because you can use the same mouse to look at the same... Ariel Gomez:               Right. So the beauty of that is that, Hirofumi, by doing that, he got rid of any genetic variation between the mice. Because you are doing the high and low pressure in the same mouse. Maria Luisa Sequeira-Lopez:  And another question I can think that we have said was when, if you calculate the number of cells that increase in one kidney and decreases in the other one, if you add them, it ends up being the number of cells in a non-aortic coarctation mouse. So it looks like- Cindy St. Hilaire:         It's a literal seesaw. That's beautiful. At least the math works out in your favor in the end. That's great. Maria Luisa Sequeira-Lopez:  And that's something that Luis Tovian didn't see, because what he did is he increased the perfusion pressure in an isolated kidney and what he observed was less granulation. So it was an indirect method to find less renin in those kidneys. But with a low pressure, he didn't observe an increase in renin, or increase in granulation. What we know that really happens. Cindy St. Hilaire:        So you mentioned smooth muscle cells in the beginning of our discussion and my training has been in smooth muscle cells, vascular smooth muscle cells, mostly though focused on the aorta, especially in mice. A lot of times we just say smooth muscle cells, but people are really talking about the aortic smooth muscle cells in the mice. And in humans, in the coronaries. But we use the mouse aortic smooth muscle cells as the model, which you can obviously see when you frame it out like that, some issues. And one of the things we talk about at least in athero is the cell plasticity and this phenotype switching from the contractile quiescent state to one that's associated with disease processes. Cindy St. Hilaire:        And we've really evolved on what we've known about that. It used to be just about the migration and proliferation. Now it's about the actual phenotypic switching into different kinds of cells. Macrophage-like cells, for one. And yours really was the first to bring to my eyes that there's probably many more regarding that. So could you maybe expand a little bit on these renal smooth muscle cells or renin-like cells maybe, and what's happening in that disease process? And do we know the point at which it can switch and make renin and go back versus switches and doesn't return? Is that part of the disease process? Ariel Gomez:               We describe the plasticity of the smooth muscle cells from the renal arterioles long time ago. I mean, I think, I would say that even at the beginning of my career. And at that time people didn't use that term so much, plasticity. We didn't know how to call it because it was a switch back and forth from a smooth muscle contractile phenotype to endocrine without, at the moment, without causing disease. And the cells were able to come back to be smooth muscle cells. But the period of the stimulation was only a week or so. So during that time, the cells can go back and forth. And now we know that they do that. But if you create a persistent stimulation, and this is another paper that we are working with Hirofumi and Maria Luisa, if you create a knockout renin or knockout of angiotensin receptors or so forth, the stimulation doesn't stop because there is no angiotensin. Ariel Gomez:               And so under those conditions, the cells reach a point in which they become very aggressive, almost embryonic-like. They are constantly stimulated. They are attempting to reestablish the phenotype and in doing so, they create these concentric vascular hypertrophy. And I don't know whether we are going to send the paper to Circulation Research or to where, but we are still writing it. After that, we don't know whether they can come back because they are so seriously sick. And we know that they are responsible for this, but this is another paper. Maria Luisa Sequeira-Lopez:  Another thing that I wanted to add is that these cells have been extremely difficult to study. Ariel has been developing many, many tools that allow him to dissect them and cover many secrets of the cells. But if you... First because they are very, very few in the kidney. And there were no markers to isolate them. And if you put them in culture, now that we can have them live with a person marker, they stop expressing renin and making renin within 24-48 hours. So it's difficult to study. So that's why Hirofumi [inaudible 00:19:21] how the system works. Stimulating them with cyclic AMP, they go back like renin. If not, they differentiate into vascular smooth muscle cells. It looks like that's their default pathway. So they need to sense that there is a need for renin to increase the blood pressure and electrolyte homeostasis. So that's one of the characteristics of the cells. But if you stimulate constantly, as Ariel said, then they may be hard to… They cannot come back. Cindy St. Hilaire:        It's over the tipping point a bit. Maria Luisa Sequeira-Lopez:  Yes. Cindy St. Hilaire:        In your discussion you mentioned another study from your group that kind of took more of a developmental angle. And you mentioned that you had identified unique chromatin structures of renin-producing cells, and you also identified what are called super enhancers that help dictate the differentiation of these running progenitor cells into renin producing cells. And then in your mechanical stimuli experiments, you mentioned identifying similar chromatin signatures. And I was wondering what this might suggest in regards to the disease pathogenesis. And I guess I'm thinking about it in terms of in many diseased states, we see this activation of developmental programs that either are not stopped or just go on and are even higher expressed than in developmental programs. And is that you think is happening in these renin cells? A developmental program gone awry? Ariel Gomez:               Yeah, definitely. I definitely think so. I think we all, the three of us think that way. Yeah. I think it's an exaggeration of a developmental program. One thing that we didn't mention and why the vessels get so sick is because during development, these cells contribute to the formation of the vasculature. And so when they regress so much trying to make renin... And they make it. I mean, they go from 20,000 units to 2 million of renin, right? And they never stop. But when they regress so much, they regressed on embryonic stage and they think that they need to make more blood vessels to actually increase the flow and the oxygenation of the tissue. But in doing so, they create more pathology. So maybe, Hirofumi, I don't know if you're going to ask him, but one of those super enhancers is the Lamin A/C gene. And he has studied that in this Circulation Research paper that we are talking about. Maria Luisa Sequeira-Lopez:  I just wanted to add that they also make lots of angiogenic factors to make the vessels. Cindy St. Hilaire:        Got it. So developmentally, they're activating more production of renin but they're also producing these pro angiogenic cytokines and really driving that… Ariel Gomez:               BGF. They produce a type of BGF or angiopoietins. Cindy St. Hilaire:        Interesting. Ariel Gomez:               Yeah. And things like that. Cindy St. Hilaire:        I really liked reading about this magnetic bead experiment that you used as the mechanical stimuli. Frankly, I saw the picture and I brought it to my lab and said, "Guys, figure out how to do this." Can you explain a little bit about it? It seemed really nice, really elegant and very tuneable. So I'm excited. I'm sure many more people are excited to hear about it. Hirofumi Watanabe:   So we applied coated magnetic beads to the cultured ring cells. Then we placed a magnet above the cells so we can pull the cells by magnetic force. Cindy St. Hilaire:        How strong is the magnet that it doesn't just rip everything up? Hirofumi Watanabe:   Yeah. We cannot observe the shapes of the cells, but yeah, I hope it's just stretch. Cindy St. Hilaire:        Yeah. Well, it certainly elicited an effect. So, in terms of future translational potential, what do you think about these findings that suggest either potential future therapies or even targets that we can use to develop therapies? Is there a future therapeutic angle to these really interesting biomechanical findings? Ariel Gomez:               Discovering or knowing the structure of these pressure sensing mechanism, I think we'll eventually have many applications because it will be applicable to hypertension, of course. And maybe we can begin to think... Not yet because it's really a fundamental discovery, it's not yet at that stage. But eventually the information can be used to start thinking about treatments that are addressing those particular structures that are involved from the beta one, integrating all the way to the nucleus. And little by little people started developing epigenetic therapies, right? And we are testing some of these compounds in our lower authority. Not with this model, with other models. But I think eventually we will be able to do what was the dream. It was really a dream years ago, was to do molecular therapy, right? And so a small compound development will play an important role. And eventually driving the molecules to the exact place in the genome is... So it would be not only patient-oriented, personalized medicine, but local specific. That should be the goal of medicine in the future. I won't be there when we get there. Cindy St. Hilaire:        I don't know. CRISPR is moving things rather fast, so that's great. Ariel Gomez:               Oh, yeah. You're right. You're right. You're right about that. Okay. Cindy St. Hilaire:        So what's next in this project? What do you think is the next low hanging fruit? Now that you've identified this baroreceptor or maybe a component of a larger baroreceptor family, what do you think is the next most important question? Maria Luisa Sequeira-Lopez:  We want to know what is in-between. And the bigger one integrating and the Lamin A/C. And also, we want to see how fast this reacts. So we'll be doing experiments with the constriction for just a few hours, and harvest both kidneys and we will try to do single cell RNA-seq and a from those vials. Hirofumi Watanabe:   I think we want to study how Lamin A/C regulates renin expression in renin cells, so chromatic modification initiated by changes in particle pressure more. Ariel Gomez:               And I think the... What I've been now pushing a little bit is to remember that there is another cell in there that is in between the pressure and the JG cells. And that is the endothelium cell. Right? And so, they are communicating with one another. So we are going to engage some... In fact, it's already happening. A member of the lab is already working with the same model that Hirofumi used, looking at endothelial cells label also using aninterfering promoter linked to a fluorescent protein. So we want to know what happens to the endothelial cells, because they are receiving the brunt of the pressure. And we don't know how they sense. We described the mechanosensing capability of the JG cells, the renin cells, but the whole system is probably a lot more complex than what we think. Cindy St. Hilaire:        I think that's the lesson of renin angiotensin signaling. It's always more complex. Ariel Gomez:               Yeah. Exactly. Cindy St. Hilaire:        Well, thank you all so much for joining me today. This is a beautiful study, very elegant. And I liked the new kind of in vitro models with this bead system. And congratulations on a whole lot of work. The amount of mice was probably a lot. I look forward to your future studies and learning what's happening at this endothelial renin cell junction. Maria Luisa Sequeira-Lopez:  Thank you. And we feel honored that you chose us. Ariel Gomez:               Yeah. Well, so I want to thank you for interviewing us. But I want to say that Hirofumi spent three years in the lab and he did a magnificent amount of work. Cindy St. Hilaire:        Wow. Yeah. I would have guessed a lot longer. Ariel Gomez:               Yeah. So he did a lot of work. And I'm very, very proud of what he has accomplished. Maria Luisa Sequeira-Lopez:  Yes. And I would like to add also that we were very lucky to have an expert in integrins, Dr DeSimone, who is the chair of Cell Biology at UVA and when we went and told him that we thought that this could be part of a mechanism sensing receptor, he started collaborating with us and opened his lab for us and trained Hirofumi with some experiments. It was really highly collaborative. Cindy St. Hilaire:        That's it for the highlights from our June 25th and July 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 #DiscoverCircRes. Thank you to our guests, Dr Hirofumi Watanabe, Dr Ariel Gomez, and Dr Maria Luisa Sequeira-Lopez. Cindy St. Hilaire:        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 the 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 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 of the American Heart Association. For more information, please visit ahajournals.org.  

This month on Episode 14 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the July 3 and July 17 issues of Circulation Research. This episode also features an in-depth conversation with Dr. Brenda Ogle and Drs. Molly Kupfer and Wei-Han Lin regarding their study, In Situ Expansion, Differentiation and Electromechanical Coupling of Human Cardiac Muscle in a 3D Bioprinted, Chambered Organoid.     Article highlights:   Wei, et al. Palmitoylation Cycling and Endothelial Maturity   van Ouwerkerk, et al. Functional Variant Elements in Atrial Fibrillation Models   Ibarrola, et al.  Aldosterone in MVP   Sharma, et al. Atherosclerosis Regression Requires Regulatory T Cells   Cynthia 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. Today I'm going to share with you four articles selected from our July issues of Circulation Research, as well as have a discussion with Dr. Brenda Ogle and the first authors, Molly Kupfer and Wei-Han Lin, regarding their study, In Situ Expansion, Differentiation and Electromechanical Coupling of Human Cardiac Muscle in a 3D Bioprinted, Chambered Organoid. So first, the highlights. The first article I want to share with you is titled, "Endothelial Palmitoylation Cycling Coordinates Vessel Remodeling in Peripheral Artery Disease." The first author is Xiaochao Wei, and the corresponding author is Clay Semenkovich from Washington University, St. Louis. Peripheral artery disease, or PAD for short, is a vascular occlusive disease of the lower extremities. It affects more than 2 million individuals globally, and its prevalence is ever increasing as our population ages. While statin therapy can be useful for combating coronary artery disease in peripheral artery disease patients, it does not prevent or reduce PAD patients' rates of lower extremity amputation. So looking to gain insights into the mechanisms underlying PAD, this team focused on the findings that circulating fibronectin and the dietary saturated fatty acid, palmitate, are associated with peripheral artery disease. They found this interesting as lipid modification proteins has been implicated in infections, premature aging, cancer and diabetes. One such protein modification is palmitoylation, which is the formation of a thioester bond between palmitate sand cysteine. Acyl-protein thioesterase 1, or APT1, is a depalmitoylase enzyme, which removes the fatty acid palmitate from protein. Using mouse models with inactivated endothelial APT1, as well as cell systems in arterial samples from humans with end stage peripheral artery disease, they tested whether deficiencies in palmitoylation cycling promotes endothelial instability, which is a hallmark of chronic arterial occlusive diseases. They discovered that as many as 10% of all proteins are palmitoylated. They found deficiency of APT1 in endothelial cells disrupts vascular homeostasis, in part by altering the intracellular trafficking of the small GTPase R-Ras. Impaired R-Ras membrane trafficking was rescued by modifying the palmitoylated R-Ras molecule to promote dissociation from membranes. These observations identify palmitoylation cycling as a potential therapeutic target in the treatment of peripheral vascular disease. The second article I want to highlight is titled, "Identification of Functional Variant Enhancers Associated with Atrial Fibrillation." The first author is Antoinette van Ouwerkerk, and the corresponding authors are Antoine de Vries and Vincent Christoffels, And they're from UMC Amsterdam. As we heard in our podcast last month with our interview with Dr. David McManus, atrial fibrillation, or AFib, is the most common form of arrhythmia, and is a major risk for heart failure, dementia, and stroke, and sudden death. Genome-wide association studies have revealed more than a hundred genetic loci linked to this condition, and many of these loci are found in non-coding regions, which are enriched for transcription factor binding sites and epigenetic modification sites, suggesting that these loci could potentially have gene regulatory roles. To test this idea, they use the method called self-transcribing active regulatory region sequencing, or STARR-seq, which is a method used to identify the sequences that act as transcriptional enhancers in a direct quantitative and genome-wide manner. They use STARR-seq to screen 12 of the strongest AFib linked regions of the genome, which contain more than 1600 individual aphid linked genetic variance, and they did this in cultured rat atrial monocytes. From this screen, they found approximately 400 regulatory elements, of which 24 exhibited variant-specific differences in regulatory activity. For one of these elements, upstream of the gene HCN4, deletion of the orthologous element in mice caused diminished transcriptional activity of the gene. Moreover, these variant-containing mice had brachycardia and sinus node dysfunction, both components of arrhythmia. This proof of principle study confirms that such a regulatory element screen could provide insight into the consequences of variants associated with AFib, or for that matter, many other diseases. The next article I want to share with you is titled, "A New Role for the Aldosterone/Mineralocorticoid Receptor Pathway in the Development of Mitral Valve Prolapse." The first author is Jaime Ibarrola, and the corresponding author is Natalia López-Andrés, and their work was completed at Sanitaria de Navarra in Pamplona, Spain. Mitral valve prolapse is a condition where blood leaks back into the left atrium of the heart, and it is the most common form of heart valve defects. The underlying pathology includes an overabundance of cells in the valve leaflet, so-called valve interstitial cells, or VICs. These activated VICs overproduce extracellular matrix protein, and the combination of increased numbers of VICs and increased amounts of extracellular matrix proteins contributes to the impairment of the structural integrity of the valve leaflet. The increase in VICs is due to excess proliferation, but also transformation of valve endothelial cells, so the cells that line the leaflet, valve endothelial cells, into mesenchymal like VICs. As a driver of endothelial to mesenchymal transition, aldosterone was suspected to play a role. Aldoesterone increased expression of VIC activation markers in cultured valve endothelial cells and increased production of certain extracellular matrix protein components. Spironolactone, an aldosterone inhibitor, prevented these effects, and importantly, prevented valve remodeling in a mouse model of mitral valve prolapse. The team showed that valve tissue from mitral valve prolapse patients taking aldosterone receptor inhibitors displayed less evidence of VIC activation and lower production of disease-regulated extracellular matrix components, than those not taking the drugs. These exciting results suggest aldosterone antagonists, already used for certain patients with heart failure or high blood pressure, may also benefit those with mitral valve prolapse. The last article I want to share before we switch to our interview, is titled, " Regulatory T Cells License Macrophage Pro-Resolving Functions During Atherosclerosis Regression." The first author is Monika Sharma, and the corresponding author is Kathryn Moore, and they're from New York University. Atherosclerosis is a chronic inflammatory condition characterized by the buildup of fatty deposits in the artery walls, and monocytes and macrophages can infiltrate into these fatty deposits and contribute to the formation of plaque. Cholesterol-lowering drugs, like statins, promote the reduction of low-density lipoproteins in the blood, which can help to slow plaque growth, but they do not reverse disease progression. One possibility for changing the course of the disease is to develop therapies that can reduce plaque inflammation, and therefore, progression. With that goal in mind, this team investigated how the immunosuppressive activity of regulatory T cells, or Tregs, may influence the functions of plaque monocytes and macrophages. Using mouse models in which the disease can be reversed through aggressive lipid lowering, they found that depletion of the Treg population caused an increase in the numbers of monocytes and macrophages in the plaques, and resulted in poorer plaque regression. Indeed, these monocytes and macrophages proliferated more, remained in the plaques longer, and were less likely to adopt an anti-inflammatory pro-plaque resolving M2-like phenotype than plaque macrophages in mice with normal Treg numbers. Together, these results highlight the importance of Tregs for promoting plaque regression, and suggest future therapies aimed at boosting these cells, or indeed, M2 macrophages may enable atherosclerosis remission. Okay, so now we're going to switch over to the interview portion of our podcast. I have with me Dr. Brenda Ogle, who is a professor of biomedical engineering, and first authors Molly Kupfer and Wei-Han Lin, and they're from the University of Minnesota. And today we're going to be discussing their manuscript titled, "In Situ Expansion, Differentiation and Electromechanical Coupling of Human Cardiac Muscle in a 3D Bioprinted, Chambered Organoid." So thank you all for joining me today.   Brenda Ogle: Thank you. Molly Kupfer: Thanks for having us. Wei-Han Lin: Thank you. Cynthia St. Hilaire: Great. I'm glad we can all do this remotely and nice and safe for COVID. So Dr. Ogle, you're the PI of the group, but Molly and Wei-Han, what stages of career are you at? Molly Kupfer: I just recently completed my PhD, so this work is sort of the culmination of that. Cynthia St. Hilaire: Oh, congratulations! Molly Kupfer: Yeah. Thank you. Cynthia St. Hilaire: Well done. Circ Research is a great thesis publication. Congratulations. Molly Kupfer: Thank you. Cynthia St. Hilaire: Wei-Han, how about you? Wei-Han Lin: So I'm a BME PhD student at the University of Minnesota. And I got my master degree in chemical engineering, but in Taiwan, and now I'm working with professor Brenda Ogle on cardiac tissue engineering stuff. Cynthia St. Hilaire: Excellent. So this is a beautiful paper. It's stunning. It has all sorts of wonderful parts, biological, biomechanical, great imaging, and essentially you created a 3D bio-ink that can be used to print and make a living pump, kind of a heart in a dish. And it's something that you're calling this human chambered muscle pump, or ChaMP, which I think is a great name. Can you please describe exactly what that is and why did you want to go about trying to make it? Molly Kupfer: Yeah, it might help if I give a little bit of context to this. So since the beginning, one of the central questions that the lab has been exploring is how do the cells of the heart interact with their environment, or the extracellular matrix, as we call it? We know that these interactions that occur at the cellular level are absolutely critical for cardiac function, both at the tissue and the organ level. And based on years of research studying how the extracellular environment modulates cellular function, we have now sought to apply what we've learned in order to engineer functional human cardiac tissues by recapitulating those very critical interactions in vitro. And actually, back in 2017, we published another study in Circulation Research, where we generated these contractile patches of cardiac tissue using a form of light-based 3D printing that allowed us to fabricate scaffolds with really high resolution micron-level features that were distributed in a way that mimics the native extracellular environment. And what we found is that by organizing the extracellular matrix in that way, we enabled the cells to organize themselves in the scaffold and form connections with each other and with the scaffold itself. And this was critical to achieving synchronous electromechanical function of the tissue as a whole. But these were very small millimeter scale tissues, and so for this new study, we sought to create something on a larger scale where you could incorporate some new geometric features such as chambers and the capacity for perfusion. And as you mentioned, using our knowledge of the interactions between cells and the extracellular matrix, we developed this unique bio-ink that could be used as a vehicle to 3D print these centimeter scale chambered tissue structures that are based on the geometry of the human heart. And so the tissues that resulted from this, the human chambered muscle pumps, or hChaMPs, exhibit thick, contiguous muscularization. They demonstrate electrical connectivity and pump function. And notably, this is the first time that this level of function and muscularization has been achieved in an engineered cardiac tissue of this level of geometric complexity. Cynthia St. Hilaire: So can you maybe talk a little bit about what do you mean by an ink, exactly? Is it actually printed? Is this like a printer that I could buy on Amazon? Obviously there's a huge biological component, but what are the actual technical things that you had to develop to make this chamber happen?  Molly Kupfer: Yes. So we did use an extrusion-based 3D printing, which is similar to probably what people normally think about with 3D printing. Traditionally, it's been with plastics. In this case, we're printing with a bio-ink, which is essentially a formulation of proteins and other materials that we encapsulate the cells in, and then after that, we extrude it from a nozzle in a specific formulation or shape in order to create the structure. Cynthia St. Hilaire: So that's interesting. So in this mix, the cells are already in there as opposed to, I guess, some other things that people tend to call scaffolds where you kind of print that and then seed it?  Molly Kupfer: Mm-hmm (affirmative). And in the example of the paper I discussed from 2017, that was an example where we printed a scaffold and put the cells in. But in this case, for such a large and complex structure, we actually mix the cells in prior to printing, and then we create the structure. Cynthia St. Hilaire: Wow. What's the timeframe of that? Like the cells, you got to digest them and mix things up and then print it. The cells, are they happy?  Molly Kupfer: Yeah, that's a good question. So the actual printing process is quite fast, maybe a couple of minutes for this particular scale. We have to prepare, culture, the cells in advance and we're working with human-induced, pluripotent stem cells, so it takes time to grow them up, and then yes, we do detach them and singularize them, and we then mix them with the components. But overall, the actual printing process is relatively quick. Then it's a matter of maintaining the structure and culturing it and doing the differentiation as we did. And that takes weeks to do over time. But the actual process of making it, initially, is quite quick. Brenda Ogle: Challenging thing about this project was the fact that mature cardiac muscle does not transfer well. Meaning when you move it from a dish to an ink and then print it and ask it to start beating again, it doesn't typically happen. And that is because cardiomyocytes don't proliferate well, or make more of each other, and they also don't move well, or migrate. And so the premise on which most of this paper relies is on printing the stem cells first, letting them expand, sort of like they do with development, and then encouraging them to specify into cardiac cell types. Cynthia St. Hilaire: What's the bigger good that can come out of this? Why do we want to be able to do this in vitro, or even ex vivo heart in a dish? Brenda Ogle: The value is pretty tremendous because, suddenly we have a human model system in which we can perfuse volume, so volume can go in and come out, in which the cells experience those volume metric and fluid-induced forces that we haven't been able to study human cells in this way ever before. In the context of human disease, this is the first time we'll be able to look at onset of a particular disease, what was happening with onset, and then progression. And I think that is what is going to transform this field. Cynthia St. Hilaire: So what was the first one like? I'm thinking back to my graduate school and also my postdoc where I was involved in some disease discovery and I have a very vivid memory of the Western blot that proved the mutation that we found. And I literally ran down the hall holding the film. I'm imagining, maybe I'm projecting too much, but what was seeing that first one beat like? Molly Kupfer: You're not projecting. I feel like that well describes my experience. We had some early experiences where we would start to see beating areas under the microscope, but I think the moment, for me, was, I think there was one night I was working in the lab and I had some plates out, I was looking at stuff under the microscope going through just the mundane lab tasks, and I think I sort of saw it at the corner of my eye in the dish, something was moving. And that was the first time. Like I had watched parts of these things beat under a microscope all the time. I spent years looking at cardiomyocytes under a microscope, but that was the first time, for these hChaMPs, where I could actually see it moving just by my eye. Cynthia St. Hilaire: Wow. Molly Kupfer: And that was a really cool moment. Wei-Han Lin: Yeah. I was mostly working on the printing side, so the first time I realized the heart started beating, it's more like a shock to me, because I'm always printing the models or just the mold. But then really seeing those cells, or the whole structure, start to beat, was quite amazing.   Cynthia St. Hilaire: Could you please tell me a bit about the 3D printing aspect of it? Is it like a shell like the outside of a balloon, or does it have an interior structure that helps dictates where the cell go? Can you explain what the printing is? Wei-Han Lin: So the structure we are printing is derived from MRI image stacks on a real human heart. And the image stack was segmented and reduce the size by 10 times, and then we convert the stack into the STL file, which is the standard operating format. And then we modify the model a little bit to make it into two chambers and with two vessels, and two connected chambers with two openings. And this is the heart we are using for the study. Cynthia St. Hilaire: Got it. So it's got kind of the big picture items of the heart. It's got two tubes going in and it's got two chambers and the fluid can flow between all of those aspects in a specific flow pattern. Wei-Han Lin: Exactly. Cynthia St. Hilaire: You said you have to differentiate them in a dish and you're adding different factors to do that. Do the cells like being in that scaffold, or do they want to seep out of that structure or is there something about the bio-ink that they're happy there? Molly Kupfer: You know, I think this bio-ink was, to a certain extent, optimized or designed such that the cells would be able to continue to attach and grow and remodel. So basically, for the most part, these components are biological materials. Some of them are just proteins. Some of them are proteins that have been modified with photo cross-linkable elements, but they still have these moieties that the cells can attach to. And over time we do see some remodeling and some extracellular matrix gets degraded and some gets deposited. Cynthia St. Hilaire: So have you gone to the next steps of something like single cell seq and trying to see what kind of cells you're getting in this? Or even maybe inputting different, the scaffold is getting one differentiation protocol, but are you possibly able to prime IPS cells such that they're maybe halfway to a vascular cell, or halfway to a cardiomyocyte cell, and then put them in the bio-ink? Brenda Ogle: That's a really interesting idea. I'm going to take that one. Cynthia St. Hilaire: Give me an acknowledgment. Brenda Ogle: So we've been thinking about that, the context of if expansion of IPS cells is the best way, for many cell types, how do we get multiple cell types and organize them? And you can imagine even just printing in specific areas, different cell types. Cynthia St. Hilaire: Oh, sure. Brenda Ogle: But the other thing we've thought about is delivering differentiation factor spatially. So almost printing a cell, but then printing that. depot of a factor, in the area that we wanted or in an arrangement that we want, and then releasing it when we want. And it's challenging for stem cell differentiation, because you really need no release, and then basically zero order release for two or three days, and then no release again. Cynthia St. Hilaire: Right. Brenda Ogle: So it's a challenging drug delivery problem, but we've been thinking a lot about it. Now priming the cells beforehand is another interesting approach. Cynthia St. Hilaire: Well, that's wonderful. I just want to congratulate you all again. Brenda Ogle: Thank you so much for having us. Cynthia St. Hilaire: Yeah, thank you so much. Wei-Han Lin: Thank you so much. Cynthia St. Hilaire:  That's it for our highlights from the July issues of Circulation Research. Thank you so much for listening. Please check out the Circulation Research Facebook page and follow us on Twitter and on Instagram with the handle @CircRes and #discovercircres. Thank you to our guests, Dr. Brenda Ogle, Dr. Molly Kupfer and Wei-Han Lin. This podcast is produced by Rebecca McTavish and Ishara Ratnayake, 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. 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.  

Jane Ferguson:                  Hi, everyone. Welcome to episode 35 of Getting Personal: Omics of the Heart, the podcast from Circulation: Genomic and Precision Medicine. I'm Jane Ferguson, an assistant professor of medicine at Vanderbilt University Medical Center, and an associate editor at Circulation: Genomic and Precision Medicine. This episode is first airing in December 2019. Let's see what we published this month.                                                 Our first paper is an “Integrated Multiomics Approach to Identify Genetic Underpinnings of Heart Failure and Its Echocardiographic Precursors: The Framingham Heart Study” from Charlotte Anderson, Ramachandran Vasan and colleagues from Herlev and Gentofte Hospital, Denmark and Boston University.                                                 In this paper, the team investigated the genomics of heart failure, combining GWAS with methylation and gene expression data, to prioritize candidate genes. They analyzed four heart failure related and eight echocardiography related phenotypes in several thousand individuals, and then identified SNPs, methylation markers, and differential gene expression associated with those phenotypes. They then created scores for each gene, based on the rank of statistical significance, aggregated across the different omics analysis.                                                 They examined the top ranked genes for evidence of pathway enrichment, and also looked up top SNPs for PheWAS associations in UK Biobank, and examined tissue specific expression in public data. While their data cannot definitively identify causal genes, they highlight several genes of potential relevance to heart failure pathogenesis, which may be promising candidates for future mechanistic studies.                                                 The next paper is “Genetic Determinants of Lipids and Cardiovascular Disease Outcomes: A Wide-Angled Mendelian Randomization Investigation” and comes from Elias Allara, Stephen Burgess and colleagues, from the University of Cambridge and the INVENT consortium. While it has been established, therapies to lower LDL cholesterol and triglycerides lead to lower risk of coronary artery disease, it remains less clear whether these lipid lowering efforts can also reduce risk for other cardiovascular outcomes. The team set out to address this question using Mendelian randomization. They generated genetic predictors of LDL cholesterol and triglycerides using data from the Global Lipids Genetics Consortium, and then assessed whether genetically predicted increased LDL and triglycerides associated with risk of cardiovascular phenotypes using UK Biobank data. Beyond CAD, they found that higher LDL was associated with abdominal aortic aneurysm and aortic valve stenosis. High triglyceride levels were positively associated with aortic valve stenosis and hypertension, but inversely associated with venous thromboembolism and hemorrhagic stroke.                                                 High LDL cholesterol and triglycerides were also associated with heart failure, which appeared to be mediated by CAD. Their data suggests that LDL lowering may have additional cardiovascular benefits in reducing aortic aneurism and aortic stenosis, while efforts to lower triglycerides may reduce the risk of aortic valve stenosis, but could result in increased thromboembolic risk.                                                 Next up is a paper from Steven Joffe, G.L. Splansky and colleagues, from the University of Pennsylvania and Boston University, on “Preferences for Return of Genetic Results Among Participants in the Jackson Heart Study and Framingham Heart Study”. There has been increasing discussion and concern about how to handle genetic data, and whether genetic results should be returned to participants, and under which circumstances. In this study, the teams that had to assess what participants themselves think. They query participants in the Jackson Heart Study, the Framingham Heart Study and the FHS Omni cohort, presenting them with potential scenarios that varied by five factors including phenotype severity, actionability, reproductive significance and relative of the absolute risk of the phenotype.                                                 Across all scenarios, 88 to 92% of respondents said that they would definitely or probably want to learn their result. In Jackson Heart Study respondents, factors increasing the desire for results included a positive attitude towards genetic testing, lower education, higher subjective numeracy, and younger age. The five pre-identified factors did not affect desire to receive results in Jackson Heart Study. Among Framingham Heart Study respondents, desire for results was associated with higher absolute risk, presentability, reproductive risk and positive attitudes towards genetic testing. Among FHS Omni respondents, desire for results was associated with positive attitudes towards genetic testing and younger age. Overall, these data show that across a variety of studies, there a high level of interest in receiving genetic results and that these are not necessarily linked to the phenotype or clinical significance of the results themselves.                                                 The next paper concerns “Peripheral Blood RNA Levels of QSOX1 and PLBD1 Are New Independent Predictors of Left Ventricular Dysfunction after Acute Myocardial Infarction” and this comes from Martin Vanhaverbeke, Peter Sinnaeve and colleagues, from University Hospital Leuven. They were interested in understanding whether they could identify subsequent left ventricular dysfunction in patients who suffered an acute myocardial infarction. They obtained blood and performed RNA-Seq at multiple time points in 143 individuals, following acute MI, to identify transcripts that were associated with subsequent LV dysfunction. They validated candidate gene transcripts in a validation sample of 449 individuals, confirming that expression of QSOX1 and PLBD1 at admission, were associated with LV dysfunction at follow-up. Adding QSOX1 to a model, consisting of clinical variables and cardiac biomarkers, including NT proBNP, had an incremental predictive value. They took their findings to a pig model and found that whole blood expression of both genes was associated with neutrophil infiltration in these ischemic myocardium. This study suggests that expression of QSOX1 and PLBD1 following MI, may have utility in predicting development of LV dysfunction and may be markers of cardiac inflammation.                                                 The next paper is a research letter from Hanna Hanania, Denver Sallee and Dianna Milewicz, from the University of Texas Health Science Center, and Emory University School of Medicine. Who set out to answer the question, “Do HCN4 Variants Predisposed to Thoracic Aortic Aneurysms and Dissections?” Previous work has suggested that rare variants in HCN4 associated with thoracic aortic disease, including ascending aortic dilation, left ventricular noncompaction cardiomyopathy, and sinus bradycardia. However, the evidence for disease segregation was relatively weak. The team set out to explore these potential associations using exome sequencing data from 521 individuals, from 347 unrelated families with heritable thoracic aortic disease, as well as 355 individuals with early onset sporadic aortic dissections, but no family history of disease. They identified a missense variant G482R, which segregated with disease in four unrelated families, was absent from the nomad database and was predicted to disrupt protein function and have deleterious effects. Their data support the evidence that HCN4 rare variants can cause heritable thoracic aortic disease with left ventricular noncompaction cardiomyopathy and bradycardia.                                                 Our final paper is a white paper from H. Li, X. J. Luo and colleagues, from the National Heart, Lung and Blood Institute at the NIH, and will likely interest anybody who applies for NIH grants, which I'm assuming is most of you listening to this podcast. Their paper on, “Portfolio Analysis of Research Grants in Data Science Funded by the National Heart, Lung, and Blood Institute”, delves into the type of data science research funded by NHLBI between fiscal year 2008 and fiscal year 2017. They identified 630 data science focused grants, funded by NHLBI, using keywords for bioinformatics and computational biology. They then analyzed the distribution of these grants across different disease areas and compared the results to data science grants funded by other NIH institutes or centers. Around 64% of funded grants were for cardiovascular disease with 22% in lung and airway disease, 12% in blood disease and 2% in sleep.                                                 NHLBI's investment in data science research grants averaged about 1% of its overall research grant investment, and this remained constant over the 10-year period. However, this proportion does not include other large scale investment by NHLBI in building data science platforms through other mechanisms. Of relevance to our listeners across all institutes, most funded data science research grants were related to genomics and other omics data. In this paper they include lots of graphs breaking down grant distributions across different categories, so it's worth a look as you plan your next grant application.                                                 That's all for December and the final episode of 2019. Thanks for listening and happy holidays to all who celebrate. I'm excited to be back in 2020, to kick off the next decade of exciting advances in genomic and precision cardiovascular medicine.                                                 This podcast was brought to you by Circulation: Genomic and Precision Medicine, and the American Heart Association Council on Genomic and Precision Medicine. This program is copyright American Heart Association 2019.  

Jane:                                     Hi, everyone. Welcome to Episode 18 of Getting Personal: Omics of the Heart. I'm Jane Ferguson, and this podcast is brought to you by the Circulation: Genomic and Precision Medicine Journal and the American Heart Association Counsel on Genomic and Precision Medicine. It is July 2018, which means that the best possible place to be listening to this episode is at the beach, but failing that I can also recommend listening on planes, during your commute, while exercising or while drinking a nice cup of tea.                                                 So before I get into the papers we published this month, I want to ask for your help. If you're listening to this right now, hi, that means you, we're a year and a half into podcasting and I would love to know what content you like and where we could improve things. We have a poll up on Twitter this week, and I would really appreciate your input. If you're listening to this a little bit later and miss the active voting part of the poll, you can still leave suggestions.                                                 Okay, so what I would like you to do right now is to go to Twitter. You can find us as Circ_Gen and locate the poll. If you don't already follow us on Twitter, go do that now too. We want you to let us know what content we should focus on and what is most useful to you, so go ahead and pick your favorites from the options and also please reply or tweet at us with other thoughts and suggestions.                                                 Options include giving summaries of the recent articles like I'm about to do later this episode, conducting interviews with authors of recently published papers, interviews with people working in cardiovascular genomics, broader topics. For example, to get their insight on career paths and lessons learned along the way.                                                 And something we have not done yet on the podcast but are considering, would be to record podcasts that focus on particular topics in genomics and precision medicine. These could give some background on an emerging field or technology and we could talk to experts who are leading particular innovations in the field. So, if that sounds good to you, let me know! If you're not on Twitter, I don't want to exclude you, so you can email me at jane.f.ferguson@vanderbilt.edu and give me your thoughts that way. I'm looking forward to hearing from you.                                                 Okay, so on to the July 2018 issue of Circ.: Genomic and Precision Medicine. First up is a PhWAS from Abrahim Rao, Eric Ingelsson, and colleagues from Stanford. The discovery of the PCSK9 gene as a regulator of cholesterol levels has led to a new avenue of LDL lowering therapies through PCSK9 inhibition. However, some studies suggest that long term use of PCSK9 inhibitors could have adverse consequences. Because of the long follow-up time required, it will take many more years to address this question through clinical studies. However, genetic approaches offer a fast and convenient alternative to address the issue.                                                 In this paper, entitled: "Large Scale  Phenome-Wide Association Study of PCSK9 Variants Demonstrates Protection Against Ischemic Stroke," the authors use genetic and phenotype data from over 300,000 individuals in the UK BioBank to address whether genetic loss of function variants in PCSK9 are associated with phenotypes including coronary heart disease, stroke, type II diabetes, cataracts, heart failure, atrial fibrillation, epilepsy, and cognitive function.                                                 The missense variant RS11591147 was associated with protection against coronary heart disease and ischemic stroke. This SNP also associated with type II diabetes after adjustment for lipid medication status. Overall, this study recapitulated the associations between PCSK9 and coronary disease, and revealed an association with stroke.                                                 Previous studies suggested use of LDL lowering therapies may increase risk of cataracts, epilepsy, and cognitive dysfunction, but there was no evidence of association in this study. Overall, this study provides some reassurance that the primary effect of PCSK9 is on lipids and lipid related diseases, and that any effects on other phenotypes appear to be modest at best. While a PhWAS can't recapitulate a clinical trial, what this study indicates is that PCSK9 inhibition is an effective strategy for CVD prevention, which may confer protection against ischemic stroke and does not appear to convey increased risk for cognitive side effects.                                                 Next up we have a manuscript form Jason Cowan, Ray Hershberger, and colleagues from Ohio State University College of Medicine. Their paper, "Multigenic Disease and Bilineal Inheritance in Dilated Cardiomyopathy Is Illustrated in Non-segregating LMNA Pedigrees," explored pedigrees of apparent LMNA related cardiomyopathy identifying family members who manifested disease, despite not carrying the purported causal LMNA variant. Of 19 pedigrees studies, six of them had family members with dilated cardiomyopathy who did not carry the family's LMNA mutation. In five of those six pedigrees, the authors identified at least one additional rare variant in a known DCM gene that was a plausible candidate for disease causation.                                                 Presence of additional variants was associated with more severe disease phenotype in those individuals. Overall, what this study tells us is that in DCM, there is evidence for multi-gene causality and bilineal inheritance may be more common than previously suspected. Future larger studies should consider multi-genic causes and will be required to fully understand the genetic architecture of DCM.                                                 Yukiko Nakano, Yasuki Kihara, and colleagues from Hiroshima University published a manuscript detailing how HCN4 gene polymorphisms are associated with tachycardia inducted cardiomyopathy in patients with atrial fibrillation. Tachycardia induced cardiomyopathy is common in subjects with atrial fibrillation, but the pathophysiology is poorly understood. Recent studies have implicated the cardiac hyperpolarization activated cyclic nucleotide gated channel gene, or HCN4, in atrial fibrillation and ventricular function.                                                 In this paper, the authors enrolled almost 3,000 Japanese subjects with atrial fibrillation, both with and without tachycardia-induced cardiomyopathy, as well as non-AF controls. They compared frequency of variants in HCN4 in AF subjects with or without tachycardia-induced cardiomyopathy, and found a SNP, RS7164883, that may be a novel marker of tachycardia-induced cardiomyopathy in atrial fibrillation.                                                 Xinyu Yang, Fuli Yu, and coauthors from Tianjin University were interested in finding causal genes for intracranial aneurysms, and report their results in a manuscript entitled, "Rho Guanine Nucleotide Exchange Factor ARHGEF17 Is a Risk Gene for Intracranial Aneurysms." They sequenced the genomes of 20 Chinese intracranial aneurysm patients to search for potentially deleterious, rare, and low frequency variants. They found a coding variant in the ARHGEF17 gene which was associated with associated with increased risk in the discovery sample, and which they replicated in a sample of Japanese IA and in a larger Chinese sample.                                                 They expanded this to other published studies, including individuals of European-American and French-Canadian origin and found a significantly increased mutation burden in ARHGEF17 in IA patients across all samples. They were interested in further functional characterization of this gene and found that Zebra fish ARHGEF17 was highly expressed in blood vessels in the brain. They used morpholinos to knock down ARHGEF17 in Zebra fish, and found that ARHGEF17 deficient Zebra fish developed endothelial lesions on cerebral blood vessels, and showed evidence of bleeding consistent with defects in the vessel. This study implicates ARHGEF17 as a cerebro-vascular disease gene which may impact disease risk through effects on endothelial function and blood vessel stability.                                                 Sumeet Khetarpal, Paul Babb, Dan Rader, Ben Voight, and colleagues from the University of Pennsylvania used targeted resequencing to look at determinants of extreme HDL cholesterol in their aptly titled manuscript, "Multiplexed Targeted Resequencing Identifies Coding and Regulatory Variation Underlying Phenotypic Extremes of HDL Cholesterol in Humans." Stay tuned because we're gonna hear more about this paper from the first author Dr. Sumeet Khetarpal later this episode.                                                 Rounding out this issue we have a Perspective article from Chris Haggerty, Cynthia James, and coauthors from Geisinger and Johns Hopkins Medical Center entitled, "Managing Secondary Genomic Findings Associated With Arrhythmogenic Right Ventricular Cardiomyopathy: Case Studies and Proposal for Clinical Surveillance." In this paper the authors discuss the challenges for returning findings from clinical sequencing for arrhythmogenic right ventricular cardiomyopathy, presenting case studies exemplifying these challenges. They also propose a management approach for returning clinical genomic findings, and discuss new innovations in the light of precision medicine.                                                 We also published a review article by Pradeep Natarajan, Siddhartha Jaiswal, and Sekar Kathiresan from MGH on "Clonal Hematopoiesis Somatic Mutations in Blood Cells and Atherosclerosis", which discusses recent advances in our knowledge on the role of somatic mutations in cardiovascular disease risk.                                                 Finally, we have an update on some pharmacogenomics research into CYP2C19 Genotype-Guided Antiplatelet Therapy by Craig Lee and colleagues which we published a few months ago. Dr. Lee was also featured on Podcast episode 15 in April of this year.                                                 Jernice Aw and colleagues from Khoo Teck Puat Hospital, Singapore shared from complimentary data from their sample of 247 Asian subjects which found the risk for major adverse cardiovascular events was over 30-fold greater for poor metabolizers, as defined by CYP2C19 genotype on clopidogrel, as compared to those with no loss of function allele.                                                 You can read that letter and the response from Dr. Lee and colleagues online now. And, as usual, all of the original research articles come with an editorial to help give some more background and perspective to each paper. Go to circgenetics.ahajournals.org to find all the papers and to access video summaries and more.                                                 Our interview is with Dr. Sumeet Khetarpal who recently completed his MD-PhD training at the University of Pennsylvania, and is currently a resident in Internal Medicine at Massachusets General Hospital. Sumeet kindly took some time out from his busy residency schedule to talk to me about his recently published paper, and to explain how molecular inversion probe target capture actually works.                                                 So I am here with Dr. Sumeet Khetarpal who is co-first author on a manuscript entitled, "Multiplexed Targeted Resequencing Identifies Coding and Regulatory Variation Underlying Phenotypic Extremes of High-Density Lipoprotein Cholesterol in Humans."                                                 Welcome Sumeet, thanks for taking the time to talk to me. Dr. Khetarpal:                    Thank you so much Dr. Ferguson, it's really a pleasure to talk to you today. Jane:                                     Before we get started, maybe you could give a brief introduction on yourself and then how you started working on this paper. Dr. Khetarpal:                    Sure, so this work actually was a collaboration that came out at the University of Pennsylvania that I was involved with through my PhD thesis lab, my mentor was Dan Rader, and also a lab that is a somewhat newer lab at Penn, Benjamin Voight's lab which is a strong sort of computational genomic lab.                                                 This work actually highlights the fun of collaborating within your institution. We had, for some time, been interested in developing a way to sequence candidate genes. Both known genes and also new genes that have come out of genome-wide association studies that underlie the extremes of HDL cholesterol, namely very high cholesterol versus low HDL cholesterol. We've been looking for a cost-effective and scalable way to do this.                                                 Independently, Ben, who is very interested in capturing the non-coding genome, was interested in developing a method to better understand the non-coding variation, both common and rare variation that may be present at all of these new loci that have come out for complex traits such as HDL.                                                 We, at some Penn event several years ago, were talking about our common interest and Ben had actually identified this work that had come out of J. Shendure's lab at the University of Washington. A paper by the first author, Brian O'Rouke, in Science in 2012 in which they had developed an approach that involved molecular inversion probes, or MIPs, to capture regions of the genome related to target the gene that they were interested in studying for autism-spectrum disorders.                                                 They had applied this largely to coding regions of, I think, almost 50 genes and almost 2,500 patients with the feedback to do deep, targeted sequencing. So our thought was, well, we could try to apply this approach and adapt it to capture non-coding regions, and also see if we can expand the utility of this approach to study the phenotypic extremes of a complex trait such as HDL cholesterol. Jane:                                     Yeah, that's really cool. I love how you saw this method in a totally different application and then realized that there was expertise at Penn that you could bring together to apply this in a different way.                                                 I'd love to hear more about this MIP, the molecular inversion probe. How does it work? How difficult is it to actually do? Is it very different from normal library preparation for sequencing or is it something that's actually relatively easy to apply? Dr. Khetarpal:                    These MIP probes are oligonucleotide probes that capture your region of interest by flanking them and capturing by gap filling. There's a method to capture parts of the genome in a library-free way. They do ultimately involve barcoding the way traditional library-based target capture does and then deep sequencing.                                                 But the most impressive feature about them is just that they're very scalable. I think in the original paper by O'Rouke and colleagues they were able to sequence their set of genes and their set of samples at about a sample preparation cost of $1 per sample, and we were actually able to do about the same for our study.                                                 The main utility of the approach is just the economic scalability, and the ability to customize your panel to capture several regions of the genome that are adjacent to each other. Jane:                                     Right, so how many genes or regions can you multiplex at the same time? Is it just one prep, like you just design all of your oligos, you put them all together in one reaction, or are you doing separate reactions for each region? Dr. Khetarpal:                    We're actually doing all of our oligos together. In our case, I think it ended up being around the order of almost 600 oligos together to capture our ultimately 50kB of genomic territory that we wanted to capture. Really, our study was kind of a pilot experiment where we picked a few genes or regions of high interest to us, both known genes that effect HDL and also those that have been implicated in genome-wide association studies that were of high interest to our labs.                                                 I think that this approach could actually be expanded to capture much more genomic territory in a single capture reaction. We sort of touched the surface probably of what we could do. Jane:                                     Wow, that's cool! And then for sequencing it, I guess it's really just a function of how many samples you wanna multiplex and how much you want to sequence from each region. So I suppose the way you did it, you had about 50kB and then you had over 1,500 participants and you were able to do those on a single HiSeq run, right? Dr. Khetarpal:                    Right. Jane:                                     So I suppose if you'd done more genetic regions, you would've had fewer people and vice versa so you can balance that out depending on if you're having more samples or more genomic regions to sequence. Dr. Khetarpal:                    Exactly, in certain ways the design of our experiment we had a limited sample size that did afford us some luxury in terms of knowing that we would have deep coverage of the region that we were targeting. I think that's always a critical question in sort of targeted or just sequencing in general. The balance between the number of regions that you want to sequence and the number of samples you want to sequence is going to dictate what your sequencing depth with be. Jane:                                     Right, okay so I guess if we go on to what you actually found, how'd you pick this? You picked seven regions which encompasses eight candidate genes for HDL, so how did you select those? Dr. Khetarpal:                    The population that we were studying, the samples we were looking to sequence were largely individuals which fall into two bins if you will. One was extremely high HDL cholesterol which we're defining as the greater than the 95th percentile, but really there was a range within that population that spanned individuals with probably greater than the 99th percentile of HDL.                                                 We were hoping as a proof of principle effort to identify variation in genes that were known causes of high HDL cholesterol in prior studies of Mendelian genes for HDL. So genes such as LIP gene which encodes endothelial lipase or CETP or SCARB1, these 3 genes are, at this point, well-known genes that loss of function mutations are associated with extremely high HDL. We thought that capturing some of those genes would potentially both provide a level of validation for the approach, hypothesizing that individuals with high HDL would be enriched with these genes, but also may allow us to find new variants in these genes or also non-coding variants which has not previously been studied before.                                                 Some of the genes came out from that line of thinking, then some of the other genes happened to be genes that in the Rader laboratory we had a vested interest in understanding the genetic variation that might link the genes to HDL, which may not have necessarily come out before.                                                 For example, the gene GALNT2 is one of the first g-loss implicated novel genes for HDL, novel as in the earliest g-loss study for plasma lipids had identified that gene as associated with HDL but it never had come out before as being so. Our laboratory was very interested in better understanding the genetic relationship between genes such as GALNT2 and several of the others such as CCDC92 and ZNF664 with HDL.                                                 It ended up being a hodge-podge or a sampling of genes that had at some level been implicated with HDL, but really it's just a proof of principle that this method could work for both identifying variation in known genes and also less studied ones. Jane:                                     You validated the MIP genotyping by exome genotyping, and then saw concordance of over 90%, is that lower than you were expecting? Was it about what you were expecting based on these two different methods of genotyping? Dr. Khetarpal:                    Yes, I think we were expecting somewhere on the order of 90 plus percent. It's hard to know why we just hit that, we likely would've benefited from being able to genotype all of the individuals by the exome chip that we had sequenced as well, where we were able to validate in about two-thirds of those individuals.                                                 It's hard to know exactly what the cause of the about 10% discordance rate might be, whether it's just in certain samples the genotyping quality was perhaps on the border of being valid or the sequencing quality. Jane:                                     Right, I'm wondering sort of with the MIP, what's the gold standard? Is the XM chip genotyping still the gold standard and the MIP maybe is more error-prone, or perhaps the other way around? Or is it you can't tell at this point which is the true genotype and which is an error potentially for those discordant ones? Dr. Khetarpal:                    Certainly whenever there's a new sequencing methodology that is proposed I think it's critical to have some sort of validation. We happened to cover regions that would span the genome enough that we had XM chip genotyping in a large subset, that that might be the best approach. But if you had a limited number of regions or variance that you were interested in one could imagine also doing Sanger sequencing as the tried and tested validation approach. Of course it becomes not so scalable at a certain point.                                                 Certainly we would say that the MIPs, while the method has been developed and expanded by the Shendure lab, our hope is that through our studies maybe it will be applied further. It's still very much a new approach and so validation is key. Jane:                                     Very important. What do you think was the most exciting finding that came out of this, after you analyzed the data, what were you most excited about seeing? Dr. Khetarpal:                    The critical finding for us, which I think implies the utility of the approach, was just the validation of four of the loci that we had studied. Validation in our cohort of known genome-wide significant associations for HDL that had been published previously in almost 200,000 individuals in terms of sample size, in our experiment involving just about 1,500 people we were able to find consistent associations of those same variants that segregated with low versus high HDL. Directionally consistent with the large genome-wide association studies.                                                 I think the value of this finding is really just to emphasize the utility of the case control design in these phenotypic extremes, in addition to the overarching goal of our study, which was in a way that perhaps provides the most validation of the approach in terms of concordance with prior known studies. Jane:                                     So if somebody was listening to this and was trying to decide should they use MIP for a study they have in mind, should they use another technique? Based on your experience, what would you recommend? Dr. Khetarpal:                    I think in our current stage it's a very exciting time because we're just seeing whole genome sequencing really take off and being used at scale to ask critical questions about non-coding variation as it relates to both disease and complex traits. I don't think we're quite there yet with being able to apply that approach in a cost effective manner. The ability to annotate and analyze that data is still at it's infancy. The utility of the MIPs is that it provides a very cheap alternative.                                                 I can say from my experiences actually doing the capture and preparation from sample to sequencer stage that it's a very easy to use methodology that is very fast and cheap. That if one is really interested in a handful, or more than a handful, of candidate genes and their non-coding regions as it relates to a trait or disease of interest, it may not be the era for going full on with whole genome sequencing, especially at the current cost. That's where I think the MIPs really come in to be very useful. Jane:                                     It sounds great, is there anything else that you'd like to mention? Dr. Khetarpal:                    Just to say that we recognize it's a relatively small study as our pioneer approach with this method but that the Rader lab and Voight labs are actively pursuing larger applications of this to study, not only HDL, but other complex traits, such as diabetes, in much larger populations. I can't overemphasize how easy of a method it is to apply, but also that I think a bigger take home of this study for me as a very recent graduate student working in a very collaborative institution the ability of two laboratories to come together with different sets of expertise to try to tackle a problem that I think goes beyond the individual science. For any human geneticist how to find the variation you're interested in and not break the bank is kind of at the core of what we do, and so I think it was very fun to be part of this collaboration and our hope is that the outcome of it is a method that can be useful for many people, both in our field and beyond. Jane:                                     I think it's great and I'm hoping this will inspire a lot of other people to try this method and see if it can work for them. So, congratulations on the study, it's really nice work. Dr. Khetarpal:    Thank you so much!                                                                                                                                       Jane:                                     That's all I have for you for July, thanks for listening. Send me your thoughts on the podcast via Twitter or email, or leave us a review in Itunes. I look forward to talking to you next month.  

Dr. Paul Wang:           Welcome to the monthly podcast On the Beat for Circulation Arrhythmia and Electrophysiology. I'm Dr Paul Wang, editor-in-chief, with some of the key highlights for this month's issue. We'll also hear from Dr. Suraj Kapa reporting on new research from the latest journal articles in the field.                                                 In our first article, Barry Maron associates report on the long term clinical course of hypertrophic cardiomyopathy patients following ICD therapy for ventricular arrhythmias. They studied a cohort of 486 high-risk hypertrophic cardiomyopathy patients with ICDs from eight international centers. Of these 486 patients over 6.4 years, 94 patients or 19% experienced appropriate ICD interventions, terminating VT or VF. Of the 94 patients receiving appropriate ICD therapy, 87 were asymptomatic or only mildly symptomatic at the time of appropriate ICD interventions. Of these 87 patients, 74 or 85% remained in classes one or two without significant change in clinical status of the subsequent 5.9 years up to 22 years. Among the 94 patients, there was one sudden death in three patients who died from non arrhythmic hypertrophic cardiomyopathy related processes. Post ICD intervention, freedom from hypertrophic cardiomyopathy, mortality was 100% at one year, 97% at five years, and 92% at 10 years, distinctly lower than the risk of ischemic or non ischemic cardiomyopathy in ICD trials.                                                 Hypertrophic cardiomyopathy patients with ICDs interventions reported the heightened anxiety and expectation of future shocks. However, they did not affect general psychological well-being or quality of life. The authors concluded that in hypertrophic cardiomyopathy, unlike ischemic heart disease, prevention of sudden death with ICD therapies unassociated with a significant increase in cardiovascular morbidity and mortality, nor transformation into heart failure deterioration, ICD therapy does not substantially impair overall psychological and physical well-being. In our next article, Abdulla Damluji and associates examined the cost of hospitalizations for cardiac arrest using the US nationwide inpatient sample from 2003 to 2012. Using the log transformation of inflation adjusted costs the authors examined 1,387,396 patients who were hospitalized after cardiac arrest. They had a mean age of 66 years. Inpatient procedures included coronary angiography in 15%, PCI in 7%, intra-aortic balloon pump in 4.4%, therapeutic hypothermia in 1.1%, and mechanical circulatory support in 0.1% of patients.                                                 Notably the rates of therapeutic hypothermia increased from 0 in 2003 to 2.7 in 2012, p less than 0.001. Both hospital charges inflation adjusted costs linear increased over time. In a multi-variant analysis predictors of inflation adjusted costs included large hospitals size, urban teaching hospital, and length of stay. Among co-morbidities, atrial fibrillation or fluid and electrolytes imbalance were the most common associated with cost. The authors found that during the period between 2003 and 2012 post cardiac arrest, hospitalizations had a steady rise and associated healthcare costs likely related to increase length of stay, medical procedures and systems of care.                                                 In our next paper, Peter Huntjens and associates examined intrinsic interventricular dyssynchrony as a predictor of human dynamic response to cardiac resynchronization. The authors use a cardiovascular computational model CircAdapt to characterize the isolated effect of intrinsic interventricular or intraventricular activation on resynchronization therapy response that is the change in LV dP/dt max. The simulated change in LV dP to dt max had a range of 1.3 to 26.5% increased considerably with increasing inter ventricular dyssynchrony. In contrast, the isolated effect of intra ventricular dyssynchrony was limited with the change in the LV dP/dt max range and the left ventricle from 12.3 to 18.3% in the right ventricle from 14 to 15.7%.                                                 Secondly, electrocardiographic imaging derived activation characteristics of 51 CRT candidates were used to create individual models of ventricular activation in CircAdapt. The model predicted change in LV dP/dt max was close to the actual value in left bundle branch block patients with 2.7% difference between measured and simulated when only intrinsic interventricular dyssynchrony was personalized. Among non left bundle branch block patients a change in LV dP/dt max was systematically over predicted by CircAdapt with a 9.2% difference between measured and simulated. Adding intra ventricular activation to the model did not improve the accuracy of response prediction. The authors found that computer revealed intrinsic interventricular dyssynchrony is the dominant component of the electrical substrate driving the response to CRT.                                                 In the next paper Kenji Kuroki and associates examined the use of voltage limit adjustment of substrate mapping and fast Fourier transform analysis of local ventricular bipolar electrograms during sinus rhythm to predict VT isthmuses. They performed these studies and nine post infarction patients who underwent catheter ablation for total of 13 monomorphic ventricular tachycardias. Relatively higher voltage areas on electroanatomical map or defined as high voltage channels, which were further classified as full or partial if the entire or more than 30% of the high voltage channel was detectable. 12 full high voltage channels were identified in seven of nine patients. Relatively higher fast Fourier transform areas were defined as high frequency channels, which were located on seven of 12 full high voltage channels. Five VT isthmuses or 71% were included in the seven full high voltage channels positive in high frequency channel positive sites.                                                 While no VT isthmuses were found in five full high voltage channel positive but high frequency channel negative sites, high frequency channels were identical to 9 out of 16 partial high voltage channels. Eight VT isthmuses or 89% were included in nine partial high voltage channel positive in high frequency channel positive sites, whereas no VTs isthmuses were found in the seven partial high voltage channel positive and high frequency channel negative sites.                                                 All high voltage channel positive in high-frequency channel positive sites predicted VT isthmus with a sensitivity of 100% and specificity of 80%. The authors concluded that based on this small series that combined use of voltage, limited adjustment and fast Fourier transform analysis may be useful method to detect VT isthmuses.                                                 In the next study, John Whitaker and associates examined the use of lesion index, LSI index, a proprietary algorithm combining contact force, radio-frequency application duration, and RF current. Cardiac CT was used to assess atrial tissue thickness. Ablation lines two to three per animal were created in the right atrium in seven mini pigs with point lesions using 25 watts of energy. Two weeks after the ablation, serial sections of targeted atrial tissue or examine histologically to identify gaps and transmural ablation. LSI guidelines had a lower incidence of histological gaps. Four gaps in the 69 catheter moved or 5.8% compared to ablation using LSI plus two millimeter lines in which there is seven gaps in 33 catheter moves or 21.2% and using LSI plus four millimeter lines in which there are 15 gaps in 23 moves or 65.2% p less than 0.0. The change in LSI was calculated retrospectively is a distance between two adjacent lesions above the mean LSI of the two lesions. Changing LSI values of 1.5 or less were associated with no gaps in transmural ablation.                                                 The authors concluded that in this mod of chronic atrial ablation delivery of uninterrupted transmural linear lesions may be facilitated using LSI to guide catheter movement. When change in LSI between adjacent legions is 1.5 millimeters or lower, no gaps in atrial linear lesions should be expected.                                                 In our next paper, Matthew Bennett and associate examined whether their response to antitachycardia pacing in patients with ICD could further discriminate ventricular from super ventricular arrhythmias in patients receiving ATP in the RAFT trial. The RAFT trial randomized 1,798 patients with New York Heart Association class two or three heart failure, left ventricular ejection fraction less than or equal to 30%, in QRS duration 120 millisecond or greater, to an ICD plus or a minus cardiac resynchronization. Beginning with 10,916 ATP attempts for 8,150 tachycardia episodes in 924 patients, the author's excluded tachycardias where ATP terminated the episode or were the specific etiology tachycardia was uncertain. In this study, they analyzed 3,676 ATP attempts delivered to 2,046 tachycardia episodes in 541 patients. The authors found that a shorter difference between the post pacing interval is PPI minus TCL, was more likely to be associated with VT than SVT, mean of 138.1 milliseconds for VT and 277.4 milliseconds for SVT p, less than 0.001. A PPI minus TCL value of less than or equal to 300 milliseconds had a sensitivity in 97.4% and a specificity of 28.3% for VT.                                                 The authors concluded that specifically the PPI minus TCL following antitachycardia pacing may help distinguish ventricular from supraventricular arrhythmias.                                                 In the next study, Shailee Shah and Amr Barakat and associates examined the outcomes after repeat AF ablation. The authors examined 137 patients out of a total of 10,378 patients undergoing Afib ablation who had had initial long-term success defined from recurrent arrhythmias for greater than 36 months off anti-arrhythmic drugs in subsequent underwent repeat ablation for recurrent atrial fibrillation. The median arrhythmia free period that define long-term success was 52 months. In redo-ablations reconnection of at least one of the pulmonary veins was found in 111 or 81% of patients. Additional non PV ablations were performed in 127 or 92.7% of patients. After a mean follow-up of 17 months, 103 patients or 75% were arrhythmia-free, 79 off anti-arrhythmics, and 24 on arrhythmics. The authors found that repeat ablations with re-isolation to the point of veins and modifying the atrial substrate had a good success rate.                                                 In the next article Qiongling Wang and associates hypothesized that genetic inhibition of CaMKII oxidation in a mouse model of Duchenne muscular dystrophy can alleviate abnormal calcium homeostasis thus preventing ventricular arrhythmias. The authors tested whether the selective loss of oxidation of the CaMKII effects ventricular arrhythmias in the mouse model of Duchenne muscular dystrophy. Genetic inhibition of ox-CaM kinase II by knocking replacement of the regulatory domain methionines with valines, which we'll call MMVV, prevented ventricular tachycardia in the mdx mice. Confocal calcium imaging of ventricular myocytes, isolated from the mdx MMVV mice revealed normalization of intra-calcium release events compared to myocytes from the mdx mice. Abnormal action potentials as assessed by optical mapping mdx were also alleviated by genetic inhibition of ox-CaMK II. Knockout of the NADPH oxidase regulatory sub-unit P 47 Fox normalized elevated ox-CaMK II, repaired intracellular calcium hemostasis and rescued inducible ventricular arrhythmias in the mdx mice. The authors concluded that inhibition of ROS or ox-CaMK II protects against pro-arrhythmic intracellular calcium handling, preventing ventricular arrhythmias in a mouse model of Duchenne muscular dystrophy.                                                 In the next article, Kyohei Marume and Teruo Noguchi and associates examined whether the combination of QRS duration of 120 milliseconds or greater in late gadolinium enhancement is a precise prognostic indicator for the primary endpoint of all cause death and a composite of sudden cardiac death or aborted sudden cardiac death in 531 patients with dilated cardiomyopathy. They also analyzed the association between the combination of late gadolinium enhancement and increased QRS duration in these end points among patients with a class one indication for implantable defibrillator. The author's divided study patients in three groups according to late gadolinium enhancement in QRS duration. Two negative indices that is late gadolinium enhancement negative and narrow QRS, one positive index with either late gadolinium enhancement positive or wide QRS or two positive indices late gadolinium positive and wide QRS and followed them for 3.8 years. Multiple variable Cox regression analysis identified to positive indices as significant predictors of all cause death. A hazard ratio of 4.29 p equals 0.026. Among the 317 patients with a class one indication for ICD, the five year event rate of sudden cardiac death or aborted sudden cardiac death was lowest in the two negative indices groups, 1.4%. With propensity score matching cohorts the two negative indices group had a significant lower event rate of sudden cardiac death or aborted sudden cardiac death than to two other groups hazard ratio 0.2, p equals 0.046.                                                 The authors concluded that the combination of late gadolinium enhancement in wide QRS provides additional prognostic stratification compared to late gadolinium enhancement status alone.                                                 In the next study, Matthew Sulkin and associates examined whether a novel local impedance measurement on an ablation catheter identifies catheter tissue coupling and is predictive of lesion formation. The author's first studied explanted hearts, 10 swine, and then in vivo 10 swine, using an investigational electro anatomical mapping system that measures impedance from an ablation catheter with mini electrodes incorporated into the distal electrode. Rhythmia and Intellanav, Boston Scientific.                                                 Explanted tissue was placed in a warmed 37 degree celsius saline bath mounted on a scale, and the local impedance was measured 15 millimeters away from the tissue to five millimeters of catheter tissue compression at multiple catheter angles. Lesions were created for 31 and 50 watts from 5 to 45 seconds for an N of 70. During in vivo valuation of the local impedance measurements of the myocardium 90 and blood pool 30 were guided by intracardiac ultrasound while operators were blinded to the local impedance data. Lesions were created with 31 and 50 watts for 45 seconds in the ventricle with an n of 72. The local impedance of myocardium, which was 119.7 ohms, was significantly greater than in blood pool 67.6 ohms the p of less than 0.01. Models that incorporate local impedance drop to predict lesion size had better performance that models incorporate force time integral r squared of 0.75 versus r squared of 0.54 and generator impedance drop r squared of 0.2 versus r squared of 0.58. Steam pops displayed a significantly higher starting local impedance and a larger change in local impedance compared to successful RF applications, p less than 0.01.                                                 The authors concluded that local impedance recorded for miniature electrodes provides a valuable measure of catheter tissue coupling and the change in local impedance is predictive of lesion formation during RF ablation.                                                 In the next paper, Boaz Avitall and associates found that the rising impedance recorded from a ring electrode placed two millimeters from the cryoballoon signifies ice formation covering the balloon surface and indicates ice expansion. The authors studied 12 canines in a total of 57 pulmonary veins, which were targeted for isolation. Two cryoapplications were delivered per vein with a minimum of 90 and a maximum 180 second duration. Cryoapplications was terminated upon reaching a 500 ohm change from baseline. Animals recovered 38 plus or minus six days post procedure, and the veins were assessed electrically for isolation. Heart tissue was histological examined. Extra cardiac structures were examined for damage. Pulmonary vein isolation was achieved in 100% of veins if the impedance reached 500 ohms in 90 to 180 seconds. When the final impedance was between 200 and 500 ohms within 180 seconds of freeze time, pulmonary vein isolation was achieved in 86.8%. For impedance of less than 200 ohms pulmonary vein isolation was achieved in 14%. No extra cardiac damage was recorded. The authors found that impedance rise of 500 ohms at less than 90 seconds with a freeze time of 90 seconds resulted in 100% pulmonary vein isolation.                                                 In our final papers Sally-Ann Clur and associates examined left ventricular isovolumetric relaxation time as the potential diagnostic marker for fetal Long QT Syndrome. Left ventricular isovolumetric contraction time, ejection time, left ventricular isovolumetric relaxation time, cycle length, and fetal heart rate were measured using pulse doppler wave forms in fetuses. Time intervals were expressed as percentage of cycle length, and the left ventricular myocardium performance index was calculated. Single measurements were stratified and compared between Long QT Syndrome fetuses and controls. Receiver operator curves were reformed for fetal heart rate in normalized left ventricular isovolumetric relaxation time. A linear mixed effect model including multiple measurements was used to analyze fetal heart rate, the left ventricular iso volume metric relaxation time, and the left ventricular myocardial performance index. There were 33 Long QT fetuses in 469 controls. In Long QT fetuses the left ventricular isovolumetric relaxation time was prolonged in all groups, p less than 0.001, as was the left ventricular isovolumetric relaxation time.                                                 The best cutoff to diagnose Long QT syndrome was the normalized left ventricular isovolumetric relaxation time greater than equal to 11.3 at less than or equal to 20 weeks, giving a sensitivity in 92% and a specificity of 70%. Simultaneous analysis of the normalized left ventricular isovolumetric relaxation time and fetal heart rate improved the sensitivity and specificity of Long QT Syndrome, AUC of 0.96. The normalized left ventricular isovolumetric relaxation time, the left ventricular myocardial performance index, and fetal heart rate trends differed significantly between Long QT Syndrome fetuses and controls throughout gestation.                                                 The authors concluded that left ventricular volumetric relaxation time is Prolonged QT fetuses. Findings of a prolonged normalize left ventricular isovolumetric relaxation time, and sinus bradycardia can improve the prenatal detection of fetal Long QT Syndrome.                                                 That's it for this month, but keep listening. Suraj Kapa will be surveying all journals for the latest topics of interest in our field. Remember to download the podcasts On the Beat. Take it away Suraj. Suraj Kapa:                          Thank you, Paul and welcome back to On the Beat were we will be summarizing hard-hitting articles across the entire electrophysiologic literature. Today we'll be starting within the realm of atrial fibrillation where we're review an article within the realm of anticoagulation and stroke prevention. Quon et al. published in last month's issue of JACC cardiac electrophysiology on anticoagulant use and risk of ischemic stroke and bleeding in patients with secondary atrial fibrillation. It is well known that use of anticoagulation in atrial fibrillation can reduce overall thromboembolic outcomes. However, its role in secondary atrial fibrillation is unclear. Thus, the authors sought to evaluate the effects anticoagulant use on stroke and bleeding risk. Amongst those where atrial fibrillation occurred in the setting of acute coronary syndrome, pulmonary disease, or sepsis. Amongst around 2300 patients evaluated retrospectively there was no evidence of a lower incidence of ischemic stroke among those treated with anticoagulants compared to those who are not.                                                 However, anticoagulation was associated with a higher risk of bleeding in those with new onset AF associated with acute pulmonary disease. The authors suggest as a result that there is unclear overall benefit for long-term anticoagulation in patients with presumed secondary atrial fibrillation. The difficulty in assessing this is how to define secondary atrial fibrillation. However, in many studies patients who developed in the setting of acute illness still had a high risk of developing quote unquote clinically significant AF in long-term follow-up. However, this was not necessarily absolute as many patients not necessarily develop AF that could be considered clinically significant. Thus, the clinical question that arises is: how long should we treat a patient with anticoagulation when they have presumed secondary atrial fibrillation. These data seem to suggest that there may be no net overall benefits. In other words, all-comers with secondary atrial fibrillation should not necessarily be forever treated with anti-coagulation. However, this slightly requires clinical trials to evaluate further.                                                 Next we delve into the realm of cardiac mapping and ablation where we view an article by Gaita et al. entitled 'Very long-term outcome following transcatheter ablation of atrial fibrillation. Are results maintained after 10 years of follow-up?', published in Europace last month. While pulmonary vein isolation is a widely accepted approach for treatment of atrial fibrillation, most reported studies review outcomes in terms of freedom of AF over a relatively short time period, generally two to five years. However longer term follow up is inconsistently reported. Gaita et al. sought to review 10 year outcomes amongst 255 patients undergoing ablation in a single center. They noted 52% remainder arrhythmia-free amongst a mixed cohort of both paroxysmal and persistent patients while 10% progressed to permanent atrial fiBrillation. They found that absence of increases in blood pressure, BMI, and fasting glucose was protective against an arrhythmia recurrence.                                                 These findings suggest that in a relatively small cohort of patients limited to a single center that even long-term outcomes after pulmonary vein isolation are generally quite good, exceeding 50%. However, future freedom from atrial fibrillation is heavily tied to control of other risk factors. In other words, if a patient is going to have poor control of diabetes, blood pressure, or gain weight, the benefit of their pulmonary vein isolation over long-term follow-up is likely less. These data thus highlight both the potential long-term benefit of PVI, but also the importance of counseling patients regarding the need for continued management and control of future and existing risk factors.                                                 Staying within the realm of atrial fibrillation we next review an article by Weng et al. entitled 'Genetic Predisposition, Clinical Risk Factor Burden, and Lifetime Risk of Atrial Fibrillation' published in last month's issue of circulation. The probability of detecting atrial fibrillation in patients based on clinical factors and genetic risk is unknown. Weng et al. sought to clarify whether a combination of clinical and polygenic risk scores could be used to predict risk of developing atrial fibrillation over long-term followup in the Framingham Heart Study. Amongst 4,600 individuals, 580 developed incident atrial fibrillation and had an overall lifetime risk of developing atrial fibrillation of 37%. Those are the lowest risk tertile based on clinical risk factor burden and genetic predisposition had a lifetime risk of 22% versus 48% in the highest. Furthermore, a lower clinical risk factor burden was associated with delayed atrial fibrillation onset. In order to identify patients with atrial fibrillation, before negative sequelae such as stroke occur, patient and physician understanding of risk and monitoring needs is necessary. The fact is that it will be great to identify every single patient who has atrial fibrillation before they have a negative sequela of that atrial fibrillation such as ischemic stroke.                                                 However, performing continuous monitoring of all patients with potential negative sequelae of atrial fibrillation is extraordinarily difficult. The reason is it's excessively costly. We cannot monitor the entire population irrespective of whatever the risk factors are. However, if we're able to identify the highest risk cohorts early on before the atrial fibrillation onsets, this may offer opportunities for use of newer cheaper monitors. The work by Weng et al. suggests one such possible approach combines clinical and polygenic risk scores. Actionability of these data, however, remains to be seen and further validation other cohorts is necessary to clarify generalized ability.                                                 The next article we review is published in last month's issue of the Journal of American College of Cardiology by Lopes at al. entitled 'Digoxin and Mortality in Patients With Atrial Fibrillation. Lopes et al. sought to evaluate the impact of the Digoxin on mortality in patients with atrial fibrillation and the association with the Digoxin serum concentration and heart failure status. They value this association in over 17,000 patients. At baseline 32% were receiving Digoxin. Baseline Digoxin use did not associate with risk of death, but even in these patients a serum concentration of greater than 1.2 nanograms per milliliter was associated with a 56% increase in mortality risk. For each .5 nanogram per milliliter increase in oxygen concentration the hazard ratio increased by 19% for overall mortality. This was irrespective of heart failure status. Furthermore, in patients who are newly started in Digoxin over the follow-up period, the risk and death and sudden death was higher. These data suggests a significant risk associated with Digoxin use for management of atrial fibrillation irrespective of heart failure status. Furthermore, serum valleys above 1.2 require close consideration of dose de-escalation. Whether there is any optimal dose, however, from the study is unclear. These data amongst a host of prior data strongly suggest again strategic use of Digoxin  principally for the management of atrial fibrillation.                                                 Moving on within the realm of atrial fibrillation, we review an article published in last month's issue of Circulation Research by Yan et al. entitled Stress Signaling JNK2 Crosstalk with CaMKII Underlies Enhanced Atrial Arrhythmogenesis. In this more acellular based study the mechanism underlying atrial arrhythmogenesis associated with aging was evaluated. Yan et al. sought to figure out whether the stress response JNK in calcium mediated arrhythmias might contribute to atrial arrhythmogenesis in aged transgenic mouse models. They demonstrated significant increased activity of JNK2 and aging atria, those furthermore associated with rhythmic remodeling. This association was mediated through CaMKII and ryanodine receptor channel function, with activation of the former leading to increased calcium leak mediated by the ladder. This in turn related to increase atrial fibrillation likelihood. Identifying novel targets for atrial fibrillation therapy is critical. Given atrial fibrillation is a complex disease process related to a multitude of risk factors it can be assumed that the contribution of any single factor may be mediated through distinct mechanisms.                                                 Aging in particular as well regarded, but considered to be non-modifiable risk factor for atrial fibrillation. Identifying genes or pathways, the immediate aging associated fibrillation, may take the risk of aging as no longer a non-modifiable thing. The finding of the significance of JNK2 and associate downstream effects with AF risks and aging hearts may hold potential in offering unique therapeutic targets.                                                 Finally, within the realm of atrial fibrillation, we're viewing article by Chen et al. in last month's issue of the Journal of the American Heart Association entitled Association of Atrial Fibrillation With Cognitive Decline and Dementia Over 20 Years: The ARIC-NCS Study. Multiple studies have suggested a significant association between atrial fibrillation risk of dementia. However, these studies have limited time follow-up and were often done and predominantly white patients. Thus, the authors sought to use the data from ARIC, the Atherosclerosis Risk in Communities Neurocognitive Study, to assess the risk of cognitive decline associated with atrial fibrillation. Amongst over 12,000 participants, a quarter of whom are black and half of whom are white, they noted 2100 patients developed atrial fibrillation and 1,150 develop dementia over a 20 year follow up period.                                                 There was a significantly greater risk of cognitive decline amongst those who developed atrial fibrillation. In turn incident atrial fibrillation for the follow-up period was associated with a higher risk of dementia even after adjusting for other clinical and cardiovascular risk factors such as incidents that ischemic stroke. These data further strengthened prior evidence of a direct link between atrial fibrillation and risk of cognitive decline and dementia. Understanding this long-term risk raises the need to additionally identify approaches to prevent this occurrence, which in turn is dependent on understanding the underlying mechanisms. The finding that the risk of cognitive decline dementias independent of ischemic stroke events raises concern that either subclinical micro-embolic events or other factors may be playing a role in this risk and in turn raises question as to how best to prevent them. Until better understood, however, the question of whether the association is causal remains to be seen.                                                 Changing gears yet again, we now delve into the realm of ICDs, pacemakers and CRT. Published in last month, issue of Heart Rhythm Tarakji et al. published a paper entitled 'Unrecognized venous injuries after cardiac implantable electronic device transvenous lead extraction.' Overall risk of transvenous lead extraction includes that of potentially fatal venous laceration. The authors sought to evaluate the incidence of venous injury that may be unrecognized based on microscopic study of extracted leads. Amongst 861 leads obtained from 461 patients they noted 80 leads or almost 9%. Amongst 15% of patients showed segments vein on the lead body, most of which were transmural including the tissue layer. However, in terms of clinical significance, only 1% had need for emergent surgical intervention for clinically significant venous laceration. Risk factors for having the entire vein on the lead included age of lead, ICD leads, and the use of the laser sheath.                                                 These findings suggest that there may be a high incidence of subclinical venous injury after lead extraction though rarely resulting clinically apparent sequelae. As would be expected, venous injury, including transmural removal of portions of the vein traversed by the lead, was more common amongst older leads, which generally more often require laser sheets and ICD leads. The question is however, whether this carries any direct clinical implications. One they may be considered is the potential additive risk of an advancing new lead through the same venous channel, particularly in the setting of potential transmural venous injury that already exists.                                                 Next in last month's issue of Heart Rhythm we review an article by Sharma at al. entitled 'Permanent His-bundle pacing as an alternative to biventricular pacing for cardiac resynchronization therapy: A multicenter experience.' The use of resynchronization therapy for treatment of patients with heart failure and wide QRS has been shown to offer morbidity and mortality benefits. However, many patients maybe non-responders, and recent studies on His bundle pacing of suggested potential clinical benefits. His bundle pacing essentially only requires one pacing catheter attached within the region of the His bundle Sharma et al. sought to evaluate the safety and success rates of His bundle pacing for patients who have either failed standard resynchronization therapy or in whom most tried as a primary intervention. They noted His bundle pacing was successful in 90% of patients with reasonable myocardial and His bundle capture thresholds. Patients in both groups exhibits significant narrowing of QRS morphology and improvement in left ventricular ejection fraction from a mean of 30 to 43%. However, a total of seven patients had lead related complications.                                                 These database on a retrospective analysis of two types of patients, those failing standard biventricular therapy, and those on whom his bundle pacing was attempted as a primary modality suggest overall safety and efficacy in a handful of experienced centers. The promise of His bundle pacing is that a may allow for more effective resynchronization than standard approaches. The high rate of success suggests that His bundle pacing maybe both safe and reasonable to pursue. However randomized trials across more centers are needed to fully prove its benefit, particularly as a primary modality of treatments.                                                 Next we review ICDs and chronic kidney disease. In last month's issue of JAMA cardiology by Bansal at al. entitled 'Long-term Outcomes Associated With Implantable Cardioverter Defibrillator in Adults With Chronic Kidney Disease.' While the benefit of ICDs in patients with low EF is widely recognized, modifying factors that may increase risk of death are not as well defined. These include things like advanced age and chronic kidney disease. Bansal et al. sought to evaluate long-term outcomes and ICD therapy in patients with chronic kidney disease. In retrospective study of almost 5,900 ambulatory patients amongst whom 1550 had an ICD, they found no difference in all cause mortality. However, ICD placement was associated with an increased risk of subsequent hospitalization due to heart failure or any cause hospitalization.                                                 In light of recent studies such as DANISH the robust sense of ICD benefit is being questioned. One of the thoughts for the absence of similar benefit to prior studies lies in the improving care of ambulatory heart failure patients. In patients with chronic kidney disease several questions rises to the risk with ICD, including infectious risk in dialysis patients and the concomitant mortality risk with renal dysfunction. The author suggested in retrospective study, no incremental benefit of ICDs in patients with chronic kidney disease and perhaps some element of added risk is related to hospitalization. However, this study has several limitations. It is retrospective and many patients received ICDs may have been perceived to be sicker in some way. Thus care must be taken in interpretation, but consideration of randomized studies to adjudicate benefit are likely necessary.                                                 Finally, within the realm of devices, we reviewed an article by Tayal et al. entitled "Cardiac Resynchronization Therapy in Patients With Heart Failure and Narrow QRS Complexes.' publishing the Journal of American College of Cardiology last month. Several parameters have been stressed to identify benefit of resynchronization therapy in patients with wide QRS include cross correlation analysis with tissue doppler imaging. However, many patients may have evidence in mechanical dyssynchrony even in the absence of an apparent wide QRS thus Tayal et al. sought to evaluate the benefit of resynchronization therapy amongst 807 patients with heart failure and a narrow QRS mean criteria in a randomized study. Of the 807 46% had delayed mechanical activation. Those without delay mechanical activation had underwent we standardization therapy and were associated with worse overall outcomes likely due to new delayed mechanical activation potentially related to CRT pacing. These data support the absence of a role for resynchronization therapy in patients with a narrow QRS. This is expected as resynchronization therapy likely offers the most benefit in patients with mechanical dyssynchrony that results from electrical dyssynchrony.                                                 Since by its very nature resynchronization therapy relies on non physiologic cardiac pacing thus compared to normal cardiac activation the nature of resynchronization pacing is desynchronization. These data support the absence of a role for resynchronization therapy in patients with heart failure and narrow QRS complexes.                                                 Moving on to cellular electrophysiology we review an article by Kozasa et al. published in last month's issue of Journal of Physiology entitled 'HCN4 pacemaker channels attenuate the parasympathetic response and stabilize the spontaneous firing of the sinoatrial node.' Heart rate is controlled by an interplay between sympathetic and parasympathetic components. In turn HCN4 abnormalities have been implicated in congenital sick sinus syndrome. The authors sought to clarify the contribution of HCN4 to sinus node autonomic regulation. They created a novel gain-of-function mouse where the HCN4 activity could be modulating from zero to three times normal. They then evaluated ambulatory heart-rate variability and responsive heart rate to vagus nerve stimulation. They found HCN4 over-expression did not increase heart rate, but attenuated heart-rate variability. It also attenuated bradycardic response to vagus nerve stimulation. Knockdown of HCN4 in turn lead to sinus arrhythmia and enhanced parasympathetic response. These data suggest HCN4 attenuates sinus node response to vagal stimuli thus stabilizing spontaneous firing of the node. The clinical application of this remain to be seen but are maybe important in that they highlight a mechanism for a heretofore poorly understood mechanism for how exactly HCN4 abnormalities may lead to sick sinus syndrome.                                                 Within the realm of ventricular arrhythmias we highlighted a number of articles published this past month. The first article we review was published in last month's issue of JACC clinical electrophysiology, entitled characterization of the electrode atomic substrate and cardiac sarcoidosis: correlation with imaging findings of scarring inflammation published by [inaudible 00:41:40] et al. In patients with cardiac sarcoidosis one of the questions is how to define the electronic atomic substrate, particularly before we entered the electrophysiology laboratory. Both active inflammation and replacement fibrosis maybe be seen in patients. The authors evaluated in 42 patients with cardiac sarcoidosis, the association between an abnormal electrograms and cardiac imaging findings including PET and Computed Tomography, as well as Cardiac MRI. They noted that amongst these 40 patients, a total of 21,000 electrograms were obtained, and a total of 19% of these were classified as abnormal. Most of the abnormalities occurred in the basal paravalvular segments and intraventricular septum. They further noted that many of these abnormalities in terms of electrograms were located outside the low voltage areas, particularly as it relates to fractionation. In about 90% of patients they notice late gadolinium enhancements and they noted abnormal FDG uptakes suggesting active inflammation in about 48%.                                                 However, it should be noted that only 29 of the 42 patients underwent cardiac imaging. Segments with abnormal electrograms tended to have more late gadolinium enhancement evidence scar transmurality, and also they noted that the association of abnormal PET scan did not necessarily occur with abnormal electrograms. Thus, they concluded that in patients with cardiac sarcoidosis and ventricular tachycardia pre-procedural imaging with cardiac MRI could be useful in detecting electroanatomic map abnormalities that may in turn be potential targets for substrate ablation. However, they were more likely associated with more scar transmurality and lower degrees of inflammation on PET scanning. These data are important in that they highlight potential non-invasive means by which to understand where substrate might occur in patients with the cardiac sarcoidosis. It is well recognized that cardiac sarcoidosis is associated with increased risk of ventricular arrhythmias. These risks have increased ventricular arrhythmias, might be targetable with ablation. Newer therapies might even offer non invasive means by which to perform ablation in patients best. Thus if we could identify non based on mechanisms of identifying the substrate, this will be even more critical.                                                 The critical findings of this particular paper lie in noting that most of the abnormalities still is in intra ventricular sePtum in basal segments, and also that it is MRI in late gadolinium enhancement and associates more with the abnormal electrograms. Interestingly, the absence of inflammation correlating with the presence of more abnormal electrograms suggests that it is not so much the act of inflammation as being reflected in the endocardial map, but the existence of scar.                                                 Next, again within JACC clinical electrophysiology we review an article by Porta-Sánchez et al. entitled 'Multicenter Study of Ischemic Ventricular Tachycardia Ablation With Decrement Evoked Potential Mapping With Extra Stimulus.' The authors sought to conduct a multicenter study of decrement evoked potential base functional tech ventricular tachycardia substrate modification to see if such mechanistic and physiologic strategies could result in reduction in VT burden. It is noted that really only a fraction of the myocardium in what we presume to be substrate based on the presence of low voltage areas are actually involved in the initiation and perpetuation of VT. Thus if we can identify the critical areas within the presumed substrate for ablation, this would be even a better way of potentially honing in on our targets. They included 20 consecutive patients with ischemic cardiomyopathy. During substrate mapping fractionated late potentials were targeted and an extra stimulus was provided to determine which display decrements. All patients underwent DEEP focus ablation with elimination being correlated with VT non-inducibility after radio-frequency ablation. Patients were predominantly male, and they noted that the specificity of these decrement evoked potentials to detect the cardiac isthmus for VT was better than that of using late potentials alone. They noted 15 of 20 patients were free of any VT after ablation of these targets over six months of follow-up, and there was a strong reduction in VT burden compared to six months pre ablation.                                                 They concluded that detriment evoked potential based strategies towards ablation for ventricular tachycardia might identify the functional substrate and those areas most critical to ablation. They in turn regarded that by its physiologic nature it offers greater access to folks to ablation therapies.                                                 This publication is important in that it highlights another means by which we can better hone in on the most critical regions for substrate evaluation in patients with ventricular tachycardia. The fact is more extensive ablation is not necessarily better and might result in increased risk of harm if we think about the potential effects of longer ablations or more ablation lesions. Thus if we could identify ways of only targeting those areas that are most critical to the VT circuits, we could perhaps short and ablation procedural time, allow for novel ways of approaching targeted ablation with limited amounts of ablation performed, or perhaps even improve overall VT outcomes by knowing the areas that are most critical to ensure adequate ablation therapy provided. However, we need to understand that this is still a limited number of patients evaluated in a non randomized manner. Thus whether or not more extensive ablation performed might have been better is as of yet unclear                                                 Staying within the realm of ventricular tachycardia we review an article published in last month's issue of Heart Rhythm by Winterfield et al. entitled the 'Impact of ventricular tachycardia ablation on healthcare utilization.' Catheter ablation of atrial tachycardia has been well accepted to reduce recurrent shocks in patients with ICDs. However, this is a potentially costly procedure, and thus effect on overall long-term health care utilization remains to be seen. The authors sought to evaluate in a large scale real world retrospective study the effect of VT ablation on overall medical expenditures in healthcare utilization. A total 523 patients met study inclusion criteria from the market scan database. After VT ablation median annual cardiac rhythm related medical expenditures actually decreased by over $5,000. Moreover the percentage of patients with at least one cardiac rhythm related hospitalization an ER visit decreased from 53 and 41% before ablation respectively, to 28 and 26% after ablation. Similar changes we're seeing in number of all cause hospitalizations and ER visits. During the year before VT ablation interestingly there was an increasing rate of healthcare resource utilization, but a drastic slowing after ablation.                                                 These data suggests that catheter ablation may lead to reduced hospitalization in overall healthcare utilization. The importance of these findings lies in understanding why we do the things we do. We can provide a number of therapies to patients, but we seek two different effects. One is the individual effect of improving their particular health. The second thing is trying to avoid increasing healthcare expenditures on a population level and making sure resources are utilized. If we can reduce recurrent hospitalizations and overall healthcare expenditure in patients by providing a therapy in addition to provide individual benefit, this is the optimal situation. These data suggests that VT ablation might provide such a benefits, that in fact it reduces overall healthcare utilization while improving overall outcomes.                                                 Next and finally within the realm of ventricular arrhythmias, we review more on the basic side the role of Titin cardiomyopathy leads to altered mitochondrial energetics, increased fibrosis and long-term life-threatening arrhythmias, published by Verdonschot et al. in last month's issue of European Heart Journal. It is known now that truncating Titin variants might be the most prevalent genetic cause of dilated cardiomyopathy. Thus, the authors sought to study clinical parameters and long term outcomes related to Titin abnormalities in dilated cardiomyopathy. They reviewed 303 consecutive and extensively phenotype dilated cardiomyopathy patients who underwent cardiac imaging, Holter monitoring, and endomyocardial biopsy and in turn also underwent DNA sequencing of 47 cardiomyopathy associated genes. 13% of these patients had Titin abnormalities. Over long-term followup they noted that these patients had increased ventricular arrhythmias compared to other types of dilated cardiomyopathy, but interestingly, they had similar survival rates. Arrhythmias in those Titin abnormal patients were most prominent in those who were subjected to an additional environmental trigger, including viral infection, cardiac inflammation, other systemic disease or toxic exposure. They also noted the cardiac mass was relatively reduced in titan admirable patients.                                                 They felt that all components of the mitochondrial electron transport chain we're simply up-regulated in Titin abnormal patients during RNA sequencing and interstitial fibrosis was also augmented. As a result, they concluded that Titin variant associated dilated cardiomyopathy was associated with an increased risk of ventricular arrhythmias, and also with more interstitial fibrosis. For a long time we have reviewed all non ischemic cardiomyopathy as essentially equal. However, more recent data has suggested that we can actually hone in on the cause. In turn, if we hone in on the cause, we might be able to understand the effects of specific therapies for ventricular arrhythmias based on that underlying cause. Patchy fibrosis might not be as amenable, for example, to ablation as discreet substrate that we might see in infarct related VT. Understanding the relative benefit in very specific types of myopathies might hold benefit in understanding how to, one, risk stratify these patients, and two, understand what type of therapy, whether pharmacologic or ablative, might result in greatest benefit to the patients.                                                 Changing gears entirely now to the role of genetics, we review multiple articles in various genetic syndromes published this past month. First, we reviewed an article by Providência et al. published in the last month's issue of heart entitled 'Impact of QTc formulae in the prevalence of short corrected QT interval and impact on probability and diagnosis of short QT syndrome.' The authors sought to assess the overall prevalence of short corrected QT intervals and the impact on diagnosis of short QT syndrome using different methods for correcting the QT interval. In this observational study they reviewed the sudden cardiac death screening of risk factors cohorts. They then applied multiple different correction formulae to the ECGs. They noted that the prevalence of individuals with the QTc less than 330 and 320 was extremely low, namely less than .07 and .02% respectively. They were also more frequently identified using the Framingham correction. The different QTc correction formulae could lead to a shift of anywhere from 5 to 10% of individuals in the cohort overall.                                                 They further noted, that based on consensus criteria, instead of 12 individuals diagnosed with short gut syndrome using the Bazett equation, a different number of individuals would have met diagnostic criteria with other formulae, 11 using Fridericia, 9 with Hodges, and 16 using the Framingham equation. Thus, they noted that overall the prevalence of short QT syndrome exceedingly low and an apparently healthy adult population. However, reclassification as meeting criteria might be heavily dependent on which QT correction formula is used. The importance of these findings is that not all QTs are created equal.                                                 Depending on how you compute the QT interval in which formula to use may affect how you actually risk characterize a patient. Unfortunately, these data do not necessarily tell us which is the right formula, but this highlights that it might be relevant to in the future evaluate the role of different formulae and identifying which is the most necessary to classify a patient.                                                 Moving on to an article published in last month's issue of the journal of clinical investigation by Chai et al. we review an article entitled 'Physiological genomics identifies genetic modifiers of Long QT Syndrome type 2 severity.' Congenital Long QT Syndrome is a very well recognized, inherited channelopathy associated life-threatening arrhythmias. LQTS type 2 is specifically caused by mutations in casein to encoding the potassium channel hERG. However, even with the mutation not all patients exhibit the same phenotype. Namely some patients are more at risk of life threatening arrhythmias in spite of having the same mutation as others who do not exhibit the same severity phenotype. The authors sought to evaluate whether specific modifiable factors within the remaining genetic code might be modifying the existing mutation. Thus, they sought to identify contributors to variable expressivity in an LQT 2 family by using induced pluripotent stem cell derived cardiomyocytes and whole exome sequencing in a synergistic manner.                                                 They found that patients with severely effected LQT 2 displayed prolonged action potentials compare to sales from mildly effected first-degree relatives. Furthermore, stem cells derived from patients were different in terms of how much L-type calcium current they exhibited. They noted that whole exome sequencing identified variants of KCNK17 and the GTP-binding protein REM2 in those patients with more severe phenotypes in whom greater L-type calcium current was seen. This suggests that abnormalities or even polymorphisms in other genes might be modifying the risk attributed to by mutations in the primary gene. This showcases the power of combining complimentary physiological and genomic analysis to identify genetic modifiers and potential therapeutic targets of a monogenic disorder. This is extraordinarily critical as we understand on one level that when we sequence a monogenic disorder that there might exist variants of uncertain significance, namely they have not been classified as disease causing, but could be. In turn, we also recognize that mutations in a family might effect different relatives differently. However, why this is has been relatively unclear.                                                 If we can understand and identify those patients who are most at risk of dangerous abnormal rhythms, this will be useful in how much to follow them, and what type of therapy to use in them. The fact that other genes might modify the risk even in the absence of specific mutations, suggests that novel approaches to characterizing the risk might help for the risk modified patients classification in general. Clinical use, however, remains to be seen.                                                 Moving on from long QT, we evaluate 'The Diagnostic Yield of Brugada Syndrome After Sudden Death With Normal Autopsy' noted in last month's issue of the Journal of American College of Cardiology and published by Papadakis et al. It is well known, the negative autopsies are not uncommon in patients, however, families might be wondering how at risk they are. Thus, the authors sought to assess the impact of systematic ajmaline provocation testing using high right precordial leads on the diagnostic yield Brugada syndrome in a large cohort of Sudden Arrhythmic Death syndrome families. Amongst 303 families affected by Sudden Arrhythmic Death Syndrome evaluation was done to determine whether or not there was a genetic inherited channelopathy cause. An inherited cardiac disease was diagnosed in 42% of the families and 22% of relatives Brugada syndrome was the most prevalent diagnosis overall amongst 28% of families. Ajmaline testing was required, however, to unmask the Brugada Syndrome in 97% of diagnosed individuals. Furthermore, they use of high right precordial leads showed a 16% incremental diagnostic yield of ajmaline testing for diagnosing Brugada syndrome.                                                 They further noted that a spontaneous type 1 regard or pattern or a clinically significant rhythmic event developed in 17% of these concealed regardless syndrome patients. The authors concluded the systematic use of ajmaline testing with high right precordial leads increases the yield of Brugada Syndrome testing in Sudden Arrhythmic Death Syndrome families. Furthermore, they noted that assessments should be performed in expert centers or patients could also be counseled appropriately. These findings are important and one of the big questions always becomes how aggressively to test family members of patients or of deceased individuals who experienced sudden arrhythmic death. Many of these patients have negative autopsies, and genetic autopsy might not be possible due to lack of tissue or blood products that can be adequately tested.                                                 The data here suggest that amongst a group of 303 sudden arrhythmic death, families that Brugada Syndrome is by far the most frequent diagnosis. If an inherited cardiac disease was identified. In turn, it is not ECG alone or echo alone that helps identify them, but requires drug provocation testing in addition to different electrode placements. Whether or not this will consistently offer benefit in patients in general or my result in overcalling remains to be seen next within the realm of genetic predisposition.                                                 We view an area where we don't know if there's a genetic predisposition in article published by Tester et al. entitled Cardiac Genetic Predisposition in Sudden Infant Death Syndrome in last month's issue of the journal of american college of cardiology. Sudden Infant Death Syndrome is the leading cause of post-neonatal mortality and genetic heart diseases might underlie some cases of SIDS. Thus the authors sought to determine the spectrum and prevalence of genetic heart disease associated mutations as a potential monogenic basis for Sudden Infant Death Syndrome. They study the largest cohort to date of unrelated SIDS cases, including a total of 419 individuals who underwent whole exome sequencing and targeted analysis for 90 genetic heart disease susceptibility genes. Overall, 12.6% of these cases had at least one potentially informative genetic heart disease associated variants. The yield was higher in those mixed European ancestry than those of European ancestry.                                                 Infants older than four months were more likely to host a potentially informative gene. Furthermore, they noted that only 18 of the 419 SIDS cases hold a [inaudible 01:01:26] or likely pathogenic variant. So in other words, only 4% of cases really had a variant that they could say was distinctly pathogenic or likely pathogenic. Thus, overall, the minority of SIDS cases have potentially informative variant in genetic heart disease susceptibility gene, and these individuals were mostly in the 4 to 12 month age group. Also, only 4% of cases had immediately clinically actionable variance, namely a variant, which is well recognized as pathogenic and where we could actually say that a specific therapy might have had some effect. These findings can have major implications for how best to investigate SIDS cases in families. It might suggest that SIDS cases where the individual was older, nearly 4 to 12 months of age might have a greater yield in terms of identifying variance.                                                 While this might not affect the deceased in fit, it might affect, families are planning on having another child in whom a variant can be identified.                                                 Finally, within the realm of genetics, we review an article published in last month's issue of Science Advances by Huang. et al. entitled 'Mechanisms of KCNQ1 Channel Dysfunction in Long QT Syndrome Involving Voltage Sensor Domain Mutations'. Mutations that induce loss of function of human KCNQ1 underlie the Long QT Syndrome type 1. While hundreds of mutations have been identified the molecular mechanism by which they result in impaired function are not as well understood. The authors sought to investigate impact of 51 specific variants located within the voltage sensor domain and emphasized effect on cell surface expression, protein folding, and structure. For each variant efficiency of trafficking of the plasma membrane, impact of proteasome inhibition, and protein stability were evaluated. They noted that more than half of the loss of function mutations were seen to destabilized structure of the voltage sensor domain, generally accompanied by mistrafficking and degradation by the proteasome.                                                 They also noted that five of the folding defective Long QT Syndrome mutant sites were located in the S0 helix, where they tend to interact with a number of other loss of function mutation sites in other segments of the voltage sensor domain. They suggested these observations reveal a critical role for the S0 helix as a central scaffold to help organize and stabilized KCNQ1 overall. They also note the importance of these findings is that mutation-induced destabilization of membrane proteins may be a more common cause of disease functioning in humans. The importance of these findings lies in better understanding why specific mutations lead to appa

Commentary by Dr. Valentin Fuster

Commentary by Dr. Valentin Fuster

Fri, 16 Dec 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16587/ https://edoc.ub.uni-muenchen.de/16587/1/Scharr_Andreas.pdf Scharr, Andreas ddc:540, ddc:500, Fakultät f

Seit der ersten Charakterisierung des Ih-Stroms wird dessen Funktion im Herzen, insbesondere während der langsamen spontanen Depolarisation im Sinusknoten kontrovers diskutiert. 1998 gelang es mehreren Arbeitsgruppen unabhängig voneinander, die entsprechenden Gene zu identifizieren, die für die Ih-Kanäle kodieren. Diese HCN-Genfamilie umfasst 4 Isoformen, wobei jede Isoform ein spezifisches Expressionsmuster besitzt. Im Säugetiersinusknoten konnte auf Transkriptebene gezeigt werden, dass HCN4 die dominante HCN Isoform darstellt. Um die physiologische Funktion von HCN4 aufzuklären wurden in der vorliegenden Arbeit Mäuse generiert und analysiert, die für diese HCN Isoform defizient sind. Es konnte gezeigt werden, dass HCN4 für die Funktion des sich entwickelnden kardialen Reizleitungssystems essentiell ist. Im Wildtyp Embryo wird HCN4 Transkript und Protein in der Region exprimiert, in der sich der Sinusknoten entwickelt. Mäuse die für den HCN4-Kanal ubiquitär oder herzspezifisch defizient sind, sterben zwischen ET 10,0 und 11,5. Histologische Untersuchungen an den HCN4-defizienten Tieren zeigten keine offensichtlichen morphologischen Defekte. Im Durchschnitt ist Ih in den Knock-out Kardiomyozyten um 85% reduziert. Die Herzen der HCN4-defizienten Mäuse schlagen signifikant langsamer als die von Wildtypen und können durch cAMP nicht stimuliert werden. Sowohl in Wildtypen als auch in HCN4-/--Mäusen konnten Kardiomyozyten mit einem "primitiven" Schrittmacherpotential detektiert werden. Hingegen sind Zellen mit einem ausgereiften Schrittmacherpotential, die im Wildtyp ab ET 9,0 auftreten, nicht im Knock-out zu finden. Deshalb ist der HCN4-Kanal für die Bildung von Schrittmacherpotentialen im sich entwickelndem Sinusknoten essentiell. Im adulten Tier wird HCN4 ausschließlich in Herzregionen exprimiert die spontane Aktivität aufweisen. Proteinexpression wurde sowohl im ganzen Sinusknoten als auch in isolierten Sinusknotenzellen, im AV Knoten und auf den Herzklappen nachgewiesen. Wegen der embryonalen Letalität des globalen HCN4 Knock-outs wurden mit Hilfe des Cre loxP Systems herzspezifische HCN4-defiziente Tiere hergestellt. Nach genauer Analyse von fünf verschiedenen Cre Transgenen konnte schließlich mit Hilfe der induzierbaren MerCreMer Maus die HCN4 Expression im Sinusknoten um etwa 90% reduziert werden. Mit diesen Tieren sollte es möglich sein, die Rolle von Ih im Herz zu untersuchen. Vorläufige in-vivo EKG Messungen ergaben aber Bemerkenswerterweise bisher noch keine Unterschiede zwischen Wildtyp und der herzspezifischen HCN4-MerCreMer-KO Maus.