Podcasts about cav1

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.09.548255v1?rss=1 Authors: Li, G., McLaughlin, D. W., Peskin, C. S. Abstract: Synaptic plasticity (long term potentiation/depression (LTP/D)), is a cellular mechanism underlying learning. Two distinct types of early LTP/D (E-LTP/D), acting on very different time scales, have been observed experimentally -- spike timing dependent plasticity (STDP), on time scales of tens of ms; and behavioral time scale plasticity(BTSP), on time scales of seconds. BTSP is a candidate for the mechanism for rapid learning of spatial location by hippocampal place cells. Here a computational model of the induction of E-LTP/D at a spine head of a synapse of a hippocampal pyramidal neuron is developed. The single compartment model represents two interacting biochemical pathways for the activation (phosphorylation) of the kinase (CaMKII) with a phosphatase, with Ion inflow described by NMDAR, CaV1, and Na channels. The biochemical reactions are represented by a deterministic system of differential equations. This single model captures realistic responses (temporal profiles with the differing timescales) of STDP and BTSP and their asymmetries for each (STDP or BTSP) signaling protocol. The simulations detail several mechanisms underlying both STDP and BTSP, including i) the flow of Ca^2+ through NMDAR vs CaV1 channels, and ii) the origin of several time scales in the activation of CaMKII. The model also realizes a priming mechanism for E-LTP that is induced by Ca^2+ flow through CaV1.3 channels. Once in the spine head, this small additional Ca^2+ opens the compact state of CaMKII, placing CaMKII "in the ready" for subsequent induction of LTP. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.24.546368v1?rss=1 Authors: Hackett, J., Nadkarni, V., Singh, R. S., Carthy, C. L., Antigua, S., Hall, B. S., Rajadhyaksha, A. M. Abstract: Impairments in social behavior are observed in a range of neuropsychiatric disorders and several lines of evidence have demonstrated that dysfunction of the prefrontal cortex (PFC) plays a central role in social deficits. We have previously shown that loss of neuropsychiatric risk gene vCacna1cv that codes for the Cav1.2 isoform of L-type calcium channels (LTCCs) in the PFC result in impaired sociability as tested using the three-chamber social approach test. In this study we aimed to further characterize the nature of the social deficit associated with a reduction in PFC Cav1.2 channels (Cav1.2PFCKO mice) by testing male mice in a range of social and non-social tests while examining PFC neural activity using in vivo GCaMP6s fiber photometry. We found that during the first investigation of the social and non-social stimulus in the three-chamber test, both Cav1.2PFCKO male mice and Cav1.2PFCGFPGFP controls spent significantly more time with the social stimulus compared to a non-social object. In contrast, during repeat investigations while Cav1.2PFCWT mice continued to spend more time with the social stimulus, Cav1.2PFCKO mice spent equal amount of time with both social and non-social stimuli. Neural activity recordings paralleled social behavior with increase in PFC population activity in Cav1.2PFCWT mice during first and repeat investigations, which was predictive of social preference behavior. In Cav1.2PFCKO mice, there was an increase in PFC activity during first social investigation but not during repeat investigations. These behavioral and neural differences were not observed during a reciprocal social interaction test nor during a forced alternation novelty test. To evaluate a potential deficit in reward-related processes, we tested mice in a three-chamber test wherein the social stimulus was replaced by food. Behavioral testing revealed that both Cav1.2PFCWT and Cav1.2PFCKO mice showed a preference for food over object with significantly greater preference during repeat investigation. Interestingly, there was no increase in PFC activity when Cav1.2PFCWT or Cav1.2PFCKO first investigated the food however activity significantly increased in Cav1.2PFCWT mice during repeat investigations of the food. This was not observed in Cav1.2PFCKO mice. In summary, a reduction in Cav1.2 channels in the PFC suppresses the development of a sustained social preference in mice that is associated with lack of PFC neuronal population activity that may be related to deficits in social reward. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.21.513252v1?rss=1 Authors: Yang, Q., Perfitt, T. L., Quay, J., Hu, L., Colbran, R. J. Abstract: Clustering of neuronal L-type voltage-gated Ca2+ channels (LTCC) in the plasma membrane is increasingly implicated in creating highly localized Ca2+ signaling nanodomains. For example, LTCC activation can increase phosphorylation of the nuclear CREB transcription factor by increasing Ca2+ concentrations within a nanodomain close to the channel, without requiring bulk Ca2+ increases in the cytosol or nucleus. However, the molecular basis for LTCC clustering is poorly understood. The postsynaptic scaffolding protein Shank3 specifically associates with one of the major neuronal LTCCs, the CaV1.3 calcium channel, and is required for optimal LTCC-dependent excitation-transcription coupling. Here, we co-expressed CaV1.3 1 subunits with two distinct epitope-tags with or without Shank3 in HEK cells. Co-immunoprecipitation studies using the cell lysates revealed that Shank3 can assemble multiple CaV1.3 1 subunits in a complex under basal conditions. Moreover, CaV1.3 LTCC complex formation was facilitated by CaV{beta} subunits ({beta}3 and {beta}2a), which also interact with Shank3. Shank3 interactions with CaV1.3 LTCCs and multimeric CaV1.3 LTCC complex assembly were disrupted following addition of Ca2+ and calmodulin (Ca2+/CaM) to cell lysates, perhaps simulating conditions within an activated CaV1.3 LTCC nanodomain. In intact HEK293T cells, co-expression of Shank3 enhanced the intensity of membrane-localized CaV1.3 LTCC clusters under basal conditions, but not after Ca2+ channel activation. Live cell imaging studies also revealed that Ca2+ influx through LTCCs disassociated Shank3 from CaV1.3 LTCCs clusters and reduced the CaV1.3 cluster intensity. Deletion of the PDZ domain from Shank3 prevented both binding to CaV1.3 and the changes in multimeric CaV1.3 LTCC complex assembly in vitro and in HEK293 cells. Finally, we found that shRNA knock-down of Shank3 expression in cultured rat primary hippocampal neurons reduced the intensity of surface-localized CaV1.3 LTCC clusters in dendrites. Taken together, our findings reveal a novel molecular mechanism contributing to neuronal LTCC clustering under basal conditions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

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 22 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the March 5 and March 19 issues of Circulation Research. This episode also features an in-depth conversation with Norberto Gonzalez-Juarbe and Maryann Platt from the J. Craig Venter Institute to discuss their study, Influenza Causes MLKL-Driven Cardiac Proteome Remodeling During Convalescence.   Article highlights:   Carnicer, et al. BH4 Prevents and Reverses Diabetic LV Dysfunction   Kyryachenko, et al. Regulatory Profiles of Mitral Valve   Mangner, et al. Heart Failure Associated Diaphragm Dysfunction   Peper, et al. Identification of McT1 as Caveolin3 Interactor       Dr Cindy St. Hilaire:        Hi, and welcome to Discover CircRes: the podcast of the American Heart Association's Journal, Circulation Research. I'm your host, Dr Cindy St Hilaire, from the Vascular Medicine Institute at the University of Pittsburgh. Today I will be highlighting four articles selected from our March 5th and March 19th issues of Circ Res. After the highlights Drs Norberto Gonzalez-Juarbe and Maryann Platt from the J. Craig Venter Institute are here to discuss their study, Influenza Causes MLKL-Driven Cardiac Proteome Remodeling During Convalescence Dr Cindy St. Hilaire:        The first article I want to share is titled, BH4 Increases nNOS Activity and Preserves Left Ventricular Function in Diabetes. The first author is Ricardo Carnicer, who is also corresponding author alongside Barbara Casadei and they're from University of Oxford in the UK. Cardiomyopathy and heart failure are common complications of diabetes, but the molecular pathology underlying this cardiac dysfunction is not entirely clear. Increased oxidative stress and reduced functioning of both mitochondria and nitric oxide synthase or nNOS have been implicated in diabetic cardiomyopathy. Tetrahydrobiopterin or BH4 is a co-factor necessary for nNOS activity. Dr Cindy St. Hilaire:        And in diabetic patients and animals oxidation of BH4 inactivates nNOS and induces vascular endothelial pathology. But, what happens in the cardiac tissue itself? This group shows that although boosting BH4 levels by genetic or pharmacological means prevented or reversed heart dysfunction in diabetic mice, the status of BH4 oxidation and nNOS function in the heart tissue of diabetic patients and mice, did not actually differ significantly from that of healthy controls. Instead through molecular analysis, they revealed that in diabetic mouse cardiomyocytes boosting BH4 promoted a nNOS dependent increase in glucose uptake, which then preserved the cell’s mitochondrial function. Regardless of the pathways involved, the fact that BH4 reversed diabetic associated cardiac dysfunction in mice suggests the potential for therapies that could be used to lower the risks of such complications in humans as well. Dr Cindy St. Hilaire:        The second article I want to share is titled, Chromatin Accessibility of Human Mitral Valves and Functional Assessment of MVP Risk Loci. The first authors are Sergiy Kyryachenko, Adrien Georges, and Mengyao Yu, and the corresponding author is Nabila Bouatia-Naji from Paris Cardiovascular Research Institute in France. The mitral valve opens and closes to direct a one-way flow of blood from the left atrium to the ventricle. If the mitral valve fails, as in the case of mitral valve prolapse or MVP, blood regurgitation, cardiac arrhythmia, and ultimately heart failure can occur. Dr Cindy St. Hilaire:        With 11 valves from MVP patients and 7 control patients, this group used a highly sensitive chromatin profiling technique called ATAC-Seq to identify regions of the genome with increased accessibility, which indicates transcriptional activity. They found that while diseased and healthy valves had similar chromatin profiles, they differed from those of other heart tissues. Valve specific open chromatin regions were enriched in binding sites for NFATC, a transcription factor known to regulate valve formation. And, specifically in MVP tissues, they found two potential causative sequence variants. These MVP-linked variants exhibited enhancer activity in cultured cells. And for one variant, the team identified the gene target of this variant. In providing the first mitral valve cell chromatin profiles and demonstrating their use and functional analysis of MVP-linked variants, this work supplies a valuable research for mitral valve prolapse evological studies. Dr Cindy St. Hilaire:        The third article I want to share is titled, Molecular Mechanisms of Diaphragm Myopathy in Humans with Severe Heart Failure. The first author is Norman Mangner, and the co-senior authors are Axel Linke and Volker Adams from Dresden University of Technology in Germany. The diaphragm is the primary muscle controlling a person's breathing. This muscle can become weakened during heart failure, which exacerbates symptoms and increases the risk of death. The pathological mechanisms underlying the diaphragm's demise are largely unclear. Studies in animals have pointed to increase reactive oxygen species as a contributing factor, but human studies have been limited. This group evaluated the histological and molecular features of human diaphragm biopsies from both heart failure patients and controls. Dr Cindy St. Hilaire:        The diaphragm samples were collected from 18 heart failure patients, who were undergoing implantation of left ventricular assist devices. And 21 control samples were obtained from patients not having heart failure bypass graft surgery. Compared with the controls, the heart failure diaphragms showed significantly reduced thickness, severe muscle fiber atrophy, increased oxidative stress in the form of protein oxidation, increased proteolysis, impaired calcium handling and mitochondrial abnormalities and dysfunction. Pathological measures also correlated with clinical severity. These data are the first insights into the pathology of heart failure related diaphragm weakness, and this work points to the molecular players that could be targeted for novel treatments. Dr Cindy St. Hilaire:        The last article I want to share before our interview is titled, Caveolin3 Stabilizes McT1-Mediated Lactate/Proton Transport in Cardiomyocytes. The first author is Jonas Peper and the corresponding author is Stephan Lehnart from the Heart Research Center, Göttingen in Germany. Caveolae are invaginations of the plasma membrane, and these structures are involved in endocytosis, signal transduction and other important cellular processes. Caveolin is the key protein component of caveolae and isoforms of Caveolin have been implicated in heart conditions. Mice lacking the isoform CAV1 develop heart failure and genome-wide association studies have been linked to human CAV1 variants with cardiac conduction disease and atrial fibrillation. Rare variants of CAV3 are known to cause hypertrophic cardiomyopathy. However, little is known about the normal or pathological actions of Caveolin in heart cells where caveolae are plentiful. To learn more, this group performed mass spectrometry, immunoprecipitation, and other analysis in cardiomyocyte, and uncovered novel CAV associated proteins, some of which turned out to be isoform specific. Dr Cindy St. Hilaire:        CAV1 interacted specifically with aquaporin while CAV3 was associated specifically with the lactate transporting McT1 protein and the iron transporting TFr1 protein. When the team knocked out the function of CAV3 in stem cells derived from human cardiomyocytes, they found that McT1 had reduced surface expression and function, and that the cells exhibited abnormal de-polarizations. Together the results set the stage for future studies of cardiomyocyte CAV biology, including how CAV variants might contribute to disease pathogenesis. Dr Cindy St. Hilaire:        Today I have with me Drs Norberto Gonzalez-Juarbe and Maryann Platt from the J. Craig Venter Institute, and they're here to discuss their study, Influenza Causes MLKL-Driven Cardiac Proteome Remodeling During Convalescence . And this is in our March 5th issue of Circulation Research. So thank you both for being with me today. Dr Maryann Platt:           Great to be here. Dr Norberto Gonzalez-Juarbe:    Thank you. Dr Cindy St. Hilaire:        So I want to start with influenza mediated cardiac complications. So what are these complications? How prevalent are they in people who catch influenza and who's most affected? Dr Norberto Gonzalez-Juarbe:    So for the last hundred years, we have known that every time there's an epidemic or pandemic from influenza, there's adverse cardiac events that come after you get the disease. During the 1918 pandemic, we could see myocardial damage and about 90% of all people that succumb to the infection, and in the latest epidemics that has been about 40% to 50%, suggesting that the more pandemic the strain of influenza is, the more virulent, the more of these adverse cardiac events we are going to see. So it seems that it is attached to severity of disease. The virus can get to the heart easy, the more severe your disease phenotype is, but it seems that some pandemic strains have a better way to get there of causing more damage than the common epidemic strengths. Dr Cindy St. Hilaire:        That was actually one of my other questions, how does it get to the heart? What's happening there? Do we know much about that? I guess, specifically for flu, but I'm sure in the back of everybody's mind, people are also thinking about SARS-CoV2 too. So how does that kind of pathway work or transportation work? Dr Norberto Gonzalez-Juarbe:    Circulation is going to be the main way it gets there for, for example, if we were to look at COVID then in the heart there's the same receptors for the epithelial cells that are in there, the ACE-2 receptor, that's also in the cardiac tissue and COVID-19 can actually infect cardiomyocytes through that receptor. In terms of influenza, it's basically similar. Some of these receptors are present on the epithelium in the lungs, are also present there and flu can actually infect cardiomyocytes. In our study we also look at some other cell types like endothelial cells and fibroblasts, and we show that there's actually some lower grade infection too. But that's why it's all of these, it starts in the severity of disease, that's the more virus is going to be in your bloodstream, the easier it's going to be to get there. And since the same receptors are present in the heart, so it's going to be easy for the virus to affect the cell. Dr Maryann Platt:           It's not necessarily dependent on age or race or anything it's dependent on how sick you are, for sure. Dr Cindy St. Hilaire:        And by sick, does that directly correlate with viral load of the patients or just their response, an overactive response or something like that? Do we know? Dr Norberto Gonzalez-Juarbe:    I think it's a double edged sword, so it's going to be related to viral load, but also the type of immune responses that you're going to be having, it's going to affect the role of the virus in their heart. In our case we studied way after you cleared the proof from the lungs. So most of the studies that have been out there for a while show, when you're really, really sick, what is happening, but that of your compounding because you have all of these immune responses happening, and the virus is doing its thing. But once you clear the virus from the lungs, your, kind of, immune system settles down. And in our study, we show that even if you clear it from the lungs, the virus is still present in the heart. Dr Cindy St. Hilaire:        So one of the mechanisms that you focused on in terms of how influenza was contributing or leading to cardiac complications, is this process called necroptosis? Can you just maybe give us a primer on what that is, and what it's doing specifically in the cardiomyocytes? Dr Maryann Platt:           Sure. So necroptosis, there's a couple of different ways that cells can die, either under normal circumstances, just maintaining the number of cells in your body or in the case of infection, trying to get rid of the infection. So most commonly, cells will undergo apoptosis, which is programmed cell death, not very inflammatory. And then necroptosis is another way that is highly inflammatory and driven by, initiated by, some of the same molecular cascades, but then affected by a different set of molecules. Dr Cindy St. Hilaire:        Interesting. And so it's really that inflammatory component that is driving pathogenesis in the cardiac tissue then. Dr Maryann Platt:           Yeah. Dr Norberto Gonzalez-Juarbe:    And evolutionarily necroptosis has been shown to help the host against viral infections. Specifically, influenza has proteins that can block apoptosis, which is kind of like the good way of dying. And then the cell has to undergo these other necrotic type of cell death to get rid of viral replication. But while some of these might interact with both pathways, necroptosis effect their molecule. MLKL is the last protein in the pathway. That's the one that actually rupture the cells. So we wanted to prevent that from happening to see if we can actually stimulate something protective by having all of the other good cascade-type molecules still there. Dr Cindy St. Hilaire:        ‘Good’in quotes (laughing). Dr Maryann Platt:           Still dying cells, less bad, not as inflammatory Dr Norberto Gonzalez-Juarbe:    Inflammatory since the heart is this type of organ that any injury will be, more or less, long lasting, and that will have detrimental effects throughout life. Dr Cindy St. Hilaire:        Got it. That's interesting. So can you maybe give us a summary of your experimental design and kind of the groups you were looking at, and a summary of the results? Dr Maryann Platt:           Sure. So we had four different groups of mice, two of them were wild type mice and two were MLKL, all knockout mice, which could not undergo necroptosis. And then each of those genotypes, we had uninfected mice or mice that were infected with flu. And then we monitored long viral titer to see how much infection was there at the lungs. And then after the infections subsided in the lungs, two days after a viral load was undetectable, we sacrificed those animals, collected their hearts. Dr Cindy St. Hilaire:        That's great. So that two day resolution, is that a similar time course with humans, in terms of a pathogenesis of developing cardiac complications? How similar, I mean, mice are never perfect models, but what's good and what's not good about using a mouse as for this model? Dr Norberto Gonzalez-Juarbe:    So, mice are not human right?. So, we are always thinking about that quote, but most of the cardiac events that occurred during these type of infections and similar things have been observed in, for example, pneumococcal infection, which is by streptococcus pneumonia. Most of these adverse cardiac events occur right after you leave the hospital. Those are a specific set of adverse cardiac events that are different from the ones that happen when you are severely infected in the hospital. And these can be arrhythmias and myocardial infarction, and some of these things that can happen up to 10 years after you recover from the pulmonary infection. Dr Norberto Gonzalez-Juarbe:    So our model was designed to see that step of the host trying to retcover. And if there was still something there in the heart, right after you get out of the hospital, that you receive your therapeutics, and you're thinking, 'Oh, I don't have any more flu in my lungs, and I'm recovering', that timeframe right after you get out, you might still have some other things happening in your body, that might determine what happens to your heart. Dr Cindy St. Hilaire:        Interesting. So you may actually be feeling pretty good, but your heart or even possibly other organs are still kind of under the weather, so to speak? Dr Norberto Gonzalez-Juarbe:    Exactly. Dr Maryann Platt:           Exactly. Dr Cindy St. Hilaire:        So in your proteomic analysis, I think you stated it was some, it was just under a hundred proteins were differentially regulated, and a majority were actually in kind of metabolic mitochondrial related pathways. Could you maybe tell us the importance about that? But then also, yes, that was a big chunk of it, but were there any other pathways that were either up or down, that were surprising in your findings? Dr Norberto Gonzalez-Juarbe:    The importance of the major mitochondrial proteins that we found, first that the MLKL knockout, so inhibiting these necrotic cell death actually promoted mitochondrial health. So that first was interesting, because that will suggest that this can be quite therapeutic target in the future. That innovation enhance some proteins that protect the mitochondria and aid in mitochondrial function. And if we think about the heart as our engine, we need energy for an engine to work and mitochondria is that energy resource that we have. And the heart is really relying on these, because if you have a metabolic breakdown in the heart, you get cardiac event. So most of the proteins that were changed upon infection had to do with these specific, important metabolic function of the heart. Some other proteins have to do with cellular signaling mechanisms and calcium homeostasis, all these other things that are important to maintaining homeostasis in the heart thus suggesting that the virus is inducing massive stress in their heart without actively replicating or causing inflammation. Dr Norberto Gonzalez-Juarbe:    And that was very important in our study that we didn’t see these antiviral effects, but at the same time, we saw all of these detrimental metabolic effects. So future studies might be also targeting what viral factors might be actually inducing these metabolic effects in the heart. But we also saw some molecules important for cell death mechanisms that were not necroptosis. Dr Norberto Gonzalez-Juarbe:    Marianne, you can describe some of those. Dr Maryann Platt:           So one third way that cells can die is called pyroptosis. And we actually saw that pyroptosis was also elevated in flu infected mice, in their hearts, suggesting that it might not just be necroptosis. All this inflammation coming from necroptosis is what's driving breakdown of heart function, but also possibly pyroptosis. Dr Cindy St. Hilaire:        The mitochondrial aspect is interesting. In heart failure normally there's the switch from fatty acid oxidation to glycolysis. Does that happen in a shorter or smaller way after flu? And in some patients they just don't recover? Is there a metabolic switch to an infected cardiomyocyte, that is more transient, and then in a subset it turns to permanent? Is that what's happening? Dr Norberto Gonzalez-Juarbe:    Yeah, that is something that we might need to follow up on, since our study was more of a snapshot of that specific time point. It will be good to do follow-up studies where we look at different time points post infection. And even maybe three months after infection, then six months after infection. We have done similar studies with pneumococcal pneumonia, and we have found that cardiac function and metabolic function, it is significantly remodeled, even three months after the pneumonia event. Dr Cindy St. Hilaire:        Interesting. So once it's actually cleared from the lungs, it's still… Dr Norberto Gonzalez-Juarbe:    The heart is still undergoing this injury recovery, which cause scarring process and these leads to reduced cardiac function. Dr Cindy St. Hilaire: So influenza actually, maybe a lot of people know this now, but it was somewhat new to me, I guess, at least a year ago when COVID first started. But influenza like SARS-CoV2 is an enveloped virus. It's a single strand RNA virus. So are these findings specific to this class of viruses, specific to RNA viruses? Or is this something that you think is operative in other types of viruses in terms of causing these cardiac complications? Dr Maryann Platt:           It's certainly possible. I'm not a virologist. (laughs). Dr Cindy St. Hilaire:        Not yet. (laughs). Dr Norberto Gonzalez-Juarbe:    Eventually you'll get there. Dr Maryann Platt:           Yeah, eventually probably. But you know, there have been reports of lots of adverse cardiac events in SARS-CoV too. So it's certainly not just unique to influenza, as far as other types of double stranded RNA viruses. I'm not sure. Dr Norberto Gonzalez-Juarbe:    Yeah, of course Coxsackieviruses viruses have shown inductionof cardiac events. And there's a Review in the New England Journal of Medicine about some of these other pneumonia causing agents, but also all other pathogens that can do some of these events, but it's all clinical observations. So, we think that our study and several others studies that are starting to come out, can induce a shift part of field to look at how some of these major respiratory viruses can induce these adverse cardiac events that we see are highly prevalent, right after the event, like during infection. And importantly, how all the pathogens may synergize. Some pathogens such as RSB, flu, COVID, have synergized with bacteria or other virus one enhancing the ability of the other to cause injury and disease. Dr Norberto Gonzalez-Juarbe:    For example, flu with pneumococcal disease, COVID with assorted grand negative pathogens, and actually influenza also has been shown to cause co-infection. So we don't know how some of these pathogens may synergize in the lungs, but also in other organs, to cause these injury that are going to be long lasting. So we are having the acute problem now with COVID and we had this with the 2009 pandemic flu, but in the next 10 years, five years, we're going to see this equivalent of disease damage, the damage associated with the disease, and we are going to have to explain why people are having these cardiac events, why people are having kidney events or liver damage problem. So we need to better understand not only how RNA viruses do this, and there's actually data shows that COVID is present in the cardiac tissue and can replicate in cardiac cells, but also how they may synergize to potentiate these effects. And how can we prevent all of these from happening? By action, therapies to antivirals, or any other way. Dr Cindy St. Hilaire:        That's a perfect segue to my last question I had. And that is, how can, what you found in the study regarding necroptosis, or even just the base proteins that are involved, is it able to be leveraged either for the development of therapies or perhaps even like a screening method, a biomarker to determine which flu patients might go on to develop cardiac phenotypes? Dr Norberto Gonzalez-Juarbe:    There might be a couple of avenues our study can help create these adjunct therapeutics to anti-virals. So one might be targeting the specific necrotic cell pathways to prevent that titrating that is long-lasting and these can be targeting necroptosis or pyroptosis, and there's FDA approved drugs that we may be able to repurpose to target some of these pathways that have these secondary effects, that can target these pathways. But also the very interesting part for me was that MLKL lesion increased this protein called NNT, which is a major factor of mitochondrial function and ATP production. So if we can improve the ability of the heart function and to protect their mitochondria, then we probably can have more roughly protective response against not only flu, but maybe COVID or other viruses that might also do similar things to the heart. Dr Cindy St. Hilaire:         Or even just other heart failures. That's pretty neat. Dr Norberto Gonzalez-Juarbe:    Exactly. Dr Maryann Platt:           Yeah, exactly. Dr Cindy St. Hilaire:        That's great. Drs Gonzalez-Juarbe and Platt. Thank you so much for joining me today. Congratulations on an excellent study and I'm really looking forward to your future, probably viral related, work. Dr Norberto Gonzalez-Juarbe:    Thank you very much. Dr Maryann Platt:           Thanks. Dr Cindy St. Hilaire:        That's it for our highlights from the March 5th and 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 Circ. Thank you to our guests, Drs Norberto Gonzalez-Juarbe and Maryann Platt. The podcast is produced by Rebecca McTavish and Ashara Ratnayaka, edited by Melissa Stoner, and supported by the Editorial Team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire and this is Discover CircRes, your on-the-go source for the most exciting discoveries in basic cardiovascular research.  

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.19.390013v1?rss=1 Authors: Tabatabaee, M. s., Kerkovius, J. K., Menard, F. Abstract: In the brain, astrocytes undergo rapid morphological changes when stimulated by the excitatory neurotransmitter glutamate. We developed a chemical probe to monitor how glutamate affects the density and distribution of astrocytic L-type voltage-gated calcium channels (LTCC). The imaging probe FluoBar1 was created from a barbiturate ligand modified with a fluorescent coumarin moiety. The probe selectivity was examined with colocalization analyses of confocal fluorescence imaging in U118-MG and transfected COS-7 cells. Living cells treated with 50 nM FluoBar1 were imaged in real time to reveal changes in density and distribution of astrocytic LTCCs upon exposure to glutamate. The selectivity of the probe was demonstrated with immunoblotting and confocal imaging of immunostained cells expressing the CaV1.2 isoform of LTCCs proteins. Applying FluoBar1 to astrocyte model cells U118-MG allowed us to measure a 5-fold increase in fluorescence density of LTCCs upon glutamate exposure. The imaging probe FluoBar1 allows the real-time monitoring of LTCCs in living cells, revealing for first time that glutamate causes a rapid increase of LTCC membranar density in astrocyte model cells. It may help tackle previously intractable questions about LTCC dynamics in cellular events. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.18.253120v1?rss=1 Authors: Colon-Cruz, L., Rodriguez-Morales, R., Santana-Cruz, A., Castres-Velez, J., Torrado-Tapias, A., Yudowski, G., Kensler, R., Marie, B., Burgess, S. M., Renaud, O., Varshney, G. K., Behra, M. Abstract: The role of the cannabinoid receptor 2 (CNR2) is still poorly described in sensory epithelia. We found strong cnr2 expression in hair cells (HCs) of the inner ear and the lateral line (LL), a superficial sensory structure in fish. Next, we demonstrated that sensory synapses in HCs were severely perturbed in larvae lacking cnr2. Appearance and distribution of presynaptic ribbons and calcium channels (Cav1.3) were profoundly altered in mutant animals. Clustering of membrane-associated guanylate kinase (MAGUK) in post-synaptic densities (PSDs) was also heavily affected, suggesting a role for cnr2 for maintaining the sensory synapse. Furthermore, vesicular trafficking in HCs was strongly perturbed suggesting a retrograde action of the endocannabinoid system (ECs) via cnr2 that was modulating HC mechanotransduction. We found similar perturbations in retinal ribbon synapses. Finally, we showed that larval swimming behaviors after sound and light stimulations were significantly different in mutant animals. Thus, we propose that cnr2 is critical for the processing of sensory information in the developing larva Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.12.248120v1?rss=1 Authors: Lauffer, M., Wen, H., Myers, B., Plumb, A., Parker, K. L., Williams, A. J. Abstract: L-type voltage-gated calcium channels (LVGCCs) are important regulators of neuronal activity and are widely expressed throughout the brain. One of the major LVGCC isoforms in the brain is CaV1.3. Mice lacking CaV1.3 (CaV1.3 KO) have impairments in fear conditioning and depressive-like behaviors, which have been linked to the role of CaV1.3 in hippocampal and amygdala function. Genetic variation in CaV1.3 has been linked to a variety of psychiatric disorders, including autism and schizophrenia, which are associated with motor, learning, and social deficits. Here, we explored whether CaV1.3 plays a role in these behaviors. We found that CaV1.3 KO mice have deficits in rotarod learning despite normal locomotor function. Deletion of CaV1.3 is also associated with impaired associative learning on the Erasmus Ladder. We did not observe any impairments in CaV1.3 KO mice on assays of anxiety-like, depression-like, or social preference behaviors. Our results suggest an important role for CaV1.3 in neural circuits involved in motor learning and concur with previous data showing its involvement in associative learning. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.29.123695v1?rss=1 Authors: Tigaret, C. M., Lin, T.-C. E., Morrell, E., Sykes, L., O'Donovan, M. C., Owen, M. J., Wilkinson, L. S., Jones, M. W., Thomas, K. L., Hall, J. Abstract: Genetic variation in CACNA1C, which encodes the alpha-1 subunit of CaV1.2 L-type voltage-gated calcium channels, is strongly linked to risk for psychiatric disorders including schizophrenia and bipolar disorder. To translate genetics to neurobiological mechanisms and rational therapeutic targets, we investigated the impact of altered Cacna1c dosage on rat cognitive, synaptic and circuit phenotypes implicated by patient studies. We show that rats hemizygous for Cacna1c harbor marked impairments in learning to disregard non-salient stimuli, a behavioral change previously associated with psychosis. This behavioral deficit is accompanied by dys-coordinated network oscillations during learning, pathway-selective disruption of hippocampal synaptic plasticity, attenuated Ca2+ signaling in dendritic spines and decreased signaling through the Extracellular-signal Regulated Kinase (ERK) pathway. Activation of the ERK pathway by a small molecule agonist of TrkB/TrkC neurotrophin receptors rescued both behavioral and synaptic plasticity deficits in Cacna1c+/- rats. These results map a route through which genetic variation in CACNA1C can disrupt experience-dependent synaptic signaling and circuit activity, culminating in cognitive alterations associated with psychiatric disorders. Our findings highlight targeted activation of neurotrophin signaling pathways with BDNF mimetic drugs as a novel, genetically informed therapeutic approach for rescuing behavioral abnormalities in psychiatric disorder. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.13.093336v1?rss=1 Authors: Chang, J.-H., Greene, C., Futter, C., Nichols, B. J., Campbell, M., Turowski, P. Abstract: The blood-brain barrier (BBB) is a multifactorial and multicellular vascular interface separating the systemic environment from the central nervous system (CNS). It gates cerebral penetration of circulating molecules and cells and is the principal reason for low accumulation of many therapeutics in the brain. Low dose methamphetamine (METH) induces fluid phase transcytosis across the BBB in vitro and could therefore be used to enhance CNS drug delivery. Here we show, that low dose intravascular METH induced significant leakage exclusively via caveolar transport at the intact BBB in rodents ex vivo. Notably, METH-induced leakage was suppressed at 4C and in Caveolin-1 (CAV1) knockout mice. Furthermore, METH strongly enhanced brain penetration of therapeutic molecules, namely doxorubicin (DOX), a small chemotherapeutic agent, and aflibercept (AFL), a ca. 100 kDA recombinant protein. Lastly, METH improved the therapeutic efficacy of DOX in a mouse model of human glioblastoma (GBM), as measured by a 25% increase in median survival time (p = 0.0024). Collectively, our data indicated that METH can facilitate preclinical assessment of novel experimental treatments and has the potential to enhance drug delivery to the diseased CNS. Copy rights belong to original authors. Visit the link for more info

Johannes Hell and Manuel Navedo explain that modification of a particular serine residue affects the activity of the L-type calcium channel Cav1.2 in the brain and vasculature, but not in the heart.

Caveolin-1 (Cav1) is a scaffold protein and pathogen receptor in the mucosa of the gastrointestinal tract. Chronic infection of gastric epithelial cells by Helicobacter pylori (H. pylori) is a major risk factor for human gastric cancer (GC) where Cav1 is frequently down-regulated. However, the function of Cav1 in H. pylori infection and pathogenesis of GC remained unknown. We show here that Cav1-deficient mice, infected for 11 months with the CagA-delivery deficient H. pylori strain SS1, developed more severe gastritis and tissue damage, including loss of parietal cells and foveolar hyperplasia, and displayed lower colonisation of the gastric mucosa than wild-type B6129 littermates. Cav1-null mice showed enhanced infiltration of macrophages and B-cells and secretion of chemokines (RANTES) but had reduced levels of CD25+ regulatory T-cells. Cav1-deficient human GC cells (AGS), infected with the CagA-delivery proficient H. pylori strain G27, were more sensitive to CagA-related cytoskeletal stress morphologies ("humming bird") compared to AGS cells stably transfected with Cav1 (AGS/Cav1). Infection of AGS/Cav1 cells triggered the recruitment of p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1) to Cav1 and counteracted CagA-induced cytoskeletal rearrangements. In human GC cell lines (MKN45, N87) and mouse stomach tissue, H. pylori down-regulated endogenous expression of Cav1 independently of CagA. Mechanistically, H. pylori activated sterol-responsive element-binding protein-1 (SREBP1) to repress transcription of the human Cav1 gene from sterol-responsive elements (SREs) in the proximal Cav1 promoter. These data suggested a protective role of Cav1 against H. pylori-induced inflammation and tissue damage. We propose that H. pylori exploits down-regulation of Cav1 to subvert the host's immune response and to promote signalling of its virulence factors in host cells.

Wed, 6 Jul 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13312/ https://edoc.ub.uni-muenchen.de/13312/1/Domes_Katrin.pdf Domes, Katrin ddc:540, ddc:500, Fakultät fü

Thu, 12 Nov 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/10959/ https://edoc.ub.uni-muenchen.de/10959/1/Griessmeier_Kristina.pdf Grießmeier, Kristina ddc:540, ddc:500, Fakultät für Chem

The ability of neurons to regenerate in the adult mammalian central nervous system (CNS) is often poor, leading to persistent deficits after injury. Failure of axon regeneration in the CNS has been attributed to the presence of an extrinsic inhibitory environment and to an intrinsic limitation to support growth. Remarkably, in adult primary sensory neurons of the dorsal root ganglia (DRG), a peripheral lesion primes neurons to grow and to override the inhibitory environment. Under this condition not only their peripheral axons regrow, but also their injured central axons coursing in the spinal cord regenerate. However, the nature of the signal that is sensed by the cell upon peripheral lesion to initiate the regenerative response is poorly understood. This study started from the hypothesis that electrical silencing caused by peripheral deafferentiation is an important signal to trigger axon regrowth in adult DRG neurons. I first examined the effect of electrical activity on axon growth of cultured DRG neurons. I found that either chronic depolarization or electrical field stimulation strongly inhibits axon outgrowth in cultured DRG neurons. The inhibitory effect depends on Ca2+ influx through L-type voltage-gated calcium channels and involves transcriptional changes. Consistently, after a peripheral lesion, L-type current is diminished and the L-type pore-forming subunit Cav1.2 is downregulated. To determine whether the lack of L-type channels is sufficient to promote axon growth, mice lacking the pore-forming subunit of L-type channel, Cav1.2, in the nervous system were generated. Neurons isolated from adult Cav1.2 knockout (KO) mice grew more extensively than those from their control littermates. Taken together, these data provide evidence that electrical activity is a limiting factor for axon growth in adult DRG neurons and that releasing this “brake” is sufficient to induce axon growth. My results further suggest that electrical silencing might promote axon regeneration in vivo. Consequently, I have attempted to apply this knowledge to a model of spinal cord injury. However, these in vivo experiments have been so far hampered by technical limitations. Further endeavors are currently in progress.

Die glatte Muskulatur steuert eine Vielzahl wichtiger physiologischer Funktionen, wie beispielsweise den Blutdruck, die Magen-Darm-Motilität und die Harnblasen Entleerung. Die Kontraktion des glatten Muskel wird nach hormoneller Stimulation G-Protein-gekoppelter Rezeptoren vor allem durch L-Typ Ca2+-Kanäle vermittelt. Dies konnte mit Untersuchungen an genetisch veränderten Mauslinien, die glattmuskelspezifisch eine Deletion des Cav1.2 Ca2+-Kanals aufweisen (SMACKO Mäuse), belegt werden. Die Ansteuerung des L-Typ Ca2+-Kanals nach hormoneller Stimulation ist jedoch noch nicht vollständig geklärt. Das Ziel der vorliegenden Arbeit ist daher, den Kopplungsmechanismus sowie eventuell beteiligte Linkerproteine zwischen dem M3 Rezeptor und dem L-Typ Ca2+-Kanal in der Harnblase zu untersuchen. Die Experimente wurden sowohl an Harnblasen von Wildtyp und SMACKO Mäusen, als auch an Schweineharnblasen durchgeführt. Die funktionellen Untersuchungen legten eine cholinerge Modulation des Cav1.2 Ca2+-Kanal über die PKC nahe. So bewirkte der Einsatz von PKC- und Cav1.2 Ca2+-Kanal-Antagonisten eine Hemmung der cholinergen Kontraktion. Eine Aktivierung der PKC durch den Phorbolester PdBu hingegen führte zu einer Verstärkung der cholinergen Kontraktionsantwort mit einer gleichzeitigen Erhöhung des intrazellulären Ca2+-Signals. Den Beleg einer direkten Assoziation von PKC und Cav1.2 Ca2+-Kanal lieferten die biochemischen Untersuchungen. Die BN Page Versuche zeigten die Anwesenheit der PKC im Cav1.2 Proteinkomplex. Eine Mitfällung der PKC mit dem Cav1.2 Antikörper bestätigten die Hypothese einer direkten Interaktion beider Proteine. Die Phosphorylierungsversuche belegten, dass die α1C-Untereinheit und im Schwein auch die β3-Untereinheit des Cav1.2 Ca2+-Kanals verstärkt nach cholinerger Stimulation phosphoryliert werden. Die Ergebnisse zeigten die Beteiligung eines PKC/ Cav1.2 Signalkomplexes bei der cholinerg induzierten Kontraktion der Harnblase von Maus und Schwein.

Thu, 20 Dec 2007 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/7848/ https://edoc.ub.uni-muenchen.de/7848/1/Lemke_Toni.pdf Lemke, Toni Franziska ddc:500, ddc:54

Tue, 27 Nov 2007 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/7741/ https://edoc.ub.uni-muenchen.de/7741/1/Peter_Lenhardt.pdf Lenhardt, Peter ddc:

This study provides novel insights to the function and regulation of Cav1.4 LTCCs. In the first part of the sudy the basic biophysical and pharmacological properties of Cav1.4 have been characterized. To this end Cav1.4 was cloned from murine retinal cDNA. The full-length cDNA comprises 6111bp and contains an open reading frame encoding for a protein of 1984 amino acids. Cav1.4 was functionally expressed in HEK 293 cells. Like in the case of other LTCCs the coexpression of alpha2delta and beta subunits was necessary to get measurable currents. The electrophysiological properties of Cav1.4 found in patch clamp experiments distinguish these channels from other LTCCs. Activation kinetics were very fast, the activation threshold was relatively low and the time course of inactivation was extremely slow. Also the pharmacological properties were different from those of classical LTCCs. Cav1.4 channels show a much lower sensitivity for LTCC blockers compared to Cav1.2b channels. The most important findings of this study are the novel insights on the regulation of CDI. Surprisingly, no CDI was observed in Cav1.4 LTCCs in electrophysiological experiments. CDI is a negative feedback mechanism by which Ca2+ limits its own influx into the cell. This feedback inhibition is essential for many cell types to prevent excessive and potentially toxic Ca2+ levels and is widespread among HVA calcium channels. The sequences conferring CDI are conserved throughout the whole HVA calcium channel family and also in Cav1.4 raising the question of how this channel manages to switch off CDI. We identified an autoinhibitory domain in the distal C- terminus of Cav1.4 that serves to abolish CDI. This domain (ICDI, inhibitor of CDI) uncouples the molecular machinery conferring CDI from the inactivation gate by binding to the EF hand motif in the proximal C-terminus. Deletion of ICDI completely restores Ca2+-calmodulin mediated CDI in Cav1.4. CDI can be switched off again in the truncated Cav1.4 channel by coexpression of ICDI indicating that it works as an autonomous unit. Furthermore, replacement of the distal C-terminus in the Cav1.2b LTCC by the corresponding sequence of Cav1.4 is sufficient to block CDI. This finding suggests that autoinhibition of CDI can be principally introduced into other Ca2+ channel types. The novel mechanism described is also of great physiological impact. In vivo, Cav1.4 is expressed in photoreceptors and bipolar cells of the retina. In these cells the lack of CDI is of great physiological importance since it is required to generate a sustained Ca2+ influx and, hence, to mediate tonic glutamate release from synaptic terminals. Mutations in the gene coding for the Cav1.4alpha1 subunit in humans are linked to a disease called congenital stationary nightblindness type 2 (CSNB2). Some of these mutations lead to truncated channels nearly identical to channel mutants analyzed in this study that show CDI. Thus, the phenotype of these mutations can be explained by the recovery of CDI.