Cynthia St. Hilaire, PhD & Milka Koupenova, PhD
September 2019 Issue
This month on the Discover CircRes podcast, host Cindy St. Hilaire highlights five featured articles from recent issues of Circulation Research and talks with Matthew Stratton, Rushita Bagchi, and Tim McKinsey about their article on Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation. Article highlights: Vincentz, et al. HAND1 Enhancer Variation Impacts Heart Conduction Zhuang, et al. EC-Klf2-Foxp1-Nlrp3 Regulates Atherogenesis Quintanilla, et al. Robust Targets for Persistent AF Ablation Lambert et al. Characterization of Kcnk3-Mutated Rats Myagmar et al. Gq Mediates Cardioprotection Transcript Cindy St. H: Hi, welcome to Discover CircRes, the monthly podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St Hilaire, and I'm an assistant professor at the University of Pittsburgh. My goal as host of this podcast is to share with you highlights from recent articles published in the August 30th and September 13th issues of Circulation Research. We'll also have an in-depth conversation with Drs. Matthew Stratton, Rushita Bagchi, and Tim McKinsey, who are the lead authors of one of the exciting discoveries presented in the September 13th issue. Cindy St. H: The first article I want to share with you is titled, "Variation in a Left Ventricle–Specific Hand1 Enhancer Impairs GATA Transcription Factor Binding and Disrupts Conduction System Development and Function." The first author is Joshua Vincentz and the corresponding author is Anthony Firulli, and this work was conducted in the Departments of Pediatrics, Anatomy, and Medical and Molecular Genetics at Indiana Medical School in Indianapolis, Indiana. Cindy St. H: The heart's ventricular conduction system, or VCS, is composed of specialized muscle cells that propagate electrical signals through the working myocardium of the ventricles to coordinate the rhythmic contractions of the heart chambers. Disorders of the VCS can lead to certain types of arrhythmia. Genome-wide association studies have identified a number of single nucleotide polymorphisms, or SNPs, that appear to increase the risk of VCS-mediated arrhythmias. Two such SNPs are located in the upstream region of a gene encoding for Hand1. And Hand1 is a transcription factor that is involved in left ventricle development. Conditional cardiac Hand1 ablation during embryogenesis leads to ventricular septal defects and hyperplastic arterial ventricular valves, and a reduction in Hand1 expression could lead to morphological, and therefore functional defects. Vincentz and colleagues hypothesized that these SNPs might reside in an enhancer element, and that's a region of DNA and a promoter that allows for the increased expression of a gene. The region containing the SNPs is highly conserved from mammals to reptiles and includes two sequences that allow for the binding of GATA transcription factors. And GATA transcription factors are well known to drive cardiac development. So this team used CRISPR-Cas9 technology to show that the deletion of the enhancer impaired normal VCS morphology and therefore function. And they did this in a mouse model and in the in vitro electromobility shift assay (which frankly was one of my favorite love-to-hate experiments of my PhD). So this group did their own electromobility shift essay and showed that GATA-4 binds to these enhancer sites. And together, these results support a role for Hand1 in the formation and function of the VCS and offer insights to possible arrhythmia etiologies. And what I really love about this paper is that they could actually go from a SNP in a GWAS to a functional role of a protein, which is great. A lot of times with GWAS studies, you have no clue what the heck is going on. So this was a beautiful study where they actually could link a single nucleotide polymorphism to differential expression of a gene. Cindy St. H: The next article I'd like to highlight is titled, "Endothelial Foxp1 Suppresses Atherosclerosis via Modulation of Nlrp3 Inflammasome Activation." The first authors are Tao Zhuang and Jie Liu, and the corresponding authors (there's three of them) are Zhongmin Liu, Muredach Reilly, and Yuzhen Zhang. The Liu and Zhang teams are from the Key Laboratory of Arrhythmias of the Ministry of Education of China, and the Research Center for Translational Medicine at Shanghai East Hospital, which is part of Tongji University School of Medicine in Shanghai. And the Reilly team is from the Cardiology Division in the Department of Medicine and the Irving Institute for Clinical and Translational Research at Columbia University in New York, New York. And I have to say my good friend Rob Bauer is also a coauthor on this article. So Rob, I hope you're listening. Cindy St. H: Chronic inflammation contributes to atherosclerotic disease and is a major pathological mechanism contributing to the dysfunction of the vascular endothelium. So leukocytes, which are inflammatory cells that float around in your blood, leukocytes can adhere to the endothelial layer, and then they can migrate through the endothelial wall into the wall of the vasculature. And it's this activity, along with the uptake of oxidized LDL and the formation of a little fatty streak, that is the start of atherosclerosis. And now Zhuang and colleagues have identified that the transcription factor Foxp1 is a potential regulator of vascular endothelial health. So first they showed that while healthy arteries express Foxp1 robustly, atherosclerotic endothelium from both mice and humans exhibits reduced expression of this transcription factor. The team then generated atheroprone mice that either lacked Foxp1 or overexpressed Foxp1 specifically in the endothelium. The mice lacking Foxp1 were shown to have exacerbated athero with much larger plaque sizes and increased macrophage infiltration into the vessels, while overexpression of Foxp1 had largely the opposite effect. It actually curtailed progression of atherosclerotic disease. The team went on to examine the atherosclerosis-suppressing mechanism of Foxp1, showing that the factor suppressed expression of the inflammasome components in the endothelial cells. Cindy St. H: So all together, these results highlight that Foxp1-mediated regulation of the inflammasome is a potential targetable pathway for atherosclerotic treatments, and having a new targetable pathway is important, as the CANTOS trial, which provides proof of concept of the inflammation hypothesis of atherosclerosis in humans, showed robust effects in only a small subset of the population tested. Thus, there is a need to identify other means, a plan B if you will, by which we can control the inflammation that contributes to atherosclerosis. Cindy St. H: The next paper I want to highlight is titled, "Instantaneous Amplitude and Frequency Modulations Detect the Footprint of Rotational Activity and Reveal Stable Driver Regions as Targets for Persistent Atrial Fibrillation Ablation." The first author is Jorge Quintanilla, who is also a corresponding author alongside David Filgueiras-Rama, and they are from the National Center for Cardiovascular Research and the Center for Biomedical Research in Cardiovascular Diseases Network in Madrid, Spain. Uncoordinated contractions of the atria to the ventricles of the heart is called atrial fibrillation, or AFib, and AFib causes symptoms such as heart palpitations, dizziness, and taken to the extreme, AFib can actually cause death. To correct such rhythm problems, doctors can ablate certain regions of the heart suspected to be driving this misfiring. In an ablation procedure, a catheter is inserted through the blood vessels and into the heart. An electrophysiologist then identifies the locations of the heart that are sending abnormal electrical impulses, and with either delivery of tiny pulses of painless, low-level energy or using a catheter that has a cold tip to freeze the misfiring areas, the electrophysiologist can ablate and hopefully stop AFib. The problem is that this approach often fails, and AFib still occurs or can reoccur after a length of time. So Quintanilla and colleagues wanted to develop a more personalized medicine approach to treating AFib. So to do this, they wanted to make it something simple, something affordable, and something that hospitals currently have access to. So they used the standard electroanatomical mapping system to track the amplitude and also the frequency modulations of the electrical signals from the hearts with AFib. And they found that regions with high and stable instantaneous frequency signals were the drivers of fibrillation in the hearts. When these regions were ablated in pigs with persistent AFib, the misfiring stopped in almost all cases and was sustained. The team went on to test the system in three patients with Afib, and two of the three remained arrhythmia-free without drugs for at least 16 months. So with further development and testing, this frequency mapping could potentially replace systems that are currently in use, and more importantly, this could provide a more accurate and patient-tailored way to find and ablate the drivers of AFib. Cindy St. H: The next paper I want to highlight is titled, "Characterization of Kcnk3-Mutated Rat, a Novel Model of Pulmonary Hypertension." Oh, now that was a nice title. That was nice and short. The first author is Mélanie Lambert, and the corresponding author is Fabrice Antigny, and they are from the INSERM Hôpital Marie Lannelongue in Le Plessis Robinson, France. Cindy St. H: Pulmonary hypertension is a rare but life-threatening condition where the adverse remodeling of the pulmonary arteries causes an increase in the blood pressure that's needed to push the blood through the lungs, and this high blood pressure causes the heart to work harder, and it leads ultimately to right ventricular hypertrophy and heart failure. So genome-wide association studies have identified a number of mutations that have been linked to pulmonary hypertension and these include several loss-of-function mutations in the gene encoding for a potassium channel, and that's a protein that can release potassium from a cell to the extracellular environment. And the particular one that has been found to be mutated in pulmonary hypertension patients is Kcnk3. And this channel regulates the resting membrane potential of pulmonary artery smooth muscle cells. To date, it is not known how the loss of Kcnk3 contributes to pulmonary hypertension. To start to unravel this mystery, Lambert and colleagues created a full-body knockout of Kcnk3 in rats, and they used rats because that's a much more robust model for studying pulmonary hypertension than some of the murine models available. These knockout animals exhibited an increased pulmonary artery pressure. They also had faster heart rates and they were more susceptible than their wild-type counterparts to both pharmacological or hypoxia-induced pulmonary hypertension. These Kcnk3 knockout rats also had evidence of remodeled pulmonary vasculature, and this vasculature showed signs of endothelial dysfunction, altered vaso transcription, and altered neomuscularization. In in vitro studies, they used pulmonary artery smooth muscle cells that they isolated from these knockout rats, and these cells showed increased activation of proliferation markers, which is another signature of pulmonary hypertension. And this was also mirrored in human pulmonary artery smooth muscle cells that were treated with a Kcnk3 inhibitor. So together, this work starts to uncover the role of Kcnk3 in pulmonary hypertension pathogenesis. And it also provides the field with a novel model system from which people can learn more about the role of membrane potential of pulmonary artery smooth muscle cells in pulmonary hypertension. Cindy St. H: The last paper I want to highlight before our interview is titled, "Coupling to Gq Signaling Is Required for Cardioprotection by an Alpha-1A-Adrenergic Receptor Agonist." The first author is Bat-Erdene Myagmar, and the corresponding author is Paul Simpson from the VA Medical Center in San Francisco, California. So like their name says, G-protein coupled receptors interact with G-protein subunits to propagate the signal when a ligand binds. The protein G alpha q has long been considered a key mediator of cardiac hypertrophy. And that's because in mice, when this Gq protein was overexpressed, it induced hypertrophy, myocardial apoptosis, and contractile failure. However, this sub unit Gq can interact with a multitude of G-protein coupled receptors that themselves bind a variety of ligands. So which receptor or which signaling pathway specifically is responsible for the hypertrophic phenotype? Recent studies by others had shown that stimulation of the alpha-1A adrenergic receptor prevents cardiotoxicity and heart failure. So Myagmar and colleagues asked whether this cardio-protective alpha-1A stimulation is dependent on the alpha q subunit. So using mice with a mutant version of alpha-1A that allows the binding of the ligand but does not couple with the Gq subunit, the team found that alpha-1A induced cardioprotection was absent. The mutant animals were more likely to die than their wild-type counterparts when hypertrophy was induced pharmacologically or surgically. And furthermore, in the mutant myocytes themselves, the group observed that alpha-1A induced ERK signaling, which is essential for the receptors cardioprotective activity, was impaired. So together these results showed that alpha-1A-induced cardioprotection is dependent on alpha q, and actually it showed that alpha q signaling is not always maladaptive. Cindy St. H: Now we're going to move to our interview with Drs. Matthew Stratton, Rushita Bagchi and Tim McKinsey and we're going to talk about their great paper titled "Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation." Cindy St. H: Okay, so now we're going to have our interview with Drs. Stratton, Bagchi, and McKinsey on their paper titled, "Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation." So welcome, everyone. Dr Tim M: Thank you. Dr Rushita B: Thank you. Dr Matt S: Thank you. Cindy St. H: I was wondering if you could just all maybe go around and introduce yourselves. Dr Tim M: Sure. I'm Tim McKinsey. I'm a professor in the Division of Cardiology at the University of Colorado Anschutz Medical Campus. I also direct a newly formed fibrosis center on campus. It's called the CFReT, the Consortium for Fibrosis Research and Translation, and our goal is to understand new mechanisms that regulate fibrosis and develop new therapies to treat scarring, or fibrosis, in organs. Dr Rushita B: I'm Rushita Bagchi. I'm currently a postdoctoral fellow in Dr McKinsey's lab. I grew up in India, and that's where I did my undergrad and master's degrees. Then I moved to Canada to do my PhD focusing on transcriptional regulation of cardiac fibrosis under the supervision of Dr Michael Czubryt. After that, I transitioned to Dr McKinsey's lab here in Denver to enhance or add to my expertise of transcription by studying epigenetics, and especially trying to find the underlying mechanisms that cause cardiovascular disease. The nice thing about this position for me has been that I have been able to constantly build up on my experience studying tissue fibrosis, but at the same time, Tim has been very generous and has let me develop projects of my own as well. Cindy St. H: You're lucky. That's awesome. Thank you for joining us. And Dr Stratton. Dr Matt S: I'm Matt Stratton. I'm an assistant professor in the Department of Physiology and Cell Biology at Ohio State University. I did my graduate training at Colorado State University in neurodevelopment and neuroendocrinology and then moved to Tim's lab for a postdoc and assumed my current position this past December. Cindy St. H: Wow. How's it going? Dr Matt S: It's going well. Starting a lab is a lot of fun and a lot of stuff going on. Cindy St. H: Yeah, I'm four years in now and at the same time you feel brand new and excited and then, oh my God, what am I doing? So that's great. Well thank you all for joining me. So I really like this paper, mostly because I'm also a vascular biologist. I kind of focus more on the heart valves, but I have a real interest in cell phenotype transitioning and cell shifting, and so when you started to talk about chromatin remodeling and bromodomain protein, I was really interested and wanted to hear more. So maybe we can start by telling everyone what is the clinical need that your paper at base is trying to address? Dr Tim M: Well before we get into that, could I start by saying that we're honored to have our work published in Circulation Research. We're really grateful for that. I also want to point out that this is the result of a very detailed collaborative effort involving at least six other labs, including the labs of Charles Lan at Baylor College of Medicine, Jun Qi at the Dana-Farber Cancer Institute, Kunhua Song and Maggie Lam here in Colorado, as well as Sap Haldar and Deepak Srivastava at the Gladstone Institutes in San Francisco. Without this collaborative effort, none of this would have been possible. Cindy St. H: That's great to hear and I'm really happy you mentioned that. Team science is so important, and I feel like we almost can't get these big, groundbreaking papers unless we really work as a good team, so thank you for highlighting that. Dr Tim M: So we're really interested in fibrosis, which is a hallmark of heart failure. Fibrosis can actually be a good thing for the heart. If you have a myocardial infarction, you need a strong scar to form to prevent the ventricle from rupturing. But in response to chronic stress like hypertension and other things, you can get this longstanding fibrosis that results in cardiac dysfunction. That's because fibrosis is essentially a scarring process and one of the things that that does is to create a stiff in the left ventricle that can't relax effectively. Unfortunately, despite the well-known roles of fibrosis in cardiac disease, there are no targeted anti-fibrotic therapies for the heart, and that's really our focus in the lab. We've had a long-standing interest in epigenetic regulation of heart failure and cardiac fibrosis, and we've known for some time that inhibitors have a family of epigenetic reader proteins called the bromodomain and extraterminal proteins, the BET proteins. Inhibitors of those BET proteins can block cardiac fibrosis in rodent models and improve cardiac function. What we knew going into this work is that systemic delivery of those compounds was efficacious. But as you know, the heart is made up of many different cell types. So we really wanted to understand if the efficacy of these compounds was related to effects in resident cardiac fibroblasts. Cindy St. H: Excellent. So what is the role of a cardiac fibroblast in a healthy cell, and where does that go awry? Dr Matt S: So in a undiseased heart, fibroblasts are necessary to provide structure, right? They lay down the extracellular matrix that really holds the heart together. Without them, you would not have a good pump function. Where they go awry, I mean, that's one of the things that we're trying to study, right? They become proliferative, they become contractile, and they secrete, or we call them super-secretors, of extracellular matrix. So TGF-beta is really a known signaling molecule that kicks the fibroblasts into this activated or myofibroblast state. We use that in the paper as a agonist for our cultured cells. Cindy St. H: Great, thank you. So what was the hypothesis you were testing in this paper? Dr Matt S: So what we wanted to know, if BRD4 and BET proteins are important for this activation of cardiac fibroblasts? So going from a quiescent fibroblast to a proliferative and super-secretor of extracellular matrix fibroblast in the heart. And those experiments hit right away. I mean, we did those experiments, and it was quite dramatic that if you use JQ1 to inhibit these BET proteins, you completely blocked this myofibroblast differentiation. We went in and did some siRNA and shRNA work to show that really BRD4 appears to be the main culprit of the BET protein families. Cindy St. H: Rushita, could you tell us a little bit about what a bromodomain protein is and what maybe specifically BRD4 is in relation to the other bromodomain proteins? Dr Rushita B: Sure. So when we talk about the chromatin, there are various players in there that are known as, in general, chromatin modifiers. So you have enzymes that add acetylation mark on lysine residues on histone tails, which is basically DNA is wound around these histones and those histones have lysine tails, but you have the big acetyl group sitting. Now when you have this acetyl group sitting, this makes it more accessible for the transcriptional machinery and allowing gene transcription to happen. Those enzymes are known as histone acetyltransferase, the ones that add the acetyl mark there. The ones that take it away, which is what our lab has been studying for a long time, and Tim is a known world expert in the field, those are known as histone deacetylases, or HDACs, which basically remove those acetyl marks and compact the chromatin, thereby suppressing gene expression. This BET proteins or bromodomains are transcriptional coactivators. So this bromodomain is actually in charge or takes up the duty of identifying these acetyl marks on the lysine residues and therefore, tells the transcription machinery to come in and allow gene transcription to happen. There are a few BET proteins. Of them, BRD4 has been studied extensively in cancer as well as in the heart. But as Tim mentioned, the role of BRD4 has been studied vastly in the heart in terms of the cardiac myocytes, but not so much in the non-myocyte population, which is where our work stands out really well and starts highlighting the role of this specific chromatin modifier protein in activation or control of profibrotic gene expression. Cindy St. H: Yeah. So correct me if I'm wrong, but my understanding is, you're going to need a little bit of the cardiac fibroblasts remodeling in the early phase. But where it is really detrimental is when that overcompensates and overproliferates and throws down too much matrix and then is bad. So do you see your study as a way to kind of target that window of where a potential treatment might be applicable? Dr Tim M: Yeah, we think that BRD4 is a nodal regulator of cardiac fibrosis and therefore, an excellent therapeutic target. The challenge will be developing selective BRD4 inhibitors that are safe, as well as effective. We know that BRD4 is not only expressed in cardiac fibroblasts-it's all over the body. But we think our work provides an entry point to the development of highly selective BRD4 inhibitors for fibrotic indications, including heart failure. Cindy St. H: So that's one of the things I was wondering, how specific your drug is to BRD4 versus the other ones, but also you mentioned the myofibroblast versus the immune cells infiltrating the heart. Do we know what BRD4 is doing in those cells in this system? Dr Tim M: BRD4 is definitely pro-inflammatory, and BET protein inhibitors like JQ1 are anti-inflammatory, that's for sure. Interestingly, there's a BET family inhibitor called Apabetalone, RVX-208, that's in Phase III clinical testing for people with atherosclerosis. So if that's successful, it will provide proof of concept that you can target this family of epigenetic readers to treat cardiovascular disease. I also wanted to point out that JQ1 was initially discovered by Jay Bradner's lab, in particular Jun Qi, who is a coauthor and collaborator on this paper. Cindy St. H: Oh, very nice. Okay, good conflict of interest too, I guess. So maybe you guys can talk a little bit about how you managed to get this huge team of scientists together efficiently, and what were any hang-ups? Matt is laughing a bit, but you two are the lead authors, Matthew and Rushita. How did you two kind of lead the way on this and divvy up this huge project? Dr Matt S: So it is definitely a project management-style approach I think you have to take. I mean, there's a lot of communication, really a lot of communication with bioinformatics, analysts, and getting the right sequencing done, and that was fun, but it took a lot of effort. And once you get this big data, how do you present it in an intelligible story and how do you pick things out that may lead to new discoveries, right? So we highlight Sertad4 in here as a gene that's very much BRD4-dependent. And I think this is a proof of concept for using this genomics, and particularly BRD4, as kind of a molecular string to pull on to unwind this puzzle. So that was a lot of fun. And you know, Rushita was super awesome in helping with this project. Dr Rushita B: Yeah, I think having stared at cardiac fibroblasts for six years during my PhD definitely gave me the confidence that I could step up to the plate and deliver what was necessary. And like Matt said, there was a lot of omics-based stuff that we did in the paper. And that is actually one of the key highlights, because we see papers or manuscripts that are published that have RNA-Seq, ChIP-Seq and proteomics, but I believe the strength in our article is the combination of all three. So we were actually able to do overlapping ChIP-Seq and RNA-Seq experiments, and then there was proteomics involved. So we are looking at it at the genomic transcriptome and protium-wide changes that are happening all together, put in one manuscript. And the beauty of this work is it has now created data sets that people can mine and get more information out of. And this is something that will definitely continue to drive our future studies in the lab as well. Cindy St. H: Can you maybe expand on that? Could you maybe describe briefly for the audience what ChIP-Seq is and what RNA-Seq is, and really the power that is created when you can couple those techniques with the same samples? Dr Matt S: Sure. So BRD4 was the center point of the paper, right? So we did BRD4 ChIP-Seq and RNA Pol II ChIP-Seq in fibroblasts treated with TGF-beta or not. So in ChIP-Seq, you basically immunoprecipitated your target protein, and that brings with it, if it's bound to chromatin, that brings with it the DNA that it's bound to. And then you can sequence the DNA that comes out of your immunoprecipitation and map that to the genome, and you get a very nice picture of where is BRD4 enriched, and where does it go after stimulation like TGF-beta, when the fibroblast becomes a myofibroblast. So you can line all these up and you can pick out what gene changes we think are directly dependent on BRD4. That's something that we like, because we now know that BRD4 is a good target, right, so that kind of pulls it together. Cindy St. H: Great. Thank you. What else do you want to bring up? Dr Matt S: I think understanding how signals get translated to changes in gene expression is obviously something that the field is very much interested in. And because BRD4 is basically a step away from RNA polymerase II, it gives you a little bit more specificity in knowing that that's a disease-activated pathway, right? So trying to figure out what directs BRD4 to new locations in the chromatin and cause it to be removed from previous locations in the chromatin is really an interesting area of research. So we did a pathway screen basically using inhibitors, and we use Sertad4 as the readout, right. And we found that a p38 inhibitor was able to block the ability of TGF-beta to induce Sertad4. And we were able then to show that p38 had a role in targeting BRD4 specifically to the Sertad4 locus. Dr Tim M: I wanted to say, you know, one of the challenges with this project is that fibroblasts are difficult to work with. You would think that they would be easier to work with than a myocyte. But when a fibroblast hits a plastic cell culture dish, it rapidly transforms into an activated cell, because that plastic has a very high tensile strength. So it took a lot of optimization to figure out methods to culture these cells to maintain them in a quiescent state. Cindy St. H: What did you do? What was that trick? Dr Tim M: I mean, it involves changing cell density, changing the constituents of the medium, and doing other things. Cindy St. H: Science magic. Dr Tim M: Yeah. Dr Rushita B: And I'll just add to that. The nice thing about being able to contribute to a study like this is also that, like Tim said, fibroblasts, they change phenotype rapidly. You take them out of a biological system, whether it's a heart or any other tissue, you plate them out in cell culture, they start changing. The nice thing about the in vivo study, the RNA-Seq that was done using the in vivo study with JQ1, was that we used a very simple pressure overload model known as the TAC model, which is a very well-established and accepted model worldwide in the field of cardiovascular disease, treating animals with JQ1. So we isolated fibroblasts, but the time from the isolation of cells to the time an RNA was prepared was an hour or two. So we made sure that we minimally exposed them to culture conditions in the lab, so we retained their biology. So what we did on plastic dishes before, although they were plated on plastic, and we had RNA-Seq done on those cells, like Tim said, we did optimize the conditions. And then being able to similarly treat or use the cells that come from an animal directly and both of them contributing to a similar cohort of genes or pathways that we can look at, that has definitely given immense strength to this manuscript. Cindy St. H: And that's why it's in Circ Research, so it's a beautiful paper. Very well done. So I can't imagine all these hearts that you had to isolate and get single cells of and culture. What kind of days were you pulling? What was the actual boots on the ground of getting this done? How did that work? Dr Tim M: It wasn't uncommon for me to get emails from Matt and Rushita at very odd hours of the night or early in the morning. Dr Rushita B: Yeah, it was like we had the animals being sacrificed, hearts taken, and running to the cell culture room to do everything under sterile conditions. Most important thing- I think what worked out really well is we made sure we had all the reagents prepared ahead of time, so that once the heart is out, it's weighed, because we were also looking at hypertrophy because of the TAC model. We weighed the heart and it goes into your BST right away. Cindy St. H: I try to teach that to my lab. It's like the cooking idea of mise en place. I make them lay out everything in the cell culture hood ahead of time, and it's all in the order and you just boom, boom, boom, boom. Dr Rushita B: And a lot of our experiments were done later in the evening, so the nice thing was we had access to multiple centrifuges, which is usually a huge plus. And I still remember Matt being on one side, I'd be on the other. And then we had help from members of the lab as well. They were running between the cell culture room and the centrifuge. So it was actually quite fun. It turned out really well. Cindy St. H: I'm picturing like those old water brigades to put out a fire where like a bucket is just passed. Is that what this was? Dr Rushita B: That was very similar to the situation you just talked about. Cindy St. H: That's great. It sounds kind of painful, but also kind of fun. I guess lastly, maybe one of you can end with telling us what are the bigger picture results of this, and what are the next steps in terms of maybe possibly translating this to the clinic? Dr Tim M: Well as I mentioned, one of the things we're trying to do is to selectively inhibit BRD4. We're also trying to inhibit it only in cardiac fibroblasts with the hope that we'll be able to improve the therapeutic index of BRD4 inhibition. So create a situation where patients can tolerate this anti-fibrotic therapy better than if it was delivered systemically. We're also looking at other regions of BRD4. BRD4 contains the bromodomains, and those are the targets of JQ1, but there are other interesting domains on BRD4 that we're actively pursuing. Cindy St. H: Thank you. And Matthew, what are you doing in your new lab, or is it just set up right now? Dr Matt S: Well I have a K Award from the National Institute of Aging. Cindy St. H: Congratulations. Dr Matt S: Thank you. To look at BRD4's role in the heart and cardiac aging. And I also have a couple projects based on some of the mining that we've done from these datasets. So hopefully those lead to good publications and follow-on grants. Cindy St. H: Well, if this is a good start, I'm sure they will. And Rushita, what are your next plans? How long have you been with Tim? Dr Rushita B: So I've been here with Tim for almost four years now, so I'm pretty much in the final leg of my postdoctoral training. So I'm still continuing to work on tissue fibrosis projects, including the heart. But I have been able to develop a new field of interest and something that Tim has entrusted me to carry on in the lab in the field of cardiometabolic disease, but definitely with an epigenetic focus. So hopefully in a year's time I see myself having an independent academic scientist position. My dream job will be to be at an academic institute where I can lead a research team which focuses on deciphering or trying to even find the most basic molecules that define the underlying mechanisms of tissue fibrosis and cardiometabolic disease. Cindy St. H: That sounds like a great plan. Very best luck to you. Dr Rushita B: Thank you. Cindy St. H: Do you guys want to add anything else? Dr Tim M: The field of cardiovascular epigenetics is in its infancy and we still have a lot to learn. Cindy St. H: And I'm sure all of you will do your parts in moving that field forward. So with that, we're going to end our interview with Drs. Stratton, Bagchi, and McKinsey. Thank you all for joining me and thank you to the listeners for listening. Have a great day. Dr Tim M: Thank you. Dr Rushita B: Thank you. Dr Matt S: Thank you. Cindy St. H: That's it for highlights from the August 30th and September 13th issues of Circulation Research. Thank you for listening. This podcast is produced by Rebecca McTavish, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Copy text for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St Hilaire, and this is Discover CircRes, your source for the most up-to-date and exciting discoveries in basic cardiovascular research.
Duration: 37 min