Mar 17, 2022
This month on Episode 34 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the March 4 and March 18th issues of Circulation Research. This episode also features a conversation with Dr Mireille Ouimet and Sabrina Robichaud from the University of Ottawa Heart Institute to discuss their study, Autophagy is Differentially Regulated in Leukocyte and Non-Leukocyte Foam Cells During Atherosclerosis.
Article highlights:
Pauza, et al. GLP1R in CB Suppress Chemoreflex-Mediated SNA
Lim, et al. IL11 in Marfan Syndrome
Hohl, et al. Renal Denervation Prevents Atrial Remodeling in CKD
Liu, et al. Smooth Muscle Cell YAP Promotes Arterial Stiffness
Cindy St. Hilaire: Hi and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to be highlighting articles from our March issues of Circulation Research. I'm also going to speak with Dr Mireille Ouimet and Sabrina Robichaud from the University of Ottawa Heart Institute, and they're with me to discuss their study, Autophagy is Differentially Regulated in Leukocyte and Non-Leukocyte Foam Cells During Atherosclerosis.
The first article I want to share is titled GLP1R Attenuates Sympathetic Response to High Glucose via Carotid Body Inhibition. The first author is Audrys Pauza, and the corresponding authors are Julian Paton and David Murphy at the University of Bristol.
Cindy St. Hilaire: Hypertension and diabetes are risk factors for cardiovascular disease. And yet, for many patients with these two conditions, lowering blood pressure and blood sugar is insufficient for eliminating the risk. The carotid body is a cluster of sensory cells in the carotid artery, and it regulates sympathetic nerve activity. Because hypertension and diabetes are linked to increased sympathetic nerve activation, this group investigated the role of the carotid body in these disease states. They performed a transcriptome analysis of crowded body tissue, from rats with and without spontaneous hypertension. And they found among many differentially-expressed genes that the transcript encoding glucagon-like peptide-1 receptor or GLP1R, was considerably less abundant in hypertensive animals.
Cindy St. Hilaire: This was of particular interest because the gut hormone GLP-1 promotes insulin secretion and tends to be suppressed in Type 2 diabetes. Moreover, GLP1R agonists are already used as diabetic treatments. This group showed that treating rat carotid body with GLP1R agonist suppresses sympathetic nerve activation and arterial blood pressure, suggesting that these drugs may provide benefits in more than one way. Perhaps the carotid body could be a novel target for lowering cardiovascular disease risk in metabolic syndrome.
Cindy St. Hilaire: The second article I want to share is titled Inhibition of IL11 Signaling Reduces Aortic Pathology in Murine Marfan syndrome. The first author is Wei-Wen Lim, and the corresponding author is Stuart Cook and they're from the National Heart Center in Singapore. People with the genetic connective tissue disorder Marfan syndrome, are typically tall and thin with long limbs and are prone to skeletal, eye and cardiovascular problems, including a life-threatening weakening of the aorta. While Marfan syndrome patients commonly take blood pressure-lowering treatments to minimize risk of aortic aneurysm and dissection, there's currently no cure for Marfan syndrome or targeted therapy.
Cindy St. Hilaire: The cytokine IL11 is strongly induced in vascular smooth muscle cells upon treatment with the growth factor TGF-beta, which is over activated in Marfan syndrome patients. And TGF-beta is also considered a key feature of the syndrome’s molecular pathology. This study found that IL11 is strongly upregulated in the aortas of Marfan syndrome model mouse, and that genetically eliminating IL11 in these animals protected them against aortic dilation, fibrosis, inflammation, elastin degradation and loss of smooth muscle cells. Treating Marfan syndrome mice with anti-IL11 neutralizing antibodies exhibited the same beneficial effects. These results suggest that perhaps inhibiting IL11’s activity could be a novel approach for protecting the aortas of Marfan syndrome patients.
Cindy St. Hilaire: The next article I want to mention is titled Renal Denervation Prevents Atrial Arrhythmogenic Substrate Development in Chronic Kidney Disease. The first authors are, Mathias Hohl, Simina-Ramona Selejan and Jan Wintrich, and the corresponding authors also Mathias Hohl, and they're from Saarland University. People with chronic kidney disease have a two to three fold higher risk than the general population of developing atrial fibrillation, which is a common form of arrhythmia that can be life-threatening. Chronic kidney disease is associated with activation of the sympathetic nervous system, which can be damaging to the heart. Thus, this group examined myocardial tissues from atrial fibrillation patients with and without chronic kidney disease to see how they differ. They found that atrial fibrosis was more pronounced in patients with both conditions than in patients with atrial fibrillation alone, suggesting that chronic kidney disease perhaps exacerbates or even drives arterial remodeling.
Cindy St. Hilaire: Sure enough, induction of chronic kidney disease in rats led to greater atrial fibrosis and incidence of atrial fibrillation than seen in the control animals. Renal denervation is a treatment in which the sympathetic nerves are ablated, and it's a medical procedure that's used for treating uncontrolled hypertension, and it has also been shown in animals to reduce atrial fibrillation. Performing renal denervation in the rats with chronic kidney disease reduced atrial fibrosis and atrial fibrillation susceptibility. This study not only shows that chronic kidney disease induces atrial fibrosis and in turn atrial fibrillation, but also suggests that renal denervation may be used in chronic kidney disease patients to break this pathological link and prevent potentially deadly arrhythmias.
Cindy St. Hilaire: The last article I want to highlight is titled YAP Targets the TGFβ Pathway to Mediate High-Fat/High-Sucrose Diet-Induced Arterial Stiffness. First author is Yanan Liu and the corresponding author is Ding Ai from Tianjin Medical University. Metabolic syndrome is characterized as a collection of conditions that increase the risk of cardiovascular diseases, such as obesity, hypertension and diabetes. Among the tissue pathologies associated with metabolic syndrome is arterial stiffness, which itself is a predictor of cardiovascular disease incidence and mortality. To specifically investigate how arterial stiffness develops in metabolic syndrome, this group fed mice a high-fat, high-sugar diet, which is known to induce metabolic syndrome and concomitant arterial stiffness.
Cindy St. Hilaire: After two weeks on the diet, the animals’ aorta has exhibited significant upregulation of TGF-beta signaling, which is a pathway known for its role in tissue fibrosis, and the aorta has also exhibited increased levels of yes-associated protein, or YAP, which has previously been implicated in vascular remodeling, collagen deposition and inflammation. YAP gain and loss of function experiments in transgenic mice revealed that while knockdown of protein in the animals’ smooth muscle cells attenuated arterial stiffness, increased expression exacerbated the condition.
Cindy St. Hilaire: The team went on to show that YAP interacted with and prevented the activation of PPM-1 B, which is a phosphatase that normally inhibits TGF-beta signaling and thus fibrosis. Together the results suggest that targeting the YAP, PPM-1 B pathway, could be a strategy for reducing arterial stiffness and associated cardiovascular disease risk in metabolic syndrome.
Cindy St. Hilaire: Today, Sabrina Robichaud and Dr Mireille Ouimet from University of Ottawa Heart Institute are with me to discuss their study Autophagy is Differentially Regulated in Leukocyte and Non-Leukocyte Foam Cells During Atherosclerosis, which is in our March 18 issue of Circulation Research. So thank you both for joining me today.
Sabrina Robichaud: Thank you so much for having us. It's a pleasure.
Mireille Ouimet: Thank you for having us.
Cindy St. Hilaire: Yeah, and congrats on the study. So we know that LDL particles contain cholesterol and fats, and these are the initiating factors in atherosclerosis. And it's also really now appreciated that inflammation in the vessel wall is a secondary consequence to this lipid accumulation. Macrophages are an immune cell that, in the context of the plaque, gobble up this cholesterol to the point that they become laden with lipids and exhibit this foamy appearance, which we now call foam cells. And these foam cells can exhibit atheroprotective properties, one of them called reverse cholesterol transport, and that's really one of the focuses of your paper. So before we dig into what your paper is all about, could you give us a little bit of background about what reverse cholesterol transport is in the context of the atherosclerotic plaque? And maybe introduce how it links to this cellular recycling program, autophagy, which is also a big feature of your study.
Mireille Ouimet: Yes, so the reverse cholesterol transport pathway is a pathway that's very highly anti-atherogenic. It's linked to HDL function and the HDL protective effects, in that HDL can serve as a cholesterol acceptor for any excess cholesterol from arterial cells or other cells of the body and return this excess cholesterol to the liver for excretion into the feces. There is also trans-intestinal cholesterol efflux that can help eliminate any excess bodily cholesterol.
Mireille Ouimet: So reverse cholesterol transport is a way that we can eliminate excess cholesterol from foam cells in the vascular wall, and that's why we're really interested in the process. But the rate-limiting step of cholesterol efflux out of foam cells in plaques is actually, they have to be mobilized in the form of free cholesterol to be pumped out of the cells through the action of the ATP-binding cassette transporters. And so the rate-limiting step of the process is the hydrolysis of the cholesterol esters and the lipid droplets, because that's where the excess cholesterol is stored in foam cells.
Mireille Ouimet: And so for years, people investigated the actions of cytosol like lipases in mobilizing free cholesterol from lipid droplets, although the identity of those lipases are not well-known and in macrophage themselves, but our recent work showed a role for autophagy in the catabolism of lipid droplets. And in fact, in macrophage foam cells, 50% of lipid droplet hydrolysis is attributable to autophagy while the other half is mediated by neutral lipases, which makes it really important to investigate the mechanisms of autophagy-mediated lipid droplet catabolism.
Cindy St. Hilaire: That is so interesting. I guess I didn't realize it was that significant a component in that kind of rate-limiting step. That's so cool. So really, a lot of the cholesterol efflux studies, and maybe this is just limited to my knowledge of a lot of these cholesterol efflux studies, but to my knowledge, it's been really focused on the foam cell itself, the macrophage foam cell. However, there's been a lot of recent work that has now implicated vascular smooth muscle cells in this process. So could you share some of the research specific to smooth muscle cells and smooth muscle-derived foam cells that led you to want to investigate the contributions of smooth muscle cell-derived foam cells in cholesterol efflux?
Mireille Ouimet: Yeah, so you're right in the sense that macrophages have always been the culprit foam cells in the atherosclerotic plaques but pioneering work from several groups, including Edward Fisher and Gordon Francis, they've shown that the smooth muscle cells can actually acquire a macrophage-like phenotype becoming lipid-loaded and foamy. And there's been work specifically looking at the ABC transporters, and their ability to efflux cholesterol from these vascular smooth muscle cell-derived foam cells, because as they trans-differentiate into macrophage-like cells, they acquire the expression of ABCA1, but this is to a lower extent, as compared to their macrophage counterparts.
Mireille Ouimet: And the efflux is defective because there's an impairment in liposomal cholesterol processing of the lipoproteins that's really important to activate a like cell, and the expression of the ABC transporters, so vascular smooth muscle cell-derived foam cells are very poor effluxes.
Sabrina Robichaud: There's very few studies that look at the vascular smooth muscle cell foam cells, and the very few that did look at it mostly focused on the ABCA1 transporters, and did show that they were poor effluxes. And as we all know, ABC1 is not the only cholesterol transporters that can transport cholesterol out of cells, there's also ABCG1 which is also one of our major findings in our paper.
Cindy St. Hilaire: Can you tell us a little bit about the models you chose in the study and why you picked them? And also maybe a step back in terms of, what are the pros and cons of using mouse models in atherosclerotic studies?
Sabrina Robichaud: So we chose to use the GFP-LC3 reporter mouse model because it allows us to track in lifestyle the movement of LC3, which is the main component of the autophagosome which is involved in pathology. So by using this reporter model, we could infer whether or not the cells had high autophagy or low autophagy. And to induce atherosclerosis in these mice, instead of backcrossing them to either an LDLR knockout or an ApoE knockout, we chose to do the adeno-associated virus that encode the gain of function PCSK9 instead to kind of minimize the time for breeding. It did have the effect that we needed in terms of raising plasma cholesterol to induce the atherosclerosis. So that was one of the models that we used in our paper.
Mireille Ouimet: There's not very many good mouse models to study autophagy flux in vivo and GFP-LC3 is kind of the main one currently. We're working on developing some other tools to track lipophagy in vivo, but these things take time to put in place. So in the future, we hope to have some better tools to track lipophagy in real-time in vivo.
Cindy St. Hilaire: How difficult is it to measure autophagy flux in vivo? I know there's certain part like LC3 or P62, a lot of people use a western blot and it's like, oh, it's high, it must be active, but it's a flux. So it's a little bit more... There's more subtleties to that, dynamic than that. So how difficult is it to really measure this flux in in vivo tissues?
Mireille Ouimet: Yes, so now there are more recent mouse models that have been developed more recently to replace kind of the GFP-LC3 is the Rosella LC3. So it has both a red and a green tag, and so two LC3, so when autophagosomes are fused to lysosomes and are degraded, then there's preferential quenching of the GFP first, and then you have the red appearance that predominates so we know that then it's kind of like it a live flux measurements. Because we use the GFP-LC3 mouse, Sabrina treated her cells ex vivo. When we dissected out the aortic arches, digested the cells then we divided those into two components and added bafilomycin so that we can inhibit lysosome acidification to see the changes in the flux. And that's really to get the differences in untreated versus bafilomycin-treated.
Mireille Ouimet: When we inhibit the lysosome, then we're sure that it is a functional flux or not. But it's kind of an indirect way of measuring it, and it reads very complex when we're talking about P62 and LC3 degradation with or without lysosome inhibition, but you really need that lysosomal inhibition, to show that if you block the degradation of the autophagosomes that fuse in with a lysosome, then you get an increase in the LC3 and the P62, and that's when you know that the flux is you intact.
Mireille Ouimet: Because you could get an increase in LC3, that's just related to a defect in the breakdown of the autophagosome. But in our study, we've used phosphorylated ATG16L1, which is a now better marker of active autophagy. And I would recommend researchers to begin to use that rather than the combination of P62 and LC3 together with or without a lysosome inhibitors such as-
Cindy St. Hilaire: Oh, interesting. So let's repeat that, phosphorylated ATG-
Mireille Ouimet: 16L1, yes. So there's been an antibody that was developed by a colleague at the University of Ottawa, Dr Ryan Russell, and it's commercially available through cell signaling now, and it really has been a great tool to track active autophagy.
Cindy St. Hilaire: That's great. I remember my lab was looking at that at one point, and I was trying to explain the flux as... I don't know if people are going to remember this, but there's this amazing, I Love Lucy skit, where her and Ethel are working on a chocolate factory conveyor belt, and it picks up speed. And because she can't get it all done quick, she starts stuffing them in her mouth. And it's like, if you just took a snapshot of that, you would not know whether it's going too fast, or not functioning properly. And so I equate the flux experiments to that. Which are probably aging myself a lot on so.
Cindy St. Hilaire: All right, so sticking to kind of the autophagy angle, what were the differences you found in autophagy in early and late atherosclerotic plaques? Because I know you looked at those two time points, but also, importantly, between the macrophage foam cells and the smooth muscle cell-derived foam cells?
Sabrina Robichaud: So surprisingly, there weren't that big of a difference between each time point when we were looking at the individual cell type by themselves. Surprisingly, we did find that the macrophages did have a functional autophagy flux, even at the later stages of atherosclerosis, which was kind of interesting in itself. But when we looked at the vascular smooth muscle cell foam cells, though, that was a whole other story, and we found that these were actually defective at a very early stage and stayed defective up until the very late stage of atherosclerosis.
Cindy St. Hilaire: And what is the very early stage like? What's that definition with the smooth muscle cell?
Sabrina Robichaud: So we did a six-week time points in terms of our atherosclerosis study, and then a 25-week time point. So there are far apart, which shows like the very early, early stage and what would be considered the most effective autophagy at that point with the necrotic core and everything. So surprisingly, the two phenotype were quite similar at early and both late stages for both cell types, but were functional in the macrophages but dysfunctional in the smooth muscle cells.
Cindy St. Hilaire: So you mentioned at one point in the discussion that you observed inconsistent lipid loading of the smooth muscle cells, and you mentioned that a lipase, which is excreted from the foam cells can then be internalized by, I assume kind of neighboring or in the vicinity, smooth muscle cells. And so the question I had it's kind of one of those chicken-and-egg question, and it's, is the smooth muscle cell-derived foam cell an independent process? Does it happen alone or de novo as a function of a smooth muscle-mediated process? Or is it really dependent first on this macrophage foam cell providing this lipid that is efflux that is then internalized by a smooth muscle cell that kind of goes on to become a foam cells. It's kind of a question of like the continuum of an atherosclerotic plaque and what do you think is happening, either based on your data or just kind of a hunch?
Mireille Ouimet: That's an excellent question. And there's no doubt that macrophages really drive the initiating events of atherosclerosis. So I don't think that without the macrophage there would ever be a vascular smooth muscle cell, or there would be minimal vascular smooth muscle cell-derived foam cells. Definitely the inconsistencies that we observed in our study, were if we added like aggregated LDL on its own to a primary mouse vascular smooth muscle cell, we would get poor lipid loading and a very low percentage of those cells that would become foamy, relative to treating them with cyclodextrin complex cholesterol, for instance.
Mireille Ouimet: So free cholesterol, that’s cell permeable, will go into the vascular smooth muscle cell, no problem, and generate the foaminess and then allow that cell to acquire the macrophage-like phenotype. But aggregated LDL on its own in our hands, just gave very poor loading. And when we treated the vascular smooth muscle cells with aggregated LDL along with macrophage-derived condition media, we got some improvements, but it was still kind of inconsistent. But then we thought if we treat the vascular smooth muscle cells with aggregated LDL in the presence of conditioned media from macrophage foam cells that were preloaded with the aggregated LDL, would that promote their foaminess to a greater extent? And it did.
Mireille Ouimet: So, there have been studies from Gordon Francis's lab that showed that adding recombinant lysosomal acid lipase to vascular smooth muscle cells that contained aggregated LDL, promoted the lysosomal hydrolysis of the aggregated LDL and to generate the foamy macrophages and allow the lysosomal processing. So we know that that vascular smooth muscle cells take up lysosomal acid lipase, and we know that macrophages undergo lysosome exocytosis and they can secrete lysosome acid lipase and acidify the extracellular milieu.
Mireille Ouimet: So work from Fred Maxfield group has shown the presence of these cell surface connected compartments that are acidified, containing macrophage-derived lysosomal acid lipase, that even hydrolyze extra cellularly-aggregated LDL for macrophages. So we're not sure whether there's probably a local production of free cholesterol in the plaque by macrophages, this free cholesterol could be taken up by the vascular smooth muscle cell. And also the vascular smooth muscle cells do express some scavenger receptors, whether the expression of these scavenger receptors like LRP or CD36 even goes up when they've taken up a little bit of the free cholesterol. And then that allows the aggregated LDL to come in and then there would be some lysosomal acid lipase secreted by the macrophage foam cells that would promote the lysosomal processing of this aggregated LDL. All of those are very complex questions that will require some addressing in vivo models.
Cindy St. Hilaire: You also mentioned in the paper that studies... There's a handful of them now. Studies have shown that between 30% and 70% of the cells that are staining positively for macrophage markers, meaning they’re foam cells, are of the smooth muscle cell lineage. And so I believe people have seen that in mouse plaques with lineage tracing, but they've also used newer techniques to really see this also in human atherosclerotic plaques. So we know it's not just from a mouse, we know that smooth muscle cells can turn into a macrophage-like foam cell, and it's 30% to 70%, which is a huge range.
Cindy St. Hilaire: So do we know the factors that dictate whether a specific plaque is going to have more or less smooth muscle cell derived foam cells? And I guess more important to what you found in your paper is, how important would it be to know whether a plaque is on the 30% end or on the 70% end in terms of therapeutic strategies?
Sabrina Robichaud: Yeah, most of these studies, the range can be attributed to the different time points at which these studies have been collected early on will be a little bit more macrophage understanding would be at a later time point. Now of course in terms of therapeutics, as we saw in our paper, metformin actually will positively increase cholesterol efflux in the vascular smooth muscle cell foam cells, but not in the macrophages. So obviously, being able to know at which point there's a majority of macrophages versus vascular smooth muscle cells, definitely going to determine which therapeutic we're going to be able to use.
Sabrina Robichaud: Ideally, we would be able to find a therapeutic that would work in both foam cell, but from what we've seen, the mechanistic behind the autophagy dysfunction between both cell types are so different, that I'm not entirely sure that that would be possible, we would need some sort of combination therapy. But again, we need to be a little bit more targeted depending on the percentage of the foam cells that are comprising the plaque at that particular moment in time.
Cindy St. Hilaire: Yeah, so you mentioned there's a function of time there. If you look earlier, there's more macrophage, if you look later, the percent of smooth muscle cell-derived foam cell increases. Is there a point in a very advanced atherosclerotic plaque where it's just mostly smooth muscle cells? Or do those macrophage foam cells stay, and it's just the increasing number of smooth muscle cell-derived foam cells? Do we know?
Mireille Ouimet: This is an excellent question, and I was going to bring up the topic of clonal expansion of the vascular smooth muscle cells. So it's a very heterogeneous population and understanding that might be some of the differences that we see in different studies. It could be the model has one type of a smooth muscle cell that's expanding more than another, what are the factors that govern that? Does one clone take over at the later stages versus the earlier stages? We don't know.
Mireille Ouimet: But we were surprised in our studies to see that the macrophages that are present at least on the lumen of the plaques were very active in autophagy. They had the highest staining for the phospho-ATG16L1 in that late stage. So we're not sure if it's newly-recruited macrophages that come in, that are more active and in autophagy, and then have good lysosomal capacity that keeps degrading the lipid present in the plaque and tries to ingest it, but also as a consequence keeps releasing some of the degraded cholesterol into the milieu where the smooth muscle cells that are proliferating are internalizing it and becoming more foamy. So these are really great open questions that need to be addressed in the field.
Cindy St. Hilaire: So drug-eluting stents are coated with rapamycin or the various chemical compositions that are derived from rapamycin. And rapamycin itself induces autophagy. So while the thought behind using this coating on stents was to prevent smooth muscle cell proliferation, and thus restenosis or ingrowing of the stent, your study suggests that this could also help to promote autophagy in the cells underlying the stent. So has anyone gone in and looked at plaques that have been stented and either failed or not, and investigated the foam cell content or markers for autophagy activity?
Mireille Ouimet: Not to my knowledge, and this has been something we've definitely... We think that this is what's happening. Some of the protective effects of these drug-eluting stents that have everolimus or sirolimus or the rapamycin or rapamycin analogs, we do believe that some of their protective effect can be attributed to autophagy activation, but this remains to be demonstrated. We think that autophagy activation locally would promote reverse cholesterol transport and would be one of the processes that prevents restenosis because we can promote the efflux of cholesterol out.
Cindy St. Hilaire: Great. So I guess stemming from my question on the stents, what are the other translational implications of the findings of your study? And what would you like to see come out of this?
Mireille Ouimet: So one of the things is, as Sabrina mentioned, would be to target both foam cell populations because it seems as though the vascular smooth muscle cell foam cells are very much defective in their autophagy capacity, and they're very poor effluxes, but we could potentially restore autophagy in the cell population to promote reverse cholesterol transport.
And looking at prevention of atherosclerosis is a bit different than looking at regression, because regression is at a later stage where the plaques are more advanced. And if they're mostly vascular smooth muscle cell-derived, maybe then those drugs that we're considering that protect against the development of atherosclerosis are effective on the macrophage themselves early on, but might not be mimicking what we would see in the clinic where the patients that present are older.
Cindy St. Hilaire: Yeah, it's kind of really reminiscent of like the CANTOS trial and like, where do we want to target the therapy? It's going to be very different if it's an early smaller plaque, versus a late-stage possibly pro close to rupturing type of plaque. Well, Sabrina Robichaud and Dr Ouimet, thank you so much for joining me today. Congratulations again on a wonderful study, and I'm really looking forward to hearing more about this from your group.
Sabrina Robichaud: Thank you.
Mireille Ouimet: Thank you very much. And we also want to thank all the co-authors on the study, specifically also Adil Rasheed, who is co-first author on the work and Katey Rayner’s group for all the support and involvement in this study.
Cindy St. Hilaire: That's it for the highlights from the March issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @circres and #DiscoverCircRes. Thank you to our guests, Sabrina Robichaud and Dr Mireille Ouimet Sabrina. This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles was provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire and this is Discover CircRes, you're on-the-go source for the most up-to-date and exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2022, The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.