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Discover CircRes

May 21, 2020

This month on Episode 12 of the Discover CircRes podcast, host Cindy St. Hilaire highlights three featured articles from the May 8 issue of Circulation Research and gives listeners an inside scoop of the cutting edge ideas in the May 22nd Compendium on Obesity. This episode also features an in-depth conversation with Dr Eduardo Marbán concerning COVID-19 and its effects on the heart.


Article highlights:

Roberts et al.  LYN Regulates Monocyte Heterogeneity and Lifespan

Lu, et al. Acute Hyperglycemia Activates CaMKII-ROS Pathway

Yan, et al. Epicardium and Atrial Cardiomyopathy

Dr Cindy St. Hilaire: Hi. Welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, from the Vascular Medicine Institute at the University of Pittsburgh. Today, I'm going to share with you articles selected from the May 8th issue of Circulation Research as well as give you a hint at the cutting-edge ideas in the May 22nd Compendium on Obesity. We'll also have discussion with Dr Eduardo Marbán from the Smidt Heart Institute at Cedar Sinai Medical Center about his Review on COVID-19 and its effects on heart. So, first the highlights.

The first article I'm sharing with you is titled Deep Phenotyping by Mass Cytometry and Single Cell RNA Sequencing reveals LYN Regulated Signaling Profiles Underlying Monocyte Subset Heterogeneity and Lifespan. The first authors are Morgan Roberts and Maunish Barvalia and the corresponding author is Kenneth Harder and they're from the University of British Columbia. Monocytes can be separated into two main groups, conventional monocytes which enter tissues from the bloodstream and differentiate into macrophages, and patrolling monocytes, which developed from conventional monocytes but tend to remain in the blood vessel walls where they can scavenge cells and tissue debris.

It's thought that patrolling monocytes help to prevent a range of diseases like atherosclerosis by helping to clean up the vessel walls. Studies in mice harboring genetic mutations in a gene called Nr4a1 cause mice to have less than normal numbers of patrolling monocytes. In these mice, the development of atherosclerosis is exacerbated.

In addition to Nr4a1, this group has now identified another factor regulating the survival of patrolling monocytes, the tyrosine kinase LYN, L-Y-N. Genetic deficiency of LYN caused the upregulation of Nr4a1 and other genes involved in patrolling monocytes development and survival. This in turn led to the accumulation of patrolling monocytes in the blood, also in the bone marrow, spleen, and the aorta. Loss of LYN was also protective against atherosclerosis in mouse models of this disease. These results not only provide novel insights into patrolling monocyte biology, but also suggest that targeting LYN could offer novel treatments for diseases like atherosclerosis, where boosting the patrolling monocyte numbers could be beneficial.

The second article I want to highlight is titled Hyperglycemia Acutely Increases Cytosolic Reactive Oxygen Species via O-linked GlcNAcylation and CaMKII Activation in Mouse Ventricular Myocytes. The first author is Shan Lu and the corresponding author is Don Bers, and they're from the University of California, Davis. Diabetes affects more than 400 million people worldwide and puts these individuals at a higher risk for developing heart failure. When heart failure does occur, the outcomes for these patients with diabetes are likely to be far worse than for individuals without the diabetic condition. Both heart failure and diabetes have been associated with excessive production of reactive oxygen species and also with increased activation of a protein kinase in the cells of the heart called CaMKII.

Both ROS and CaMKII are induced by hypoglycemia, where there is an increased amount of extracellular glucose levels in the blood. This study shows that reactive oxygen species in CaMKII are causally linked. When CaMKII was inhibited or genetically deleted in mouse cardiomyocytes, high extracellular glucose levels were unable to induce reactive oxygen species production, which is what would normally occur. The team also discovered that O-GlcNAcylation post-translational modification of CaMKII is induced by the extracellular glucose and this modification is necessary for the enzyme's reactive oxygen species- boosting effects.

Lastly, they found that the enzyme NADPH oxidase 2 or NOX2 was the source of this CaMKII induced reactive oxygen species. This work uncovers the molecular pathway linking hyperglycemia, cardiomyocyte-damaging reactive oxygen species production, and it helps explain why heart failure pathology is exacerbated in diabetic patients.

The next article I want to share with you is Reactivation of the Epicardium at the Origin of Myocardial Fibro-Fatty Infiltration During the Atrial Cardiomyopathy. The first author is Nadine Suffee and the corresponding author is Stéphane Hatem and they're from Inserm in Montpellier, France. Fatty tissue surrounding the heart is linked to an increased risk for atrial fibrillation, which is the most common form of arrhythmia. It seems that a combination of fat cells, which are called adipocytes and the fibroblast localized within the heart's epicardium, builds up and expand into the subepicardial layers, and this is a feature that is called fibro-fatty infiltration.

These fibro-fatty infiltrations cause disturbances to the electrical rhythms that regulate the heart beating. Although generally quiescent in the adult heart, epicardial cells possess the ability to proliferate and have been shown that they harbor the ability to differentiate into adipocytes and fibroblast. This team hypothesized that the epicardial cells were the source of the damaging fibro-fatty infiltrations. Sure enough, when they looked at human heart sections, they found that within the epicardial layer, there were cells that were expressing fibroblast and adipocyte progenitor cell markers.

In culture, these epicardial cells with fibroblast progenitor markers could be differentiated into fibroblasts by treatment with angiotensin II and cells with the adipocyte progenitor markers could be differentiated into adipocytes by treatment with atrial natriuretic peptide. The team also showed that these epicardial fibro-fatty infiltrations occurred in a mouse model of atrial cardiomyopathy. Together this work highlights the pathogenesis of epicardial fibro-fatty infiltrations and suggest a novel model in which to study its progression to AFib.

The last thing I want to share with you before we switch to our interview with Dr Marbán is that the May 22nd issue of Circulation Research is our Obesity Compendium. Obesity is a major threat to cardiovascular health worldwide. While early studies focused on body mass index as a generalized measure of obesity and focused on the BMIs relation to cardiovascular disease, studies within the last decade have now tried to more fully understand adipose tissue physiology and the overall impact of obesity on cardiovascular disease.

The articles in this compendium are obesity phenotypes, diabetes and cardiovascular diseases, basic mechanisms of diabetic heart disease, leukocyte heterogeneity and adipose tissue including obesity, an eclectic cast of cellular actors orchestrates innate immune responses and the mechanisms driving obesity and the metabolic perturbation, metabolic inflammation and insulin resistance in obesity, genetic insights into the relationship between Type 2 diabetes and coronary heart disease, metabolomics and proteomics in Type 2 diabetes, metabolic and molecular imaging in diabetic cardiomyopathy and treatment of obesity and mitigating metabolic risk.

This compendium reflects the collective work of leading investigators in the space of diabetes, cardiometabolic disease, and cardiovascular disease with the ultimate goal of providing a summary of selected aspects of obesity and metabolic physiology central to cardiovascular disease development.

So, I have with me here today, Dr Eduardo Marbán, the founder of the Smidt Heart Institute at Cedars-Sinai Medical Center in Los Angeles, California. He's a leading physician scientist in the fields of electrophysiology, cardiac progenitor cells, and next generation cell-free therapeutics. Dr Marbán, thank you very much for taking the time out of your busy schedule to speak with us today about your article COVID-19 and the Heart, which is now freely available on the Circulation Research webpage.

Dr Eduardo Marbán: It's my pleasure to talk to you Cynthia.

Dr Cindy St. Hilaire: First off, how are you and how are things at your hospital center in LA?

Dr Eduardo Marbán: We seem to have dodged the bullet here in the sense that we were pretty progressive in terms of quarantine and stay at home orders. Given that, we seem to have peaked at a level that is very manageable in terms of our surge capacity. So, we feel for those who are worse off, but at least knock on wood here, we seem to be surviving so far.

Dr Cindy St. Hilaire: Yeah, that's similar to how we are in Pittsburgh. We shut down about the same time that Philadelphia, who was already surging was shutting down. So, we are feeling safe but still prepared. So, I was extremely excited to read this article because as we know, cardiac injury is happening in between 20% to 30% of the COVID-19 patients and cardiac injury is also the cause of about 40% of the COVID-19 related deaths. So, my first question is, what are the types of cardiac injuries or events that you're seeing in these COVID-19 patients and are there any particular characteristics that the subpopulation of patients shares that's different from non-cardiac injury COVID patients?

Dr Eduardo Marbán: What seems to be extremely common in COVID-19 patients is elevations of circulating biomarkers, things like troponin I, troponin T, BNP as an indicator of heart failure, but what's much less certain is whether these biomarker elevations have any clinical significance. At the level of isolated case reports, there's fulminant myocarditis, ventricular tachycardia, arrhythmias, occasional acute coronary syndromes, but there seems to be a disconnect between the almost ubiquitous nature of the circulating biomarker elevations and the relative rarity of clinical events.

Dr Cindy St. Hilaire: So, do these patients, do a majority of them have a history of cardiovascular disease or is this all new developments? Do we know?

Dr Eduardo Marbán: Underlying cardiovascular disease, diabetes, hypertension, and recently obesity and, of course age, have all been implicated as general risk factors for being critically ill with COVID, but there's no specific indication epidemiologically yet that those with underlying cardiovascular disease have a particular predilection to manifesting worse heart symptoms or signs during COVID-19. It makes sense that that would be the case, but so far, the epidemiology is somewhat more general.

Dr Cindy St. Hilaire: When you were first writing this article, I'm sure between then and now we even have more epidemiological data points that are constantly changing.

Dr Eduardo Marbán: Since the article was published online on April 7th, I've given four updated versions of the webinar to various audiences. Every time we do so, the slides need to change subtly. It's a very rapidly evolving field.

Dr Cindy St. Hilaire: Yeah, that's amazing. In the first SARS outbreak, which was in 2002-2003, scientists discovered that this type of Coronavirus enters the cell by binding to angiotensin converting enzyme II as a receptor. So, ACE2 as it's called. It's not a receptor in the canonical sense of the word, but it's a cell surface enzyme and it's involved in the renin angiotensin aldosterone system, which regulates a handful of cardiovascular homeostatic processes and is quite frankly, rather complicated. So, I don't want to talk specifically about that, but I'm wondering if you could tell us a little bit about what ACE2 is, what cells it's found on, and what that might mean for the implications of this virus and its effects on the cardiovascular system?

Dr Eduardo Marbán: Well as you correctly stated, ACE2 is central to cardiac physiology in the sense that it creates the bioactive form of angiotensin. In so doing, its regulation is central to that of blood pressure, human dynamics. What is less appreciated and to me was a bit of a revelation is the fact that it's expressed fairly richly on the surface of epithelial cells of the lung and the SARS-CoV virus family seems to have co-opted the presence of that in order to create a handy sort of hook to get into the cells in the first place. Whether there are broader ranging implications of ACE2 other than the particular mode of entry into the cell for a viral infection is a topic of great speculation at this point.

Dr Cindy St. Hilaire: Yeah. In some of my preparation for this and also just my curiosity regarding this virus and the vascular system, when you look at things like the human protein atlas, you can see that ACE2 is highly expressed, not only on the lung epithelial like you say, but they're also expressed on cardiovascular cells in nearly all of the tissue. I'm thinking of cells like the smooth muscle cell and the endothelial cell. Is the virus binding to ACE2 positive cells part of the reason for the cardiac events or these cardiac events secondary to systemic toxicity? So, I guess the real question is, do we know anything about the direct versus the indirect effects of the virus on the heart?

Dr Eduardo Marbán: No question in vitro that SARS-CoV can infect cardiac myocytes and most surely almost any other cell that expresses these two on its surface. In vivo, how frequently that happens as opposed to triggering secondary cardiac damage due to the systemic inflammation is uncertain, but I can tell you from the various case reports that have actually analyzed human tissue either at autopsy or an endomyocardial biopsy in cases of fulminant myocarditis, the frequency of direct viral infection seen either by culturing viral particles or more frequently by electron microscopy and visualization of inclusion bodies within cells points to perhaps a third of the cases being due to direct infection and two thirds of the cases likely being due to some bystander effect of systemic inflammation.

Dr Cindy St. Hilaire: Interesting. So, are the phenotypes different between those patients where it seems to be direct versus indirect? Does the myocarditis appear similar or the cytokine profiles, anything like that?

Dr Eduardo Marbán: There are too few patients to make really good conclusions about whether or not the phenotypes differ greatly when there's direct versus indirect cardiac involvement, but certainly from the literature as it exists now, there's no reason to believe that we could outsmart the clinical picture. They all look pretty much the same from the bedside.

Dr Cindy St. Hilaire: So from the first SARS outbreak, do we know anything about the long-term effects of this type of viral infection on the cardiovascular system or on the heart specifically?

Dr Eduardo Marbán: Yeah. COVID-19 of course the follow-up is limited to a few months since the first cases probably didn't emerge until late October early November and weren't really recognized as such until late '19 early 2020, but for SARS from the 2002-2003 epidemic, some of the long lasting sequelae are unanticipated and include hypertension, hyperlipidemia, pulmonary fibrosis, avascular necrosis. So, it seems that even when a patient is out of the woods, perhaps they're not really out of the woods in terms of long-term sequelae. We need to be watchful for long-term sequelae in COVID-19 survivors. They're going to be many more of them than there were from the SARS epidemic.

Dr Cindy St. Hilaire: So, one of the things that's come out recently, which I've been really mulling about because my background is vascular biology and specifically smooth muscle cells and endothelial cells, but one of the findings is about the later stage or more sick patients. These are patients who are going on ventilators and about 50% of them going on the ventilators are dying and/or just not responding to ventilator therapy as doctors expect. So, just to give a little background about ventilators, they're normally used when a patient's blood oxygen level drops too low.

So, normal levels are between 95% and 100%. However, patients with pneumonia or acute respiratory symptoms are put on ventilators sometimes when their oxygen drops below 90%, but some of the COVID-19 patients are exhibiting blood oxygen levels at 70% or sometimes even lower, but they don't have outward signs of distress and they can still hold conversations. So, I'm wondering if you can give me any insight into possibly what's going on there with the lens of vascular remodeling, what might be happening to the vasculature in the lung that is unique to this ventilator response and COVID response?

Dr Eduardo Marbán: The observation you described is common that sometimes a patient will be profoundly hypoxemic but chatting away or surfing the internet as if nothing were happening. We're not used to seeing this in other cases of ARDS or viral sepsis where the patient usually is in extremis by the time the blood oxygen levels get that low. It begs the question as to whether perhaps there's something about the cerebral circulation, and this is complete and rampant speculation. Whether there's something about the cerebral circulation that makes it somewhat resistant to the effects of systemic hypoxia, perhaps there's a compensatory vasodilation that occurs that compensates for the otherwise deadly systemic hypoxemia. It would be quite interesting to monitor oxygen tensions within the cerebral parenchyma to test that, but all I can say with any certainty right now is that the clinical observation is robust. We see this not infrequently in patients who in the sort of clinical jargon have no right to look that good.

Dr Cindy St. Hilaire: Yeah. Yeah. It's like your numbers, you really have those numbers? Yeah. There's just so many questions. It's really unprecedented. So, I guess we've been talking a lot about the disease itself and the symptoms and the pathogenesis, but I want to switch to ask about potential therapies. There's been several therapies that have been suggested by a variety of people and there's, I don't even know how many clinical trials. I looked a week ago and there's really a great response of pharmaceutical companies and university hospital systems trying what they can with the tools they have. So, things like antivirals, HIV protease inhibitors, inhibitory antibodies, and even antimalarial drugs have been suggested that they could possibly work. So, I'm wondering if you could give us some insight from a cardiovascular standpoint, what are the potential implications or potential adverse side effects of using these different therapies off label and what might that mean for the heart in addition to treating the viral infection?

Dr Eduardo Marbán: You're correct in the explosion of clinical trials in this area or at least, clinical interventions. At our IRB, as of today, there are 56 active COVID protocols. Imagine nobody even cared about COVID until mid-February, right?

Dr Cindy St. Hilaire: That's just at Cedars-Sinai.

Dr Eduardo Marbán: Yeah. Now, we have 56 active protocols. So, not all of those are interventional. Some of them are epidemiological or biomarker studies, but still there's an incredible plethora. You're right, the approaches of targeted anything from the viral infection to the viremia to the downstream consequences of viral infection including the hyper inflammation and cytokine storm. The rationale for anti-malarials is actually fairly thin and resides on in vitro observations that actually were just from February that SARS-CoV-2 infection in vitro is somewhat retarded by exposure to hydroxychloroquine. This didn't come out of the blue. There had been an extensive literature and quite controversial literature, I should say, that anti-malarials might be useful in influenza and other infections.

In a very general sense, there was a lot of hype created by early in vitro studies, which turned out to be neutral or in some cases even harmful clinically, but this has led to an almost universal adoption of hydroxychloroquine in patients with COVID-19 coupled sometimes with the antibacterial agent azithromycin for which the rationale is even thinner. There's no reason to believe that an antibacterial per se would help in a viral infection, but azithromycin is said to have antioxidant properties, which may or may not potentiate the effects of hydroxychloroquine, but for sure what they do together is prolong repolarization of the heart and lead to a clinical syndrome known as prolonged QT, which is a known substrate for toxic arrhythmias like polymorphic ventricular tachycardia. So, in prescribing some of these agents, one needs to weigh the uncertain benefits against the very certain risk that they entail.

Dr Cindy St. Hilaire: Yeah. I think that's a really important point. I think one of the scary things that has the potential of happening during this crisis is too quick of a jump to conclusions. While there is a need for as rapid a response as possible, we still need to make sure that we're taking in all the scientific information we have and that that science is good and strong. I think one of the things that you mentioned in the Review is the lack of power in some of those initial anti-malarial studies. I think it's really important thing I want to emphasize that it's an emergency, but we still need to make proper good scientific decisions.

Dr Eduardo Marbán: Well, one of the problems is that hydroxychloroquine and other agents in some cases, remdesivir and you know, you choose, have gotten so popular and hyped that there's almost no possibility of being an ethical clinical trial because the patients want to be on them. So, it may be easier in some settings than in others, but it's certainly not going to be a trivial thing to sort out the true risk benefit ratio of these drugs in this illness.

Dr Cindy St. Hilaire: So right now, doctors and scientists, we're all in crisis mode, but once things settled down, we could really start to sit down and think about more mechanistic questions that might be able to be tested that will really help us flush out our understanding of COVID-19 disease pathogenesis and its effects on the cardiovascular system. So, what do you see after this initial crisis is under control, what do you see as the immediate next questions that basic scientists and translational scientists need to address that can help the next time that this comes again?

Dr Eduardo Marbán: First of all, it's quite clear that we've all become consumed by COVID-19 and SARS-CoV-2. We can't think of anything else often. It's really hard to even focus on work from the laboratory that doesn't have to do with SARS-CoV-2 and COVID. It's so ubiquitous in public perception and the way we're living our lives that it just makes it incredibly difficult to think about anything else. I think there's going to be a correction in which we're going to get frankly tired of SARS-CoV-2 and COVID and want to think about other things, but among the lasting questions and the ones that will have greater biological merit above and beyond how to deal with this particular virus and this particular pandemic are the following. What is the role of ACE2 in human biology? Clearly here, there's an experiment of nature in which this surface enzyme has been co-opted for viral entry and a tremendous amount of speculation surrounds the question of whether high ACE2 values are protective and detrimental and ACE inhibitors and angiotensin receptor blockers might be detrimental or beneficial.

All of these fundamental mechanisms need to be sorted out and now there's motivation to do so because of the epidemic. Some of this work is easier than others and those institutions that happen to have a BSL-3 level facility for being able to directly study the effects of the virus on various tissues should do so with alacrity because it's a limited resource right now where the number of questions really far exceed the ability to answer them just physically. Another question which I think is going to be motivated by our experience with COVID-19 is that of the mechanisms of cytokine storm and hyper thrombotic states. These are things that characterize the critically ill patient with COVID-19.

Dr Cindy St. Hilaire: Can you just explain what is a cytokine storm? What does that exactly mean?

Dr Eduardo Marbán: So, patients who are critically ill with COVID-19 manifest a late stage of the illness, which is often fatal, in which circulating levels of various inflammatory biomarkers, interleukin 6, C-reactive protein, ferritin being among them, but basically anything that goes up in an inflammatory state. And some of these appeared not to just be markers of inflammation. Something like C-reactive protein is probably just a biomarker of inflammation, but interleukin 6 for example, is a highly bioactive cytokine that itself probably causes tremendous tissue injury and there's some enthusiasm for the use of anti IL6 antibodies and anti IL6 receptor antibodies to treat the critically ill with some anecdotal dramatic success I should say. So perhaps the cytokine storm isn't just a marker of those who are critically ill, perhaps it's causative.

If that presumption is real, then it makes good sense to target the cytokine storm, but from a scientific point of view, what causes it in the first place? How does a viral infection lead to massive production of cytokines and inflammatory biomarkers and how can that be mitigated? One of the ways of dealing with that is by understanding precisely how it happens in the first place and there's not that much literature on it. There's a recent study which I found quite provocative that glucose metabolism and the whole process known as O-GlcNAcylation might actually be a trigger in the production of cytokines during viral infections like COVID-19, but I think understanding how it happens will lead to much more targeted therapeutics and perhaps enable us to eventually divorce the infection from the overreaction. Really what's happening is friendly fire. The body's immune system is turning against itself in a sort of vain effort to control the virus. Sometimes the viremia is actually almost gone by the time that these inflammatory biomarkers increase, and the cytokine storm surges.

Dr Cindy St. Hilaire: So, it's almost like the inflammatory response reaches some point beyond which it doesn't need virus anymore. It is just full force feeding forward and causing more damage by itself.

Dr Eduardo Marbán: Yeah, exactly. It's almost as if there's an eroding cliff and even though the river may be back down to normal levels, the cliff is still unstable and the whole hillside could come crashing down.

Dr Cindy St. Hilaire: Are there long terms effects of that? I wonder how long that would last after the infection or is it only during a viral titer in the system?

Dr Eduardo Marbán: Well, you raised yet another interesting question to the extent that patients who have survived SARS- CoV-2 infection develop long-term sequelae, what's the mechanism of those long-term sequelae? Why should patients who are previously well develop hyperlipidemia and hypertension after the infection, if in fact they do, so are any of these related to micro thrombotic events? It's quite conceivable.

Dr Cindy St. Hilaire: Great. Well, thank you so very much for taking the time to speak with me today. I don't think I found a ton of answers. I found a lot more questions, but hopefully as this develops and we get it under control, maybe we can talk again and talk about some of those new mechanistic findings and potential therapies.

Dr Eduardo Marbán: Absolutely. You're welcome, and I hope you and all the listeners stay safe during this pandemic.

Dr Cindy St. Hilaire: You too, and your clinical team.

That's it for highlights from the May 10th and May 22nd Obesity Compendium issues of Circulation Research. Thank you so much for listening. Please check out the Circulation Research Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DscoverCircRes. Thank you to our guest, Dr Eduardo Marbán. This podcast is produced by Rebecca McTavish, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on-the-go source for the most up-to-date and exciting discoveries in basic cardiovascular research.