March 2021 Discover CircRes - a podcast by Cynthia St. Hilaire, PhD & Milka Koupenova, PhD
from 2021-03-18T18:00
This month on Episode 22 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the March 5 and March 19 issues of Circulation Research. This episode also features an in-depth conversation with Norberto Gonzalez-Juarbe and Maryann Platt from the J. Craig Venter Institute to discuss their study, Influenza Causes MLKL-Driven Cardiac Proteome Remodeling During Convalescence.
Article highlights:
Carnicer, et al. BH4 Prevents and Reverses Diabetic LV Dysfunction
Kyryachenko, et al. Regulatory Profiles of Mitral Valve
Mangner, et al. Heart Failure Associated Diaphragm Dysfunction
Peper, et al. Identification of McT1 as Caveolin3 Interactor
Dr Cindy St. Hilaire: Hi, and welcome to Discover CircRes: the podcast of the American Heart Association's Journal, Circulation Research. I'm your host, Dr Cindy St Hilaire, from the Vascular Medicine Institute at the University of Pittsburgh. Today I will be highlighting four articles selected from our March 5th and March 19th issues of Circ Res. After the highlights Drs Norberto Gonzalez-Juarbe and Maryann Platt from the J. Craig Venter Institute are here to discuss their study, Influenza Causes MLKL-Driven Cardiac Proteome Remodeling During Convalescence
Dr Cindy St. Hilaire: The first article I want to share is titled, BH4 Increases nNOS Activity and Preserves Left Ventricular Function in Diabetes. The first author is Ricardo Carnicer, who is also corresponding author alongside Barbara Casadei and they're from University of Oxford in the UK. Cardiomyopathy and heart failure are common complications of diabetes, but the molecular pathology underlying this cardiac dysfunction is not entirely clear. Increased oxidative stress and reduced functioning of both mitochondria and nitric oxide synthase or nNOS have been implicated in diabetic cardiomyopathy. Tetrahydrobiopterin or BH4 is a co-factor necessary for nNOS activity.
Dr Cindy St. Hilaire: And in diabetic patients and animals oxidation of BH4 inactivates nNOS and induces vascular endothelial pathology. But, what happens in the cardiac tissue itself? This group shows that although boosting BH4 levels by genetic or pharmacological means prevented or reversed heart dysfunction in diabetic mice, the status of BH4 oxidation and nNOS function in the heart tissue of diabetic patients and mice, did not actually differ significantly from that of healthy controls. Instead through molecular analysis, they revealed that in diabetic mouse cardiomyocytes boosting BH4 promoted a nNOS dependent increase in glucose uptake, which then preserved the cell’s mitochondrial function. Regardless of the pathways involved, the fact that BH4 reversed diabetic associated cardiac dysfunction in mice suggests the potential for therapies that could be used to lower the risks of such complications in humans as well.
Dr Cindy St. Hilaire: The second article I want to share is titled, Chromatin Accessibility of Human Mitral Valves and Functional Assessment of MVP Risk Loci. The first authors are Sergiy Kyryachenko, Adrien Georges, and Mengyao Yu, and the corresponding author is Nabila Bouatia-Naji from Paris Cardiovascular Research Institute in France. The mitral valve opens and closes to direct a one-way flow of blood from the left atrium to the ventricle. If the mitral valve fails, as in the case of mitral valve prolapse or MVP, blood regurgitation, cardiac arrhythmia, and ultimately heart failure can occur.
Dr Cindy St. Hilaire: With 11 valves from MVP patients and 7 control patients, this group used a highly sensitive chromatin profiling technique called ATAC-Seq to identify regions of the genome with increased accessibility, which indicates transcriptional activity. They found that while diseased and healthy valves had similar chromatin profiles, they differed from those of other heart tissues. Valve specific open chromatin regions were enriched in binding sites for NFATC, a transcription factor known to regulate valve formation. And, specifically in MVP tissues, they found two potential causative sequence variants. These MVP-linked variants exhibited enhancer activity in cultured cells. And for one variant, the team identified the gene target of this variant. In providing the first mitral valve cell chromatin profiles and demonstrating their use and functional analysis of MVP-linked variants, this work supplies a valuable research for mitral valve prolapse evological studies.
Dr Cindy St. Hilaire: The third article I want to share is titled, Molecular Mechanisms of Diaphragm Myopathy in Humans with Severe Heart Failure. The first author is Norman Mangner, and the co-senior authors are Axel Linke and Volker Adams from Dresden University of Technology in Germany. The diaphragm is the primary muscle controlling a person's breathing. This muscle can become weakened during heart failure, which exacerbates symptoms and increases the risk of death. The pathological mechanisms underlying the diaphragm's demise are largely unclear. Studies in animals have pointed to increase reactive oxygen species as a contributing factor, but human studies have been limited. This group evaluated the histological and molecular features of human diaphragm biopsies from both heart failure patients and controls.
Dr Cindy St. Hilaire: The diaphragm samples were collected from 18 heart failure patients, who were undergoing implantation of left ventricular assist devices. And 21 control samples were obtained from patients not having heart failure bypass graft surgery. Compared with the controls, the heart failure diaphragms showed significantly reduced thickness, severe muscle fiber atrophy, increased oxidative stress in the form of protein oxidation, increased proteolysis, impaired calcium handling and mitochondrial abnormalities and dysfunction. Pathological measures also correlated with clinical severity. These data are the first insights into the pathology of heart failure related diaphragm weakness, and this work points to the molecular players that could be targeted for novel treatments.
Dr Cindy St. Hilaire: The last article I want to share before our interview is titled, Caveolin3 Stabilizes McT1-Mediated Lactate/Proton Transport in Cardiomyocytes. The first author is Jonas Peper and the corresponding author is Stephan Lehnart from the Heart Research Center, Göttingen in Germany. Caveolae are invaginations of the plasma membrane, and these structures are involved in endocytosis, signal transduction and other important cellular processes. Caveolin is the key protein component of caveolae and isoforms of Caveolin have been implicated in heart conditions. Mice lacking the isoform CAV1 develop heart failure and genome-wide association studies have been linked to human CAV1 variants with cardiac conduction disease and atrial fibrillation. Rare variants of CAV3 are known to cause hypertrophic cardiomyopathy. However, little is known about the normal or pathological actions of Caveolin in heart cells where caveolae are plentiful. To learn more, this group performed mass spectrometry, immunoprecipitation, and other analysis in cardiomyocyte, and uncovered novel CAV associated proteins, some of which turned out to be isoform specific.
Dr Cindy St. Hilaire: CAV1 interacted specifically with aquaporin while CAV3 was associated specifically with the lactate transporting McT1 protein and the iron transporting TFr1 protein. When the team knocked out the function of CAV3 in stem cells derived from human cardiomyocytes, they found that McT1 had reduced surface expression and function, and that the cells exhibited abnormal de-polarizations. Together the results set the stage for future studies of cardiomyocyte CAV biology, including how CAV variants might contribute to disease pathogenesis.
Dr Cindy St. Hilaire: Today I have with me Drs Norberto Gonzalez-Juarbe and Maryann Platt from the J. Craig Venter Institute, and they're here to discuss their study, Influenza Causes MLKL-Driven Cardiac Proteome Remodeling During Convalescence . And this is in our March 5th issue of Circulation Research. So thank you both for being with me today.
Dr Maryann Platt: Great to be here.
Dr Norberto Gonzalez-Juarbe: Thank you.
Dr Cindy St. Hilaire: So I want to start with influenza mediated cardiac complications. So what are these complications? How prevalent are they in people who catch influenza and who's most affected?
Dr Norberto Gonzalez-Juarbe: So for the last hundred years, we have known that every time there's an epidemic or pandemic from influenza, there's adverse cardiac events that come after you get the disease. During the 1918 pandemic, we could see myocardial damage and about 90% of all people that succumb to the infection, and in the latest epidemics that has been about 40% to 50%, suggesting that the more pandemic the strain of influenza is, the more virulent, the more of these adverse cardiac events we are going to see. So it seems that it is attached to severity of disease. The virus can get to the heart easy, the more severe your disease phenotype is, but it seems that some pandemic strains have a better way to get there of causing more damage than the common epidemic strengths.
Dr Cindy St. Hilaire: That was actually one of my other questions, how does it get to the heart? What's happening there? Do we know much about that? I guess, specifically for flu, but I'm sure in the back of everybody's mind, people are also thinking about SARS-CoV2 too. So how does that kind of pathway work or transportation work?
Dr Norberto Gonzalez-Juarbe: Circulation is going to be the main way it gets there for, for example, if we were to look at COVID then in the heart there's the same receptors for the epithelial cells that are in there, the ACE-2 receptor, that's also in the cardiac tissue and COVID-19 can actually infect cardiomyocytes through that receptor. In terms of influenza, it's basically similar. Some of these receptors are present on the epithelium in the lungs, are also present there and flu can actually infect cardiomyocytes. In our study we also look at some other cell types like endothelial cells and fibroblasts, and we show that there's actually some lower grade infection too. But that's why it's all of these, it starts in the severity of disease, that's the more virus is going to be in your bloodstream, the easier it's going to be to get there. And since the same receptors are present in the heart, so it's going to be easy for the virus to affect the cell.
Dr Maryann Platt: It's not necessarily dependent on age or race or anything it's dependent on how sick you are, for sure.
Dr Cindy St. Hilaire: And by sick, does that directly correlate with viral load of the patients or just their response, an overactive response or something like that? Do we know?
Dr Norberto Gonzalez-Juarbe: I think it's a double edged sword, so it's going to be related to viral load, but also the type of immune responses that you're going to be having, it's going to affect the role of the virus in their heart. In our case we studied way after you cleared the proof from the lungs. So most of the studies that have been out there for a while show, when you're really, really sick, what is happening, but that of your compounding because you have all of these immune responses happening, and the virus is doing its thing. But once you clear the virus from the lungs, your, kind of, immune system settles down. And in our study, we show that even if you clear it from the lungs, the virus is still present in the heart.
Dr Cindy St. Hilaire: So one of the mechanisms that you focused on in terms of how influenza was contributing or leading to cardiac complications, is this process called necroptosis? Can you just maybe give us a primer on what that is, and what it's doing specifically in the cardiomyocytes?
Dr Maryann Platt: Sure. So necroptosis, there's a couple of different ways that cells can die, either under normal circumstances, just maintaining the number of cells in your body or in the case of infection, trying to get rid of the infection. So most commonly, cells will undergo apoptosis, which is programmed cell death, not very inflammatory. And then necroptosis is another way that is highly inflammatory and driven by, initiated by, some of the same molecular cascades, but then affected by a different set of molecules.
Dr Cindy St. Hilaire: Interesting. And so it's really that inflammatory component that is driving pathogenesis in the cardiac tissue then.
Dr Maryann Platt: Yeah.
Dr Norberto Gonzalez-Juarbe: And evolutionarily necroptosis has been shown to help the host against viral infections. Specifically, influenza has proteins that can block apoptosis, which is kind of like the good way of dying. And then the cell has to undergo these other necrotic type of cell death to get rid of viral replication. But while some of these might interact with both pathways, necroptosis effect their molecule. MLKL is the last protein in the pathway. That's the one that actually rupture the cells. So we wanted to prevent that from happening to see if we can actually stimulate something protective by having all of the other good cascade-type molecules still there.
Dr Cindy St. Hilaire: ‘Good’in quotes (laughing).
Dr Maryann Platt: Still dying cells, less bad, not as inflammatory
Dr Norberto Gonzalez-Juarbe: Inflammatory since the heart is this type of organ that any injury will be, more or less, long lasting, and that will have detrimental effects throughout life.
Dr Cindy St. Hilaire: Got it. That's interesting. So can you maybe give us a summary of your experimental design and kind of the groups you were looking at, and a summary of the results?
Dr Maryann Platt: Sure. So we had four different groups of mice, two of them were wild type mice and two were MLKL, all knockout mice, which could not undergo necroptosis. And then each of those genotypes, we had uninfected mice or mice that were infected with flu. And then we monitored long viral titer to see how much infection was there at the lungs. And then after the infections subsided in the lungs, two days after a viral load was undetectable, we sacrificed those animals, collected their hearts.
Dr Cindy St. Hilaire: That's great. So that two day resolution, is that a similar time course with humans, in terms of a pathogenesis of developing cardiac complications? How similar, I mean, mice are never perfect models, but what's good and what's not good about using a mouse as for this model?
Dr Norberto Gonzalez-Juarbe: So, mice are not human right?. So, we are always thinking about that quote, but most of the cardiac events that occurred during these type of infections and similar things have been observed in, for example, pneumococcal infection, which is by streptococcus pneumonia. Most of these adverse cardiac events occur right after you leave the hospital. Those are a specific set of adverse cardiac events that are different from the ones that happen when you are severely infected in the hospital. And these can be arrhythmias and myocardial infarction, and some of these things that can happen up to 10 years after you recover from the pulmonary infection.
Dr Norberto Gonzalez-Juarbe: So our model was designed to see that step of the host trying to retcover. And if there was still something there in the heart, right after you get out of the hospital, that you receive your therapeutics, and you're thinking, 'Oh, I don't have any more flu in my lungs, and I'm recovering', that timeframe right after you get out, you might still have some other things happening in your body, that might determine what happens to your heart.
Dr Cindy St. Hilaire: Interesting. So you may actually be feeling pretty good, but your heart or even possibly other organs are still kind of under the weather, so to speak?
Dr Norberto Gonzalez-Juarbe: Exactly.
Dr Maryann Platt: Exactly.
Dr Cindy St. Hilaire: So in your proteomic analysis, I think you stated it was some, it was just under a hundred proteins were differentially regulated, and a majority were actually in kind of metabolic mitochondrial related pathways. Could you maybe tell us the importance about that? But then also, yes, that was a big chunk of it, but were there any other pathways that were either up or down, that were surprising in your findings?
Dr Norberto Gonzalez-Juarbe: The importance of the major mitochondrial proteins that we found, first that the MLKL knockout, so inhibiting these necrotic cell death actually promoted mitochondrial health. So that first was interesting, because that will suggest that this can be quite therapeutic target in the future. That innovation enhance some proteins that protect the mitochondria and aid in mitochondrial function. And if we think about the heart as our engine, we need energy for an engine to work and mitochondria is that energy resource that we have. And the heart is really relying on these, because if you have a metabolic breakdown in the heart, you get cardiac event. So most of the proteins that were changed upon infection had to do with these specific, important metabolic function of the heart. Some other proteins have to do with cellular signaling mechanisms and calcium homeostasis, all these other things that are important to maintaining homeostasis in the heart thus suggesting that the virus is inducing massive stress in their heart without actively replicating or causing inflammation.
Dr Norberto Gonzalez-Juarbe: And that was very important in our study that we didn’t see these antiviral effects, but at the same time, we saw all of these detrimental metabolic effects. So future studies might be also targeting what viral factors might be actually inducing these metabolic effects in the heart. But we also saw some molecules important for cell death mechanisms that were not necroptosis.
Dr Norberto Gonzalez-Juarbe: Marianne, you can describe some of those.
Dr Maryann Platt: So one third way that cells can die is called pyroptosis. And we actually saw that pyroptosis was also elevated in flu infected mice, in their hearts, suggesting that it might not just be necroptosis. All this inflammation coming from necroptosis is what's driving breakdown of heart function, but also possibly pyroptosis.
Dr Cindy St. Hilaire: The mitochondrial aspect is interesting. In heart failure normally there's the switch from fatty acid oxidation to glycolysis. Does that happen in a shorter or smaller way after flu? And in some patients they just don't recover? Is there a metabolic switch to an infected cardiomyocyte, that is more transient, and then in a subset it turns to permanent? Is that what's happening?
Dr Norberto Gonzalez-Juarbe: Yeah, that is something that we might need to follow up on, since our study was more of a snapshot of that specific time point. It will be good to do follow-up studies where we look at different time points post infection. And even maybe three months after infection, then six months after infection. We have done similar studies with pneumococcal pneumonia, and we have found that cardiac function and metabolic function, it is significantly remodeled, even three months after the pneumonia event.
Dr Cindy St. Hilaire: Interesting. So once it's actually cleared from the lungs, it's still…
Dr Norberto Gonzalez-Juarbe: The heart is still undergoing this injury recovery, which cause scarring process and these leads to reduced cardiac function.
Dr Cindy St. Hilaire: So influenza actually, maybe a lot of people know this now, but it was somewhat new to me, I guess, at least a year ago when COVID first started. But influenza like SARS-CoV2 is an enveloped virus. It's a single strand RNA virus. So are these findings specific to this class of viruses, specific to RNA viruses? Or is this something that you think is operative in other types of viruses in terms of causing these cardiac complications?
Dr Maryann Platt: It's certainly possible. I'm not a virologist. (laughs).
Dr Cindy St. Hilaire: Not yet. (laughs).
Dr Norberto Gonzalez-Juarbe: Eventually you'll get there.
Dr Maryann Platt: Yeah, eventually probably. But you know, there have been reports of lots of adverse cardiac events in SARS-CoV too. So it's certainly not just unique to influenza, as far as other types of double stranded RNA viruses. I'm not sure.
Dr Norberto Gonzalez-Juarbe: Yeah, of course Coxsackieviruses viruses have shown inductionof cardiac events. And there's a Review in the New England Journal of Medicine about some of these other pneumonia causing agents, but also all other pathogens that can do some of these events, but it's all clinical observations. So, we think that our study and several others studies that are starting to come out, can induce a shift part of field to look at how some of these major respiratory viruses can induce these adverse cardiac events that we see are highly prevalent, right after the event, like during infection. And importantly, how all the pathogens may synergize. Some pathogens such as RSB, flu, COVID, have synergized with bacteria or other virus one enhancing the ability of the other to cause injury and disease.
Dr Norberto Gonzalez-Juarbe: For example, flu with pneumococcal disease, COVID with assorted grand negative pathogens, and actually influenza also has been shown to cause co-infection. So we don't know how some of these pathogens may synergize in the lungs, but also in other organs, to cause these injury that are going to be long lasting. So we are having the acute problem now with COVID and we had this with the 2009 pandemic flu, but in the next 10 years, five years, we're going to see this equivalent of disease damage, the damage associated with the disease, and we are going to have to explain why people are having these cardiac events, why people are having kidney events or liver damage problem. So we need to better understand not only how RNA viruses do this, and there's actually data shows that COVID is present in the cardiac tissue and can replicate in cardiac cells, but also how they may synergize to potentiate these effects. And how can we prevent all of these from happening? By action, therapies to antivirals, or any other way.
Dr Cindy St. Hilaire: That's a perfect segue to my last question I had. And that is, how can, what you found in the study regarding necroptosis, or even just the base proteins that are involved, is it able to be leveraged either for the development of therapies or perhaps even like a screening method, a biomarker to determine which flu patients might go on to develop cardiac phenotypes?
Dr Norberto Gonzalez-Juarbe: There might be a couple of avenues our study can help create these adjunct therapeutics to anti-virals. So one might be targeting the specific necrotic cell pathways to prevent that titrating that is long-lasting and these can be targeting necroptosis or pyroptosis, and there's FDA approved drugs that we may be able to repurpose to target some of these pathways that have these secondary effects, that can target these pathways. But also the very interesting part for me was that MLKL lesion increased this protein called NNT, which is a major factor of mitochondrial function and ATP production. So if we can improve the ability of the heart function and to protect their mitochondria, then we probably can have more roughly protective response against not only flu, but maybe COVID or other viruses that might also do similar things to the heart.
Dr Cindy St. Hilaire: Or even just other heart failures. That's pretty neat.
Dr Norberto Gonzalez-Juarbe: Exactly.
Dr Maryann Platt: Yeah, exactly.
Dr Cindy St. Hilaire: That's great. Drs Gonzalez-Juarbe and Platt. Thank you so much for joining me today. Congratulations on an excellent study and I'm really looking forward to your future, probably viral related, work.
Dr Norberto Gonzalez-Juarbe: Thank you very much.
Dr Maryann Platt: Thanks.
Dr Cindy St. Hilaire: That's it for our highlights from the March 5th and 19th issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and Circ. Thank you to our guests, Drs Norberto Gonzalez-Juarbe and Maryann Platt. The podcast is produced by Rebecca McTavish and Ashara Ratnayaka, edited by Melissa Stoner, and supported by the Editorial Team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire and this is Discover CircRes, your on-the-go source for the most exciting discoveries in basic cardiovascular research.
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