April 2022 Discover Circ Res

This month on Episode 35 of Discover CircRes, host Cynthia St. Hilaire highlights two original research articles featured in the April 1 issue of Circulation Research, as well as highlights from the Stroke and Neurocognitive Impairment Compendium in the April 15th issue.  This episode also features a conversation with Dr Shubing Chen and Dr Yuling Han from Weill Cornell Medical College to discuss their study, SARS-CoV-2 Infection Induces Ferroptosis of Sinoatrial Node Pacemaker Cells.   Article highlights:   Pabel, et al. Effects of Atrial Fibrillation on the Ventricle   Pattarabanjird, et al. P62-Mediated B1b Cell Atheroprotection   Iadecola, et al. Introduction to the Compendium on Stroke and Neurocognitive Impairment   Cindy St. Hilaire:        Hi and welcome to Discover CircRes, the podcast of the American Heart Association's Journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, from the Vascular Medicine Institute at the University of Pittsburgh. And today I'm going to be highlighting articles from our April issues of Circulation Research.                                     I'll also speak with Dr Shubing Chen and Dr Yuling Han from Weill Cornell Medical College, and they're with me to discuss their study, SARS-CoV-2 infection induces ferroptosis of Sinoatrial node pacemaker cells.   Cindy St. Hilaire:        The first article I want to share is titled, Effects of Atrial Fibrillation on the Human Ventricle. The first author is Steffen Pabel and the corresponding author is Samuel Sossalla and they're from Regensburg University. Atrial fibrillation, or AFib, is the most common form of heart arrhythmia. Patients with AFib may experience shortness of breath, dizziness and weakness. And they're also at risk for more life-threatening complications, such as clot-induced stroke and heart failure. Focusing on heart failure, this study investigated how disruptions to rhythm in the atria might lead to changes in the ventricular myocardium. The team studied ventricular muscle tissue from 24 patients with AFib and 31 without AFib. While the levels of fibrosis were equivalent in ventricular myocytes from both the AFib and the non AFib patients, other cellular features were distinct. For example, patients with AFib had reduced systolic calcium release, prolonged action potential duration and increased oxidative stress, compared with the non AFib patient controls. These differences were largely recapitulated in ventricular myocytes derived from human induced pluripotent stem cells that had been electrically stimulated to either mimic AFib or normal sinus rhythm. The results indicate that AFib affects the ventricles just as well as the atria and might therefore be best studied and treated with the whole heart in mind.   Cindy St. Hilaire:        The second article I want to share is titled B-1b Cells Possess Unique bHLH-Driven P62-Dependent Self-Renewal and Atheroprotection. The first author is Tanyaporn Pattarabanjird and the corresponding author is Colleen McNamara, from the University of Virginia.   Atherosclerosis is a complex and dynamic chronic inflammatory condition. However, not all immune cells exacerbate this disease. Some immune cells are actively dampening the inflammation. B-1 cells are such cells that do this, and they produce IgM antibodies that bind cholesterol, preventing its uptake into macrophages and therefore limiting macrophage driven inflammatory responses. Increased number of B1 cells, therefore, might be atheroprotective. In mice, deletion of the transcription factor ID3 leads to a boost in B-1 cell IgM production.   Cindy St. Hilaire:        In this work the authors investigated the molecular mechanism underlying this effect and found that upon deletion of ID3 in mice B-1b cells, the level of P62 protein was increased. B-1b cell proliferation was found to be dependent on P62 and over expression of P62 in mouse B-1b cells increased cell numbers, raised plasma IgM levels and importantly, ameliorated diet-induced atherosclerosis in animals. The team went on to show that people with an ID3 mutation had an unusually high level of serum IgM and B-1b cell P62. This suggests that results from mice may hold true for humans, and if so, could inform the development of immunomodulatory treatments for atherosclerosis.   Cindy St. Hilaire:        So the April 15th issue of Circulation Research is our Stroke And Neurocognitive Impairment Compendium. The last Circulation Research Compendium on Stroke was published about five years ago. In this year Dr Costantino Iadecola, Dr Mark Fisher and Dr Ralph Sacco focused this update on advances made over the past five years, with a focus on topics that were not addressed in the previous compendium, that best reflect the leading edge of basic in clinical science related to cerebral vascular diseases. Seemant Chaturvedi, Brian Mac Grory and colleagues provide an overview of preventative strategies according to stroke mechanism, including stroke of unknown cause. And the challenges of stroke prevention with antithrombotic therapy and subjects with increased hemorrhage risk are also considered.   Cindy St. Hilaire:        Stéphanie Debette and Hugh Markus provide an account of the most recent developments in the genetics of cerebrovascular diseases. The gut microbiota is another factor that has recently been linked to stroke risk and Pedram Honarpisheh, Louise McCullough and colleagues provide a comprehensive overview of the microbiology and the microbiota, and the influence that stroke risk factors exert on its composition and homeostatic relationship with mucosal surfaces. Karin Hochrainer and Wei Yang provide a systematic review of the large amount of data and stroke proteomic from animal models and human patients. Matthias Endres and colleagues cover the dramatic effect that innate and adaptive immunity exert on stroke risk and on acute brain damage and post stroke sequelae, such as post-stroke cognitive impairment and depression.                                     Cindy St. Hilaire:        Manuela De Michele, Alexander Merkler and colleagues discuss the cerebral vascular diseases that have emerged as a frequent manifestation of the maladaptive immune response to severe SARS-CoV-2 infection. Jessica Magid-Bernstein and Lauren Sansing review the current concepts on epidemiology, risk factors in etiology, clinical features, as well as the medical and surgical interventions for cerebral hemorrhage. Yunyun Xiong and Marc Fisher cover the progress that has been achieved in the treatment of acute ischemic stroke and Natalie Rost and Martin Dichgans and colleagues address the long term impact of stroke on cognitive function, which is becoming a significant healthcare challenge in the world's aging population.   Cindy St. Hilaire:        So today I have Dr Shubing Chen and Yuling Han from Weill Cornell Medical College. And they're with me to discuss their study SARS-CoV-2 infection induces ferroptosis of Sinoatrial node pacemaker cells. And this article is in our April 1st issue of Circulation Research. So thank you both for joining me today.   Shubing Chen:             Thank you. It's really nice to join the program, and it's really a great honor.   Cindy St. Hilaire:        It's a really great article. I'm so excited to talk about. So there's a lot of research happening regarding SARS-CoV-2 virus and the patients who are infected and have COVID-19. And this paper is focusing on the impact of viral infection on the heart and specifically on the sinoatrial node, which is the primary cardiac pacemaker that keeps our hearts beating. So I was wondering if you could tell us what led you to focus on this particular aspect of COVID-19 symptoms, and also how early in the pandemic did you start this?   Shubing Chen:             Yeah, so we started working on SARS-CoV-2 through back to early 2020 when very unfortunately, New York City was a pandemic center and we had a lot of patients in the hospital unit, and also postdoc students working very hard in the lab. So that's the time we start working on SARS-CoV-2. And I was trained as a stem cell biologist. And what we're really interest is to set up a platform to basically understand which type of cells can be infected by SARS-CoV-2 and if they can, how they respond to SARS-CoV-2 infection. Not only for SARS-CoV-2, we sent it as like a viral infection platform, but SARS-CoV-2 is one of the virus we study now. And it's kind of very surprising. We have a pretty broad platform. We have a lung organoid, we have colon organoids, we have pancreas, we have cardiomyocytes, pacemaker cells. And as expected, we see lung can be infected like colon and because patient had GI tract, liver can be infected, but very surprisingly we see very high cardiomyocytes infection as well as pacemakers.                                       So as we'll know that still big controversy in the field, whether we can detect SARS-CoV-2 like viral protein or viral RA in the heart, in particular, cardiomyocytes. But I think now everyone agree that the cardiomyocytes really can be very well infected actually. Because it's very difficult to get the pacemaker tissue and the sinoatrial tissue from the COVID patient. So we collaborate with Dr Ben Andora’s lab at NYU to get this hamster model. So we basically take SA tissue from hamster and then other colleagues basically did the section imaging, and we confirm that the hC4 polymerase cells can be infected by SARS-CoV-2. And at that time we start to learn a more clinical studies they report the COVID patient, they develop arrhythmia, or some other problem, not only with cardiomyocyte, as well as the conduction system. So at that time, that's the time that we say maybe we should do something on the pacemaker and focus on that. So that's how the project was developed.   Cindy St. Hilaire:        That is so interesting. And so I know humans infected, like you just said with SARS-CoV-2, they can develop arrhythmias. What's that timeframe? Is there a common timeframe that this happens? Does it normally happen very close to the infection or only in later stage? What's that window of when these arrhythmias are happening?   Shubing Chen:             At least based on the clinical study we show right now, actually the patient can develop acute arrhythmia. So it can be very soon after they developed symptom for COVID.   Cindy St. Hilaire:        Wow. That's amazing. So you mentioned this, your study utilized a hamster model, which you actually don't see a lot of. Most studies use a lot of rats or most studies I'm familiar with, especially in Circulation Research, they use more rats or more mouse models. So what advantages does that hamster model have and why were you interested in using it?   Shubing Chen:             Yeah, that's actually really specific for SARS-CoV-2. As SARS-CoV-2 mainly use ACE2 as a key entry factor to enter the cells. Of course, there's additional receptor, like neutrophils is one. Like all this enzyme involved, but human and mouse ACE2, they have very different structure. So the SARS-CoV-2 virus combine with human ACE2 very well but not mouse ACE2. So from the beginning, the rat and mouse was not used as a very good model to study SARS-CoV-2 infection. Of course there are other models, like knockin human ACE2 in the mouse and also like ACE2 transgenic mice. That's how different mouse model use. But hamster you don't need any modification, but they are very promising to SARS-CoV-2 infection. And so that's a reason we decide to use that as an animal model to basically run in parallel with our human stem cell model.   Cindy St. Hilaire:        We joke in my lab, mice are not little humans, but it's really true in a lot of cases, they're beautiful models in so many ways, but then when they don't work, they really don't work.   Shubing Chen:             Yeah. Before COVID every time when we try to talk about our human stem cell, derived cells, organoids as a disease model. People always ask, why do you want to work on human organoids? Right? It's that we have all these beautiful animal models like as you mentioned, mouse or rats, that's very broadly used. And we have to find different reasons. And now when we start working on SARS-CoV-2, which is very clear example, that mouse are not identical to human. Yeah.   Cindy St. Hilaire:        Yeah. That's great. I love finding additional models to use that are the best one for the question. So in order to investigate, I guess kind of the mechanism of how this was happening in the SAN cells, the sinoatrial node cells, you had to develop a new differentiation protocol that took the human embryonic stem cells, I think it was the H9 line you used, and essentially differentiate that cell line into a sinoatrial node-like cell. So I was wondering if you could tell us a little bit about A) how did you figure out that protocol and B) how does it work?   Shubing Chen:             So it's actually a long story to cell line.   Cindy St. Hilaire:        We can condense it. Let's get-   Shubing Chen:             At least based on the clinical study we show right now, actually the patient can. Let's condense it. But it's as you can imagine, we did not develop this cell line only for this particular project. Actually, we start working on this cell line back to maybe six, seven years ago. The first postdoc we have who basically knockin the mCherry, Myh6. Which basically label the atrial cardiomyocytes. And another postdoc, Zanir, he basically put a GFP in the SARS2 locus. So now we have this duel reporter line we can visualize the SA nodal cells. And we really spend a lot of time on that because we think that unfortunately in our hand, there is not really no good antibody for SARS2. We think it's very, very important that you can see these cells. So after developing these lines and because my lab run a lot of chemical screening, where we run Zanir, we run several chemical screening to develop the protocol.                                       And Jialing Zhu, another postdoc in the lab, also pick up the project to further develop the protocol. And there is several years’ work. We do have this good protocol to make pretty efficiently to make the cells. And it's not only our work. I want to say that. For example, Dr Sean Wu from Stanford, they did this beautiful study on the single cell RNC mouse conduction system and Dr Gordon Keller and many other labs also basically published protocol in the field. We are very excited about this duel reporter line. I think they gave us a lot of new opportunity and we are very happy to share this line. Yeah. So if anyone in the field are interested in that, just contact us.   Cindy St. Hilaire:        Yeah. Anyone listening. That's great. So were you surprised to find the entry factors that SARS-CoV-2 uses to get into a cell, were you surprised to find them on these sinoatrial node cells? And I guess in the context of comparing these particular cells to other cells in the heart, are those entry factors higher in the sinoatrial node cells?   Shubing Chen:             So it can be either surprised or not surprised let's say this way. So because one, we see the cardiomyocytes that can be infected, we were kind of surprised. And then we find actually several type of cells in the heart can be infected, like endothelial cells. I will say that the ACE2 expression of like ACE2 aminophenol in pacemaker cell, it's not significantly higher than cardiomyocytes. So we are not really saying, or seeing that SA nodal cells are more permissive to SARS-CoV-2 infections compared to cardiomyocytes, even in the petri dish, but they can be infected.   Cindy St. Hilaire:        So you found SARS-CoV-2 infection in these sinoatrial nodal cells induces a process called ferroptosis. So Yuling, I was wondering if you could tell us what is ferroptosis and what is it doing in these pacemaker cells?   Yuling Han:                 For the ferroptosis, they was surprised so far that its by the RA sequencing of the SARS-CoV-2 infection make our cells. And the first process is mainly caused by the-   Shubing Chen:             Error in iron.   Yuling Han:                 Yes. So more intake of the iron error and induced the RA's pathway and caused the cell deaths. So by our RA sequencing, we found the key factor involved in ferroptosis pathway is the GPS score was checked after the SARS-CoV-2 infection. So we focused on the ferroptosis pathway and found other key factors or checked after the infection makes in the pacemaker cells.   Cindy St. Hilaire:        What is the ferroptosis doing that disrupts the SNA cells?   Shubing Chen:             Ferroptosis is a type of cell death mechanism. So eventually it will cause cell death. And we think something that is really surprising, but we think it's very interesting, is we only see ferroptosis in the SARS-CoV-2 infected general atrial cells. So SA cells, we actually, as Yuling mentioned, when we develop this platform, we see different type of cell can be affected. And we are very curious what happened. So we see that we run a sequence on each individual cells we can see infection and along, we can see cell death like apoptosis in cardiomyocytes. We see apoptosis and only in SA nodal cells, we actually see the ferroptosis pathway as we come up.   Cindy St. Hilaire:        Why do you think that is in that cell type versus in another? Do you have any ideas about why?   Shubing Chen:             No, we don't have any idea yet to be honest, but we are working on that. But at least I think that it gave us some clue that we really need to use different type of whole cells to study the whole cell response. Because traditionally when we study viral infection and when we see lung, we always say, oh, the cell died. It's fairly simple. But now if we really study the details and we think it's maybe over simplified way to think about how cells can respond to viral infection, not only to SARS-CoV-2 infection. So it gives us the motivation, very strong motivation to now really study how different host tissues response to viral infection.   Cindy St. Hilaire:        I thought that was really interesting, not all cell death is the same.   Shubing Chen:             Yeah. And another thing is kind of a little bit surprising is we actually did a very careful comparison between the SA nodal cells and the cardiomyocyte. We only see ferroptosis come up as SA nodal cell, but not cardiomyocyte. Again, we don't understand why as maybe some host factor that is specific, we're working on that.   Cindy St. Hilaire:        So in addition to working out this mechanism of what is going wrong when these cells are infected with the virus, you also used this embryonic stem cell like tool for a drug screen. So can you walk us through that process in terms of what you did to do that? Did you focus in on one specific type of drugs or was it just kind of an unbiased screen?   Yuling Han:                 For the sinoatrial pacemaker cells, we focus on the antiviral drugs screening. And we also did several other projects, like lot of night or some neuron cells. For the [they did drug screening to find some drugs to inhibit the SARS-CoV-2 entry. And for the dominic neuron, we found SARS-CoV-2 infection can cause neuro cells synapses. So we focus on the synapses associated drug screening, but for the pacemaker cells, they only did the antiviral drug screen.   Cindy St. Hilaire:        And you came up with two drugs that you wrote about in the paper, deferoxamine and imatinib. So what are the mechanisms of action of those drugs? Are they targeting the same thing or are they targeting slightly different things?   Yuling Han:                 For the imatinib, we also found this drug inhibit SARS-CoV-2 entry and we did several other screenings, like the lung organoids and neuro cells. We also found this drugs. And the six drug, the mechanism is kept and the spec protein of SARS-CoV-2. And this was found by several other groups and published some paper this year. And we found this in 2020 maybe. And we published this paper before and we found this mechanism. And for another drug, we checked the RA sequencing data of SARS-CoV-2 affect the peacemaker cells. And we did several run of RA sequencing. And we compared the key factors, involved in SARS-CoV-2 entry. Several key factors like CTSL and like TMPS2 and among several run of RA sequencing. We only found the drug can decrease the expression of CTSL. So we also did PTR immunostaining, and then we found the drug decrease the expression level of CTSL.   Shubing Chen:             Yeah. So actually the other drug, it's also an antiferroptosis drug. So we did the mechanism study and it's very nice to see, we also identify the drug from an unbiased chemical screen. And for the chemical screening, we actually have a pretty large platform and we have around 1200 FDA approved drugs. We have like a 2000 anatrofin amino acid that signal pathway regulators for most of the SARS-CoV-2 screening, as you did mention, we have multiple screening platform. We focus on FDA approved drug. So it's more like for the drug repurposing and for other screening we also write larger skills.   Cindy St. Hilaire:        So we got a mechanism, we got a super specific cell type and we now have some drugs. So what are the translational implications of these findings? And I guess I'm thinking about that in terms of the time course of when a patient gets infected, has symptoms, has arrhythmia, like where could you possibly target this ferroptosis pathway? Meaning if someone already is exhibiting AFib as a result of the infection, is that actually too late? Or can you start to treat it to reverse it or prevent it from getting worse? Like what do you see as a therapeutic potential for using these drugs?   Shubing Chen:             That's a very good question. I will say this way, I think when we identify all these drugs, it's very, very exciting. But for antiviral drug development perspective, we definitely want a drug that show broader spectrum. So for COVID patient, of course we want to protect their heart, but we also want to protect their lungs.   Cindy St. Hilaire:        Exactly. Protect everything.   Shubing Chen:             Exactly. Exactly. So for the real drug that can clinical use, I think the lack of broad spectrum antiviral drug, I think that will be the way to go for drug development and for the cardioprotective respective. So if the patient do have very severe cardio symptom, particularly like arrhythmia symptom, I think that can be considered. But I don't want to really say this is the drug to treat the COVID patient. I don't think that's a way to go, particularly for ferroptosis is a cell type. This is a phenotype, very specific for the pacemaker. And I think for us, as a basic scientist, is very, very important that we understand the biology and we can identify these normal chemical tools that we can manipulate the system that can facilitate the future drug development.   Cindy St. Hilaire:        So do you think your findings and I mean findings at multiple levels, that a viral infection can induce apoptosis in one cell, but ferroptosis in another cell, but also the findings of viral infection in general, sufficient enough to drives sinoatrial node cell dysfunction. Do you think this is specific to SARS-CoV-2 and corona viruses or do you think this is something that is more broad with other viruses that maybe we just haven't recognized possibly because we don't have the tools yet?   Shubing Chen:            That's a great question. I will say some other type of virus can also infect heart, at least cardiomyocyte, like a Coxsackie virus, regular virus three. And there's actually a lot of study on the viral infection on the cardiomyocytes. And for us, the most exciting part is we really have now in serious, limited starting materials to get these pacemaker cells. Like I SA nodal cells. So we can use this as a platform to study how other virus infect, how the viral infection in general cause cell dysfunction. Because in the study we also do the calcium blocks assay, we can monitor their beating and then we can do RN-seq to monitor their transcription changes. Because this we have this still reporting system, we can purify cells, we can even run larger scale, like epigenetic level, how they change. So that's a very useful tool to study how cell responds to viral infection. I'm very excited about that.   Cindy St. Hilaire:        That's great. Well, Dr Chen and Dr Han, thank you so much for joining me today. Congratulations on a beautiful story. And I look forward to hearing more out all these different organoid and cell models you have.   Shubing Chen:            Cindy, thank you. Thank you for so much for having us.   Cindy St. Hilaire:        That's it for the highlights from the April issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Dr Shubing Chen and Dr Yuling Han. This podcast was produced by Ishara Rantikac 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 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 our information visit ahajournals.org.