Discover CircRes

This month on Episode 28 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the August 20th and September 3rd issues of Circulation Research. This episode also features an in-depth conversation with Dr Scott Cameron from the Cleveland Clinic and Dr Milka Koupenova from the University of Massachusetts Medical Center about their study, SARS-CoV-2 Initiates Programmed Cell Death in Platelets.

Article highlights:

Gupta, et al. Electronic Cigarettes and Oxidized Lipids

Bartosova, et al. Glucose Derivative Induced Vasculopathy in CKD

Atmanli, et al. DMD Correction Attenuates Cardiac Abnormalities

Ma, et al. Length Dependent Activation in Porcine Myocardium

Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast for 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 will be highlighting articles presented in our August 20th and September 3rd issues of Circulation Research. I also will speak with Dr Scott Cameron from the Cleveland Clinic and Dr Milka Koupenova from the University of Massachusetts Medical Center about their study, SARS-CoV-2 Initiates Programmed Cell Death in Platelets.

Cindy St. Hilaire: The first article I want to share is titled Electronic and Tobacco Cigarettes Alter Polyunsaturated Fatty Acids and Oxidative Biomarkers. The first author is Rajat Gupta and the corresponding author is Jesus Araujo from UCLA. E-cigarettes have surged in popularity in the last decade and while many people switching from traditional cigarettes to smokeless ones view the latter as a safe alternative to smoking tobacco, emerging data shows that E-cigarettes cause adverse effects such as oxidative stress, inflammation and endothelial dysfunction in users. The aerosols produced during vaping contain similar levels of reactive oxygen species, also called ROS, as the vapors of tobacco smoke. However, data on the extent to which E-cigarettes, E-cigarette ROS, influences cardiovascular health is lacking.

Cindy St. Hilaire: To address this, this group recruited 32 chronic users of E-cigarettes, 29 chronic tobacco smokers, and 45 individuals that used neither and they measured their plasma levels of oxidative biomarkers. The team found both similarities and differences between the E-cigarettes and the tobacco users. For example, both smoking groups had increased plasma antioxidant capacity and decreased levels of oxidized linoleic acid compared with the levels seen in non-users, while arachidonic acid levels were raised in tobacco smokers and reduced in E-cigarette users. Overall, however, the biomarker levels were deemed to be intermediate for E-cigarette users between the non-users and the tobacco users. This study suggests that while E-cigarettes carry a lower health risk than tobacco, they are by no means safe.

Cindy St. Hilaire: The second article I want to share is titled Glucose Derivative Induced Vasculopathy in Children on Chronic Peritoneal Dialysis. The first author is Maria Bartosova and the corresponding author is Claus Schmitt and they're from the University of Heidelberg. Diabetes, high blood pressure and obesity are risk factors for both cardiovascular disease and chronic kidney disease. Worse still, loss of kidney function and even dialysis itself are thought to exacerbate cardiovascular issues. In the case of dialysis, it's thought that high levels of glucose degradation products, or GDPs, in the dialysis fluids can promote the addition of sugar moieties to vascular proteins and lipids causing vascular damage. To investigate this theory, Bartosova and colleagues studied vascular tissue from children with chronic kidney disease receiving dialysis fluids with either high levels or low levels of glucose degradation products and compared these to tissues from children not on dialysis at all.

Cindy St. Hilaire: Proteome and transcriptome analysis of the vessel tissues revealed that compared with patients or no to low GDP fluids, patients receiving high GDP fluids had higher levels of damaging glycation, increased transcription of genes involved in cell death, and decreased transcription of genes involved in cell survival and cytoskeletal reorganization. In line with these findings, vessels from high GDP patients displayed considerable evidence of damage, such as markers of apoptosis, skeletal disintegration and thickened intimas. The results confirmed GDPs can cause vasculopathy and suggest low GDP fluids should be used for dialysis patients.

Cindy St. Hilaire: The next article I want to share is titled Cardiac Myoediting Attenuates Cardiac Abnormalities in Human and Mouse Models of Duchenne Muscular Dystrophy. The first author is Ayhan Atmanli and the corresponding author is Eric Olson from UT Southwestern. Duchenne Muscular Dystrophy, or DMD, affects one in 5,000 baby boys and is caused by mutations in gene for dystrophin, an architectural protein essential for muscle cell integrity. Patients display profound muscle degeneration and weakness, with respiratory and heart muscle dysfunction being a major cause for death. With the recent improvements in respiratory medicine that extend the lives of patients, this group now focused on heart dysfunction and specifically, whether gene editing could mitigate it. The team created induced pluripotent stem cells, or iPSCs, from Duchenne Muscular Dystrophy patient and his healthy brother and showed that gene editing from the DMD cells enabled their development into normal-looking cardiomyocytes with normal contractile function and calcium handling, equivalent to that seen in healthy control cells. The unedited DMD cells, by contrast, did not develop normally. For great clinical relevance, the team edited DMD cells after cardiomyocyte differentiation showing that this reduced their propensity for arrhythmia, compared with that of unedited cells.

Cindy St. Hilaire: Lastly, the team provided evidence to suggest gene editing may improve heart abnormalities in mice with the same mutation. All together the results are proof of principle and support of the development of gene editing therapy as treatment for DMD.

Cindy St. Hilaire: The last article I want to share is titled The Super-Relaxed State and Length Dependent Activation in Porcine Myocardium. The first authors are Weikang Ma and Marcus Henze and the corresponding author is Thomas Irving and they're from the Illinois Institute of Technology. Myofilament length-dependent activation or LDA is the fundamental mechanism coupling the force of the heart's contraction to it's proceeding diastolic volume. In other words, LDA ensures that the more the heart fills, the stronger it contracts. Studies of rodent hearts have given insights into LDA mechanics. However, how it operates in large mammalian hearts is unknown. Using structural and biochemical analysis of pig myocardial fibers, this group found that compared with small stretches of the fibers which were equivalent to small diastolic volumes, long stretches induced greater ATP turnover and greater numbers of cross bridges between myosin and actin filaments which are critical contractile machinery proteins.

Cindy St. Hilaire: Myosin motors can be found in three stages, engaged with actin, unengaged in a disordered, relaxed state but ready to engage, or super-relaxed state where they are essentially switched off. The team showed that as muscle stretch increased, the amount of super-relaxed myosin motors diminished with more myosin motors becoming engaged to enable a stronger contraction. When the fibers were treated with a myosin motor inhibitor, these stretch effects were impaired. In revealing the mechanisms of myofilament length-dependent activation, this study provides a platform for studying cardiomyopathies in which this system goes awry.

Cindy St. Hilaire: So today, Dr Scott Cameron from the Cleveland Clinic and corresponding author of the paper, Dr Milka Koupenova from the University of Massachusetts Medical Center, are both with me to discuss their study, SARS-CoV-2 Initiates Programmed Cell Death in Platelets. And this article is in our September 3rd issue of Circ Research and for full disclosure, the editor of Circ Res, Dr Jane Freedman is also an author on this manuscript. And for full double disclosure, I know Dr Koupenova quite well as we were both graduate students together back in the Ravid Lab at Boston University. However, the full Editorial Board selects these articles, not just me alone and this one is timely, novel, and an amazing story. So thank you both for joining me today.

Milka Koupenova: Thank you for having us.

Scott Cameron: Privileged to be here.

Cindy St. Hilaire: So before we jump into the story that is your paper, can you give us a little bit of background about platelets? I know for years, I guess certainly before Katya's lab, I just thought of platelets as little nucleus-free particles that clot. But we know they are so much more than that. So why are they so important? And how do they function to do more than just stop a bleed?

Milka Koupenova: So this is a great question, Cindy, and I am happy that you alluded exactly to the anucleated nature of platelets. So platelets are cell fragments. They're precursors in the bone marrow, the megakaryocyte. They are the second most abundant blood component after the red blood cells. And traditionally, platelets have been known, as what you pointed out, as these little units that change their conformation once there is some form of a problem with either the vascular, which we have a cut, they come together, they form this clot, and bleeding is prevented. But as we have learned perhaps in the past 20 years that platelets have a profound immune role during various immune processes and infections for different kind of microbes. And particularly relevant to this paper is that we understand that platelets have clearly a role responding to the viruses and activating the immune system.

Cindy St. Hilaire: Yeah, and that was actually my next question. You and Jane are the world-leading experts on platelets and viral responses. So what was known about that interaction, I guess before we started looking at SARS-CoV-2, what was known about that platelet virus or even type of virus interaction?

Milka Koupenova: So SARS-CoV-2 is a RNA virus--respiratory virus that we actually thought similarly to influenza that it mostly stays in the lower respiratory tract where it becomes problematic. However, from our work with influenza, when we saw that in certain patients you actually can detect the virus in platelet. In the beginning of the pandemic, we hypothesized that perhaps, in some people, the virus crosses over into the circulation. And based on our previous studies with influenza, we wanted to see if that indeed is the case. Hence we initiated a study here at UMass with the department head who is also on the paper, Dr Finberg, who is a leading expert in influenza and novel virus and we collected platelets from people to see if we can detect it. And so in the beginning, we were not able to detect SARS-CoV-2 in platelets. So we collected platelets from 17 patients and by qPCR with the primers that the CDC has, for whatever reason I couldn't detect anything. And I was really frustrated because previous reports have shown that about 25%, in some people even 35% of the study population, SARS can be detected. So very interesting observations.

Milka Koupenova: I could see it by immunofluorescence but I couldn't detect the RNA. And the story goes, that I attended a seminar on SARS-CoV-2 and the person was actually referencing a company that started from University of Pitt where you are.

Cindy St. Hilaire: Oh, very nice.

Milka Koupenova: And they do specific, it's called amplicon ARTIC v3 sequencing so they enrich for the SARS-CoV-2 RNA and screen by sequencing. And when we did that, we were able to detect it in all patients. So I freaked out and I said, "Oh my gosh, something is wrong."

Milka Koupenova: And so I sent plasma, and I sent controls, and actually RNA from the virus and you can see beautifully that it's only in platelets. Four of the 17 people actually had RNA in the plasma, but what you can observe in all these people is that the virus is fragmented, meaning it's not infectious. And in a way what this tells us, it suggests that platelets are super important in the removing it from the circulation and they probably serve as a dead-end for the virus because you cannot find virus coming out of platelets and the RNA is chopped off. So what I would say, is that platelets are these amazing little units that serve as removal of the viral RNA for these particular viruses, respiratory viruses that are RNA viruses.

Cindy St. Hilaire: I think that is so interesting. So essentially, they're almost like little composters that are chewing it up and preventing it from spreading in the organism.

Milka Koupenova: Yes, and as a result there is a response.

Cindy St. Hilaire: Scott, probably the most common thing that people know with SARS is that loss of smell, or taste, and things like that, but really that doesn't send anybody to the hospital. So really what are the symptoms of COVID-19 patients that tie in with platelets specifically? I feel like that's a lot of things that we maybe in the public, or on Twitter, and things didn't hear as much about. So really what are those big symptoms linking COVID and platelets and what are the implications of platelet death in the pathogenesis of COVID?

Scott Cameron: So certainly I think several investigators are in the world of now showing that platelets are hyperactivated, Robbie Campbell and Matt Rondina put a really nice paper in Blood last year showing that platelets are hyperactive and there are other investigators who found something similar. And so the question is, what are the symptoms of hyperactive platelets in the SARS-CoV-2 patient? So what most of them would find is shortness of breath or dyspnea, and when they present to the emergency department, and certainly we saw this, the oxygen saturation which should be in the mid to high 90s on room air on an average person, was quite often low. It was in the 80s or 70s, sometimes even the 60s.

Scott Cameron: And the real surprising thing was those are patients that would normally immediately be on a ventilator, but yet they could still be talking to you. And so if you have a platelet that's activated in a hyperthrombotic condition, like SARS-CoV-2, COVID-19, and then that forms a blood clot, you have a situation where the amount of oxygen the patients taking in and the amount of oxygen you're measuring in the artery is quite discrepant and we call that the alveolar arterial or oxygen gradient. So if you've got lots of platelet plugs through the microvasculature, it's going to take up some space the oxygen should be using for diffusing in. And so that would be manifested as shortness of breath and that's certainly one of the biggest tip-offs that a patient might have a blood clot, particularly in the lung.

Cindy St. Hilaire: Some of these symptoms of COVID-19 are really worse in patients with comorbidities, diabetes, obesity and heart failure. Are platelets central to kind of the pathogenesis of those disease or the symptoms of those diseases? I guess the root of my question is, why do the comorbidities of diabetes, obesity, and heart failure make COVID worse? Is it something about those disease states themselves or is there a role for platelet?

Scott Cameron: That's a brilliant question, no one's ever asked that before. And as Dr Koupenova said, I'm a little bit biased too because I firmly believe that in different disease states, the disease educates the platelets so you've got a different platelets phenotype. So focusing on diabetes, we know the platelet phenotype is different in diabetic patients. We know that platelet reactivity seems to be higher through the P2Y12 receptor. In terms of obesity, it is true, we know that, and this has been published also, and we know that the platelet phenotype is hyperactive in a patient with obesity and so that tells me that, that's a comorbidity that might affect platelet function and also vice versa for that case. And then in terms of why is it affecting males more prominently and more severely than females, well one of the beefs, I guess, that I had is that we treat diseases in women the same as we do in men assuming that the platelet phenotype in disease must be the same, but that's absolutely not true. And that's actually a theme that we have in our lab right now, we know that the behavior of platelets, and how platelets are educated in diseases is not all the same in women as in men and I think it's a huge disservice that we really had to have a pandemic that would make that quite clear to us.

Cindy St. Hilaire: You kind of hit onto something that's really, I think it's now becoming more recognized certainly in the cardiovascular field and that is so many studies are really only on male mice, or only younger or older men, and we are missing not only a huge patient population, but probably some really interesting biology that is distinct.

Milka Koupenova: So expanding on that, we know that in platelets, the toll-like receptors, and we've looked at the expression of all 10 in a study that we published in ATVB in 2015, actually, significantly if you look at Farmingham Heart Study data and the expression of these toll-like receptors they are increased in women versus men. And also, an interesting observation that never got published, once upon a time when I was doing studies with TLR7 mice is that if you inject TLR7 agonists, male mice would have a higher level of reduced platelet count than female mice at the same time points, right? And at that time it wasn't published. Definitely there are differences, but I also want to extrapolate a little bit on what was said at the beginning. We have to understand that when it comes to these comorbidities, everything affects a unit that doesn't have a nucleus, right? And diabetes and obesity have the so called profound, chronic inflammation of cytokines, such as IL6, that keep circulating. These things have effect on platelets. So we have two responses, we have the environment that affects platelets and we have the direct response of the virus that affects platelets. And that cumulative response truly can exhaust them and once they become exhausted, once they release their contents, as we show in this paper, then you're compromising their function and you will be compromising taking out the virus from one side and from the other side you're going to be compromising the environment because all of the content that comes out from a unit that already has free form proteins, it exhibits a true insult on what's being surrounded. So these clots that form in the lung or the platelets that circulate they no longer can be resolved properly.

Cindy St. Hilaire: Yeah.

Milka Koupenova: It's a balance.

Cindy St. Hilaire: Yeah, so really it's like destroying the platelet not only are you destroying the vacuum that has to suck up those particles, you're then just dumping a whole bunch of pro-inflammatory things on all of the endothelial cell vasculature that those platelets are nearby.

Cindy St. Hilaire: Actually that was one thing that I thought you spent a decent portion of the discussion on, and that is the method by which the blood is collected really impacts the outputs you observe in quote unquote platelets. Can you talk about the importance of that because I think that's one thing, certainly as a PhD who's just like, "Oh, yeah. I'm just going to collect blood from my mice and do this thing," how critical is that point in the experiment, in the blood collection?

Milka Koupenova: So I am very adamant when it comes to platelets for the blood to be drawn in citrate. And I have to say that a lot of the studies that you would see in the literature are done using EDTA blood or serum. They all have their importance. I'm not going to dismiss it, but if you want to truly measure what's inside in plasma, versus what's inside in platelets, or what's inside in any cell for that matter, you got to go for citrate. You have to be very careful not to shake the blood. You have to be very careful not to cool down the blood. So the nurses probably hated me because often I would be like, "You can't do this. You can't put it on ice. You can't warm it up to above certain degrees. Everything has to be controlled and done correctly."

Milka Koupenova: And so I had done in the past studies in which I would take plasma from the same patient in EDTA, in citrate and then isolate the RNA, have my tech isolate the RNA, and we send it to a fragment analyzer, and you can see how much more RNA you will get in the EDTA plasma. I'm not even talking about serum.

Milka Koupenova: Serum is a very different thing, then you're definitely going to get platelet content in it, in the serum, right? So it's important to distinguish that perhaps when you're getting EDTA plasma you are looking at a content that could have been inside in platelet and I can't stress enough that when it comes to these particular studies, citrate, dextrose, phosphate is your place to go and be.

Cindy St. Hilaire: So in terms of translational potential, what do your findings suggest about future therapies or targets to investigate as therapy? And is modulating platelets a potential for combating viral infections or mitigating their severity?

Milka Koupenova: Well, Scott and I actually talk a lot about that.

Scott Cameron: That's right.

Milka Koupenova: I personally would say, control the inflammation, never let it go to platelet. Let me back up a little bit, if you have to, you have to, right? But your go to method should be inflammation, if you don't get to the point that you need to control platelets then you're in a better place because it becomes very fickle. From everything that you hear me say, you push it to one side and the balance is destroyed. You deactivate platelets or inhibit platelets well, are they now not able to pick up the virus and then you're now having the virus circulating somewhere. Now, if you don't treat platelets that's also not good. So you're in the very fickle situation if you get to the point that you need to control the activation of platelets and there are trials currently that are trying to look at those things. Scott, I'm going to refer this a little bit more to you because you have done some interesting things with that particular point.

Scott Cameron: No, it's a great question, Milka, and I think that as platelet biologists, nobody more than I wanted it to be true that platelets would be the ultimate target. I mean, clearly patients with SARS-CoV-2 have thrombosis, clearly platelets are activated, so should we inactivate them? That was the whole point of the RECOVERY trial and one of the benefits I'll tell you before I sort of go into that is, working in a large organization like the Cleveland Clinic and we have access to data and lots of it extremely quickly, and so because of that I of course could see how many patients were coming into our hospital with thrombotic events. And I could see what the independent predictors of thrombotic events was and it wasn't the platelet count, sometimes platelet count was low, sometimes it's high in the SARS-CoV-2 patient. And if you took those individuals that were on aspirin, comparing them to those that are not in a propensity match study, one of the things that we find is that aspirin doesn't seem to affect or improve mortality or the number of blood clots in the patient with SARS-CoV-2.

Scott Cameron: We compared that to all non-steroidal anti-inflammatory medications that patients may have been taking also in a propensity match study just in case it was the mechanism action of the drug, rather than the drug itself, and we found that NSAIDs not only did not protect patients, but they were not necessarily harmful either, which was one of the things that came out at the start of the pandemic. Among, I'll add, the absence of evidence based medicine and a lot of cases where naturally people, including clinicians, were scared and so they were going off label and they were trying a lot of different medications with really not a shred of randomized controlled data.

Scott Cameron: But now that we're 18 months into it, the first and biggest study that came back was the RECOVERY trial, which we were all waiting on, where patients were given aspirin and short term mortality was examined over an observational period of one month. And just like we found in a propensity match study, which is as close as you'll get to a clinical trial in a retrospective manner, the prospect of RECOVERY trial actually showed the curves were almost super imposeable, those that got aspirin versus those that didn't. So I think low dose aspirin clearly is not going to be enough for those patients, but I'll also add that over the observational period of one month they also didn't see a higher incidence of death in those patients. And I think Milka's point is really well taken that you have to remember that as well being an entity of thrombosis, platelets are immunological entities and so you've got to really consider should we be inhibiting them and if you are inhibiting them, I think the time point at which you should inhibit them is what we should examine, not just an all or nothing, inhibited or not.

Milka Koupenova: It's just in our linear brains we prefer to think of it as one straight, linear pathway, but it isn't, and I think platelets are actually a great example of how many pathways are feeding into one tiny fragment and that particular blood cell is inducing this profound response during these infections.

Cindy St. Hilaire: I think most people have heard that angiotensin-converting enzyme 2, also called ACE2 is the receptor of SARS-CoV-2. The virus itself uses it to bind and become internalized into the cell, but there's been some discussion or even some discrepancy of data as to whether platelets truly express ACE2 and if that is the means for the virus to enter the platelets. So can you share with us what is the current state of knowledge about that?

Scott Cameron: Yeah, just as a segue of some of the things that Milka said, I think the preparation of your sample is part of the answer. If you draw in the incorrect tube, if you the tube is not completely filled, and the ratio of citrates to whole blood isn't correct you're going to have discrepant results. If you biomechanically activate the platelets by drawing through a short needle, in a small-bore needle for example, that's going to activate the platelets. If you cool them, it's going to activate them. But then also, depending on how you decide to separate them, we always washed platelets in my lab, we wash them two or sometimes three times, and I can tell you if you use flow cytometer we get one white blood cell for every 12,000 platelets.

Scott Cameron: And some investigators might go one step further and they'll a CD45 depletion set, which is certainly important if you're studying RNA. But one of the issues, as you well know, a CD45 is also on the surface of platelets, so if you start with a low expressing protein and you CD45 deplete them, you are actually going to get a decrease in your platelet yields. I've seen it, I think Milka's seen it, various other investigators have, and you might find yourself at the threshold of what your antibody can detect. It's also variably expressed. If you look at even healthy individuals, some of them have almost none. So if you look at 10 individuals, you might actually find none, but then if you look at another 10, the amount of expression that we see is kind of all over the place. It's not like other receptors where one tends to express a certain amount and that's the way it is in health. ACE2 doesn't seem to be that way for whatever reason.

Milka Koupenova: We were able to detect in some of the people by qPCR, but what was interesting is that from the three primers that I used there was never the same person who we were able to detect all three primers with for that receptor. That tells you that maybe they are changes of one base that is not enough for the primer to detect it, right? That becomes another possibility of not being able to detect.

Milka Koupenova: And so I go to confocal microscopy where I use 100 lens and tons of hours in the microscope room, and Scott is completely right, it's really hard to see it particularly in healthy people. And it starts to pick a little bit more in people with cardiovascular disease or people with COVID that are old. So it's a bit complicated, but the important thing here is, besides the fact that we are detecting ACE2 and we're detecting proteins and I use controls, biological controls to prove that this is the case and it's not just an antibody problem, is that the virus will get picked up by platelets even if you don't have ACE2. That is the take home message from this paper is that the platelet has evolved various mechanisms by which is utilizes getting it inside. It is that important for this virus. This type of virus is not recirculating. In this case, what we observed is that the virus is attached to microparticles that are of platelet origin for that matter.

Cindy St. Hilaire: So really what you're saying, what I'm hearing is the platelet is the superhero of the body.

Milka Koupenova: Definitely. Absolutely. No bias, absolutely.

Cindy St. Hilaire: Unbiasedly, it is a superhero. Well, Dr Cameron and Dr Koupenova, thank you so much not only for this amazing discussion, but for really an elegant, elegant paper that is really bringing to light the complex interaction between SARS-CoV-2 and platelets. So thank you so much for joining me and keep publishing amazing stories like this.

Milka Koupenova: Thank you for having us.

Scott Cameron: Thank you, an honor to be here. Thanks again.

Cindy St. Hilaire: That's it for the highlights from August 20th and September 3rd 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 Scott Cameron and Dr Milka Koupenova. 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 the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cynthia St. Hilaire, and this is Discover CircRes, your on-the-go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association, 2021. 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, please visit ahajournals.org.

This month on Episode 27 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the July 23rd and August 6th issues of Circulation Research. This episode also features an in-depth conversation with Drs Ana Gomez and John Pierre Benitah, from INSERM and the Paris-Saclay University, about their study, Impaired Binding to Junctophilin 2 and Nanostructural Alterations in CPVT Mutation.

Article highlights:

Glasenap, et al. Imaging Inflammation and Fibrosis in Heart Failure

Shi, et al. Cardiomyocyte Pyroptosis Aggravates MI/R Injury

Koenis, et al. SPM Temper Phagocyte Responses in COVID-19

Zhang, et al. Common Origin of Heart and Extraembryonic Lineages

Cynthia St. Hilaire: Hi, and welcome to Discover CircRes, the podcast to the American Heart Association's journal, Circulation Research. I'm your host, Dr Cynthia St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'll be highlighting articles presented in our July 23rd and August 6th issues of Circulation Research. I also will speak with Drs Ana Gomez and John Pierre Benitah, from Inserm and the Paris-Saclay University, about their study, Impaired Binding to Junctophilin 2 and Nano-structural Alterations in CPVT Mutation.

Cynthia St. Hilaire: The first article I want to share comes from the July 23rd issue of Circ Res, and it's titled Molecular Imaging and Inflammation and Fibrosis in Pressure Overload Heart Failure. The first author is Aylina Glasenapp and the corresponding author is James Thackeray, and they're from Hanover Medical School in Germany. After a heart attack, inflammation and fibrosis of the heart alter cardiac contraction and can lead to its failure. Currently, for ischemic heart failure, doctors use imaging techniques such as positron emission tomography, and cardiac magnetic resonance imaging, to measure the inflammation and fibrosis to provide a prognosis.

Cynthia St. Hilaire: However, whether these imaging techniques are useful for non-ischemic heart failure was unknown. To find out, this group performed transverse aortic constriction on mice, which is a commonly used method to model non-ischemic heart failure, and then they analyzed the animal's hearts with positron emission tomography to assess the inflammation and cardiac magnetic resonance imaging to quantify scar tissue. Compared with Sham-operated animals, those that underwent TAC exhibited increased heart inflammation for at least three weeks and significant fibrosis for at least six weeks. The degree of scarring and inflammation was inversely correlated with heart function. The team also found that reversal of TAC led to reduced inflammation and fibrosis over time. Together, the results confirm that these imaging modalities are valuable for monitoring fibrosis and inflammation in non-ischemic heart failure, and they could potentially be useful for assessing the effectiveness of interventions.

Cynthia St. Hilaire: The second article I want to share is titled GSDMD Mediated Cardiomyocyte Pyroptosis Promotes Myocardial Ischemia Reperfusion Injury. The first author is Huairui Shi and the corresponding author is Junbo Ge, and they're from Fudan University in China. After myocardial infarction, restoring blood flow is essential to saving muscle function. However, restoration of flow itself causes damage by inducing inflammation and cell death. This study found that the cell death aspect of a reperfusion injury occurs via a process called pyroptosis, which is a controlled form of necrosis that is due to excessive inflammation.

Cynthia St. Hilaire: The team developed an in vitro model of reperfusion injury, where cultured cardiomyocytes are starved and then resupplied with oxygen. Using this model, they found that cells exhibited features of pyroptosis, including the release of inflammatory factors, increased production of the pyroptotic factor gasdermin D and cell death. Cardiomyocytes lacking gasdermin D did not display signs of pyroptosis under these same conditions. The team went on to show that gasdermin D was significantly increased in the hearts of mice following ischemia reperfusion. And compared with control animals, mice whose cardiomyocytes were engineered to lack gasdermin D, suffered less necrosis and smaller reperfusion injuries in their hearts. Together, these findings provide insights into the mechanisms that should be targeted to minimize pyroptosis and subsequent ischemia reperfusion injury, following myocardial infarctions.

Cynthia St. Hilaire: The next article I want to share is titled Disruptive Resolution Mechanisms Favor Altered Phagocyte Responses in COVID-19. The first authors are Duco Steven Koenis, Issa Beegun and Charlotte Camille Jouvene, and the corresponding author is Jesmond Dalli. And they're from Queen Mary University of London. Inflammation is essential in the early stages of battling and invading pathogen, but at the same time, inflammation can become damaging to the host if it is not resolved in a timely manner. Prolonged and unresolved inflammation is responsible for the hospitalizations and deaths of many COVID-19 patients. An excess of circulating pro-inflammatory cytokines is one of the key features of severe COVID-19. And now, Koenis and colleagues show that certain pro-resolving factors are out of balance in these severe patients.

Cynthia St. Hilaire: Blood samples from patients with mild COVID-19 showed an increase in specialized pro-resolving lipid mediators. However, blood from patients with severe COVID-19 had lower levels of these pro-resolving lipid factors. Expression of specialized pro-resolving lipid mediator receptors on phagocytes was also higher in patients with mild disease than those with severe COVID-19. And, in line with this, the proportion of activated pro-inflammatory phagocytes was higher in patients with severe disease.

Cynthia St. Hilaire: When patients were treated with the steroid dexamethasone, they subsequently inhibited the increased levels of the specialized pro-resolving lipid mediators in the blood. Together, these results reveal specialized pro-resolving lipid mediators are dysregulated in severe cases of COVID-19, and the findings suggest increasing these pro-resolving lipid mediators could promote resolution of out-of-control inflammation.

Cynthia St. Hilaire: The last article I want to share is titled Unveiling Complexity and Multi Potentiality of Early Heart Fields. The first authors are Qinqguan Zhang and Daniel Carlin, and the corresponding authors are Sylvia Evans, Joshua Bloomekatz, and Neil Chi, and they're from UC, San Diego. The developing heart is thought to originate from two populations of cells; the first and the second heart fields. And these are first identifiable at stages E 7.5 in the mouse, or on day 15 in the human embryo. Genes controlling the development of these fields have been linked to congenital heart defects, but interestingly, congenital heart defects are also sometimes linked to placental abnormalities. However, the mechanisms underlying this link have been unclear. Now this study has gone on to discover an unexpected link between the first heart field and extra embryonic tissues, which give rise to the yolk sack and the placenta.

Cynthia St. Hilaire: Through lineage tracing experiments and single cell transcriptomics, the team discovered that the first heart field consists of two sources of mesoderm progenitor cells, one source that is embryonic in nature and the other source arises from the interface between the extra embryonic and the embryonic tissue of the early gastrula. This latter population of progenitor cells, which is defined by the expression of the transcription factor hand one, gives rise to extra embryonic mesoderm cells in addition to the two Hartfield cell populations. The discovery of this shared source of mesodermal progenitors not only blurs the lines between the embryo and its supporting tissue but may also explain the link between placental abnormalities and congenital heart defects.

Cynthia St. Hilaire: Today I have with me Drs Ana Gomez and Jean-Pierre Benitah, and they're from Inserm and the Paris-Saclay University. And today we'll discuss their study Impaired Binding of Junctophilin 2 and Nano-structural Alterations in CPVT Mutation. And this article is in our July 23rd issue of Circulation Research. So thank you both very much for joining me today.

Jean-Pierre Benitah: Thank you.

Ana Gomez: Thank you.

Cynthia St. Hilaire: You're in Paris, so we're trying to match it so we're all meeting our normal workday on a Friday. So I very much appreciate you taking the time to meet with me. So this study is investigating a rare disease called Catecholaminergic Polymorphic Centricular Tachycardia, or CPVT. So can you describe to us what is CPVT and how does this disease present in patients?

Ana Gomez: Okay, so CPVT stands for Catecholaminergic Polymorphic Centricular Tachycardia. So it is a genetic disease that appears mainly in childhood and youth with sudden death. So the patients don't have any remarkable problem, either in the electrocardiogram or arteries, or in the cardiac structure by echocardiography, and they seem healthy. But when they have stress, it can be emotional or it can be physical, so during exercise, it presents with syncope or sudden cardiac arrest. So the problem is that, many of the times, the first symptom is the death of a child playing soccer or doing exercise and then the only treatment that they, so far, it's beta blockers, to avoid this stress, and also flecainide and propafenol. But these treatments are still not completely efficacious, or sometimes the people need to get implant defibrillator. It's a big cost and it's also stressful because if the patient feels that they have to recharge, that supposes stress, and this stress is bad for them.

Cynthia St. Hilaire: Right, so it's like if they feel a flutter, it makes them more stressful, which can exacerbate. That is terrifying. And so the goal, I guess, regarding gaps in knowledge that are leading to your investigation, what was known about this disease before you started your study? And where did you leap off from that?

Jean-Pierre Benitah: Up to now, what we know about the disease is an alteration of the calcium homeostasis in cardiac myocyte. That could induce trivial activity, and then arrhythmia and cardiac sudden death. So mainly the mutation related to an intracellular calcium channel called Ryanodine receptor. So it's up to 60% of the patient with this mutation, but also you have a mutation related also to proteins that are in-buried in the control of the Ryanodine receptor activity, priadine, calmodulin.

Cynthia St. Hilaire: Yeah, that was actually going to be my next question. So I know this cardiac Ryanodine receptor 2, or RYR2, it's obviously the channel component that helps to release that calcium signal, but it's part of a larger complex. I believe it's called the Calcium Release Unit. Can you talk about what is in that unit in terms of proteins and then where those other genetic mutations fit into that?

Ana Gomez: Yeah, so the Calcium Release Unit is formed by a cluster of Ryanodine receptors. So in the reticular cardiomyocytes, these are mostly in the junction of sarcoplasmic reticulum that is very close to the sarcoplasmic reticulum membrane inside the cardiomyocyte, inside the cell. So the channel is internal. But it's very close to the sarcolemma in the T-tubule invaginations where the L-type calcium channels are located. So this is... The channels are very important to activate contraction, so it's heartbeat. The calcium entry through the attached calcium channel on the surface makes some calcium get into these very restricted spaces, like 20 nanometers, and in this space this calcium activates the Ryanodine receptor. So the Ryanodine receptor is activated by calcium and these release much more calcium than is needed for the contraction. So the problem of the CPVT is that the channels may release calcium during diastole, so when calcium should be low because they had to relax.

Ana Gomez: For your new question, which proteins? So the main proteins are the Ryanodine receptor. But Ryanodine receptors are a very big macro complex. They are the biggest channels that are known and they have a big cytoplasmic portion with proteins that can bind to them, and most of them just keep the channel quiet. So this may be calmodulin, FKPB 12.6, or 12, sorcin. And then there are also some other proteins that scaffold kinases, like PKA and CaM kinase. And also they have some proteins that moderate the channel from the luminal side. So, calsequestrin, triadin and junctin. And this agents to fill in that we will speak later. It's important because it binds to the L-type calcium channel and to the ryanodine receptor. So it's important to keep the dyad structure. It's not only a structural role.

Cynthia St. Hilaire: Yeah, that is so interesting. So your study focused on a very specific mutation. It's the RYR2 arginine in the 420 spot to glutamine mutation. So I guess my first question is based on the patient population, how common is this specific mutation? How common is that?

Ana Gomez: Yeah. So in fact, I'm going to say that it's very common, because normally CPVT is one mutation, one family.

Cynthia St. Hilaire: I see.

Ana Gomez: Even if they are located in hotspots, but these particular mutations, we were approached by a cardiologist working in Spain who had this family with a child that died at the age 14, playing soccer game. And so Dr Zorio in Valencia, she found this RyR2 420Q mutation. And at this time this was the first mutation in this site. I mean, not really in the site, there was already RyR2 420W that was already, so it was the same spot, but different.

Cynthia St. Hilaire: That was my next follow up question to that. My PhD was biochemistry, so this brought back having to memorize the amino acid structure. So arginine is large and positively charged to glutamine is neutral. So what were the experiments that you designed to help determine the functional causes of this mutation? You know, in addition to just, okay, obviously there's a charge change, so there's probably a structural or a binding change, but how did you determine the functional consequences of this mutation?

Ana Gomez: The structure, as you say, this has been shown. In fact, they was the first family, but then also in this region, there was another family and in Israel also there is another family. So there are three, but the structural limitations that these arginine is neutral. It has been shown by a laboratory, who works in Vancouver, in a structural and the end terminal has like three logs and these are 420. It's important to hold a chloride that in the middle and, and to hold the position. So, but this is not the functional, the functional is what we were going to analyze. So the first thing that we did is to analyze calcium sparks because calcium sparks is the functional, let's say elementary event, of calcium release to RyR2 receptors. So we start analyzing calcium sparks in the cells and we found strange things, like very long calcium sparks that was not so clear in other CPVT models, even one that we studied earlier. And so then we started to continue to know why we have longer calcium sparks and different kind of analysis. So we also collaborate with some other laboratories to do the ultrastructure of the dyad by electromicroscopy.

Ana Gomez: And then we found that the sarcoplasmic reticulum, junctional sarcoplasmic reticulum, was enlarged. So we thought, well, maybe the channel, the calcium spark is longer because locally they delayed depletion. So we did another kind of experiment changing the volume of the SR and it was not so concluded so we found that it may contribute to longer calcium sparks, but it doesn't explain for it. So then we start with to analyze different proteins candidates, also the phosphorylation of course. And then we didn't find in most of these proteins, like FKVP.

Cynthia St. Hilaire: Kind of a standard go-tos. None of them were involved. Yeah.

Ana Gomez: Yeah. And then, because there is this ultrastructural alteration, we thought of junctophilin and that is how we found that junctophilin binding was impaired.

Cynthia St. Hilaire: That's a perfect segue. You're hitting all of my next questions. So can you tell us a little bit about, what did you find regarding junctophilin and the RyR2 channel?

Jean-Pierre Benitah: So mainly, junctophilin act to us the good structural design between the ryanodine receptor and the trigger L-type calcium channel. And people say that junctophilin binds to both proteins to keep them close to each other. So mainly what we found is that we don't have activation of the expression of junctophilin, but it seems that with this mutation the junctophilin is less in contact with ryanodine receptor. But it's not the case for the L-type calcium channel. It seems that coimmunoprecipitation experiments that we've done show that junctophilin stayed still with the L-type calcium channel, but have a lower affinity to the ryanodine receptor when you have this mutation. What was really important is that we saw that not only in the mouse model where we induce this mutation, but also in cardiomyocytes derived from induced pluripotent stem cells from patients that have this mutation.

Cynthia St. Hilaire: I think that's one of the great strengths of your study. You know, I like how you took a multi-faceted approach, you know, using these IPS cells from the patients and also created a knock in model. Previous studies had used more global or whole exon deletions. So how is your knock-in able to identify additional information that built upon those former studies?

Ana Gomez: Maybe this is not an exact answer to your question, but what I think is that the strength of our study or one of the strengths of our study is that we have the patients with electrocardiograms working, we have the cells from the patients. So we have...Our IPS cell is from one of the persons that have been patient, and the control line is from his brother. So we have the two brothers. They are still living, and we have the mice and everything is in the same point mutation. So in this thing, because there is a lot of, let's say, critics to the IPS cells studies because they are not mature and they don't look like an adult cardiomyocyte. And I think that besides CPVT, we can also show that of course cardiomyocytes derive from IPS cells. They are not adult, but they are still a good model because we recapitulate the same thing.

Ana Gomez: So we can mix the human context to really have what happened in patients, because that is the important thing, but we also need to manipulate the in vivo animals and there are some things that we cannot do. We cannot get adult cardiomyocytes from patients, so for that, we have the mice and we can also analyze from in vivo to the molecular level. So I think that it's a big strong point from our study that you take compared to others, that they are only in mice or only in IPS, cannot do this correlation. Then, each mutation, we think that it may, or at least each region of the mutation, may have different mechanisms. So if we find these longer calcium sparks in these R420Q mutation, it doesn't mean that because we also have other studies in C-terminal mutation, and we don't find longer calcium sparks, we just find more. So this is not because of the design of the study, but because the mechanism of the mutation is different.

Cynthia St. Hilaire: In terms of translational potential, what do your findings suggest about either the ability to screen patients potentially for the development of CPVT or actually more importantly, you know, therapies to help treat these patients when they're identified?

Jean-Pierre Benitah: Yeah. It's one of the big problems with the CPVT, especially since when you look at the different mutations, those are different mutations that have been reported on the ryanodine receptor located on different hotspots on the ryanodine receptor. And it's seems that each hotspot could have a different type of mechanism behind that. So, for example, we show, there you see, you know, different mutations in collaboration with CPVT or 420Q mutation. So the mechanism was related to an alliteration of the sensitivity of the ryanodine receptor to the calcium. So the group of branching show that in other mutations, in other spots, hot spots, it was related in fact, to a modification of this. Also the sensitivity of calcium of the ryanodine receptor calcium, but from the luminal side.

Ana Gomez: Regarding your first question was diagnosis. I think that after our work, we may also include junctophilin, because so far there has not been any link to junctophilin for sensitivity. So when a patient has CPVT, they start screening for mutations in the ryanodine receptor, since it was found that this child was involved and then in other proteins. So I think now if they don't find in a patient, because there are still like 40% of CPVT patients that the mutation has not been found.

Ana Gomez: For therapeutic side maybe find a molecule that stimulates the binding of junctophilin to ryanodine receptor, but also maybe some smaller molecule that may interact between the N-terminal and the core solenoid because we found that in the interim molecular structure, they show tighter association between the N-terminal and the core solenoid. So maybe it's more of a tide or something that can be in between too. I mean, I don't know, but it's first line there.

Cynthia St. Hilaire: Potential, but still far off. That's wonderful. So are some of these mechanisms, I assume, they would also be relevant in non-genetic forms of tachycardia? Is that the case? Could some of your findings also perhaps be applied to the tachycardia related to heart failure or other types of disease states?

Ana Gomez: I think it's actually, for example, junctophilin binding to ryanodine receptor in heart failure. It has not been yet studied, but we want to do it. It's something because as you say heart failure, it's a very common disease. So it's also very relevant to the public health. This is something that we need to know.

Jean-Pierre Benitah: One of the things that happens in heart failure is that it seems also that you are a dissociation between the calcium channels and the ryanodine receptor because you have less tissue formation. So perhaps this is difficult to try to figure out whether it would be the same, but perhaps this activation between the communication between the two channels is one of the main points that we have in CPVT and in heart failure related to tachycardia.

Ana Gomez: Yeah. In fact, many years ago we showed that. We showed that in heart failure there is a defect in calcium channel and ryanodine receptor. So in this study it was only functional. We didn't do the structure, but of course it is something that we have to keep in mind, continue investigating.

Cynthia St. Hilaire: Yeah. Well that sounds like a great future project. Well, I want to thank you so much for joining me today and helping to discuss your paper. I love it when we take rare diseases and figure out the mechanism with hopefully applying it to more common disease states. That's what I do in my lab with vascular calcification, and so thank you so much for joining me and for this great publication. And we look forward to your future work that is hopefully in Circ Res.

Jean-Pierre Benitah: Thank you for the invitation.

Ana Gomez: Yeah, thank you very much for your time.

Cynthia St. Hilaire: That's it for the highlights from the July 23rd and August 6th issues of Circulation Research. Thank you for listening.

Cynthia St. Hilaire: Please check out the Circ Res Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, doctors Ana Gomez and John-Pierre Benitah.

Cynthia St. Hilaire: 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 the highlighted articles was provided by Ruth Williams. I'm your host, Dr Cynthia St. Hilaire, and this is Discover CircRes, your on-the-go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association, 2021. 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.

This month on Episode 26 of Discover CircRes, host Cindy St. Hilaire highlights four original research articles featured in the June 25th and July 9th issues of Circulation Research. This episode also features an in-depth conversation with Dr Hirofumi Watanabe, Dr Ariel Gomez, and Dr Maria Luisa Sequeira-Lopez from the University of Virginia about their study, The Renin Cell Baroreceptor, A Nuclear Mechanotransducer Central for Homeostasis.

Article highlights:

Mesirca, et al. Electrical Remodeling of the AV Node in Athletes

Yang, et al. Macrophage-Mediated Inflammation in COVID-19 Heart

Örd, et al. Functional Fine-Mapping of CAD/MI GWAS Variants

Akhter, et al. EC-S1PR1 Activity Directs Vascular Repair

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'll be highlighting the articles presented in our June 25th and July 9th issues of Circulation Research. I'm also going to speak with Dr Hirofumi Watanabe, Dr Ariel Gomez and Dr Maria Luisa Sequeira-Lopez from the University of Virginia about their study, The Renin Cell Baroreceptor, A Nuclear Mechanotransducer Central for Homeostasis.

Cindy St. Hilaire: The first article I want to share comes from the June 25th issue of Circ Res and is titled Intrinsic Electrical Remodeling Underlies Atrial Ventricular Block in Athletes. The first authors are Pietro Mesirca, Shu Nakao, Sarah Dalgas Nissen, and the corresponding author is Alicia D'Souza. And they're from the University of Manchester in the UK.

Cindy St. Hilaire: Endurance training has cardiovascular benefits, but when taken to extremes, it can elicit heart problems such as atrial ventricular block or AV block. AV block is the impaired conduction through the AV node. In fact, some endurance athletes require pacemakers later in life due to AV block. One hypothesis for this conundrum is that the problem stems from disruptions in the autonomic nervous system. This study shows that in fact, the intrinsic electrophysiology of the heart is to blame. They used trained race horses, as well as mice, subjected to endurance swimming as models for human endurance athletes. Electrocardiograms on the animals showed that just like human athletes, the race horses and the swim-trained mice exhibited signs of AV node dysfunction that is not seen in sedentary controls.

Cindy St. Hilaire: Because the dysfunction also persisted when the autonomic nervous system was blocked, the team examined molecular changes within the heart itself. They found that ion channels, HCN4 and Cav1.2, were less abundant in the AV nodes of trained animals than those of the controls. The team went on to identify two microRNAs regulating HCN4 and Cav1.2 production and showed that suppression of these microRNAs restored normal heart electrophysiology in the mice. If the result holds true for humans, this could pave the way for novel treatments for AV block.

Cindy St. Hilaire: The second article I want to share is titled An Immuno-Cardiac Model for Macrophage-Mediated Inflammation in COVID-19 Hearts. The first authors are Liuliu Yang, Yuling Han, Fabrice Jafre, Benjamin Nilson-Payant and Yaron Bram. And the corresponding author is Shuibing Chen. And they're from Cornell University Medical Center.

Cindy St. Hilaire: COVID-19 is primarily a respiratory disease, but cardiac complications are common and appear to be linked with worsening outcomes. Post-mortem examinations of COVID-19 patients' hearts have revealed abnormally high numbers of macrophages, suggesting that these cells have a role in the heart pathology. To investigate this possibility, this group co-cultured macrophages and cardiomyocytes, both which were derived from human induced pluripotent stem cells and infected the cultures with SARS-CoV-2 virus. Upon infection, both cell types increased their rates of apoptosis. However, the number of cardiomyocytes succumbing to the cell death process was far higher than that of macrophages. When cardiomyocytes were infected with the virus in the absence of macrophages, their rate of apoptosis dropped.

Cindy St. Hilaire: The team showed that macrophages produced large amounts of the inflammatory cytokines, IL-6 and TNF, in response to the virus and that trading the cardiomyocytes directly with the cytokines could similarly induce apoptosis. Blocking IL-6 and TNF alpha signaling prevented the macrophage-driven cardiomyocyte death. The team then identified two FDA approved drugs, ranolazine and tofacitinib, that prevented the virus-induced cardiomyocyte death in vitro and suggest that these drugs now be investigated in larger animal models.

Cindy St. Hilaire: The next article I want to share is titled Single-Cell Epigenomics and Functional Fine-Mapping of Atherosclerosis GWAS Loci. The first author is Tiit Ord, and the corresponding author is Minna Kaikkonen, from the University of Eastern Finland.

Cindy St. Hilaire: Genome-wide association studies, or GWAS studies, have identified hundreds of genetic loci associated with coronary artery disease and myocardial infarction. And many of these genes likely play a role in atherosclerotic development. However, most of these loci are located in non-coding intergenic regions of the genome. Thus, their functional effects on atherosclerosis development are not clear. Non-coding regions of the genome may contain gene regulatory elements, including cell type specific enhancers. And because such enhancer elements often have open chromatin structures, this team profiled the chromatin accessibility of single cells in human atherosclerotic plaques.

Cindy St. Hilaire: They found that many cell type-specific assessable regions overlapped with both transcription factor binding motifs, as well as GWAS-identified coronary artery disease loci. Using an algorithm called Cicero, the team was able to predict likely genes under the control of these accessible intergenic regions. They found that in more than 30 cases, they were able to confirm these intergenic regions control gene expression in in vitro assays. This work highlights the power of chromatin accessibility mapping for homing in on GWAS loci with transcriptional effects, and for identifying the likely genes they regulate.

Cindy St. Hilaire: The last article I want to share is titled Programming to S1PR1+ Endothelial Cells Promote Restoration of Vascular Integrity. The first author is Mohammed Zahid Akhter, and the corresponding author is Dolly Mehta, and they're from the University of Illinois College of Medicine.

Cindy St. Hilaire: Endothelial cells line the lumen of our blood vessels, forming a barrier that regulates the transport of nutrients, fluids and circulating cells to and from tissues. The lipid signaling molecule, sphingosine-1-phosphate, or S1P, and its receptor, S1PR1, promote endothelial barrier integrity. But how S1P and S1PR1 signaling might restore barrier function to inflammation-induced leaky vessels is unclear.

Cindy St. Hilaire: Using mice with fluorescently tagged S1PR1, this group showed that when mice are given a dose of the bacterial endotoxin, LPS, which induces lung inflammation, there's a dramatic boost in the proportion of growing lung endothelial cells. This boost in S1PR1+ endothelial cells is due to their increase in proliferation.

Cindy St. Hilaire: The authors go on to show that this proliferation is accompanied by increased production of the transcription factors involved in S1P synthesis and secretion. When they transplanted S1PR1+ cells into mice whose endothelial cells lacked the receptor, they could rescue the leaky blood vessels. By detailing the cells and molecular players responsible for vessel recovery after inflammation, this work may inform repair boosting therapies for chronic inflammatory conditions.

Cindy St. Hilaire: So today with me, I have Dr Hirofumi Watanabe, Dr Ariel Gomez and Dr Maria Luisa Sequeira-Lopez, from the University of Virginia. And they are all with me to discuss their study, The Renin Cell Baroreceptor, a Nuclear Mechanotransducer Central for Homeostasis. And this article is in our July 9th issue of Circulation Research. So thank you all for joining me today. I think we're spanning 13 time zones, so I appreciate you all making the effort.

Maria Luisa Sequeira-Lopez: It's our pleasure. Thank you.

Ariel Gomez: Thank you.

Hirofumi Watanabe: Thank you.

Cindy St. Hilaire: I won't lie, the Renin-Angiotensin-Aldosterone System is quite complex, so we're not going to try to break it all down here, but it is essential for the regulation of fluid balance and blood pressure in the body. Without it, things go quite awry. And your study is focusing on the kidney cell that produces renin in response to the minute changes in the blood pressure and the composition and the volume of the extracellular fluid in the body. So I'm wondering if, before we jump into the study, if you can give us a bit of background about these renin-producing cells and what is known about the renal pressure sensing system?

Maria Luisa Sequeira-Lopez: So in the adult mammalian kidney, renin cells are located at the tip of the afferent arterioles at the entrance to the glomeruli. So that's why they are called juxtaglomerular cells. They synthesize and release renin. This is then, as you mentioned, the rate-limiting enzyme for the renin-angiotensin system that controls blood pressure and fluid-electrolyte homeostasis. However, during early embryonic development, as demonstrated many years ago, renin cells are widely distributed along the renal arterial tree and inside the glomerulus and the interstitium. And with maturation they differentiated to vascular smooth muscle cells and they end up being located in the juxtaglomerular area.

Maria Luisa Sequeira-Lopez: But in response to a homeostatic challenge, such as hypertension, dehydration, hemorrhage, there is an increase in the number of renin-expressing cells along the renal arterial tree, resembling the embryonic counter. And this occurs mostly by re-expression of renin from vascular smooth muscle cells that descended from originally renin-expressing cells. And when the challenge passes, then they stop expressing renin and become vascular smooth muscle cells again. So renin cells are extremely plastic and they can switch back and forth from an endocrine to a contractile phenotype.

Cindy St. Hilaire: I'm really glad you mentioned the vascular smooth muscle cell angle because I actually have a question about that later on. But before I get to that question, one of the things that I love reading in studies is when a current paper references much older work that often has a really intricate or insightful observation. And in your paper you cited, I believe it was in 1957, was the first real hypothesis that there is an existence of this pressure sensing mechanism in the kidney, what we're calling this baroreceptor. Yet, that was a long time ago and the identity has really been elusive. So I was wondering why has it just been so difficult to really pin down this baroreceptor and how this pressure and fluid sensing works in these cells?

Ariel Gomez: So it was elusive, as you said. The reason is the researchers didn't have the tools to actually study it. It really requires an evolution, conceptual evolution, and scientific evolution, as well as technical development. And so we were fortunate over time, over the years. We developed ways to mark the cells endogenously with the appropriate fluorescent markers, genetically engineer, then develop models that allowed to drop the blood pressure in a consistent manner, and so forth. And we could follow the lineage of these cells and study them as they move back and forth from their phenotypes. So I think it was a matter of even Dr Tovian, who is the person that you mentioned, Lou Tovian, who I actually met a long time ago. So he even postulated that maybe it was a stretch mechanism, and that's one of the great contributions of Hirofumi who figure out how to stretch the cells using different ways of doing that.

Cindy St. Hilaire: So in your quest to identify this baroreceptor, you use several murine models. A surgical tool, but also several genetic tools. And I was wondering if you could share a little bit about that initial surgical model, that aortic constriction and maybe the pros and cons about that method?

Hirofumi Watanabe: And so we established surgical model of in mice. We created inductation between the roots of the right and left renal arteries. By the surgery, and our right kidney receives high pathogen pressure, and the left kidney receives low pathogen pressure. And this surgery model resulted in a marked difference in the expression of renin in each kidney. And by RT2 PCR and in situ hybridization, renin was decreased in the right kidneys and increased in the left kidneys.

Cindy St. Hilaire: Excellent. So it's a really powerful model because you can use the same mouse to look at the same...

Ariel Gomez: Right. So the beauty of that is that, Hirofumi, by doing that, he got rid of any genetic variation between the mice. Because you are doing the high and low pressure in the same mouse.

Maria Luisa Sequeira-Lopez: And another question I can think that we have said was when, if you calculate the number of cells that increase in one kidney and decreases in the other one, if you add them, it ends up being the number of cells in a non-aortic coarctation mouse. So it looks like-

Cindy St. Hilaire: It's a literal seesaw. That's beautiful. At least the math works out in your favor in the end. That's great.

Maria Luisa Sequeira-Lopez: And that's something that Luis Tovian didn't see, because what he did is he increased the perfusion pressure in an isolated kidney and what he observed was less granulation. So it was an indirect method to find less renin in those kidneys. But with a low pressure, he didn't observe an increase in renin, or increase in granulation. What we know that really happens.

Cindy St. Hilaire: So you mentioned smooth muscle cells in the beginning of our discussion and my training has been in smooth muscle cells, vascular smooth muscle cells, mostly though focused on the aorta, especially in mice. A lot of times we just say smooth muscle cells, but people are really talking about the aortic smooth muscle cells in the mice. And in humans, in the coronaries. But we use the mouse aortic smooth muscle cells as the model, which you can obviously see when you frame it out like that, some issues. And one of the things we talk about at least in athero is the cell plasticity and this phenotype switching from the contractile quiescent state to one that's associated with disease processes.

Cindy St. Hilaire: And we've really evolved on what we've known about that. It used to be just about the migration and proliferation. Now it's about the actual phenotypic switching into different kinds of cells. Macrophage-like cells, for one. And yours really was the first to bring to my eyes that there's probably many more regarding that. So could you maybe expand a little bit on these renal smooth muscle cells or renin-like cells maybe, and what's happening in that disease process? And do we know the point at which it can switch and make renin and go back versus switches and doesn't return? Is that part of the disease process?

Ariel Gomez: We describe the plasticity of the smooth muscle cells from the renal arterioles long time ago. I mean, I think, I would say that even at the beginning of my career. And at that time people didn't use that term so much, plasticity. We didn't know how to call it because it was a switch back and forth from a smooth muscle contractile phenotype to endocrine without, at the moment, without causing disease. And the cells were able to come back to be smooth muscle cells. But the period of the stimulation was only a week or so. So during that time, the cells can go back and forth. And now we know that they do that. But if you create a persistent stimulation, and this is another paper that we are working with Hirofumi and Maria Luisa, if you create a knockout renin or knockout of angiotensin receptors or so forth, the stimulation doesn't stop because there is no angiotensin.

Ariel Gomez: And so under those conditions, the cells reach a point in which they become very aggressive, almost embryonic-like. They are constantly stimulated. They are attempting to reestablish the phenotype and in doing so, they create these concentric vascular hypertrophy. And I don't know whether we are going to send the paper to Circulation Research or to where, but we are still writing it. After that, we don't know whether they can come back because they are so seriously sick. And we know that they are responsible for this, but this is another paper.

Maria Luisa Sequeira-Lopez: Another thing that I wanted to add is that these cells have been extremely difficult to study. Ariel has been developing many, many tools that allow him to dissect them and cover many secrets of the cells. But if you... First because they are very, very few in the kidney. And there were no markers to isolate them. And if you put them in culture, now that we can have them live with a person marker, they stop expressing renin and making renin within 24-48 hours. So it's difficult to study. So that's why Hirofumi [inaudible 00:19:21] how the system works. Stimulating them with cyclic AMP, they go back like renin. If not, they differentiate into vascular smooth muscle cells. It looks like that's their default pathway. So they need to sense that there is a need for renin to increase the blood pressure and electrolyte homeostasis. So that's one of the characteristics of the cells. But if you stimulate constantly, as Ariel said, then they may be hard to… They cannot come back.

Cindy St. Hilaire: It's over the tipping point a bit.

Maria Luisa Sequeira-Lopez: Yes.

Cindy St. Hilaire: In your discussion you mentioned another study from your group that kind of took more of a developmental angle. And you mentioned that you had identified unique chromatin structures of renin-producing cells, and you also identified what are called super enhancers that help dictate the differentiation of these running progenitor cells into renin producing cells. And then in your mechanical stimuli experiments, you mentioned identifying similar chromatin signatures. And I was wondering what this might suggest in regards to the disease pathogenesis. And I guess I'm thinking about it in terms of in many diseased states, we see this activation of developmental programs that either are not stopped or just go on and are even higher expressed than in developmental programs. And is that you think is happening in these renin cells? A developmental program gone awry?

Ariel Gomez: Yeah, definitely. I definitely think so. I think we all, the three of us think that way. Yeah. I think it's an exaggeration of a developmental program. One thing that we didn't mention and why the vessels get so sick is because during development, these cells contribute to the formation of the vasculature. And so when they regress so much trying to make renin... And they make it. I mean, they go from 20,000 units to 2 million of renin, right? And they never stop. But when they regress so much, they regressed on embryonic stage and they think that they need to make more blood vessels to actually increase the flow and the oxygenation of the tissue. But in doing so, they create more pathology. So maybe, Hirofumi, I don't know if you're going to ask him, but one of those super enhancers is the Lamin A/C gene. And he has studied that in this Circulation Research paper that we are talking about.

Maria Luisa Sequeira-Lopez: I just wanted to add that they also make lots of angiogenic factors to make the vessels.

Cindy St. Hilaire: Got it. So developmentally, they're activating more production of renin but they're also producing these pro angiogenic cytokines and really driving that…

Ariel Gomez: BGF. They produce a type of BGF or angiopoietins.

Cindy St. Hilaire: Interesting.

Ariel Gomez: Yeah. And things like that.

Cindy St. Hilaire: I really liked reading about this magnetic bead experiment that you used as the mechanical stimuli. Frankly, I saw the picture and I brought it to my lab and said, "Guys, figure out how to do this." Can you explain a little bit about it? It seemed really nice, really elegant and very tuneable. So I'm excited. I'm sure many more people are excited to hear about it.

Hirofumi Watanabe: So we applied coated magnetic beads to the cultured ring cells. Then we placed a magnet above the cells so we can pull the cells by magnetic force.

Cindy St. Hilaire: How strong is the magnet that it doesn't just rip everything up?

Hirofumi Watanabe: Yeah. We cannot observe the shapes of the cells, but yeah, I hope it's just stretch.

Cindy St. Hilaire: Yeah. Well, it certainly elicited an effect. So, in terms of future translational potential, what do you think about these findings that suggest either potential future therapies or even targets that we can use to develop therapies? Is there a future therapeutic angle to these really interesting biomechanical findings?

Ariel Gomez: Discovering or knowing the structure of these pressure sensing mechanism, I think we'll eventually have many applications because it will be applicable to hypertension, of course. And maybe we can begin to think... Not yet because it's really a fundamental discovery, it's not yet at that stage. But eventually the information can be used to start thinking about treatments that are addressing those particular structures that are involved from the beta one, integrating all the way to the nucleus. And little by little people started developing epigenetic therapies, right? And we are testing some of these compounds in our lower authority. Not with this model, with other models. But I think eventually we will be able to do what was the dream. It was really a dream years ago, was to do molecular therapy, right? And so a small compound development will play an important role. And eventually driving the molecules to the exact place in the genome is... So it would be not only patient-oriented, personalized medicine, but local specific. That should be the goal of medicine in the future. I won't be there when we get there.

Cindy St. Hilaire: I don't know. CRISPR is moving things rather fast, so that's great.

Ariel Gomez: Oh, yeah. You're right. You're right. You're right about that. Okay.

Cindy St. Hilaire: So what's next in this project? What do you think is the next low hanging fruit? Now that you've identified this baroreceptor or maybe a component of a larger baroreceptor family, what do you think is the next most important question?

Maria Luisa Sequeira-Lopez: We want to know what is in-between. And the bigger one integrating and the Lamin A/C. And also, we want to see how fast this reacts. So we'll be doing experiments with the constriction for just a few hours, and harvest both kidneys and we will try to do single cell RNA-seq and a from those vials.

Hirofumi Watanabe: I think we want to study how Lamin A/C regulates renin expression in renin cells, so chromatic modification initiated by changes in particle pressure more.

Ariel Gomez: And I think the... What I've been now pushing a little bit is to remember that there is another cell in there that is in between the pressure and the JG cells. And that is the endothelium cell. Right? And so, they are communicating with one another. So we are going to engage some... In fact, it's already happening. A member of the lab is already working with the same model that Hirofumi used, looking at endothelial cells label also using aninterfering promoter linked to a fluorescent protein. So we want to know what happens to the endothelial cells, because they are receiving the brunt of the pressure. And we don't know how they sense. We described the mechanosensing capability of the JG cells, the renin cells, but the whole system is probably a lot more complex than what we think.

Cindy St. Hilaire: I think that's the lesson of renin angiotensin signaling. It's always more complex.

Ariel Gomez: Yeah. Exactly.

Cindy St. Hilaire: Well, thank you all so much for joining me today. This is a beautiful study, very elegant. And I liked the new kind of in vitro models with this bead system. And congratulations on a whole lot of work. The amount of mice was probably a lot. I look forward to your future studies and learning what's happening at this endothelial renin cell junction.

Maria Luisa Sequeira-Lopez: Thank you. And we feel honored that you chose us.

Ariel Gomez: Yeah. Well, so I want to thank you for interviewing us. But I want to say that Hirofumi spent three years in the lab and he did a magnificent amount of work.

Cindy St. Hilaire: Wow. Yeah. I would have guessed a lot longer.

Ariel Gomez: Yeah. So he did a lot of work. And I'm very, very proud of what he has accomplished.

Maria Luisa Sequeira-Lopez: Yes. And I would like to add also that we were very lucky to have an expert in integrins, Dr DeSimone, who is the chair of Cell Biology at UVA and when we went and told him that we thought that this could be part of a mechanism sensing receptor, he started collaborating with us and opened his lab for us and trained Hirofumi with some experiments. It was really highly collaborative.

Cindy St. Hilaire: That's it for the highlights from our June 25th and July 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 #DiscoverCircRes. Thank you to our guests, Dr Hirofumi Watanabe, Dr Ariel Gomez, and Dr Maria Luisa Sequeira-Lopez.

Cindy St. Hilaire: This podcast was produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles was 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. This program is copyright of the American Heart Association, 2021. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors of the American Heart Association. For more information, please visit ahajournals.org.

This month on Episode 25 of Discover CircRes, host Cindy St. Hilaire highlights the topics covered in the June 11th Compendium on Peripheral Vascular Disease, as well as discussing two original research articles from the May 28th issue of Circulation Research. This episode also features an in-depth conversation with Drs Eric Small and Ryan Burke from the University of Rochester Medical Center about their study Prevention of Fibrosis and Pathological Cardiac Remodeling by Salinomycin.

Article highlights:

Ghosh, et al. IAP Overexpression Attenuates Atherosclerosis

Dörr, et al. Etelcalcetide for Cardiac Hypertrophy

Compendium on Peripheral Vascular Disease

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 presented in our May 28th and June 11th issues of Circ Res. I'm also going to speak with Drs Eric Small and Ryan Burke from the University of Rochester Medical Center about their study Prevention of Fibrosis and Pathological Cardiac Remodeling by Salinomycin.

Cindy St. Hilaire: The first article I want to share comes from the May 28th issue and is titled Over-Expression of Intestinal Alkaline Phosphatase Attenuates Atherosclerosis. The first author is Siddhartha Ghosh, and the corresponding author is Shobha Ghosh, and they're from VCU Medical Center. The Western diet is a colloquial term that is used to say a diet that is high in fats, sugars, refined grains, and red meat. A diet consisting of these foods can cause intestinal inflammation, which weakens the gut lining and facilitates transfer of the bacterial toxin lipopolysaccharide, or LPS. Once in the blood, LPS causes systemic inflammation.

Cindy St. Hilaire: Patients with diseases such as diabetes and atherosclerosis, in which inflammation is a major contributor, have increased levels of LPS in the blood. In the gut, the enzyme, intestinal alkaline phosphatase, or IAP, is a critical barrier for the intestine. It regulates the integrity of epithelial cell junctions and helps to detoxify LPS, both of which limit intestinal inflammation. Clinical trials of oral IAP have hinted at its potential to treat patients with ulcerative colitis. In this study, Dr Ghosh and colleagues investigated whether over-expression of IAP can reduce systemic LPS and help to prevent atherosclerosis. They fed atherosclerosis-prone mice engineered to over-express gut IAP, a Western diet for 16 weeks and found that the animals had improved gut integrity, reduced plasma levels of LPS, reduced gut lipid absorption, lower body weight, and decreased aortic plaque burden as compared to normal controls. Together, these results indicate that improving gut barrier integrity by boosting IAP, either by diet choices or pharmacologically, may help to slow atherosclerosis.

Cindy St. Hilaire: The second article I want to share is titled Randomized Trial of Etelcalcetide for Cardiac Hypertrophy and Hemodialysis. The first author is Katharina Dörr, and the corresponding author is Rainer Oberbauer, and they're from the Medical University of Vienna. In chronic kidney disease, or CKD, loss of renal function leads to systemic mineral imbalances. These imbalances trigger further physiological problems, such as the excess production of parathyroid hormone and growth factor, FGF23. The former can cause muscle and bone weakness, and the latter has been implicated in left ventricle hypertrophy. Hyperparathyroidism can be treated with calcimimetics or with vitamin D, but while both approaches lower parathyroid hormone levels, calcimimetics also lower FGF23.

Cindy St. Hilaire: This study investigated whether CKD patients treated with a calcimimetic, etelcalcitide, had any measurable improvements in left ventricle mass, as compared to patients given a vitamin D analog, alfacalcidol. In a single blind randomized study, 32 CKD patients were treated with etelcalcitide and 30 were treated with alfacalcidol for a year. At the end of the study, left ventricle mass measured by magnetic resonance imaging, was found to be significantly lower in the etelcalcitide group. FGF23 levels had also declined in this group, but had risen in the alfacalcidol group. The results indicate that calcimimetics reduce the risk of cardiac hypertrophy, as well as treating hyperthyroidism, and thus, might be a preferable treatment option in CKD.

Cindy St. Hilaire: The June 11th issue of Circulation Research is the Peripheral Vascular Disease Compendium, and in this compendium, we have 14 articles that are written by the leading experts who present an update on the state of the field of peripheral vascular disease research. They discuss current research and also current therapeutic options. Drs Nick Leeper and Naomi Hamburg serve as the guest editors of this compendium. Drs Derek Klarin, Phil Tsao, and Scott Damrauer discuss the genetic determinants of peripheral artery disease. Drs Kunihiro Matsushita and Aaron Aday present a Review on the epidemiology of peripheral artery disease and polyvascular disease.

Cindy St. Hilaire: The potential of leveraging machine learning and artificial intelligence to improve peripheral artery disease detection, treatment, and outcomes is covered by Drs Alyssa Flores, Falen Demsas, Nicholas Leeper, and Elsie Ross. The benefits of walking as exercise therapy and its benefits on lower extremity skeletal muscle is presented by Drs Mary McDermott, Sudarshan Dayanidhi, Kate Kosmac, Sunil Saini, Josh Slysz, Christiaan Leeuwenburgh, Lisa Hartnell, Robert Sufit, and Luigi Ferrucci. Drs Marc Bonaca, Naomi Hamburg, and Mark Creager discuss medical therapies currently available to improve outcomes in patients with PAD. In a similar vein, Drs Joshua Beckman, Peter Schneider, and Michael Conte cover the recent advances in revascularization for peripheral artery disease.

Cindy St. Hilaire: Racial and ethnic disparities in PAD is discussed by Drs Eddie Hackler, Naomi Hamburg, and Khendi White Solaru. Drs Tom Alsaigh, Belinda Di Bartolo, Jocelyne Mulangala, Gemma Figtree, and Nicholas Leeper present their thoughts on optimizing the translational pipeline for patients with peripheral artery disease. New directions and therapeutic angiogenesis and arteriogenesis in PAD is covered by Drs Brian Annex and John Cooke. Drs Esther Kim, Jacqueline Saw, Daniella Kadian-Dodov, Melissa Wood, and Santhi Ganesh review sex-biased arterial diseases with clinical and genetic pleiotropy, focusing in on multi-focal fibromuscular dysplasia and spontaneous coronary artery dissection, which have a much higher prevalence in women.

Cindy St. Hilaire: Drs Matthew Fleming, Ling Shao, Klarissa Jackson, Joshua Beckman, Anna Burke, and Javid Moslehi cover the vascular impact of cancer therapies and focus on how cardiac and vascular sequelae of novel targeted cancer therapies can provide insights into cardiovascular biology. Epidemiology and genetics of venous thrombosis and chronic venous diseases is presented by Drs Richard Baylis, Nicholas Smith, Derek Klarin, and Eri Fukaya. Dr Stanley Rockson reviews advances in our understanding of lymphedema and the compendium concludes with an article by Drs Yogendra Kanthi, Meaghan E. Colling, and Benjamin Tourdot, which reviews, inflammation, infection, and venous thromboembolism. This comprehensive compendium on peripheral vascular disease is found in our June 11th issue.

Cindy St. Hilaire: So today, Drs Eric Small and Ryan Burke from the University of Rochester Medical Center are with me to discuss their study Prevention of Fibrosis and Pathological Cardiac Remodeling by Salinomycin. This article is in our May 28th issue of Circ Res. So thank you both for joining me today.

Eric Small: Thanks Cindy, for having us. Excited to talk about our research with you.

Ryan Burke: Yeah, thank you very much for having us.

Cindy St. Hilaire: Absolutely. So fibrosis, it's essentially a wound healing mechanism, it's where connective tissue replaces the innate tissue of the organ system that it's happening in. It's really a component of many disease states. As far as I know, treatment options are pretty limited or really non-existent except in a couple rare cases, and in particular, your study, as it's in Circ Research, is focused on cardiomyopathy and the fibrosis related to that. But before we dig into your findings, which is really focused on a great therapeutic angle, I really want to take a step back and ask about what we know about fibrosis or the fibrotic process itself, maybe in the context of the heart, and despite why it's relatively common, it's been so difficult to target in terms of either therapies or really just understanding some of the basic processes.

Eric Small: Sure, I'd be happy to discuss this. So as you know, and you alluded to already, pathological fibrosis contributes to progression of many debilitating human diseases. So in injury response in many tissues or organs, including the heart, kidneys, lungs, even the skin, leads to a wound healing process and that wound healing process is meant to repair the tissue and that includes an inflammatory response and secretion of extracellular matrix that fortifies the structural integrity of the tissue. But you can imagine in the context of a heart, that has to beat 60 plus times per minute, any alterations to the biomechanical properties of that tissue can alter the function.

Eric Small: So extracellular matrix, which is meant to improve the structural integrity of an injury, even in the heart, ultimately can lead to reduced cardiac function. So this extracellular matrix, and in the context of disease, this extracellular matrix is called fibrosis, can reduce the contractility and the relaxation of the heart. The relaxation of the heart is actually an important aspect in insufficient relaxation called diastolic dysfunction, is becoming a more prevalent disease phenotype and it is called heart failure with preserved ejection fraction. What we're finding and what some investigators are alluding to is that fibrosis is a major component of this disease, and so understanding how extracellular matrix is secreted, why it is deposited in the context of injury, especially in the context of the heart, why does that process not stop sufficiently and revert once the injury is repaired, is a really important basic science and clinical question.

Cindy St. Hilaire: So why, specifically, has fibrosis or cardiac fibrosis been so difficult to target therapeutically?

Eric Small: From my point of view, one of the reasons that fibrosis, organ fibrosis in general, and especially within the heart, is hard to target is because I think we're understanding now that one of the major cellular sources of extracellular matrix in disease is the fibroblast. This cell type has been sort of underappreciated for many years and is coming to the forefront now of biomedical research. So fibroblasts until maybe 10 or 15 years ago were thought to be more of a structural component. Of course, they contribute to wound healing, but it was thought that they contribute mostly to structural integrity and homeostasis of the injury. It's becoming more apparent now that resident cardiac fibroblasts contribute to extracellular matrix deposition in disease. But these cell types are really plastic, phenotypically plastic cell, they respond to a lot of biomechanical stimuli, especially that are induced in the context of tissue injury or disease, and so they respond to mechanical stretch or cellular deformation, and they can respond to many secreted factors, especially the canonical factor that has been studied extensively, TGF-beta.

Cindy St. Hilaire: Which itself is extremely complicated, to say the least.

Eric Small: Absolutely, and so it does so much, and they respond to factors that are really high up on this hierarchy, that do so many things that I think obviously targeting TGF-beta is not going to be really an efficacious therapeutic option. So understanding what's more downstream and much more specifically related to the fibroblast, I think is really important to come up with new therapeutics.

Cindy St. Hilaire: So in your quest to identify novel therapeutics, or even really understanding that below the surface signaling you just talked about, you developed a high-throughput screen. I think this is a term that we often use, but we don't really know the details of that term, like what does high-throughput actually mean when you're doing it with cells and disease models?

Dr Eric Small: Sure. So I think in our case, we really let the science lead the way when it came to the high-throughput screen. So I'm not a chemical biologist, I have never, before now, developed a high-throughput screen and the science just pointed me in this direction. So the basic science research related to fibroblast plasticity and what induces fibroblasts to secrete extracellular matrix in the context of disease, all culminated in this one reporter that I thought would be good for the assay. So maybe as a way of a little bit of background, one difficulty in understanding fibrosis and fibroblast plasticity is that there are no really unique specific markers for an activated fibroblast. So most of the markers that people say are myofibroblast markers, which is the term for an activated ECM-secreting fibroblast, are expressed in other tissues or cells. Probably the most used and best characterized marker of a myofibroblast, is the smooth muscle alpha-actin gene, which encodes the smooth muscle actin protein, which is highly up-regulated in myofibroblasts, but obviously is expressed in a lot of other cell types, including smooth muscle cells.

Eric Small: So it is a good marker of a myofibroblast, but it's not unique to myofibroblasts. But, this smooth muscle alpha-actin gene allowed us to make inroads into better understanding how fibroblasts respond to different stimuli. So what we did was, in the lab, one of the earlier things that we did when I set up my lab as an independent investigator, was to try to develop a stable cell line that expressed this reporter in a way that we could easily assay. So we could do it with GFP or a luciferase reporter or something like that. We made a luciferase reporter of this smooth muscle actin myofibroblast, alpha-actin gene. So one important aspect of a screen is, especially in our screen, which we were looking for chemicals that would inhibit our reporter, that we would hope would be anti-fibrotic

Eric Small: Our hope was that this reporter would actually, in some cases, lead to an anti-fibrotic compound, but an important aspect of this screen, which was, I think the original question, was to not come up with factors that would just kill fibroblasts, but come up with factors that would specifically inhibit smooth muscle actin and myofibroblast activation without being too toxic. We don't want to inject a toxic chemical into a person; we want to inject a chemical that would be specific to an activated myofibroblast. So that was the first consideration, is to make sure that these were not toxic compounds, but were acting specifically on the smooth muscle actin report.

Cindy St. Hilaire: So with this system, you were able to screen over 2000 compounds, it was like 2300 or something like that. From that 2000 compounds screen, you zeroed in on salinomycin and two other compounds that are in the same family, I think, of chemicals like polyether ionophores they were called, I think it was the top three were all this similar class. So that's probably unsurprising that similarly-structured chemicals have a similar function or phenotype, but it's also intriguing. So I'm wondering, what's known if anything, about this class of chemicals, have they been used in therapy or is there some kind of naturopathic history to salinomycin or these other compounds that maybe if we read more carefully, we would have got a hint before?

Ryan Burke: Salinomycin has a pretty storied history in the literature, but it's an odd history. It's a veterinary antibiotic. So it's actually used primarily in livestock management and it had really no approach in human science at all. Then it was discovered that salinomycin, its earliest contribution, was that it is a compound that is actually very selectively targeting cancer stem cells. So salinomycin has a very extensive literature in cancer. It affects a lot of relevant signaling pathways, it's actually where we got a lot of our insight as to what we should be evaluating in fibroblasts. Both, in terms of ... This is probably going to be a charged statement; but there's a lot of similarities in how ... Cancer cells, when they're metastasizing and activating and moving around, there's a lot of EMT involved in that, there's a lot of things that are very analogous to how fibroblasts activate in heart failure.

Ryan Burke: I'm not saying they're the same, that's the charge portion of it, but the pathways are often conserved. What we found is that salinomycin had been studied extensively in various models of both solid and blood tumors, and it was found that it was affecting a whole ton of signaling pathways and sparing others, which was actually some of the insight that we had about AKT signaling. In the heart, it seemed very easy to just apply that and say, "Well, activation of fibroblasts is largely dependent on signaling pathways like SMADs and p38 signaling, so let's see what salinomycin does to these pathways in fibroblasts," and it turned out that that wound up being a very fruitful avenue for exploration, because it does behave very similarly in fibroblasts to the way it behaves in cancer cells. We didn't really find a lot of discordance in those results.

Ryan Burke: This study was very iterative, right? So do the high-throughput screen, find the drug, then try a preclinical model in animals. Then when it worked quite well in the angiotensin, hypertension-induced remodeling, that's a pretty mild model, right? Give the mouse an MI, see if it works in that, because that's a much more serious remodeling and when it performed well there, it's like, "Wow, you really actually probably have something here."

Cindy St. Hilaire: Yeah, and that's a perfect segue for my next question really, was I wanted to ask about these different murine models. Like you identified this compound, now you want to test it. Could you maybe give us a little brief background on why you chose the models you did and the treatment regimens that you also tried?

Ryan Burke: Sure. When we began, we began with angiotensin infusion because it's a fairly mild remodeling. You get some hypertrophic remodeling of the heart, you get some proliferation and some mild fibrosis in the mouse model. We figured this would give us the best chance to see a signal versus noise. It turned out that the results were really striking. Even the mice that were given the condition that we expected to see nothing in, is the drug with a saline infusion, even that had effects that were consistent with the effects that were seen. Consistent in direction in terms of the overall morphology and function of the heart, consistent with what you were seeing with the normalization of that hypertrophic remodeling in the angiotensin model that also got the drug. So that was really interesting to us. It was just consistent all the way through.

Ryan Burke: We wound up having a meeting about it and we were like, "All right, we've done the preventative regimen. We've preloaded them and then run them through with the drug. Now let's see if we can reverse established remodeling." So we did that study and when that worked out okay, there was yet another discussion where it was like, "All right, are we doing this?" And it was a myocardial infarction study. Myocardial infarctions, that's really extensive remodeling with huge changes, both the macro and microstructures of the heart. There's a lot more of an inflammatory component involved in that.

Ryan Burke: So we weren't sure how this would perform and it turns out that it performs exactly as it performs in pressure overload. You see normalization in physiology. I think that's part of the power of this study is that you're looking at non-ischemic and ischemic heart failure models, you're looking at preventative and interventional regimens, and it's just consistently performing at a level. We wanted to check all of our boxes, really, with this.

Cindy St. Hilaire: Sure. Yeah, maybe salinomycin's going to be the new aspirin we pop when we're over 50.

Ryan Burke: I doubt it, it's worth $7 a kilogram. I very highly doubt anyone's licensing that.

Eric Small: But I think it's interesting you say that because understanding the mechanism after you understand that it's efficacious is sort of a similar idea here. We don't necessarily know precisely what it's targeting to act as an anti-fibrotic in this case, and so there's a lot of work to be done on this compound. I'd like to reiterate something that Ryan actually said is that really interesting, at least in cells and in the animal models, that salinomycin doesn't have a huge impact on the heart or on cultured fibroblasts in the absence of, for example, TGF-beta stimulation or a disease mechanism. It's really when we have a disease that salinomycin blocks the activation of the myofibroblasts and prevents that from contributing to the disease.

Cindy St. Hilaire: Interesting. So that can really, at least in the case of maybe cardiomyopathy, would help target it to the heart.

Eric Small: That would be the hope, yeah.

Cindy St. Hilaire: Yeah, that's great. Wow. Speaking of the heart, and you mentioned this in that first answer that you had about the fibroblast being kind of the forgotten child of the heart and the focus is really more the cardiomyocyte, but did this drug have any impact on the cardiomyocytes itself that are also probably exposed to this TGF-beta signaling, in the context of an injury?

Eric Small: So this is where we have some interesting, but not anticipated, results. So we obviously performed a screen in fibroblasts to look for specific anti-fibrotic compounds and when we put this into animals into ischemic or non-ischemic models, especially in the ischemic model, we found a much better outcome than we would have expected from simply an anti-fibrotic. So for example, we saw that pretreatment of mice with salinomycin prior to myocardial infarction, almost completely abrogated, not completely, but highly significantly abrogated necrotic tissue formation. So when Ryan went back and looked at the percentage of heart that became necrotic, or ischemic, after myocardial infarction, it actually reduced the necrotic core significantly. So we do think it's acting on cell types other than the fibroblasts in the context of ischemic remodeling, and it does seem to induce potentially protective signaling pathways in cultured myocytes. So that's definitely an area that we'd be interested in pursuing in more detail.

Ryan Burke: So of course the question there is, and this is a totally fair question for people to evaluate, we're looking at an organ in which all the cell types are talking to each other. We know we've affected the fibroblasts in a certain way, and we know to a certain extent, from what we found, what we've done for the fibroblasts, and we know what that looked like as a result in myocytes, but who initiated that, right? Did we affect the myocyte and then fibroblasts changed? Or did we affect fibroblasts and myocytes changed? But those are important type questions. We've shown the changes, but how do we show the connections? I think that's the really interesting work that we're still doing. We even extended it a little bit to endothelial cells in the heart, because we were showing that there was sort of a preservation of vascularization in the MI model that was associated with salinomycin, and we wouldn't rule out that we were affecting endothelial cells as well. I mean, I think this is a subject for discussion in the field, in the future. Groundwork is there, it's time to move forward.

Cindy St. Hilaire: Yeah, that is so exciting, and it's also I guess the classic chicken and egg question of science. What's causing what? That's excellent. So what's next for this project? I mean, you just highlighted some other angles, endothelial cell, but is there plans to translate it to a clinical setting, especially because it's already used in humans, so there's all that safety data out there? What's the plan?

Eric Small: So that's a really interesting question. So our collaborators here at the University, Colin Woeller, Patricia Sime, Rick Phipps, they have been involved in the study with us and they are interested in fibrosis in other aspects as well. So they're interested in lung fibrosis, idiopathic pulmonary fibrosis, ocular eye fibrosis, and they've found that in other situations, salinomycin can inhibit fibrotic disease remodeling, for example, in the eye and in the skin. So branching out into other animal models of fibrotic disease is one area that we'd like to pursue. One area that I'm really interested in looking at salinomycin would be, for example, in models of HFpEF to see whether salinomycin might be efficacious in limiting the progression of animal model HFpEF. These are now becoming more prevalent and so it'd be great to test that there.

Eric Small: So I think probably with some of these small animal studies, it would lay the groundwork for larger animal studies as collaborations or with other investigators. Absolutely, I think that's where this could definitely go next.

Ryan Burke: Also, it's a high-throughput screen, right? It wasn't the only hit and so extending the screen outwards both ... So the screen was designed to pick up both anti and pro-fibrotic drugs. So pro-fibrotic drugs have applications in wound healing. It also gives us a hint as to if a drug has some unexpected side effects in large clinical populations, then we can look at that and say, "Oh, maybe we have mechanistic understanding of why this might be the case." I think you'll see some future explorations down that path as well in that study.

Cindy St. Hilaire: Well, I look forward to seeing all of them. This was a wonderful study. I'm more vascular biologist, but obviously being on Circ Res, I'm learning so much more about the heart, but this one, I just particularly love that you started with this crazy complex question of what the heck is going on and this high-throughput screen was just designed in such a way that it really narrowed down what was a huge amount of options to start with. So it was really elegantly done and I just love the story, so congrats to you both and I look forward to future publications.

Eric Small: Thank you. I'm especially proud of this one because as a basic scientist and as a trained in graduate school as a developmental biologist, I was following the science and when this opportunity arose to try to make this high-throughput screen work, I mean, this was as clinically relevant as I could ever have imagined my lab becoming. I'm really proud that we're able to do that.

Cindy St. Hilaire: Absolutely. I know, it's something we always talk about, and this research can be translated to humans eventually, and you're almost there. That's great. Well, congrats again. Thank you both for taking the time today and I look forward to your future studies.

Eric Small: Thank you, Cindy.

Ryan Burke: Yeah, thanks Cindy.

Cindy St. Hilaire: That's it for the highlights from the May 28th and June 11th 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, Drs Eric Small and Ryan Burke.

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 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. This program is copyright of the American Heart Association, 2021. The opinions expressed by the speakers of this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahajournals.org.

This month on Episode 24 of Discover CircRes, host Cindy St. Hilaire highlights the topics covered in the May 14th Compendium on Heart Failure, as well as discussing two original research articles and a brief overview of the Review Series on Calcific Aortic Valve Disease from the April 30th issue of Circulation Research. This episode also features an in-depth conversation with Dr David Durgan and Huanan Shi from Baylor School of Medicine about their study Restructuring The Gut Microbiota by Intermittent Fasting Lowers Blood Pressure.

Article highlights:

Vacante, et al. CARMN Regulates Atherosclerosis via SMC Modulation

Hanna, et al. Cardiac Neuronal Control of the Sinoatrial Node

Cuevas, et al. Introduction to the Aortic Valve Disease Series

Compendium on Heart Failure

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 will be highlighting topics presented in our April 30th and May 14th issues of Circ Res. I'll also speak with Dr David Durgan and his graduate student, Huanan Shi, from Baylor School of Medicine about their study Restructuring The Gut Microbiota by Intermittent Fasting Lowers Blood Pressure.

Cindy St. Hilaire: The first article I want to share comes from the April 30th issue of Circ Res and is titled CARMN Loss Regulates Smooth Muscle Cells and Accelerates Atherosclerosis in Mice. The first author is Francesca Vacante and the corresponding author is Andrew Baker, and they're from the University of Edinburgh. The increased proliferation and migration of local vascular smooth muscle cells is part of the complex pathology of atherosclerotic plaques. These proatherogenic changes to smooth muscle cells are regulated in part via two micro RNAs, miR-143 and miR-145. And these are located together on human chromosome five. In this very same genetic locus is also a gene encoding a long non-coding RNA called cardiac mesoderm enhancer-associated non-coding RNA or CARMN.

Cindy St. Hilaire: This team found that levels of CARMN and miR-143/145 RNAs in mouse and human atherosclerotic plaque decreased as the condition worsened. Mechanistic experiments showed that this decrease drove smooth muscle cell pathology. Knock down of all three RNAs promoted increased proliferation and migration of human artery smooth muscle cells with the loss of CARMN specifically and independently, triggering increased proliferation. The team went on to show that in mice, loss of CARMN accelerated the progression of induced atherosclerosis. Together, the work highlights the interplay between these noncoding RNAs and atherosclerotic disease progression.

Cindy St. Hilaire: The second article I want to share is titled Innervation and Neuronal Control of the Mammalian Sinoatrial Node, a Comprehensive Atlas. The first author is Peter Hanna and the corresponding author is Kalyanam Shivkumar from UCLA. The nervous system regulates cardiac physiology and influences pathophysiological adaptations to disease. Mapping the intrinsic neurocircuitry of the heart is necessary if we are to fully understand how neural circuits function in health and in diseases such as arrhythmia. Neural circuits from outside the heart meet up with those within the heart at ganglionated plexuses on the heart surface. One such plexus is the right atrial ganglionated plexus, RGAP. And RAGP is thought to regulate the signal inputs from the vagus nerve into the sinoatrial node or the SAN, which is the heart's pacemaker.

Cindy St. Hilaire: To develop a detailed description of the connections between the RAGP and the SAN, this group used the pig's heart as it is a close anatomical match to that in a human's. Performing a combination of tissue clearing, immunohistochemistry and 3D fluorescent microscopy, this group showed that approximately 99% of the neurons in RAGP and most of those innervating the SAN, are cholinergic neurons. In spite of this, single cell transcriptomic analysis revealed a great deal of phenotypic diversity among these RAGP neurons. Through electrophysiological and neural ablation studies, the team revealed the extent of RAGPs modulation of the sinoatrial node functions, which characterizes the RAGP as an integrative neural structure and not just a relay station within the intrinsic cardiac nervous system. This work now creates a very detailed reference atlas of the RAGP sinoatrial node conductivity and a framework for mapping other aspects of the intrinsic cardiac nervous system.

Cindy St. Hilaire: The April 30th issue of Circ Res also has a short review series on calcific aortic valve disease. Dr Rolando Cuevas and I write an introduction to this series. Dr Joy Lincoln covers genetic and developmental contributors to aortic stenosis, Dr Jonathan Butcher covers inflammatory and biomechanical drivers of endothelial interstitial interactions in calcific aortic valve disease. Dr Tom Gleason covers current therapeutic options in aortic stenosis and Dr

Elena Aikawa covers multi-ohmic approaches to define calcific aortic valve disease pathogenesis.

Cindy St. Hilaire: The May 14th issue of Circulation Research is the heart failure compendium. This features 10 articles written by the leading experts, who present an update on the state of the field of heart failure research and current therapeutic options. Dr Douglas Mann is the guest editor of this compendium, and in his introduction, he emphasized his vision that the authors of this series, "Focusing on linking disease pathophysiology with the mechanisms action of current therapies, with the hope that past successes would serve as a prologue for the development of future therapies." Together, these Reviews present the recent therapeutic advances in heart failure and is truly representative of the successful transition of bench top research to the bedside of patients.

Cindy St. Hilaire: In the first article in the compendium, Dr Veronique Rogers provides an update on heart failure epidemiology, including a focus on the role of healthcare disparities. Dr Michael Felker, and Dr Mann follow with an overview of the pathophysiology of heart failure with reduced ejection fraction and highlight how several successful heart failure trials fit or do not fit into the current conceptual translational models of heart failure. Dr Walter Paulus and Michael Zile discuss heart failure with preserved ejection fraction, with a focus on the role of systemic inflammation and myocardial stiffness, and relate this pathophysiology to distinct clinical phenotypes and tailored medical therapies.

Cindy St. Hilaire: Drs Joyce Njoroge and John Teerlink discuss what is currently known regarding pathophysiology of acute decompensated heart failure, and present a handful of new therapy developments. Drs Gary Lopaschuk, Qutuba Karwi, Rong Tian, Adam Wende, and Dale Abel discuss cardiac energy metabolism and heart failure and review several promising approaches to beneficially altering metabolism in the failing heart. Highly relevant to the long-term cardiovascular phenotype seen in patients who have had COVID-19, Drs Ray Hershberger, Jason Cowan, Elizabeth Jordan, and Daniel Kinnamon reviewed the genetic basis for dilated cardiomyopathy and discuss what is known regarding the interaction of genetic risk and environmental factors.

Cindy St. Hilaire: Drs Jan Griffin, Hannah Rosenblum and Matthew Maurer discussed cardiac amyloidosis due to light chain or transthyretin amyloidosis and cover current effective therapeutic strategies and active clinical trials. Drs Virginia Hahn, Kathleen Zhang, Lova Sun, Vivek Narayan, Daniel Lenihan, and Bonnie Ky covered the development of heart failure due to targeted cancer therapies and discuss the rationale and evidence supporting different cardiotherapeutic approaches.

Cindy St. Hilaire: The Compendium concludes with an article by Drs Daniel Burkhoff, Veli Topkara, Gabriel Sayer, and Nir Uriel that discusses the current state of left ventricular assist devices or LVADs and the structural, cellular and molecular aspects of LVAD associated reverse left ventricle remodeling. This comprehensive Compendium on Heart Failure is found in the May 14th issue of Circulation Research.

Cindy St. Hilaire: So today, Dr David Durgan and Huanan Shi from Baylor College of Medicine are here with me to discuss their study, Restructuring the Gut Microbiota by Intermittent Fasting Lowers Blood Pressure, which is in our April 30th issue of Circulation Research. So thank you both very much for joining me today.

David Durgan Pleasure to be here.

Cindy St. Hilaire: So this study is bringing together two hot fields, the gut microbiome and intermittent fasting, and it's in the context of high blood pressure, which obviously is a national and global crisis. But before we jump into the details of the paper, could you just define what is meant by gut microbiome and intermittent fasting for the purposes of the discussion?

David Durgan: Sure. So when were you referred to the gut microbiome or the gut microbiome, what we're really referring to there are all the microbes that are residing in the gut. So this can be the complex composition of bacteria, viruses, fungi. However, for the purposes of our studies, we really focus in just on the bacteria.

Cindy St. Hilaire: So how did you even come to this question? What was the premise that existed such that you wanted to ask this question? Microbiome, intermittent fasting, and hypertension?

Dr David Durgan: It really started in terms of understanding the connection between the biome and hypertension. And this actually all started in a separate model of hypertension that we developed here in our lab. And that was a model of obstructive sleep apnea. When we were first developing this and characterizing this, one of the strange observations that we found is that these animals did not have any change in blood pressure, which was contrary to what we see in patients and even what they see in the intermittent hypoxia models.

David Durgan: So when we started thinking about OSA and the patient, we started thinking about all these other co-morbidities, one of them being obesity and poor diet. So at this point we started adding in other morbidities, such as a high-fat diet. And we found that very quickly within one week, actually, when we had the combination of both apnea and high fat diet, that this was then leading to the increase in blood pressure. And really lucky, right place at the right time was that we were thinking about what the high-fat diet was doing. And there was a seminar here on campus about the gut microbiota, which we really had done nothing with up to that point. And after attending that, it quickly became obvious that our high-fat diet was going to be shifting the biota. So this is what really led us to making this connection between changes to the microbiome and blood pressure.

Cindy St. Hilaire: So can you tell me little bit about the design of your study, about the animal systems you use and the diet and the regime that you put them on?

David Durgan: Sure. So we went into this with two overall questions. So we had already shown previously in this model that the biota was disrupted and that was contributing at least to the hypertensive phenotype. So we came into this and wanted to address the questions of, one, what are the mechanisms through which the microbiota is influencing host blood pressure? And then two, is there some type of intervention that we could do to shift the makeup of the microbiota and see how that affected the hypertensive phenotype?

David Durgan: So to address those two components, we took the spontaneously hypertensive stroke prone rat, and it's normotensive parent strain, the WKY, and we put them either on a normal ad libitum food access or every other day fasting, which is just as it sounds, it was a full 24 hours of ad-lib access followed by 24 hours of no food access at all.

David Durgan: And this went on for 10 weeks with constant assessment of food intake, body weight, blood pressure. And then at the end, we isolated fecal content in order to look at the effects on the biome. And that was done with whole genome shotgun sequencing, but we also did a on-targeted metabolomics approach of both the fecal content and the plasma in order to get a real understanding of what are some of the microbial metabolites that could be influencing hosts.

Cindy St. Hilaire: Such an interesting question. And it's such a complex idea, but I thought you did a really great job winnowing it down as your paper progressed. And you did find that the every other day feeding reduced blood pressure in the hypertensive stroke prone rats, and interestingly or maybe not interestingly to you, but I thought it was interesting, is that those every other day fed animals, they certainly ate more on the days when they were allowed to eat. And obviously on the days they weren't, they were eating less. And so their overall food intake was less. And ultimately at the end of your trial period, their weight was less. So are these effects that you see on blood pressure more directly related to the weight loss or to the actual microbiome? And how did you confirm that?

Huanan Shi: So that's actually a very good question. A lot of the intermittent fasting related studies definitely can separate the effects of the fasting itself and the effects of the weight loss as intermittent fasting has been used very frequently as a method for obesity and reduce body weight. So to confirm that the effects is through intermittent fasting, to restructuring the microbiome, a sort of indirect route from the weight loss, so we collected fecal sample of these animals that have been fed either on the intermittent fasting protocol or ad libitum with food access every day. We then transferred the fecal content through our garage into germ-free rats which they do not have an established gut microbiota.

Huanan Shi: So these germ-free animals who'll receive the hypertensive HSR mode] biota with just regular feeding pattern also developed high blood pressure compared to those who received the normotensive microbiota. So interestingly is that the animals that received the microbiota from the hypertensive animal that also was fed on the fasting protocol did not develop a high blood pressure. So this study actually tells us that maybe the weight loss have some effects, but through the microbiota transplant study, we show that majority or the conjoined factors mostly from the changes in microbiota instead of the weight loss.

Cindy St. Hilaire: Yeah. It also makes me wonder how much of the microbiota changes actually influence the weight loss as well. I wonder that's probably a whole another black box to open. You did find that in the feeding regime differences, there was a difference in the actual communities of bacteria. Can you talk maybe about the implications of what that means, and also does that mean this is perhaps something that we could recapitulate with a pill, like with a probiotic pill of some sort?

David Durgan: Yeah. So some of the overall changes that we see, we do see pretty drastic changes in the beta diversity of the community. This being things like richness, evenness, the number of species that are actually present and really pretty interestingly, the way that we saw those shift was that the SHR that were undergoing the fasting protocol, their community structure overall seemed to shift more closely to resemble that of the normotensive WKY. So there were some pretty large shifts. And then when we get down to some of the genera and species levels, we again see that many of these are being normalized to look much more like the communities of the WKY.

David Durgan: In terms of taking a pill or something along that source, somewhat surprisingly, actually we found that in the hypertensive animal, a number of species that are commonly thought of as probiotics. So for instance, bifidobacteria and lactobacillus, which are two of the only FDA approved genera for probiotics, they were actually higher in our SHR control fed animals. So I think there's still a lot of work to be done to understand exactly the contribution of individual species. Maybe what's more important is understanding the functional output from the community as a whole. So what are some of the metabolites that are actually influencing the host?

Cindy St. Hilaire: So it may not be the bacteria itself, but perhaps the products that create.

David Durgan: Right. And the thing that's frustrating, but also exciting about this is that there's so much functional redundancy between different species, meaning that while you could have loss of one species, it may look very significant on paper, but it could be that other species in the community are making up for that. So they're able to make the same metabolites and thereby overcome that deficiency.

Cindy St. Hilaire: Got it. Got it. So it may not really be that big of a shift per se.

David Durgan: Yeah. We can't always go off of just what is the species change and that's why we really moved and thought it was important to move on to looking at the metabolites themselves.

Cindy St. Hilaire: So you did see that these hypertensive rats had more an inflammatory profile in certain sections of the gut. And I was trying to think about this in terms of humans. And I don't know if it's known, but do patients with IBS or with chronic diseases like Crohn's disease or some other gut inflammation phenotype, do they actually have more hypertension or develop it earlier? I guess I'm thinking of this in terms of cause and consequence, the hypertension influence the gut microbiome, or do you think the microbiome perhaps is driving the hypertension?

David Durgan: That's a great question. I've tried to look and see if there's any real conclusive evidence for inflammatory GI disorders and a concrete connection to elevated blood pressure. Personally, I've not found convincing evidence of that at this point in time.

Cindy St. Hilaire: So in terms of the metabolites, I thought it was really interesting that you found, I think it was a reduction in bile acids, and specifically you then explored further choline, that that was at play in this hypertensive state. So can you discuss what it is you exactly found and then what this might mean in terms of hypertension pathogenesis?

David Durgan: Yeah. So from our un-targeted metabolomics data, we performed random forest analysis to try and understand some of the broad pathways that were altered. First of all, just differences between our hypertensive and normotensive control animals, but then also how the fasting affected those metabolites. And there were a number of pathways of interests, which need to be followed up on, but the one that really stood out to us was primary and secondary bile acid metabolism. Now, we followed up on this by doing a targeted approach to look at a specific panel of primary and secondary bile acids. And we were really very surprised at just how different they were. We measured, I believe, 17 different bile acids. And we found that in the plasma, that 12 of these were significantly lower in a hypertensive model. So this was really exciting.

David Durgan: And the more we looked into this, it really all make sense in terms of that if you look at where bile acid receptors are located, they're present in the endothelium and smooth muscle, in brain, on inflammatory cells. So we've really, I think, just started to see the tip of the iceberg in terms of their effects systemically. In a final figure of our paper, we look at their effects on vascular function and show that by giving a TGR5 agonist, which is one of the bile acid receptors that we could improve vascular function in this hypertensive model. I mean, bile acids classically have really been looked at in regards of strictly in the liver and the GI and in the inner hepatic circulation. But just the fact that we see these receptors so widespread systemically really tells us that even though the concentrations may seem low and plasma relative to in the GI tract, that they're very likely having pretty profound effects on overall physiology.

Cindy St. Hilaire: . So do either of you follow an intermittent fasting diet and also, I guess more specifically about IF is these rats, the study, you did every other day feeding. So for humans, obviously I think right now it's Ramadan so a lot of people are almost doing that now. But for humans, that seems like a stretch. I don't think I would really want to do that regularly. So do you think any of these findings could also be similar for different forms of intermittent fasting? I know like that 8/16 hour breakdown is the popular one. What do you think about that?

David Durgan: I personally have not tried it. There have been some grad students that have come through the lab and actually one of the investigators on this paper who worked with me to develop this idea, he was very into ... I think he did the 16/8 that you're referring to.

Cindy St. Hilaire: Okay. Yeah.

David Durgan: But yeah, I mean, that's really kind of been a hindrance almost in the field is that you go to understand clinical studies on intermittent fasting, and there's just so many different protocols out there. Whether it be looking at outcomes or blood pressure or whatever effect on physiology during Ramadan, during a 16/8, during every other day. And it's really muddied the waters in terms of understanding the overall effects, but looking through all of that, it does appear that even in the small clinical studies that are out there, that there does appear to be some benefit. There is a every other day fasting. So very similar to exactly the same as our protocol in a small randomized controlled trial. And it should be said that these were healthy individuals, but even after just four weeks of EODF or every other day fasting, there was about a 5 mm decrease in blood pressure in individuals.

David Durgan: The followup papers from that group really should be very interesting. So these individuals have now gone back on a normal feeding regimen, but they plan, I believe, to look at intervals out to two years to see how long lasting these effects are.

Cindy St. Hilaire: Interesting. The other thing with humans, I mean, obviously your rats, they're eating one meal, the same meal, essentially. Humans eat a variety of things at every meal at every different day. Sometimes they have a bag of candy because it's Easter or whatever, so that doesn't help either. So what was the most challenging part of the study?

David Durgan: This required a lot of legwork by Fred in terms of some of the multi-omics analysis.

Huanan Shi: For me, two part. One part is definitely analyze these data using the machine learning protocol. A lot of things I had to learn from scratch. And eventually, it's a lot of time-consuming troubleshooting, but I'm glad everything went through pretty well. I guess something else will be since these rats are eating and fasting at the same time every day and so you have to come in every day at the same time to change cages, like food. So, yeah.

Cindy St. Hilaire: Collect poop. Well, it was a beautiful, really well done study. I thought it was super interesting. We talk about what's the next podcast going to be at all of our editorial meetings and this paper, everyone thought it was a great topic. It's just really timely with the intermittent fasting. It was really wonderful. What do you think is next? What are you going to do next on this?

David Durgan: So I think there's a lot to do next. I think that one of the most interesting ideas really I alluded to earlier, and that is the widespread distribution of these bile acid receptors. So while we've taken an initial look at vascular function, I think that there's a lot to do elsewhere. We're really interested in how this could be affecting the neurological component of hypertension. Many of these bile acids signaling pathways have been shown to be anti-inflammatory. So do we see changes in neuro inflammation, in sympathetic output? One we're capable of elevating bile acids, which are capable of passing the blood-brain barrier should be noted. So that's definitely one. And then also just beginning to look at how translational this might be. So do we see changes in bile acids in hypertensive patients as well?

Cindy St. Hilaire: You're talking about the receptors and that last figure paper figure seven where you use, I forget if it was an agonist or antagonist, but you modulated that receptor activity. Do resistance arteries, which have a bigger role in hypertension, do they have higher or different levels of expression than other vascular beds in the body? Or do we not know that yet?

David Durgan: I can think of studies that have shown similar results in terms of bile acids on vascular function, both in aorta and in mesenteric arteries. But whether the distribution is different on these receptors, I'm not really sure that's known.

Cindy St. Hilaire: Well, there's lots of super interesting questions. I mean, I came up with bunches more that I wanted to know based on the study. So I'm sure that will pan out for you, hopefully with lots more great papers like this one and funding and congrats on an excellent graduate student paper. It was a real great story. And thank you both for joining me today.

David Durgan: Thank you very much.

Cindy St. Hilaire: That's it for the highlights from the April 30th and May 14th issues of Circulation Research. Thank you for listening. Please check out the Circ Res Facebook page and follow us on Twitter and on Instagram with the handle @circres and hashtag discovercircres. Thank you to our guests, Dr David Durgan and Huanan Shi. 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 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. This program is copyright of the American Heart Association, 2021. The opinions expressed by the speakers in this podcast are their own and not necessarily those of the editors or the American Heart Association. For more information, visit ahajournals.org.

This month on Episode 23 of Discover CircRes, host Cindy St. Hilaire highlights the topics covered in the April 2nd Compendium on Hypertension issue, as well as discussing two articles from the April 16 issue of Circulation Research. This episode also features an in-depth conversation with Dr Kathryn Moore from the New York University School of Medicine, discussing her study, miR-33 Silencing Reprograms the Immune Cell Landscape in Atherosclerotic Plaques.

Article highlights:

Compendium on Hypertension

Mustroph, et al. CASK Regulates Excitation-Contraction Coupling

Ward, et al. NAA15 Haploinsufficiency and CHD

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.

Cindy St. Hilaire: Today, I'm going to be highlighting the topics presented in our April 2nd Compendium on Hypertension, as well as two articles from the April 16th issue of Circ Res. I also will speak with Dr Kathryn Moore from New York University School of Medicine about her study, miR-33 Silencing Reprograms the Immune Cell Landscape in Atherosclerotic Plaques.

So the April 16th issue of Circulation Research is a compendium on hypertension. As introduced by Rhian Touyz and Ernesto Schiffrin, there are over 10,000 articles in PubMed related to hypertension. Hypertension is a major cause of morbidity and mortality worldwide, and data trends suggest that fewer and fewer patients are able to control their blood pressure medically. Further, the recent Sprint trial showed us that lowering blood pressure to levels below previously recommended values strongly correlated with significantly reduced rates of cardiovascular events and risk of death.

Cindy St. Hilaire: As such, the April 2nd issue of Circ Res provides an extensive and expansive review on the current knowledge in the field. The series starts with an article on hypertension in low and middle-income countries by Aletta Schutte and colleagues. There they present the stark differences in the trajectory, healthcare, inequality, and established and emerging risks that are specific to low and middle-income countries.

Cindy St. Hilaire: Robert Carey and colleagues present an evidence-based update in their article titled Guideline-Driven Management of Hypertension. In Pathophysiology of Hypertension, David Harrison and colleagues present the concept of the mosaic theory of hypertension originally proposed by Dr Irvine Page in the 1940s, which proposes that hypertension is the result of multiple factors that in some, raise blood pressure and induce end-organ damage. This article further refines this theory by incorporating what is known regarding the role of things like oxidative stress, inflammation, genetics, sodium homeostasis, and the microbiome in hypertension pathogenesis.

Cindy St. Hilaire: Phil Chowienczyk and Jay Humphrey and colleagues cover the contribution of Arterial Stiffness and Cardiovascular Risk in Hypertension and identify steps required for making arterial stiffness measurements a keystone in hypertension management, and cardiovascular disease prevention as a whole. In Renin Cells, The Kidney, And Hypertension, Maria Luisa Sequeira Lopez and Ariel Gomez cover the major mechanisms that control the differentiation and fate of renin cells, the chromatin events that control the memory of the renin phenotype, and the major pathways that determine the cells' plasticity.

Cindy St. Hilaire: Meena Madhur and Annet Kirabo and colleagues penned the article, Hypertension: Do Inflammation and Immunity Hold the Key to Solving this Epidemic? In this Teview, they covered the emerging concepts of how environmental, genetic, and microbial-associated mechanisms promote both innate and adaptive immune cell activation and help lead to hypertension.

Cindy St. Hilaire: In the article, The Gut Microbiome in Hypertension. Dominik N. Müller and colleagues present insights into the host-microbiome interaction and summarize the evidence of its importance in the regulation of blood pressure and provide recommendations for ongoing and future research.

Cindy St. Hilaire: Paul Cohen, James Sowers, and colleagues cover Obesity, Adipose Tissue, and Vascular Dysfunction in which they discuss the abnormal remodeling of specific adipose tissue depots during obesity and how this contributes to the development of hypertension, endothelial dysfunction, and vascular stiffness.

Cindy St. Hilaire: Clinton Webb, Satoru Eguchi, Rita Tostes, and colleagues cover Vascular Stress Signaling in Hypertension. In this Review, they discuss common adaptive signaling mechanisms against stresses, including the unfolded protein response, antioxidant response element signaling, autophagy, mitophagy, mitochondrial fission and fusion, STING-mediated responses, and activation of pattern recognized receptors. And how all of these responses contribute to vascular stress and ultimately hypertension.

Cindy St. Hilaire: Rhian Touyz and colleagues then specifically dig into the topic of Oxidative Stress and Hypertension, focusing in on recent advances in delineating the primary and secondary sources of reactive oxygen species, the posttranslational oxidative stress modification ROS induces on protein targets important for redox signaling, their interplay between ROS and endogenous antioxidant systems, and the role of inflammation activation and endoplasmic reticular stress in the development of hypertension.

Cindy St. Hilaire: Curt Sigmund and then colleagues cover the Role of the Peroxisome Proliferator Activated Receptors in Hypertension. In this Review, they discuss the tissue- and cell-specific molecular mechanisms by which PPARs in different organ systems modulate blood pressure and related phenotypes, such as endothelial cell dysfunction. Importantly, they also discuss the role of placental PPARs in preeclampsia which is a life-threatening form of hypertension that accompanies pregnancy.

Cindy St. Hilaire: Daan van Dorst, Stephen Dobbin, and colleagues provide the Review, Hypertension and Prohypertensive Antineoplastic Therapies in Cancer Patients. Many cancer therapies have prohypertensive effects. And this Review covers some of the mechanisms by which these antineoplastic agents lead to hypertension and details the current gaps in knowledge that future clinical studies must investigate, to identify the exact pathophysiology and the optimal management of hypertension associated with anticancer therapy.

Cindy St. Hilaire: In Hypertension, a Moving Target in COVID-19, Massimo Volpe, Reinhold Kreutz, and Carmine Savoia, review available data on the role of hypertension and its management in COVID-19.

Cindy St. Hilaire: Melvin Lobo and colleagues review Device Therapy of Hypertension. In this Review, they discussed the newer technologies, which are predominantly aimed at neuromodulation of peripheral nervous system targets, and discuss the preclinical data that underpin their rationale and the human evidence that supports their use.

Cindy St. Hilaire: Last but not least, in Artificial Intelligence in Hypertension: Seeing Through a Glass Darkly, Anna Dominiczak and colleagues cover a clinician-centric perspective on artificial intelligence and machine learning as applied to medicine and hypertension. In this Review, they focus on the main roadblocks impeding implementation of this technology in clinical care and describe efforts driving potential solutions.

Cindy St. Hilaire: This is an expansive set of Reviews written by the leading experts in the field and provides an up-to-date assessment of all aspects of hypertension. The graphics, and the articles are absolutely beautiful. And I'm sure we will be seeing a lot of them in upcoming presentations. Hopefully at AHA and the other sub-meetings when we're all back in person.

Cindy St. Hilaire: In the April 16th issue, I want to highlight the article, Loss of CASK Accelerates Heart Failure Development. The first author is Julian Mustroph, and the corresponding authors are Lars Maier and Stefan Wagner from the University Medical Center in Regensburg, Germany. Despite advances in cardiovascular medicine, heart failure takes the lives of tens of thousands of Americans each year. To develop novel treatments, a better understanding of the conditions of molecular pathology is needed. One contributing factor in heart failure is increased activity of the Ca/calmodulin-dependent kinase II (CaMKII).

Cindy St. Hilaire: In this paper, the authors suggest a way to get CaMKII levels under control. Ca/CaM-dependent serine protein kinase or CASK, suppresses CaMKII neurons and the team showed that CASK is also expressed in human heart cells, where it associates with CaMKII. Next, they engineered mice to CASK specifically in cardiomyocytes, finding that when these animals are subjected to beta-adrenergic stimulation, cardiomyocyte like CaMKII activity was significantly greater than that seen in control animals. Calcium spark frequency and the propensity for arrhythmia were also increased. Furthermore, in a mouse model of heart failure, mice lacking CASK fared worse and had reduced survival compared to the wild type control animals while boosting CASK expression in wild type animals reduced the elevated CaMKII activity and calcium sparks associated with heart failure. The author suggests that increasing CASK activity might be a heart failure treatment strategy worthy of further study.

Cindy St. Hilaire: The last article I want to share from the April 16th issue is titled, Mechanisms of Congenital Heart Disease Caused by NAA15 Haploinsufficiency. The first author is Tarsha Ward, and the co-senior authors are Kris Gevaert, Christine Seidman, and JG Seidman from Harvard University in Boston, Massachusetts. A number of genetic variants are associated with congenital heart disease, including loss of function variants of the gene encoding NAA15, a sub N-terminal acetyltransferase complex called NatA, which acetylates a large portion of newly forming proteins. To find out how these variants contribute to defective heart development, the authors performed genome editing on human pluripotent stem cells to convert one or both copies of NAA15 gene into congenital heart disease linked to variants. The team then examined cardiomyocyte differentiation, protein acetylation, and protein expression in the edited and unedited cells.

Cindy St. Hilaire: They found that while NAA15 haploinsufficiency cells were able to develop into cardiomyocytes seemingly normally, the cell's contractile ability was significantly impaired. Cells homozygous for NAA15 variants failed to differentiate and had poor viability. The team also found that while only a small number of proteins had reduced end terminal acetylation in NAA15 haploinsufficiency cells, over 500 proteins had altered expression levels, four of which were encoded by congenital heart disease-linked genes. This work provides the first insights into the effects of NAA15 variants in human cells and sets the stage for analyzing other congenital heart disease-linked variants in this manner.

Cindy St. Hilaire: Today, Dr Kathryn Moore from NYU School of Medicine is with me to discuss her study, miR-33 Silencing Reprograms the Immune Cell Landscape in Atherosclerotic Plaques, which is in our April 16th issue of circulation research. So thank you so much for joining me today, Kathryn.

Kathryn Moore: My pleasure.

Cindy St. Hilaire: Atherosclerosis is the result of lipid-induced chronic inflammation, and while lipids are kind of thought to be an initial driver, therapies that target lipids alone, such as statins, they're not sufficient. They can obviously bring things down and improve things a lot, but a lot of research now is focused on uncovering the nuances of the inflammatory component of atherosclerosis to help identify new targets for therapies. One specific arm of this research has focused on resolving atherosclerotic inflammation. And my first question to you is, what exactly does resolving inflammation mean in the context of an atherosclerotic plaque? And maybe could you give us a little primer on some of those key cell types or processes involved in that.

Kathryn Moore: I'm really fascinated by the resolution of inflammation and in particular, in the atherosclerotic plaques. So inflammation used to be thought of as an active process, almost a one-way process, which in order to resolve had to stop. But actually, the pro-inflammatory and anti-inflammatory responses are a continuum. And so inflammation resolution, we now recognize is an active process, and it's not just a matter stopping the influx of immune cells but these cells take on new phenotypes and different functions. And the immune cells themselves are required for resolution of inflammation and tissue repair. And so we're really interested in looking at what those pathways are, that tip the balance between pro-inflammatory responses and pro-resolving responses and how to incite them in the plaque so that you can start to remodel the plaque to be more stable or have a more favorable phenotype, or even to regress the plaque, to shrink the plaque in size.

Cindy St. Hilaire This study specifically focused on microRNA-33, and I believe your lab was one of the very first to look at this specific, but also other micro RNAs in atherosclerosis. And the prior research that you and others have shown is that this microRNA modulates a variety of genes that control lipid metabolism. You found this in mice, but also in monkeys. And really by using anti-miRs against this microRNA, you can induce cholesterol efflux and that cholesterol will leave the liver and the macrophage cells, and it's incorporated into the protective HDL particles and excreted.

Cindy St. Hilaire: And so it has this really nice protective effect. However, the effects seen in these animal studies were suggested that microRNA-33 had HDL independent action, which I think is where your story starts. So could you tell us some of the premises or the gaps in knowledge between those first initial findings of miR-33 that led you to conduct this study and then kind of what the design of the study was?

Kathryn Moore: So, as you mentioned, we discovered miR-33 as an inhibitor of cholesterol efflux and the pathways that lead to the generation of HDL, the so-called good cholesterol. And when you inhibit miR-33 in mice and monkeys, you can raise plasma levels of HDL. But we also saw that in mice that had been fed a Western diet continuously, we saw favorable changes in the atherosclerotic plaque under conditions where we didn't see the increase in HDL. So if the mice are on a Western diet, the levels of miR-33 in the liver are very low, and inhibiting it doesn't cause the increase in HDL cholesterol. But we still saw this 25% regression in atherosclerotic plaques. And that got us thinking about the other things that miR-33 could be doing and around the same time, I was also very interested in immunometabolism and how the metabolic state of macrophages influences their function.

Kathryn Moore: And Mihail Memet, who is a former postdoc in my lab made the discovery that miR-33 could inhibit fatty acid oxidation in macrophages and that this polarized the cells to a more inflammatory phenotype. So when we give the miR-33 inhibitors, we're raising a level of fatty acid oxidation in the macrophages and they become more tissue reparative. And so we suspected that could be the mechanism going on in the plaque but those studies, those initial studies were done over five years ago. And that was before the advent of single-cell technologies, which have really revolutionized how we're studying the atherosclerotic plaque. So in this study, we were able to apply some of these more high dimensional analyses of all of the immune cells in the plaque. And really look at how inhibiting miR-33 was altering their transcriptome and their phenotype.

Cindy St. Hilaire: Yeah, so that is a perfect segue to my next question, which is you're doing this single-cell RNA-sequencing on tissue, but it's not just any tissue. It's not like a nice spleen that you can kind of pop open and all the cells fall out nicely and you can fax them or whatever. This is from an aorta, which itself is fibrous and tough on top of the atherosclerotic plaque, which is also difficult. So can you discuss maybe some of the challenges regarding doing this exact kind of analysis with this tissue and maybe some of the limitations or controls that you used to help really refine your result?

Kathryn Moore: It is a little bit challenging to learn how to digest the aorta to release the immune cells, so to isolate the CD45+ immune cellsthat then go on to the sequence that takes some trial and error to get the right conditions. But actually, once you've done that a couple of times, it's not as difficult as it seems but I think that one of the challenges of doing these types of studies is integrating the results that we get from the single-cell RNA-sequencing with the other technologies that we've used in the past to analyze atherosclerosis.

Kathryn Moore: So, previously when we were analyzing atherosclerotic plaque size or immune cell content, we are doing this through histology and immunostaining. And single-cell RNA-sequencing has identified all these new immune cell subsets based on transcriptomic signatures. And they don't really match up nicely with the protein signatures that we've used in the past.

Cindy St. Hilaire: Yeah.

Kathryn Moore: I saw this as a great opportunity to try to integrate all these techniques. And see if we could come to some middle ground. To understand how maybe the new subsets that we're identifying with single-cell RNA-seq from the aortic immune cells matched some of the things that we were able to do by looking at histology and tracing monocytes and macrophage entry and retention in the plaque.

Cindy St. Hilaire: How did it line up? What's the nice Venn diagram of this study and what we've all been doing previously?

Kathryn Moore: Well, it's a challenge, but what I thought was really really fascinating was we did monocyte-macrophage tracing experiments. Because one of the things we find when we inhibit miR-33 is we have a 50% decrease in the macrophage content of the plaque, but how is that happening? And what we found was there was an increase in the recruitment of monocytes into the plaque which may sound surprising if the plaque is shrinking, but they are the cells that are needed. They're the cleanup crew that are being introduced. But we saw a decrease in retention of macrophages and a decrease in proliferation and an increase in macrophage death and clearance of the apoptosis cells. And then through the single-cell RNA-sequencing, we were able to look at the different macrophage subsets. We had resident macrophages, Trem2hi metabolic macrophages, and MHCIIhi inflammatory macrophages.

Kathryn Moore: We were able to look at their transcriptomes and say, "Which of these subsets are most likely to be performing those functions that we saw before?" And that was fun because that was like piecing together a puzzle. And what we saw, what it leads us to believe is that the Trem2hi metabolic macrophages are the ones that are undergoing aptosis. They have an increase in aptosis genes and eat-me signals and the MHCIIhi, having an increase in athoscoertic genes like mirTK that will help them clear the dying cells and the MHCIIhi macrophages also have decreased markers of proliferation. So although we used to think about macrophages as this one big pool, now we're able to say that these different subsets are performing different functions. And to me that's really exciting.

Cindy St. Hilaire: Oh, that is exciting. And it's also extremely complicated because I was having enough trouble with just the two types of macrophages of a couple of years ago. The study showed that inhibiting this miR-33 using these anti-miR-33 oligos, and you're just kind of injecting oligos against it. And you're doing this in mice with established atherosclerosis. This helped to alter these monocyte and macrophage populations in the plaque itself.

Cindy St. Hilaire: Do you think a function of the success of this study and essentially this therapy in the mouse is really dependent on the fact that it's targeting these circulating cells that are then going to the plaque? And I guess part of that question is, do you think part of this is because it's a circulating cell that can take it up, and then change and be delivered to the location it's going to, as opposed to that oligo targeting the plaque itself and the cells that are already residing there. Do you have any sense of that?

Kathryn Moore: So it's interesting because one of the things that we did with our single-cell RNA-seq was to look at all immune cells in the plaque and say, "How many miR-33 target genes are changing in the ones from the treated mice?" And in the monocytes, you see very little change in miR-33 target genes. And that's consistent with what we know from Regulus Therapeutics who designed the anti-miR-33 antisense oligonucleotides. So we don't think that the ASO are being taken up in the circulation. I think they're actually being taken up by the macrophages in the plaque. And one of the great things about trying to target macrophages is they're very phagocytic. So they're going to be the ones that take up these ASOs, and the single-cell really allowed us to see whether it was just macrophages that were being affected or whether there were other immune cell populations that also seemed to have miR-33 induce changes. And of course it's hard from the single-cell to infer whether this is direct or indirect.

Cindy St. Hilaire: Yeah.

Kathryn Moore: But it seemed as if T-cells also were targeted by the anti-miR-33, definitely macrophages. We saw some changes in dendritic cells, very little changes in K cells, for example. And no changes in monocytes. And so it also begins to tell us how many different cell types are being affected and who's driving the bus when it comes to these changes. But by far the most miR-33 target genes change were the macrophage populations. And I think that's really due to their phagocytic ability.

Cindy St. Hilaire: So I know there's a great divergence generally in microRNAs between mice and humans or really any species, but there are homologs to this in humans. What is the same and what is different between, I guess, this particular targeting micro RNA or what we know about it in mice and humans?

Kathryn Moore: So mice have only one copy of miR-33, whereas humans and monkeys have two copies but those two copies are very similar in sequence. They differ only by two nucleotides. So you can use the same antisense oligonucleotides to target in mice and in non-human primates, for example. It's never been tried in humans.

Cindy St. Hilaire: Yeah, of course. Not yet.

Kathryn Moore: But it has been tried in monkeys, and we were able to effectively inhibit both miR-33a and miR-33b in the non-human primates. But the different variants of miR-33 have different transcriptional regulation. So they're induced under different conditions. And I think that's one way that mice and humans will really differ-the conditions where you'd have high levels of miR-33 will be different.

Cindy St. Hilaire: Got it. Yeah. And the mice has that in the SREBP gene and humans.

Kathryn Moore: And miR-33a is an SREBP-2 gene, which is SREBF2. And in humans there's an additional copy, which is SREBF. So it's in both of the SREBP genes in humans.

Cindy St. Hilaire: Interesting. So I wonder, we need to ask the evolutionary biologist. Did they segregate together? I mean, I guess they must have. That's really interesting. That's cool.

Kathryn Moore: One of the things that I love about miR-33 is that the SREBP-2 gene is turned on when cholesterol levels are low and it acts to increase the pathways involved in cholesterol synthesis and uptake. And miR-33 is transcribed at the same time. And what it does is it blocks the exits for cholesterol from the cell and from the body. And so it's just this hidden gem in the locus that sort of boosts SREBP-2 function.

Cindy St. Hilaire: Its amazing stuff works out like that. I love it. So if we were going to leverage this inflammation resolution as atherosclerotic therapy, wherein the continuum of the disease, should we target? You know, we have obviously atherosclerotic plaque does not happen overnight. Teenagers can even have evidence of a fatty streak. If we were going to leverage antisense oligos as therapy, especially specifically against miR-33, where do you think would be a good place to target? And do we know, or have the kind of imaging capabilities to maybe identify that window right now in patients?

Kathryn Moore: That's an interesting question. So lipid-lowering therapies will remain the first line of treatment for atherosclerosis, but lipid-lowering alone is insufficient to regress the plaque. It can stabilize plaques, but it doesn't really cause them to shrink. And when you think about the patient population that presents with cardiovascular disease, it's adults, for the most part. These are people in their fifties and sixties, and we've missed the chance to stop the early events. And so those are the majority of the people that are being treated. And I think there is room there to treat inflammation at the same time in the hopes of tipping that balance between pro-inflammatory events and then inflammation resolution. So we know surprisingly little about that tipping point. And now I think when miR-33 inhibition is fascinating in that it can affect both lipid metabolism and inflammation. And so I think that as an add-on therapy with lipid-lowering, it would be interesting, but of course, I'm not ready.

Cindy St. Hilaire: We're not there yet.

Cindy St. Hilaire: So I guess what's next for this line of research? What are kind of the next questions that the single-cell RNA-seq discovered for you? Was there anything kind of surprising or really exciting that you want to pursue next?

Kathryn Moore: One of the things that I thought was really interesting was that the different macrophage subpopulations had different miR-33 target genes being de repressed. And that's probably not surprising, but I didn't initially think that would happen, but of course, the subpopulations are identified based on their unique transcriptomes. So they're not all the same, which means that they'll have different levels of miR-33, and they'll have different levels of the miR-33 target genes. And so Abca1, which we think about all the time as a miR-33 target gene that's involved in cholesterol efflux, it went up in Trem2hi macrophages and the resident macrophage population, but not in the MHCIIhi. The target genes and the MHCIIhi were different than the other two populations. And I think this now gives us a chance to sort that out.

Kathryn Moore: And some of the targets in the MHCIIhi macrophages were ones that are involved in chromatin reorganization-

Cindy St. Hilaire: Oh, interesting.

Kathryn Moore: ... and inscriptional regulation. And when I looked across the other subsets, I could see that common pattern in T-cells and B-cells that were changing. And I think that's one way that miR-33 could have a broad impact. MiR-33 is a little bit of a unique microRNA. It has a very potent impact on these pathways. Other microRNAs often can change gene expression by 10 to 20%, but miR-33, when we inhibit it, we see really powerful effects. And I think that if it is involved in targeting genes that mediate chromogenic reorganization or transcriptional complex formation, that gives us a hint of how it could be having additional impact.

Cindy St. Hilaire: That's really cool. And this was an absolutely beautiful story, not only in kind of dissecting out the mechanisms at play, but you know, those beautiful tisney plots and the nice graphics of the single-cell stuff.

Kathryn Moore: The first author of the paper, Milessa Afonso, is a postdoc that just left the lab, and she worked so hard on this and did such a beautiful job.

Cindy St. Hilaire: Well, it's a wonderful story and I'm really happy we were able to publish it. So, Dr Moore, thank you so much for joining me today.

Kathryn Moore: My pleasure. Thank you.

Cindy St. Hilaire: That's it for the highlights from the April 2nd and 16th issues of Circulation Research. Thank you so much for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @circres and hashtag DiscoverCircRes. Thank you to our guest, Dr Kathryn Moore. This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the Editorial Team of Circulation Research. Copy text for the highlighted articles was 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.

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.

This month on Episode 21 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the February 2 and February 19 issues of Circulation Research. This episode also features an in-depth conversation with Konstantinos Drosatos and Ioannis Kyriazis from Temple University to discuss their study, KLF5 is Induced by FOXO1 and Causes Oxidative Stress and Diabetic Cardiomyopathy.

Article highlights:

Wittenbecher, et al. Lipidomics and Heart Failure Risk

Kryshtal, et al. Flecainide Directly Inhibits RYR2 Ca Release

Chen, et al. Klotho and Heart Aging

Grootaert, et al. SIRT6 Deacetylase Protects Against VSMC Senescence

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, and today I'll be highlighting four articles from the February 5th and 19th issues of CircRes. After the highlights, Dr Konstantinos Drosatos and Ioannis Kyriazis from Temple University will join me to discuss their study, KLF5 is Induced by FOXO1 and Causes Oxidative Stress and Diabetic Cardiomyopathy.

Dr Cindy St. Hilaire: The first article I want to share is Lipid Profiles and Heart Failure Risk: Results from Two Prospective Studies. The first author is Clemens Wittenbecher, and the corresponding author is Frank Hu from Harvard's Chan School of Public Health in Boston, Mass. Heart failure affects tens of millions of people worldwide, and as the prevalence grows, prevention strategies are becoming ever more important. While factors including age, obesity, and hypertension influence one's risk of developing heart failure, robust biomarkers that are able to pinpoint which individuals will develop heart failure are lacking. Changes in cardiac lipid metabolism predispose animal models of heart failure.

Dr Cindy St. Hilaire: This group hypothesized that blood lipid profiles might be useful to serve as a heart failure biomarker. The team examined 216 blood lipids from a cohort of individuals with various cardiovascular risk factors, but who, at the time of enrollment and blood collection, did not have heart failure. Over the observation period, which averaged out to over 12 years, 331 of the subjects developed heart failure.

Dr Cindy St. Hilaire: When compared to the baseline lipid profiles of individuals who didn't develop heart failure, the group identified two particular lipids, ceramide and phosphatidylcholine, and several networks of lipids and metabolites that were strongly predictive of developing heart failure. Importantly, the findings were corroborated in the second cohort, in which 87 individuals developed heart failure. Together, the results reveal early biomarkers for identifying at-risk individuals and point to particular lipid alterations that may yield insights into heart failure pathology and prevention.

Dr Cindy St. Hilaire: The second article I want to share is titled, RyR2 Channel Inhibition Is a Principal Mechanism of Flecainide Action in Catecholaminergic Polymorphic Ventricular Tachycardia. The first authors are Dmytro Kryshtal and Daniel Blackwell, and the corresponding author is Bjorn Knollmann, from Vanderbilt University School of Medicine in Nashville, Tennessee. Flecainide is a drug that is commonly used to treat various heart arrhythmias. Flecainide works by blocking sodium channel activity. However, the drug also has been found to reduce symptoms of catecholaminergic polymorphic ventricular tachycardia, or CPVT, a condition in which mutations affecting the function of a calcium channel ryanodine receptor, called RyR2, are to blame. In vitro studies have suggested that flecainide can in fact block RyR2 activity, but some researchers have argued that flecainide's inhibition of this receptor is too weak to be clinically relevant, and suggest its sodium channel inhibition instead provides an indirect benefit.

Dr Cindy St. Hilaire: To test that claim, this group synthesized analogs of flecainide that lack RyR2 inhibitory activity, yet retained sodium channel blocking ability. They compared the analogs with the original drug, both in vitro and in vivo. Experiments in cardiomyocytes confirmed flecainide, but not the analogs, could reduce RyR2-mediated calcium release and experiments in catecholaminergic polymorphic ventricular tachycardia model mice showed flecainide, but not the analogs, could suppress induced ventral tachyarrhythmias. These findings suggest that RyR2 inhibition is the principal mechanism of action of flecainide in treating catecholaminergic polymorphic ventricular tachycardia, and therefore, RyR2 may be a valid therapeutic target for the development of additional antiarrhythmia drugs.

Dr Cindy St. Hilaire: The third article I would like to share is titled, Klotho Deficiency Causes Heart Aging via Impairing the Nrf2-GR Pathway. The first author is Kai Chen, and the corresponding author is Zhongjie Sun, and they're from the University of Tennessee Health Science Center in Memphis, Tennessee. Age is a risk factor for many disease states, including heart failure. Even in healthy individuals, the heart size increases and its function declines with age. Aging in humans has also been associated with a decrease in circulating levels of the protein Klotho, which is thought to have anti-aging properties. Previous studies have shown, in a murine model of cardiac hypertrophy, that mice that lack Klotho fare worse than those with normal levels of the protein.

Dr Cindy St. Hilaire: This group, therefore hypothesized that Klotho decline may contribute to age-related heart changes. Similar to humans, heart function declines with age in otherwise healthy mice. Injection of Klotho into old mice reduced the size of the animal's hearts and improved cardiac function. Klotho injections also improve heart size and function in young Klotho-lacking mice with pharmacologically induced cardiac hypertrophy. The team found that Klotho induces these effects by inhibiting the accumulation of damaging reactive oxygen species, and by reducing apoptosis in aged-Klotho deficient heart cells. From these data, they suggest that perhaps boosting Klotho levels may be a strategy to prevent age-related heart failure.

Dr Cindy St. Hilaire: The last article I want to share before our interview is titled, SIRT6 Protects Smooth Muscle Cells from Senescence and Reduces Atherosclerosis. The first author is Mandy Grootaert, and the corresponding author is Martin Bennett from the University of Cambridge in Cambridge, United Kingdom. Vascular smooth muscle cells reside in the medial layer of vessels. They contribute to atherosclerotic plaque progression, as well as to the fibrous cap that helps to stave off plaque rupture. Over time, however, the increased proliferation and differentiation of plaque smooth muscle cells causes them to accumulate DNA damage, senesce, and ultimately die, leading to the destabilization of the plaque.

Dr Cindy St. Hilaire: Functional disruption of the enzyme SIRT6 has been implicated in DNA damage senescence and apoptosis, and certain polymorphisms of the SIRT6 encoding gene are linked to atherosclerosis. From these premises, the team wanted to examine the role of SIRT6 in plaque smooth muscle cells. Compared with healthy aortas, aortas from atherosclerotic mice and humans have lower levels of SIRT6 protein. Inhibiting the activity of SIRT6 and smooth muscle cells caused damage to the telomeres and induced early senescence. By contrast, overexpression of SIRT6 preserved telomeres and prevented senescence. ApoE knockout mice were then engineered to over express SIRT6, specifically in their smooth muscle cells, and these mice showed reduced severity of atherosclerosis compared to control mice. Together, these findings implicate SIRT6 suppression as a cause of plaque senescence, and suggest reversing it may in fact slow disease progression.

Dr Cindy St. Hilaire: Okay, so today we have Dr Konstantinos Drosatos and his postdoctoral fellow, Dr Ioannis Kyriazis from Temple University in Philadelphia, Pennsylvania, and they're here to discuss their study, KLF5 Is Induced by FOXO1 and Causes Oxidative Stress and Diabetic Cardiomyopathy. And this is in our February 5th issue of Circulation Research. So, thank you both so much for being with me today.

Dr K. Drosatos: Thank you for the invitation and for helping to draw attention to our study.

Dr Cindy St. Hilaire: Absolutely. And thank you for doing this at what? What is it, eight o'clock where you are?

Dr Ioannis Kyriazis: It is eight o'clock at night.

Dr Cindy St. Hilaire: Okay. Well, thank you for taking the-

Dr Ioannis Kyriazis: But it's okay, it's okay. It's quite early to be in Greece.

Dr Cindy St. Hilaire: Okay, good.

Dr K. Drosatos: Maybe we need to clarify that Ioannis is a former postdoc. I don't have a lab at Temple and in Greece.

Dr Cindy St. Hilaire: Former postdoc. Thank you for clarifying that. So I want to start with a question about cardiomyopathy. What is it and how prevalent is it? And what's the different pathogenesis of cardiomyopathy? And how does it differ from diabetic cardiomyopathy?

Dr K. Drosatos: So usually cardiomyopathy arises after heart infracts, after myocardial ischemia, and it actually reflects the reduced ability of the heart to pump blood to the rest of the body, in simple words. Diabetic cardiomyopathy has some unique features. One of those is that it's not related to coronary artery disease, so it does not start with ischemia, but it's still the heart cannot do what it is supposed to.

Dr Cindy St. Hilaire: So it's kind of its own unique driver then, the diabetic cardiomyopathy?

Dr K. Drosatos: Yeah. And there is a lot of, I wouldn't say debate, but there's a lot of discussion in the field about how to best define diabetic cardiomyopathy. It's a different kind of cardiac dysfunction. It has some certain features like oxidative stress, which is the stuff that we work with. It has fibrosis, primarily perivascular fibrosis. Diastolic dysfunction is more prevalent than systolic dysfunction. So there's a number of features that actually define diabetic cardiomyopathy.

Dr Cindy St. Hilaire: I know it's highly prevalent in patients with diabetes, but the inhibitors that people are using to try and treat the diabetic portion, I'm thinking about the sodium glucose transporter SGLT2 inhibitors, those are obviously very good at helping regulate the blood glucose, but they don't appear to alleviate the heart failure. And so what do you think about the pathogenesis or the pathophysiology between the glucose regulation and the cardiomyopathy? Is it kind of like a cliff and it gets too far and is it unrepairable?

Dr K. Drosatos: It's certainly a very trendy question. I mean, you are a scientist so you know that in science will have several trends. So SGLT2 inhibition is one of those right now. And from time to time, there are several, I would say miraculous drugs that do a number of good things, which we're not very certain about the mechanism that underlies the effect. So the SGLT2 inhibitors, which is something that we had also started in a previous paper in Circulation Research four years ago, in relation to KLF5, what it actually does, it targets a transporter in the kidney and this transporter normally returns glucose back to the bloodstream. But when it is inhibited, the extra glucose that we observe in diabetes goes out through the urine. So this is what the drug does, but it has been shown that the drug has its own effects in cardiac function, which do not necessarily pertain to the effect that the drug has in the kidney. And actually I was reading yesterday very interesting paper about using SGLT2 inhibitors in heart failure patients that do not have diabetes.

Dr Cindy St. Hilaire: Oh, interesting. So it might actually have a secondary function that we're just not aware of right now.

Dr K. Drosatos: We do believe that at least in part the beneficial effects has to do with the removal of the extra glucose from the system. My training has been in labs that work on lipid metabolism, so I believe that fatty acid oxidation is the best thing that can happen to the heart. So removing the glucose out of the system is definitely beneficial and actually, Ioannis, before he returned back to Greece, he had performed some experiments showing that removing glucose is definitely beneficial.

Dr Cindy St. Hilaire: That's great. Can you describe the study? What was the former research that the question you were asking in this study was on?

Dr K. Drosatos: That was a study that I started in the lab of Ira Goldberg at Columbia University when I was a postdoc, and we had come up with an interesting observation that in the heart, the protein interception factor, named KLF5, follows an oscillating pattern of expression. So at the early stage of the abyss, it goes down and then it goes up. So we believe that the levels of glucose in the plasma may be one of the defining factors for affecting the expression of KLF5. So this is how it started, why KLF5 goes down and then up. And at that time we observed that KLF5 is an important transcriptional regulator of cardiac PPAR-alpha, a protein that's another transcriptional factor, which seems to be a very important factor that orchestrates gene expression for fatty acid oxidation. So there is more than 20 genes that are important for fatty acid oxidation, and the expression of which has been shown to be affected by PPAR-alpha.

Dr K. Drosatos: So we started working with KLF5 and PPAR-alpha, and that was the paper we published in Circulation Research in 2016, and then Ioannis joined my lab and he works on the effect of KLF5 per se in diabetic cardiomyopathy. And one of the interesting findings from the new study is that the KLF5 has a separate effect on diabetic cardiomyopathy that does not involve PPAR-alpha.

Dr Ioannis Kyriazis: And the whole idea, when I joined Dr Drosatos' lab, the initial idea was that something is happening initially in the heart and that's why we see KLF5 goes down and we believe initially that has to do with subject utilization. And that's why KLF5 is coming down and then comes up. But after several studies, we figured out that it's actually a big tie in the transcriptional factors that act synergistically and like FOXO1, KLF5, and PPAR-alpha, and KLF5 and PPAR-alphas have distinct roles on regulating diabetic cardiomyopathy. So starting from one point of view, we transferred to a different aspect and we tried to see how KLF5 is involved in that system. And this is two stories in one, actually. And that's why we have this follow up study about glucose, that Dr Drosatos said, before I leave. We try also to make it bigger.

Dr Cindy St. Hilaire: Yeah, it's always complex, but I feel like this story got very complex as it's really interesting. You used a large amount of different mouse models. Can you talk about some of the different mouse models you used and why you had to use them? You had different drivers of CRE, but also over expression, knockout models. Can you maybe give a quick summary of all the different models you use to really test your hypothesis really thoroughly?

Dr K. Drosatos: Are you asking Ioannis why I am forcing him to do a lot of experiments?

Dr Ioannis Kyriazis: No, no.

Dr Cindy St. Hilaire: But they're really well done, so.

Dr Ioannis Kyriazis: I think the answer has to do with how the research community is able to tackle biological questions. You need to use knockout animals and conditional transgenic animals in order to answer biological questions that you are asking. So because we have in front of us a triangle of transcriptional factors that regulate the diabetic myopathy, we were obliged to use all these mouse models to answer all these questions. And we have to understand what is the driving force behind all these systems? Is it FOXO1? Is it KLF5? Is it PPAR-alpha? Do these all add together? So that's why we had two knockout-specific mouse lines for FOXO1 and KLF5. We have the global PPAR-alpha knockout mice, we have transgenic KLF5 specifically in the cardiomyocyte mouse line, and we also have gene therapy.

Dr Ioannis Kyriazis: We construct an AAV that drives KLF5 expressions, specifically in the cardiomyocytes, under the Cardiac Troponin T Promoter. So all these actually helped us combination of therapies to tackle all these biological questions that we wanted to have and to answer.

Dr K. Drosatos: So this is how it's done. So Ioannis started from this point. We were hopeful that there was a flux FOXO1 mouse. So he started working with that, and then we started making more questions. Okay, after we figured out that, yes, FOXO1 regulates KLF5, so the question then was, okay, is it an effective KLF5 through PPAR-alpha? This is where the next mouse model came. When we said, no, it's not PPAR-alpha, we said, okay, what it is then?

Dr Cindy St. Hilaire: What is it?

Dr K. Drosatos: And started thinking about different mechanisms that activate diabetic cardiomyopathy. We started with oxidative stress. I was not very ecstatic about this possibility because antioxidant therapies in diabetic patients did not really improve survival. And actually, we were right not to be so excited about this possibility because we only saw a partial improvement. So then we said, okay, what else? And this is where we started doing high throughput analysis, where we ran out of possible answers to questions. So this is when we did look at dogs and we came up with the observation that ceramides are effective.

Dr Ioannis Kyriazis: And one more thing to add is that, as Dr Drosatos said, that this study, I think, it's one of a lot of studies out there. But I think this is how we, I believe, as an early scientist, the science to know the biological systems, especially in mice. We use the transgenic models and the knockout models and we see in our study that black and white is not good. So in Kosta's, in Dr Drosatos's study in 2016, Circulation Research, he showed that KLF5 knockouts, specifically in the cardiomyocytes, actually is not good for a long time. Initially, we believed that the transgenic KLF5 mouse model will do better in diabetes.

Dr Ioannis Kyriazis: And when we saw that there actually has an accelerated cardiac dysfunction, we were like, okay, this is an interesting phenotype. We need to see how this goes, because we believe in our initial hypothesis that if we induced KLF5 in the early diabetes, then we will have something like we alleviate diabetes. But this was not the case. And I believe that the fine tuning of some proteins will be the future. It's not black, it's not white. If we knock out completely KLF5, it's not good. If we over express KLF5, it's not good. We need some physiological levels.

Dr Cindy St. Hilaire: Yeah. You need to be able to tighter it a bit and tighten it up here or loosen it up there and, yeah. Well, this is a great study to really highlight the intricate dynamics of it all. One of the interesting results, it was just one of the shorter sections, but when KLF5 was increased, you also saw a decrease in mitochondrial DNA integrity in the cardiomyocytes, which I thought was a really interesting finding. It was just a little portion of the paper, but I just thought that was really interesting, and I was wondering if you could expand on it. What does that mean? And do we know what KLF is doing to the mitochondria?

Dr K. Drosatos: We believe that this is an effect of the oxidative stress and the increase of ceramides. It's a secondary effect. But this is something we would like to pursue further because when we did... And Ioannis nicely mentioned about that. In the 2016 paper, in Circ Research, we actually saw that prolonged exhibition of KLF5 results in diabetic cardiomyopathy. So we don't want to inhibit it completely. And in that case, mitochondria number also goes down.

Dr Cindy St. Hilaire: And then, once it's down, it doesn't go back up?

Dr K. Drosatos: We believe that in the most recent papers case, it is the oxidative stress that actually targets mitochondria.

Dr Cindy St. Hilaire: I think, if I got it correctly, the time course of these events happening in the mice is about a 12 week time span you're doing your treatments for?

Dr K. Drosatos: That's correct.

Dr Cindy St. Hilaire: So obviously that's much more accelerated than humans. What do you think about these dynamics on a human scale in terms of KLF being up and then being down? Do we know how this mechanism would translate to humans or is that still kind of a black box?

Dr K. Drosatos: I think that it's a black box. If I know correctly, we still don't know how fast type one diabetes is occurred in humans. And also the majority of people that have diabetes, diabetic cardiomyopathy is not everything. So maybe their cardiac function is bad, and KLF5 is induced, but these patients do not know that they have actually diabetic cardiomyopathy. And the majority, most probably, of the samples of the research community might have is like a endpoint type 1 diabetic patients. And with the help of Professor Kyriazis, it gave us human samples that we have in our study, we saw that KLF5 is increased in isolated cardiomyocytes. So in terms of how KLF5 is induced in human samples, I think it is high, but we don't know if this 12-week timeframe that we put in the mice to have lack of an overt cardiac dysfunction is actually mimicking completely what is happening to humans.

Dr Cindy St. Hilaire: So what do you think about leveraging your findings for the development of potential therapies? What would you want to target first, or how do you think this could potentially move to the clinic setting?

Dr K. Drosatos: So regarding the previous question, first, I think it's important that in our case we observe in both type 1 and type 2 diabetes mice, that KLF5 is going now. And the result of correcting cardiac function, when we see a bit of KLF5, either genetically and specifically cardiomyocytes or pharmacologically, identifies KLF5 as a potential barrier. This is how I see.

Dr K. Drosatos: You know that in the drug discovery world, transcriptional factors are not very popular therapeutic therapies. So right now the lab is investing on identifying what do they regulate? So we are pursuing a proteomic analysis, we are pursuing sequencing analysis to see what may be happening one step earlier. This is how we envision in potential therapeutic approaches in the future. So this is how we see.

Dr K. Drosatos: For me, it's not really a black box. There is a lot of information in the last 20 years on diabetic cardiomyopathy, and you mentioned earlier the SGLT2 inhibition and we don't know how this works, but we have some ideas. I believe we are getting there. And our hope is that the piece of our work we were able to identify any important, novel points of the mechanisms, because it was actually miraculous. I mean, the experiment that excited more than any other experiment in Ioannis's paper was when he started the treatment with the KLF5 inhibitor after diabetic and after cardiac dysfunction had occurred. And the cardiac function became back normal.

Dr Cindy St. Hilaire: Do you think this KLF5 mechanism is operative in kind of traditional cardiomyopathy, kind of non-diabetic cardiomyopathy?

Dr K. Drosatos: We just published a paper in Circulation. This was the work of Matthew Hoffman and Ioannis was also a co-author in that paper. Matthew and Ioannis were working together in the lab. So Matthew showed that KLF5 is increasing ischemic cardiomyopathy as well. And this was shown both in human samples and mouse experiments. And when KLF5 was inhibited, dilated cardiomyopathy was actually the first. KLF5 was such an underappreciated transcriptional factor and when I was doing my postdoc and started working with that, I always say that I took the risk to generate the cardiomyopathy-specific mouse models because by that time there was only one study showing that it was only fibroblast KLF5 that actually protects from a pressure overload cardiomyopathy. Where they knocked out KLF5 and cardiomyocytes they did not see any protection in pressure overload-driven hypertrophy. So they said, because KLF5 has low RNA copy number, probably it's not important. And I still remember when I first presented this data to KLF meeting, and they will all say, "Yeah, but the expression is very low," but we had the results, so.

Dr Cindy St. Hilaire: Yeah. Well, good for you for sticking to your guns. And it's really, really a wonderful study and I want to congratulate you both on this. And it was a huge undertaking with all those mice.

Dr K. Drosatos: Thank you.

Dr Ioannis Kyriazis: Thank you very much.

Dr Cindy St. Hilaire: That's it for the highlights from the February 5th and February 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 @DiscoverCircRes and #DiscoverCircRes. Thank you to our guests, Kostas Drosatos and Ioannis Kyriazis. This 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.

This month on Episode 20 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the January 8 and January 22 issue of Circulation Research. This episode features an in-depth conversation with Drs Stefanie Dimmeler and Wesley Abplanalp from Goethe University in Frankfurt, Germany, regarding their study titled Clonal Hematopoiesis-Driver DNMT3A Mutations Alter Immune Cells in Heart Failure.

Article highlights:

Li, et al. FA Scaffold Genes Are Novel TAA Genes

Z Perestrelo, et al. ECM Structure and Mechanics in Heart Failure

Castranova, et al. Zebrafish Intracranial Lymphatics

Rogers, et al. Computational Phenotypes for VT/VF Risk

Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast with 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'll be highlighting four articles selected from the January 8'th and January 22'nd issues of Circ Res. After the highlights, Dr Stephanie Dimmeler and Wesley Abplanalp at Goethe University in Frankfurt, Germany will join me to discuss their study, Clonal Hematopoiesis-Driver DNMT3A Mutations Alter Immune Cells in Heart Failure.

Cindy St. Hilaire: The first article I want to share is Variants of Focal Adhesion Scaffold Genes Cause Thoracic Aortic Aneurysm. The first authors are Yang Li and Shijuan Gao, and the corresponding authors are Jie Du and Yulin Li from Beijing Institute of Heart, Blood and Lung Vessel Disease in Beijing, China. Thoracic aortic aneurysm is the localized expansion of the blood vessel. This expansion causes weakening of the vessel wall, causing it to rupture, which is a life-threatening emergency.

Although there are several genetic mutations that lead to thoracic aortic aneurysms, more often thoracic aortic aneurysms occur as an isolated event with no known cause or family history. To gain greater insight into the genetic underpinnings of isolated thoracic aortic aneurysms, Li and Gao and colleagues performed whole exome sequencing of DNA from 551 patients and 1070 healthy controls. They found that five percent of the patients screened harbored mutations in previously identified genes associated with TAA. Importantly, they identified a number of novel candidate gene variants in the remaining 95%. In four patients they discovered mutations in a gene named Testin. Testin is a scaffold protein found at focal adhesions, which are the points of connection between the extracellular matrix and the cell's intracellular cytoskeletal framework. Mice that lacked Testin, or carried a mutant version of it, had dilated aortas and impaired contractility of vascular smooth muscle cells. Moreover, the team found additional focal adhesion gene variants present in the patient cohort, suggesting weakening or dysfunction of the structural elements may be a driving force in thoracic aortic aneurysm pathology.

Cindy St. Hilaire: The second article I want to share is titled Multi-scale Analysis of Extracellular Matrix Remodeling in the Failing Heart. The first author is Ana Rubina Perestrelo, and the corresponding author is Giancarlo Forte, and they're from St. Anne's University Hospital in the Czech Republic. After a myocardial infarction, when a lack of blood supply causes injury to the cardiac muscle, the damaged muscle tissue is patched by proliferating fibroblasts and the remodeling of the extracellular matrix. This is a process that is called fibrosis. However, this fibrotic process often continues after the initial repair and itself causes progressive loss of cardiac function. To better understand how cardiac fibrosis progresses, Perestrelo and colleagues examined cardiac extracellular matrix and fibroblasts from patients with and without heart failure. Using both microscopy and mass spectrometry, they found that the extracellular matrix from heart failure patients had a larger content of collagen and other extracellular matrix proteins, as well as more compact and less elastic fibers than non-heart failure controls.

Cindy St. Hilaire: RNA analysis of fibroblasts from patient and control hearts revealed heart failure patients had increased transcription of genes involved in assembling both the extracellular matrix and focal adhesions, which, as we just learned, are the points of connection between extracellular matrix and the cell's cytoskeleton. One such gene encoded the transcription factor yes-associated protein, or YAP. In cardiac fibroblasts, high levels of YAP drove expression of extracellular matrix factors. Similarly, extracellular matrix material from heart failure patients was particularly potent in triggering YAP activity. In highlighting this positive feedback of extracellular matrix remodeling, the work suggests that blocking this YAP-driven process may be an effective strategy for slowing heart failure pathogenesis.

Cindy St. Hilaire: The third article I want to share is titled Live Imaging of Intracranial Lymphatics in the Zebrafish. The first author is Daniel Castronova and the corresponding author is Brant Weinstein from the National Institutes of Health in Bethesda, Maryland

Until recently it was believed that the mammalian central nervous system lacked a classical lymphatic system. However, that belief was overturned a few years ago when canonical lymphatic vessels were discovered in the mouse brain. The discovery has implications for the understanding of the brains inflammatory and protein clearance processes, as well as disorders associated with these processes, such as in Alzheimer's disease. Because in vivo analysis of the mammalian brain lymph system is hindered by the thickness of the skull, Castronova and colleagues turned to a recently engineered zebrafish that has practically transparent tissues. Visualizing the fish brain through the top of the animal's skull, the team found a complex network of lymphatic vessels covering much of the brain, particularly the cerebellum and the optical areas. The team confirmed the identity of the vessels with a series of lymph markers and showed that the vessels both carried out tissue drainage and contained trafficking neutrophils. The work introduces the fish as a valuable model for studying intercranial lymphatics in both health and disease states.

Cindy St. Hilaire: The last article I want to share before we switch to our interview with Dr Dimmeler is titled Machine Learned Cellular Phenotypes Predict Outcome in Ischemic Cardiomyopathy. The first authors are Albert Rogers and Anojan Selvalingam. The corresponding author is Sanjiv Narayan from Stanford University in Palo Alto, California. Sudden cardiac arrest affects over 300,000 people per year in the US alone. Individuals with reduced left ventricular ejection fraction are at an elevated risk for sudden cardiac arrest. Many of these patients qualify for implantable cardiac defibrillators. However, in the first year of implantation, these devices are rarely needed to deliver life-saving therapy, and identifying means to further risk stratify these patients has been elusive. The authors of this study hypothesized that the morphology of individual ventricular monophasic action potentials in patients with ischemic cardiomyopathy could possibly identify tissue or cellular electrophysiological phenotypes that can be identified by machine learning and then be used to predict long-term outcomes for patients.

Cindy St. Hilaire: Using 42 patients with coronary artery disease, the team recorded 5,706 ventricular monophasic action potentials and left ventricular ejection fraction during steady state pacing. Patients were then randomly allocated to independent training and testing cohorts. Support vector machines and convolutional neural networks were trained to two end points. The first, sustained ventricular arrhythmia, and the second, mortality at three years. Patient level predictions in independent test cohorts yielded a strong concordance statistic and were the most significant multivariate predictors. This machine learning of action potential recordings in patients revealed novel phenotypes for long term outcomes in ischemic cardiomyopathy. These computational phenotypes may reveal cellular mechanisms for clinical outcomes and could be applied to other conditions.

Cindy St. Hilaire: Today, Dr Stephanie Dimmeler and her postdoctoral fellow Wesley Abplanalp, from the Goethe University in Frankfurt, Germany, are here to discuss their study, Clonal Hematopoiesis-Driver DNMT3A Mutations Alter Immune Cells in Heart Failure. This article is in our January 22nd issue, the second issue of 2021. Thank you both very much for joining me here today. I know it's the evening where you are, so I appreciate you taking the time to sit with me.

Wesley Abplanalp: Of course. Thank you.

Cindy St. Hilaire: I think I want to start with the definition of clonal hematopoiesis. Just to get every listener on the same page. Our bone marrow produces billions of blood cells every day and the traditional view is that maybe 10 to 20,000 of these hematopoietic STEM and progenitor cells create all the progeny blood cells. This idea of multiple hematopoietic and progenitor cells, or HSPCs, is in contrast to this phenomenon called clonal hematopoiesis. That is where a sizable portion of the differentiated blood cells at a given time in a human has been derived from a single, kind of dominant HSPC. This idea of clonal hematopoiesis can really best, I think, be conceptualized when we think about cancers, like leukemia, but a lot of clonal hematopoiesis has been linked to what is called clonal hematopoiesis of indeterminate potential, or CHIP. I was wondering if you could kind of give us a little bit of details about what is known regarding the drivers of CHIP, this clonal hematopoiesis of indeterminate potential, and what's the genesis of exploring the role of CHIP and how it affects cardiovascular health, and specific to your study, heart failure?

Stephanie Dimmeler: Well, thank you very much. Maybe I start with the more general question, actually the CHIP refers to the occurrence of mutations in hematopoietic STEM cells, which leads to the extension of these mutated cells. Initially it was thought that this is correctly linked to cancer and the development of the leukemia, but it turned out that the occurrence of such mutation is not exclusively seen in patients with leukemia, but actually also healthy persons can acquire such mutation, and has such mutations of blood. So it's an age-dependent phenomenon and with increasing age, up to 20% of the people have such mutations.

Cindy St. Hilaire: Wow, 20%.

Stephanie Dimmeler: If they are old enough. There are a few of such mutations, particularly in these enzymes, which we are also studying in MTCA, or type two, this doesn't lead to leukemia, but still subject to such mutations die earlier. So they have a poor prognosis. As said it was not linked to leukemia or cancer, but it was shown by Eisner and colleagues and Dr Libby also that such mutation have a higher risk of dying from coronary artery disease. We have added to this information, and our group is actually working together with the hematology department, that also heart failure patients with such mutations have a very poor prognosis.

Cindy St. Hilaire: Interesting. Maybe when we're talking about clonal hematopoiesis I know in your paper you mentioned, I think it was three or four commonly found mutants. Is there something shared between these genes that are mutated? I know in this study we're focusing on DNMT3A, but what does that do normally and what are some of these other drivers of clonal hematopoiesis, and is there a similar theme to the mutations?

Wesley Abplanalp: Well, I guess I could jump in a little bit. We're looking at DNMT3A, and the other very commonly found gene that's often mutated is TED2, and I think what's really interesting about these two genes is that they can both epigenetically control gene regulation, of course. So one is a DNA methyltransferase and of course the other has the opposite effect. This is not true necessarily for all CHIP-associated mutations, but I do think it is quite interesting that these tend to be the two most abundantly found mutated genes, especially in the context of CHIP, and especially within these heart failure cohorts.

Cindy St. Hilaire: So how common are these mutations? I guess we can specifically talk about DNMT3A. How common is that in the general population as a whole? Do we know that yet?

Stephanie Dimmeler: Yeah, some studies, it's a clear age dependent phenomenon. It depends on the age. In young subjects only very few subjects have such mutations, but with increased age, like 80, for example, you have a significant number and it's even higher if you look at heart failure patients, who have up to 40% of heart failure patients with high age having such mutations. Also, of course I have to say it depends a bit how you count the mutations. It depends where you set the cut off. We, for example, set the cutoff at two percent of mutations carrying DNA in the blood, and it depends with the numbers, depending with how low or how high you set the cut off.

Cindy St. Hilaire: Wow. So really it could be quite high. I guess I didn't realize it was that high. So could you maybe walk us through the study? What did you start with and what analysis did you perform?

Wesley Abplanalp: So with this study, we enrolled subjects with chronic ischemic heart failure. We were beginning there because we'd already found that the patients who have chronic ischemic heart failure and harbored these mutations have a worse prognosis. So our question is clearly there's something in the blood that's happening that's maybe facilitating this. We wanted to know more about this. It was already understood that these cells might be associated with inflammation, but the real question is we wanted to know what the transcriptional signatures would be in these patients. We enrolled six heart failure patients with a DNMT3A mutation.

Wesley Abplanalp: We had already screened through subjects before, and another four subjects with heart failure, with no known CHIP associated mutation. So we screened for 56 mutations that are associated with CHIP or other hematological malignancies. Then this is how we began our cohort. From this, then we took the peripheral blood from these subjects and use the peripheral blood mononuclear cells. So basically it'd be immune cells, which are circulating from these subjects and then performed a droplet single cell RNA sequencing analysis on just these circulating cells. We didn't necessarily enrich any cell type. We were trying to take an unbiased approach to really capture what was happening in the landscape. Then from here, we really dove down into some of the most abundant cell types that are there. For example, the monocytes and the T cells.

Cindy St. Hilaire: That's great. I think I read you found there were no significant changes in the actual types of cells. So both mutant and control populations had similar numbers of different types of cells, but what you did find that was significantly different, was the gene expression profiles within the cells of the mutant versus the controls. Can you talk a little bit about these changes? What was the same and what was different between your groups?

Wesley Abplanalp: Right. So one thing that I think that was really striking, like you're saying, or important, we're not seeing the change in the relative shifts in the abundances of cells. So therefore we can ask what's really happening within the cell. That's where the strength of the technology really has its full effect, I guess. What we're seeing is we were able to kind of add and confirm that the hallmark inflammatory cytokines, like IL-Beta and IL-6, IL-8, for example, were upregulating, this gives us great insights for potential interventions for these subjects, for example. These were quite different. We were also seeing, so we found this in the monocytes, we were also seeing an increase in resistin. So this was at this point, unknown. In resisitin, I think is a really interesting molecule because this is a secreted protein. It's been shown that when endothelial cells are exposed to this, that they become activated.

Wesley Abplanalp: We could also take this in vitro and silence DNTM3A in monocytes, and then we could add the supernatant. So what's secreted from these monocytes and add them to otherwise naive endothelial cells. We could see indices of endothelial cells becoming activated through increases in IL-Beta and BK-1.. We are additionally showing increasing interactions between endothelial cells and monocytes, which had otherwise not been shown before. We are also kind of showing these novel interactions between monocytes and T-cells, which I think is really cool because then you wind up having this capacity for a small number of cells enriching the impact on the greater blood and population. Through the interactions with T-cells and endothelial cells, we wind up seeing strong evidence, for example, for a potential bystander effect. So that for a few rogue cells to really have a much broader impact on these cells in the greater milieu.

Cindy St. Hilaire: Yeah. I found that graphic, your graphical abstract, it was something just really neat to think about in terms of there's this clonal hematopoiesis, but it's not a hundred percent, right? Is that correct? These mutations aren't in every single one of these circulating cells. I was wondering, could you find evidence that the inflammatory, maybe this would be an in vitro study, the inflammatory activation or cytokine release or activation of endothelial cells is better, worse, the same if the monocyte has it versus the T-cell has it, versus both? Are they equal contributors? What, I guess what, in terms of a stepwise progression, where do you think these mutants are more potent?

Wesley Abplanalp: Oh, this is a good question.

Cindy St. Hilaire: If you can speculate. Maybe you can't speculate yet.

Wesley Abplanalp: Yeah, I think at this point, I think it's a lot of speculation. With the monocytes we wind up seeing, I think some of the biggest changes we wind up seeing are just in these cytokine, they turn into kind of a cytokine factory. They just really push out these cytokines. There's a much more mixed response, I think from the T-cells, but inherently the T-cells are a much more diverse population. So it begins to become a little bit difficult to compare because they all have different roles and different functions. Unfortunately, I think it's finding a good T-cell model in vitro, for example, is a little more difficult than recapitulating some of this for monocytes, for example.

Cindy St. Hilaire: I'm not an immunologist, I guess, being a vascular biologist, you're a little bit immunologist always, but what are our abilities to model this in a mouse system? Are these cell types very easily translatable between mouse and human? I know there's different cytokine profiles when we're talking macrophages and things like that, is it similar for T-cells also? Or is that more translatable?

Stephanie Dimmeler: The proton signature have been observed in type two hetero zygote STEM cell transplants in mice as well. Also, DNTM3A editing has been shown in actually, a very nice Circulation Research paper by Ken Walsh to have an effect also on heart failure. These studies suggest that at least the pro-inflammatory signature in monocytes can be recapitulated as well, can be the phenotypes that grows its development, as shown by [Ken Walsh, and the heart failure phenotypes. I think Cynthia, you've touched upon a very interesting question. Mainly, to what extent is the transcription we are seeing related directly to the mutation of the cell? To what extent can you explain and understand that so many of the cells have this changed signature? I think our data clearly suggests, at least for the monocytes, that it's not only the mutated cell alone.

Stephanie Dimmeler: They have also a channel effect on the other, non-mutated cell, because our percentage of cells which has the mutation is between maybe 2% and 10% to 30% to 40%. We have many more cells which have the inflammatory signature. In the T-cells it's a bit different maybe, but also we have excessive activation. I think we don't know yet the reason. It could be many, but we are really doing, what we are currently doing is to try to target which cell is mutated and which cell is not mutated in humans. Then we can distinguish biochemically between the mutation carriers and the biotype cells, and then we can tell more what happens directly, and what happens secondarily, because of course, if you have an inflammatory cell, this inflammation can influence the neighboring cell as well. In the STEM cell niche, the cells are also in the same environment, so] how much your environment in STEM cell niche, this may also affect the neighboring other hematopoietic STEM cells. This is what I think is the next step to do.

Cindy St. Hilaire: Yeah, that's one thing I was actually thinking about is obviously the clonal hematopoiesis aspect means there is a STEM cell harboring a mutation that is selecting or allowing that population to grow much more efficiently or faster than the non mutant cells. What is that doing to the other STEM cells in that area? Is it inflammation just in the periphery once these cells are differentiated in little packets of cytokine releasing cells, but yeah, what's happening at the level of the niche in terms of these mutations?

Stephanie Dimmeler: A very interesting question. I think it's so far understudied, at least we have no insights, but what is known is that for some heart failure, and acute myocardial infarction also had an impact on the STEM cell niche, so there is activation of the osteogenic niche, there is a change in the vascular niche. So this are maybe effects, which may link also hematopoiesis and the cardiovascular and heart disease on some level, but to know what is he and egg need some more studies to do.

Cindy St. Hilaire: Yeah, yeah. As always, I have that same problem with calcification stuff. What is the cause? What is the consequence? I guess it's ripe for funding and studies. The schematic, your graphical summary is really focusing in on this monocyte T-cell interaction on the endothelium, which is obviously not the heart tissue itself. So how do you envision, this is kind of easy to conceptualize or picture when we're talking about an atherosclerotic plaque, right? It's right at that interface where these blood cells are touching the vasculature, but what do you think is happening to exacerbate or drive the heart failure component regarding clonal hematopoiesis and specifically this mutation?

Wesley Abplanalp: So one thing that we are seeing, and I think that's particularly interesting, is perhaps this interaction between the endothelium, like you were saying, and monocytes. Ken Walsh, and I think others have shown, but Ken Walsh I think had shown that with a DNMT3A loss of function study, that they could see an increase in the extra visation, or these monocytes coming in to the myocardial tissue. That there seemed to be some kind of indices of heart failure that then accompanied this. How or why this was happening I think people didn't really know, but I think there are many ways in which this could happen. If, for example, if these cells are secreting resistin, and there's increased adhesion molecules, and of course there's increased chances for extra visation where these cells can leave the blood and then go into the heart.

Cindy St. Hilaire: Has anyone ever looked at cardiac tissue from patients harboring these mutations and seen differences in either the cardiomyocytes or the cardiac fibroblasts or anything like that? Or is that what you're doing next perhaps, or-

Wesley Abplanalp: Stephanie's smiling.

Stephanie Dimmeler: We tried to, the problem is there's difficulties in these biopsies to get enough material to study the mutation.

Cindy St. Hilaire: Of course.

Stephanie Dimmeler: So far, the problem with the patients also incomparison to mice models that we have to clean off the circulating blood, which may also be stuck in the tissue. Therefore, we have some results, but not yet for publishing because the reviewers will make us pass time to make this more confusing. So, as soon as we can detect by single cell on a sequencing or nuclear sequencing, the mutated cells and the non-mutated cells, then we have a chance to get more insights. So far, we cannot distinguish really the cardiac monocytes versus the circulating monocytes, which makes our study a bit more difficult. Really I can add one more point to your previous question.

Cindy St. Hilaire: Sure.

Stephanie Dimmeler: I think one, an interesting message of our paper would also be that if you have proinflammatory monocytes coming from the circulation into the heart, and our study will claim that they are more likely to hold through the heart tissue and then invade, because endothelial affects the capacity. Then they may replace the cardiac macrophages, which would be seen as a more physiological and protected type of cell, which is involved, as we know in electrical continuation and so on. Then we have these bone marrow cells, which are increasing in holding and this of course could aggravate all the heart failure in addition to the cytokines they are producing. I think, finally, also one shouldn't neglect the T-cell. T-cells are known to play a role in heart failure, and if we have activation of T-cells, which we have to prove now in the cardiac tissue, but at least in the circulation here, I would assume that this is the case. This could have also consequences of course, and could link hematopoiesis to heart failure phenotypes as well.

Cindy St. Hilaire: So where do you think this will go in the future? How could this knowledge, and possibly even single cell sequencing technology, be leveraged for therapies in the future?

Stephanie Dimmeler: So my wish would be that our data would be leading to a type of guided therapy. If we understand better which mutation affects which pathways, or which genes, we may use specific anti-inflammatory treatments, not global anti-inflammatory treatments, but more specified or specific treatment strategies to target patients with mutations. I think single cell sequencing is a very good way to start it. Followed by proteomics and then other -omic technologies. This is actually what I would wish we could do with it, which other people hopefully start some pilot trials with patients and try some treatments, but we could maybe get even further insights by the single cell data. Then the responses also of the treatments, the single cell sequencing, you can see, but it can normalize the phenotype, which would be, of course, the wish.

Cindy St. Hilaire: That would be amazing because we tried in the Cantos Trial to just, let's block inflammation and see what happens. There were certain populations where it seemed to have a much greater effect than others, and maybe targeting clonal hematopoiesis could help tweak or tighter those therapies. This was a great study. I want to commend you both on this excellent story and thank you so much for joining me today.

Stephanie Dimmeler: Thank you very much. Nice to see you again.

Wesley Abplanalp: Thank you.

Cindy St. Hilaire: That's it for the highlights from the January 8th and January 22nd 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, Doctors Stephanie Dimmeler and Wesley Abplanalp. This 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.

This month on Episode 19 of the Discover CircRes podcast, host Cindy St. Hilaire highlights three featured articles from the December 4 issue of Circulation Research. This episode features an in-depth conversation with Drs Mete Civelek and Redouane Aherrahrou, from the University of Virginia regarding their study titled Genetic Regulation of Atherosclerosis-Relevant Phenotypes in Human Vascular Smooth Muscle Cells.

Article highlights:

Zahreddine, et al. Tamoxifen and E2 Effects On Reendothelialization

Zheng, et al. Arterial Stiffness Preceding Diabetes

Galang, et al. ATAC-seq Identifies Novel Isl1 SAN Enhancer

Cindy St. Hilaire: Hi, welcome to Discover CircRes, the podcast to 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 will be highlighting three articles selected from the December 4'th issue of Circ Res. Drs Mete Civelek and Redouane Aherrahrou, from the University of Virginia, are here to discuss their study, Genetic Regulation of Atherosclerosis-Relevant Phenotypes in Human Vascular Smooth Muscle Cells.

Cindy St. Hilaire: The first article I want to share is titled, Tamoxifen Accelerates Endothelial Healing by Targeting Estrogen Receptor-alpha in Smooth Muscle Cells. The first author is Rana Zahreddine, and the corresponding author is John Francois Arnal and they're from INSERM and the University of Toulouse, France. For breast cancers that contain high levels of estrogen receptor, a standard treatment is to give drugs that block either estrogen production or the receptor itself, such as tamoxifen. However, estrogen can elicit beneficial vascular protective effects, so treatment with tamoxifen might increase the risk of cardiovascular disease. Depending on the tissue, tamoxifen can both antagonize or activate estrogen receptor, so it's role in cardiovascular disease is unclear.

Cindy St. Hilaire: Some evidence even suggest tamoxifen might have protective effects, such as promoting vascular endothelial healing. Zahreddine and colleagues now show that while might suffering damage to the endothelial lining of a blood vessel have improved healing when treated with tamoxifen, or with estrogen, those suffering perivascular injury, that is to say, the injury that affects both the endothelial layer as well as the surrounding smooth muscle cell layer, heal only in response to estrogen. This suggests tamoxifen's healing effects might require smooth muscle cells. In mice, lacking the estrogen receptor and smooth muscle cells, they found estrogen, but not tamoxifen, healed endovascular injuries. While in mice lacking estrogen receptor and endothelial cells alone, they found the opposite. This work reveals nuances in the molecular actions of tamoxifen that should inform further assessment of its risk and benefits for use in patients.

Cindy St. Hilaire: The second article I want to share is titled, Arterial Stiffness Proceeding Diabetes, A Longitudinal Study. The first authors are Mengyi Zheng and Xinyuan Zhang and the corresponding authors are Xiang Gao and Shouling Wu, from Pennsylvania State University and North China University of Science and Technology. As a person ages, their risk of developing diabetes and cardiovascular disease increases. Aging is also linked to increase in arterial stiffness and high blood pressure, but how all these individual conditions affect and influence each other is not entirely clear. For example, while arterial stiffness and diabetes tend to correlate, whether one increases the risk, or the other, or the risk relationship or whether the risk relationship is bi-directional, is unknown.

Cindy St. Hilaire: To assess the interplay between these disease states, Zheng and colleagues studied diabetes and arterial stiffness in a cohort of 8,956 Chinese people between 2010 and 2015, none of whom had had diabetes or cardiovascular disease at the outset of the study. With repeated measures of fasting glucose levels, which is an indicator of diabetes, and pulse wave velocity, which is a measure of arterial stiffness, the team found that participants with a higher baseline arterial stiffness were more likely to develop diabetes during the five-year period than those with lower stiffness levels. Out of the original cohort of just over 8,900 individuals, a total of 979 individuals developed diabetes during the study. Higher baseline glucose levels did not predict future arterial stiffness; this suggests a risk relationship that is a one-way street. While the results require confirmation in additional cohorts, this finding is the first to identify the pathological mechanisms linking arterial stiffness to diabetes.

Cindy St. Hilaire: The third article I want to share is titled, ATAC-Seq Reveals an ISL1 Enhancer That Regulates Sinoatrial Node Development and Function. The first author is Giselle Galang, Ravi Mandla, and Hongmei Ruan. And the corresponding author is Vasanth Vedantham, from the University of California, San Francisco. Pacemaker cells, of the sinoatrial node, establish and control the rhythmic contractions of the heart. These cells differ from regular cardiomyocytes in their transcription profiles, but how this transcriptional profile is established and maintained is not fully understood. To investigate the epigenetic landscape defining pacemaker cell fate, Galang and colleagues have employed a technique called ATAC-Seq, which identifies areas of the genome with accessible open chromatin structures, which is an indication of transcriptional activity. The team compared the genomes of pacemaker cells with atrial cardiomyocytes, and found a number of pacemaker cell-specific accessible loci that had both large numbers of transcription factor binding sequences and enhancer activity, when assayed in mice.

Cindy St. Hilaire: The team went on to specifically characterize one novel enhancer upstream of the gene, encoding ISL1, which is a key transcription factor for pacemaker cell identity. They showed that deleting the enhancer caused under development of the Sinoatrial node and arrhythmias in mice. They also noted that single cell nucleotide polymorphisms at the equivalent loci in humans, have been linked to variations in resting heart rate. The report verifies ATAC-Seq as an effective tool for identifying pacemaker enhancers and will launch future studies into how such enhancers function in heart development and disease.

Cindy St. Hilaire: Okay, so today with me is Dr Mete Civelek and Dr Redouane Aherrahrou, from the University of Virginia, and they're here to discuss their paper titled, Genetic Regulation of Atherosclerosis-Relevant Phenotypes in Human Vascular Smooth Muscle Cells. And this article is featured in our December 4th issue. So thank you both so much for being here with me today.

Mete Civelek: Great to talk to you Cindy. Thank you for choosing our paper.

Cindy St. Hilaire: Yeah, and seeing you over Zoom, I wish these were in person, but...

Redouane Aherrahrou: Thank you for having us.

Cindy St. Hilaire: Yeah. Great. So, before we dig into the details of this paper, which I think is a really nice paper, one of the things I like about it is that it couples GWAS with some functional things, which is obviously super important for figuring out what is important in that GWAS data. So, before we dig into the nitty-gritty of the paper, could you maybe explain what a GWAS study is, and what the strengths and weaknesses are, in terms of using that as an approach to figure out disease related pathophysiology?

Mete Civelek: So, we know that coronary artery disease, or these cardio-metabolic diseases, have a genetic component, and in the past we used to do linkage studies, studying families, but in the last 15 years or so, because of the developments in technology, we can do these genome-wide association studies. And essentially what they do is they look at a population and some of the people in the population will have coronary artery disease and some people will not have coronary artery disease, will be otherwise healthy. And then you study the genetic variance across the entire genome and look for frequency differences in the people with the healthy phenotype and people who have the disease. And of course you do some statistical tests to find if this frequency differences is indeed statistic, the different between these two groups and you identify essentially loci that are associated with the disease.

Mete Civelek: But I see GWAS as almost a detective work, you say something like, okay, let's say there was a murder in the United States, and then now you do GWAS of course to find the murder, right? But what that tells you is, okay, the murder occurred in, let's say Pittsburgh or Charlottesville or Washington DC, sure, it narrows down the scope of where you're going to look at, but it doesn't tell you exactly what happened and where it happened and things like that. And so after GWAS there many more questions to answer looking at the molecular mechanism of the locus, the tissue or cell type of action, the gene, which is being affected by the locus to affect the phenotype. So, it's very good at narrowing down possibilities and coming up with hypotheses, but then the real work begins.

Cindy St. Hilaire: I was wondering actually, as you were saying that, have there ever been... I guess like false discoveries, where people have really focused in, on a loci, because it came up maybe in one or multiple studies, but then maybe it didn't prove to be causative or they still can't figure it out. Are there examples of that?

Mete Civelek: The most obvious example is actually the 9p21 locus.

Cindy St. Hilaire: Interesting. That's the one I was thinking of actually.

Mete Civelek: Which has been associated with coronary artery disease susceptibility in all kinds of studies and in kinds of populations, this signal itself is real, what it's doing is been a lot of debate. Some people think that it's affecting the CDKN2A and 2B genes nearby.

Cindy St. Hilaire: Is that p21 or p16?

Mete Civelek: One is p21 and one is p16, but I can't remember which one.

Cindy St. Hilaire: Yeah, I can't either.

Mete Civelek: Right. And then there's a non-coding RNA in that region called lnRNA. Some people think it's affecting and lncRNA expression. Some people think it's affecting isoform abundance, so that's just probably the most famous locus in our field, in terms of figuring out what it's doing. Yeah.

Cindy St. Hilaire: Well, at least it's probably causing a lot of people to think of a lot of good questions to ask, so that's exciting. In your study, you state that you want to focus on the impact of coronary artery disease associated variants in atherosclerosis-relevant smooth muscle cell phenotypes, and the phenotypes you wanted to focus on were calcification, which is my personal favorite. So calcification, proliferation, and migration. So I was wondering why you wanted to focus on these phenotypes and then what kind of functional assays did you do?

Redouane Aherrahrou: So, the reason we choose those phenotypes because they are playing important role in the disease. So, for example, during the advanced stage of the disease, smooth muscles cells, they proliferate and migrate to make the fibrous cap. So the fibrous cap is actually stabilize the plaque against the rupture, and also during the advanced stage of the disease, the calcification also happening, a lot of people believe that the calcification also contribute to instability of the fibrous cap. So that's why we focus on those three phenotype, migration, proliferation, and calcification.

Cindy St. Hilaire: Interesting, and so I think you'd said you had about 150 patients in your study. Does that mean you did these functional assays in 150 different cell lines? Or how did you do that?

Redouane Aherrahrou: That's a good question. So we conducted actually, our assays from 150 healthy and multi-ethnic donors, so those people actually did die from motorcycle and car accidents, and the doctors actually use the chunk of the aorta where we'll actually isolate these cells from, and then they are actually healthy enough to use for the heart transplantation.

Cindy St. Hilaire: Wow. And so were you introducing known SNPs or SNPs that are pulled out of GWAS into the cells, or did the cells already have the SNPs available? How was the correlation done between functionality and SNPs?

Redouane Aherrahrou: That's a great question. So we actually use the natural SNPs that already exist in those donors. And we ask the question how the genetic variants of those donor affects migration, proliferation, and calcification phenotypes.

Mete Civelek: So we essentially perform a GWAS in a dish-

Cindy St. Hilaire: Yeah, that's kind of what I was thinking-

Mete Civelek: That's the bottom line, you just culture these cells and do these phenotypic characterizations, which you cannot do in healthy living human beings of course, and then just the naturally occurring genetic variation in these individuals, in these donors, to essentially calculate the association between the genetic variants and then these phenotypes.

Cindy St. Hilaire: And you were using aortic smooth muscle cells, right?

Mete Civelek: Yes.

Cindy St. Hilaire: Do you think... this is one thing I always think about, especially because kind of harping back to Mark Majesky's early work with the chick embryo and developmental origins. Do you think if you had coronary arteries from the same individuals that the smooth muscle cells would respond similarly?

Mete Civelek: This is a really good question, partially, yes and partially, no. I'll give you one specific example, for example, one of the loci that is associated with coronary artery disease is over a transcription factor called TCF21. And TCF21 is actually playing an important role in smooth muscle cell phenotypes, and that transcription factor is expressed only in coronary artery smooth muscle cells, but not in aortic smooth muscle cells.

Cindy St. Hilaire: Interesting.

Mete Civelek: This was something that Dr Tom Quertermous from Stanford showed. So presumably we are capturing some of the genetic variation that's important in coronary artery disease as some of it was probably missing because we're using aortic smooth muscle cells.

Cindy St. Hilaire: Yeah. That is so neat. I really like that heat map you had, I think it was figure 4 because you really lined up along the SNPs that were identified in these patients you looked at the effect of that SNPs on a specific function test, you did, and you did, was it 11 functional tests?

Mete Civelek: 12 different functional tests.

Cindy St. Hilaire: 12?

Mete Civelek: Yes.

Cindy St. Hilaire: It's an amazing amount of work really. Well, how long have you been working on this project?

Mete Civelek: Redouane, why don't you answer this question?

Cindy St. Hilaire: Or do you not want to talk about that?

Redouane Aherrahrou: Of course, it's not easy actually to culture and characterize 151 smooth muscle because you expect sometimes, you capture them, some of them, they will not grow, some of them they get contaminated, and you have to perform it again. And also, you cannot do the same experiment for all of them at the same time. So what we did actually, before we started the experiments, we decided to take a smooth muscle cell from one donor, and expanded many times and then we use the same donor to run each time for all the experiments, just to count for the batch and environment effects.

Cindy St. Hilaire: Yep.

Redouane Aherrahrou: So it took me actually almost one and a half year, to finish the characterization for all 151 smooth muscle cell. At that time I was also using also two incubators and then you can imagine, when you put the incubator-

Cindy St. Hilaire: It's full.

Redouane Aherrahrou: ...and I try to finish that, and then I again, start the experiment again to finish the other batch.

Cindy St. Hilaire: Oh God. Yeah, my lab also... we work only in primary human tissues from vessels, but also from valves. So my staff will certainly appreciate all your efforts for this paper.

Mete Civelek: And you can also imagine there was this group of undergraduates, trailing-

Cindy St. Hilaire: An army yeah.

Mete Civelek: Redouane wherever he goes….they were helping him out with many aspects.

Cindy St. Hilaire: Oh, sure that's amazing-

Mete Civelek: He mentored, I think maybe five, six different undergraduate students throughout this project and they're all part of the paper. Yeah.

Cindy St. Hilaire: That's excellent. So towards the end of the study, you guys really focused on a gene, MIA3. So can we talk a little bit about that? What is this gene? What its normal function? Is it known? And then what did you find out in relation to smooth muscle cells?

Mete Civelek: Well, I'll start and Redouane you can continue. So let me just walk back just a little bit to tell you how we kind of decided to focus on that. So we identified the 79 loci that are associated with smooth muscle cell phenotypes and coronary artery disease. So we wanted to show at least some kind of a validation and so we looked for loci that are not associated with lipids because we thought they will maybe not be important in smooth muscle cells. And then we then looked for loci out of those, who affect a nearby gene expression in aorta, in smooth muscle cells, but not in endothelial cells and in monocyte so we thought that will give us confidence-

Cindy St. Hilaire: So kind of enriching for this smooth muscle cell?

Mete Civelek: And this MIA3 popped up and there was only one study actually that showed that, in codes for protein that localizes to the ER exit site and affects these COPII carriers, which secrete collagen into extracellular matrix. Well, collagen as you know, is important in cell stability and what smooth cell muscle cells produced. So, that's how we decided to focus on that gene. And I will let Redouane describe I guess, what we did with that gene.

Redouane Aherrahrou: Yeah. So after the function and mutation, we come up with this gene. So the first thing we did, because we found that the genetic variety in this locus affects actually proliferation in smooth muscle cell. So to what it did at least, this is actually SNP that's affecting the proliferation of smooth muscle cell by this gene, we down-regulate this gene actually in smooth muscle cell using two different shRNA, and we found actually the downregulation of this gene lead to affect the proliferation in sense that dominant regulation of this gene will affect the proliferation.

Redouane Aherrahrou: And also we found that the same genetic variance also in this gene lead to lower periphery, migration, sorry, the expression of this gene in small as I said, and then also in the aortic tissue. And interestingly, we did not see in the monocytes macrophages or other cell type, actually in the aortic tissue suggesting that this genetic variance affecting the coronary artery disease via smooth muscle cell.

Mete Civelek: And we also collaborated with Renu Virmani's group at CVPath Institute, stained lesions, coronary artery lesions, which have these thick caps and thin caps as you very well know that is really relevant to plaque stability, and show that the thin caps have fewer smooth muscle cells which were positive for this protein MIA3, which was all in line with our genetic findings, because our genetic findings basically show that lower expression of this gene was associated with increased susceptibility to coronary artery disease.

Cindy St. Hilaire: Interesting. I wonder if it could be correlated with plaque rupture, right. If there's less smooth muscle cell, obviously, then a thin fibrous cap is nothing that anybody wants. So is the proliferation of the smooth muscle cell almost protective in a sense, that's one of the things people are starting to think about in calcification, [Alina has these amazing imaging studies, where she looks at matrix vesical accumulation and calcification mitosis. And really the field has noticed, with work by Linda Demer also, the field has noticed that a large, huge chunk of calcification seems to be much less risky, I guess, compared to the microcalcifications, and I wonder if MIA3 might be like that too, if you can have just enough smooth muscle cell proliferation to kind of keep that cap thicker, is that more protective?

Mete Civelek: I think that's a really good point that you are raising, because some of the answer is, in that figure 4 in the paper that you mentioned. What we find is that these loci, when people have the risk allele of these loci, so obviously people are at higher risk for coronary artery disease, some of them are associated with higher proliferation, but some of them associated with lower proliferation, same with calcification, same with migration. So it's really difficult to say, at least just looking at the genetic loci, yes, higher proliferation is always better.

Cindy St. Hilaire: Yeah.

Mete Civelek: There's probably this really delicate balance that allows for plaque stability.

Cindy St. Hilaire: Yeah, it's reminisent, I guess, of the IO1-beta story, right?

Mete Civelek: Exactly.

Cindy St. Hilaire: Gary Owens and colleagues have really shown that well. The role of it in early versus late plaque is different and it's complicated. That's what we learned.

Mete Civelek: I agree. And it's specific to MIA3, that locus is also associated with myocardial infarction.

Cindy St. Hilaire: Oh, interesting.

Mete Civelek: So indeed there is a possibility that really affects plaque stability.

Cindy St. Hilaire: Yeah. So Mete, say you and I are in the same faculty class in that we both started-

Mete Civelek: Yes we are. We are classmates.

Cindy St. Hilaire: 2015 we started our labs, and this is obviously a huge undertaking and really starting a project like this... when you're new, it's really a risk. You're proposing to collect 150 smooth muscle cell lines and characterize them all functionally and it turned out amazingly, but can you maybe talk about the early days in this project's development, and was there ever a moment where you're like, "What the heck am I doing? Is this going to work or..." Just kind of maybe talk to us a bit about that.

Mete Civelek: That's a really excellent question. To be totally honest with you, I was really lucky to have recruited Redouane to the lab, but he and I worked together when I was a postdoc and when he was a PhD student, as part of this little consortium. So I knew he was going to work hard on his part and he was very driven-

Cindy St. Hilaire: He had magic hands.

Mete Civelek: And he really want to do this project. So, I knew that it was going to work, but of course he and I both had these moments of, "Are we sure of what we're doing, and why are we doing this? Are we going to get something out of it?" And it was both of us kind of pumping each other, if you will say like, "Yep, this is going to work, we know it's going to work, we have faith in this," but I should also say that my lab also works on adipose tissue biology, and I already had another kind of a safe project going on in that realm.

Cindy St. Hilaire: So, that's funny, I started my lab that way too. I kind of had the project that was the direct continuation of my K grant, and then this kind of high risk, high reward project on valves and you know what, I think that's something that's smart, it's kind of you have two tracks of research and hopefully one works and then the other one will work. And if they don't work at the same time, hopefully the other one can fill in the gaps, so...

Mete Civelek: I totally agree with you, but truly, Redouane made a big difference in this project, imagine a postdoc, who's doing nothing but cell culture for two years, hoping that something is going to come out, that's a big risk for him too, it certainly paid off and it's paying off because he has two other papers in the pipeline from this project.

Cindy St. Hilaire: Wonderful. That's excellent. So what's the future for this project? What's kind of the next question you're going to ask if you don't mind sharing.

Mete Civelek: Oh no, not at all. The most obvious one is looking at gene expression. So we have cultured these cells under two distinct conditions, one is the more contractile phenotype, one is the more productive phenotype. And we did RNA-Seq from, again 150 of these individuals in both conditions and we did what is known as eQTL mapping, so looking at the effect of the genetic loci on gene expression. In a separate project, we also actually collected media from these cells and looked at secreted proteins in the media and we're also finding the genetic loci that are affecting secreted protein, because as you very well know, smooth muscle cells secrete proteins to stabilize plaque stability. So those two papers are Redouane's next projects. And he's almost finished with one and has finished the analysis of the other ones, so hopefully-

Cindy St. Hilaire: That's exciting.

Mete Civelek: ...more papers coming out in the next six months or so. Oh, I should have said the paper was chosen for the Genomic and Precision Medicine Counci; Young Investigator Award, so Redouane is competing-

Cindy St. Hilaire: Wonderful, also you are in that, excellent. Thank you both so much for joining me today. This was a lovely paper, it was actually inspiring. It made me think about some way to think about my calcification studies.

Mete Civelek: Thank you so much, Cindy. This was really wonderful.

Cindy St. Hilaire: Absolutely. Thank you.

That's it for the highlights from the December 4th issue of Circulation Research. Thank you very much 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, Drs Mete Civelek and Redouane Aherrahrou. This podcast is produced by Rebecca McTavish and Ishara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy texts for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire. And this Discover CircRes, your on the go source, for the most up-to-date and exciting discoveries in basic cardiovascular research.