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Discover CircRes


Aug 19, 2021

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.