Circulation January 10, 2023 Issue

Please join authors Loren Field and Sean Reuter, as well as Associate Editor Thomas Eschenhagen as they discuss the article "Cardiac Troponin I-Interacting Kinase Affects Cardiomyocyte S-Phase Activity But Not Cardiomyocyte Proliferation."

Dr. Greg Hundley:

Welcome listeners, to this January 10th issue of Circulation on the Run, and I am Dr. Greg Hundley, associate editor, director of the Pauley Heart Center at VCU Health in Richmond, Virginia.

Dr. Peder Myhre:

I am Dr. Peder Myhre from Akershus University Hospital and University of Oslo in Norway.

Dr. Greg Hundley:

Well, listeners, this week's feature discussion delves into the world of preclinical science and evaluates cardiac troponin I and its impact on S phase activity in cardiomyocytes, and does that relate to cardiomyocyte proliferation. But before we get to that, how about we grab a cup of coffee and Peder and I will work through some of the other articles in the issue. Peder, how about this week I go first?

Dr. Peder Myhre:

Go ahead, Greg.

Dr. Greg Hundley:

Right. So Peder, this first study evaluated whether the burden of positive coronary artery calcification on cardiovascular disease differed by multidimensional individual characteristics, and so the investigators led by Dr. Kosuke Inoue from Kyoto University sought to investigate the heterogeneity in the association between positive coronary artery calcium and incident cardiovascular disease. And so Peder, to examine this question, the authors implemented a cohort study design that included adults aged greater than 45 years, free of cardiovascular disease, from the Multi-Ethnic Study of Atherosclerosis, or MESA, and after propensity score matching in a one-to-one ratio, they applied a machine learning causal forest model to, first, evaluate the heterogeneity in the association between positive coronary artery calcium and incident cardiovascular disease and then, second, to predict the increase in cardiovascular disease risk at 10 years when the coronary artery calcium score was greater than zero, so versus is it zero at all at the individual level?

Dr. Peder Myhre:

Oh, Greg, that is so cool, so using machine learning for coronary artery calcium and risk prediction, I'm very excited. What did they find?

Dr. Greg Hundley:

Right, Peder, so the expected increases in cardiovascular disease risk when the coronary artery calcium score was greater than zero were heterogeneous across individuals. Moreover, nearly 70% of people with low atherosclerotic cardiovascular disease risk showed a large increase in cardiovascular disease risk when the coronary calcium score was greater than zero, highlighting the need for coronary artery calcium screening among such low-risk individuals. And Peder, future studies are really needed to assess whether targeting individuals for coronary artery calcium measurements based on not only the absolute ASCVD risk, but also the expected increase in CVD risk when a CAC score is greater than zero and whether that improves overall assessment of cardiovascular outcomes.

Dr. Peder Myhre:

Wow, that is so clinically relevant and very interesting. And we're actually going to stay clinically relevant with the next paper which is about anti-platelet therapy after PCI. And this paper describes the long-term results of the HOST-EXAM trial. To remind you, Greg, the HOST-EXAM trial was an investigator-initiated prospective, randomized, open label, multicenter trial done at 37 sites in Korea. They enrolled patients who had undergone PCI with DES and maintained dual anti-platelet therapy without any clinical event for a mean 12 months and then they were randomized one to-one to either clopidogrel, 75 milligrams once daily, or aspirin, 100 milligram once daily. The primary results of this trial was published in Lancet in 2021 and showed superiority of clopidogrel over aspirin in prevention of the composite of MACE and major bleeding during 24 months of followup. And then, through the current paper, this describes the results of the post trial extended followup of about five years.

Dr. Greg Hundley:

Very nice, Peder, so aspirin versus clopidogrel and looking at the maintenance of that monotherapy and cardiovascular outcomes. Wow, so what did they find?

Dr. Peder Myhre:

Yeah, Greg. They, in this extended followup study, had a total of 5.8 years median followup, and the primary endpoint occurred in 12.8% in the clopidogrel group versus 16.9% in the aspirin group, and that has a range of 0.74 with a 95% conference interval ranging from 0.63 to 0.86. So also the clopidogrel group had lower risk of the secondary thrombotic endpoint and the secondary bleeding endpoint while there was no significant difference in the incident on all caused death. So Greg, to conclude, these very interesting results from the primary analysis of the HOST-EXAM trial was consistent through the longer followup, and this support the use of clopidogrel over aspirin monotherapy from 12 months onwards after PCI.

Dr. Greg Hundley:

Very nice Peder, beautiful description and sounds like long-term clopidogrel use over aspirin was quite beneficial. Well, the next study comes to us from the world of preclinical science, and it is from the investigative group led by Dr. Yunzeng Zou from Shanghai Institute of Cardiovascular Diseases and the Zhongshan Hospital and Fudan University. Peder, the study pertains to diabetes. So diabetic heart dysfunction is a common complication of diabetes mellitus and cell death is a core event that leads to diabetic heart dysfunction. However, the time sequence of cell death pathways and the precise intervening time of particular cell death type remained largely unknown in diabetic hearts. And so, Peder, this study aimed to identify the particular cell death type that is responsible for diabetic heart dysfunction and propose a promising therapeutic strategy by intervening in this cell death pathway.

Dr. Peder Myhre:

Wow, Greg, that is really interesting. Heart dysfunction in diabetes is something that we really have to learn more about and I'm so excited to hear what these authors found, Greg.

Dr. Greg Hundley:

Right. So first, Peder, the authors identified necroptosis as the predominant cell death type at later stages in the diabetic heart. And then second, Peder, the CB2 receptor, and we'll call that CB2-R, recruits transcription factor Bach2 to repress necroptosis and protects against diabetic heart injury while hyperglycemia and MLKL in turn phosphorylates CB2-R to promote ubiquitous dependent degradation of CB2-R, thus forming a CB2-R centric feedback loop of necroptosis. And finally, Peder, cardiac CB2-R or Bach2 expression negatively correlates with both MLKL 10 expression and the extent of diabetic heart injuries in humans. And so the clinical implications of these findings, Peder, are that the CB2-R centric necrotic loop represents a promising target for the clinical treatment of diabetic heart injuries.

Dr. Peder Myhre:

So Greg, this paper that comes to us from corresponding author Amanda Paluch from University of Massachusetts Amherst, is a meta-analysis of eight prospective studies with device measured steps including more than 20,000 adults who were followed for CVD events. And the mean age of participants in this study was 63 years and 52% were women. And the participants were followed for a median of 6.2 years and 1,523 cardiovascular events occurred.

So first, Greg, there was a significant difference in the association of steps per day in cardiovascular disease between older, that is greater or equal to 60 years, and younger, that is less than 60 years adults. So for older adults that has the ratio for cardiovascular disease using Q1 as reference was 0.80 for Q2, 0.62 for Q3, and 0.51 for Q4. And for younger adults that has ratio for cardiovascular disease using Q1 as reference was 0.79 for Q2, 0.90 for Q3, and 0.95 for Q4. And in the paper, Greg, there are some beautiful, restricted cubic lines that really illustrate the association between daily steps and the risk of cardiovascular disease among older adults and in younger adults.

So the authors conclude that for older adults taking more daily steps is associated with a progressively lower risk of cardiovascular disease. And monitoring and promoting steps per day is a simple metric for clinician patient communication and population health to reduce the risk of cardiovascular disease.

Dr. Greg Hundley:

Well, Peder, we've got some other very interesting articles in this issue and how about we dive into that mail bag and discuss a few of those. So I'll go first. The first is a Perspective piece by Professor Powell-Wiley entitled “Centering Patient Voices through Community Engagement in Cardiovascular Research.” A very important topic where can those in the community actually help us design meaningful outcomes for our research initiatives? And next Peder, there is a Research Letter from Professor Evans entitled “Increasing Mononuclear deployed Cardiomyocytes by Loss of E2F7/8, and does that fail to improve cardiac regeneration post myocardial infarction?”

Dr. Peder Myhre:

Thanks, Greg. We also have an ECG Challenge by Dr. Li entitled, “What Is The Truth Behind Abnormal ECG Changes?” And this is describing a very rare and interesting cause of ST segment elevation. I recommend everyone to read that case. We also have our own Nick Murphy who gives us the Highlights from the Circulation Family of Journals where he summarizes five papers from the Circulation subspecialty journals. First, the experience with a novel visually assisted ablation catheter is reported in circulation A and E. The impact of various exercise training approaches on skeletal muscle in heart failure with preserved the F is presented in circulation heart failure. Gaps in heart failure treatment over a decade are reported in circulation cardiovascular quality and outcomes, and the associations of machine learning approaches to plaque morphology from coronary CTA with ischemia are reported in circulation cardiovascular imaging. And finally, Greg, an observational study of left main PCI at sites with and without surgical backup is reported in circulation cardiovascular interventions. Let's go on to the feature paper today describing the cardiac troponin I interacting kinase and the impact on cardiomyocyte S phase activity.

Dr. Greg Hundley:

Great, let's go.

Welcome listeners to this January 10th feature discussion. Very interesting today as we are going to delve into the world of preclinical science. And we have with us today Dr. Loren Field and Dr. Sean Reuter from University of Indiana in Indianapolis, Indiana. And our own associate editor, Dr. Thomas Eschenhagen from University Medical Center of Hamburg in Hamburg, Germany. Welcome gentlemen. Well, Loren, we're going to start with you. Can you describe for us some of the background information that went into the preparation of your study, and what was the hypothesis that you wanted to address?

Dr. Loren Field:

Sure. This study actually came about in a rather roundabout fashion. We were doing a study with Kai Wollert in Hanover, Germany, where we were looking at the impact of a CXCR4 antagonist, which is used to mobilize stem cells from the bone marrow. And we had sent our mice over to Kai's lab and we have a mouse model that allows us to track S phase activity in cardiac myocytes, so these are cells are starting to replicate. And Kai crossed them into a different genetic background. And when he sent the mice back to us to analyze the hearts, we observed that we saw things that we never saw before in our experiments here.

His injury model was different than ours and now the mouse also had a genetic background, so we had to spend about a year to figure out if it was the injury model or the background. It turned out to be the genetic background, and the phenotype was these mice had about a 15-fold elevated level of cell cycle reentry. So then it became a relatively simple genetics game where we took the progenitor mice, made F1 animals, looked for the phenotype, did backcross animals, and basically identified the gene responsible for the phenotype.

Dr. Greg Hundley:

Very nice. And so in this study moving forward, what hypothesis did you want to address?

Dr. Loren Field:

Well, the main hypothesis was to figure out what the gene was and then secondarily to figure out the degree of cell cycle progression. When the cell is proliferating, the first task is to replicate its genome, which is S phase activity that's followed by the nuclei dividing and then finally by the cell itself becoming two cells. So our task was to identify, first, the gene and secondly, how far through the cell cycles the cells progressed.

Dr. Greg Hundley:

Very nice. And how did you construct your experiment?

Dr. Loren Field:

It was, again, very straightforward. It was simply setting up the appropriate genetic crosses to produce the animals. For the past 10, 15 years, we've been developing a computer assisted assay that allows us to identify the anatomical position of S phase positive cardiac myocytes in sections of the heart. And basically, we apply that program to the different genetic backgrounds and after that it's a ball of mapping studies, QTL mapping.

Dr. Greg Hundley:

So really mechanistic understanding. Well listeners, we're next going to turn to Sean, and Sean, can you describe for us your study results?

Dr. Sean Reuter:

Yes, as Loren stated, we saw a 15-fold increase in the S phase activity within the remote zone. Now we partition the heart in three different zones after injury, so the scar, the border zone, and then the remote zone or injury. And as Loren stated, we saw a 15-fold increase in the S phase activity, cell cycle activity, in the remote zone. And it's only because we have this system in hand that we can anatomically map the S phase activity within the heart that we were able to detect and also quantify this. And I think that's the reason we discovered this particular phenotype. But in addition to that, we performed RNA-seq or Exome sequencing and discovered that TNNI3K was the responsible gene for elevated S phase activity within the remote zone and border zone, but interestingly not in the scar.

Dr. Greg Hundley:

Very interesting, Sean, and so describe for us the importance of the TNNI3K and its relationship to this S phase.

Dr. Sean Reuter:

Sure. This particular gene was first discovered around 2000, and it's been studied for a while now, but the targets of this kinase specifically expressed in the heart, and it does get elevated after injury, but the actual targets are not well described or well known. It's believed that it phosphorylates some mild filament fibers and structural proteins, but the actual mechanism and the consequence of this is not known. So when we saw this in the remote zone, the elevated S phase, our current theory is that we believe that it's probably increasing oxidative stress that would basically further out from the at-risk zone or the border zone and then it now is in the remote zone. So we think it's just causing the heart, a pathological area of the heart, basically to expand. And so that's our current theory. Other groups have published on the oxidative stress in over expression of TNNI3K as well.

Dr. Greg Hundley:

Very nice. Well listeners, next we are going to turn to our associate editor, Thomas many articles come your way and come across your desk. What attracted you to this particular article, and how do we put its results really in the context of cardiac regeneration?

Dr. Thomas Eschenhagen:

Indeed, there were several arguments. It's a cool paper and the whole field is still very important. As probably most of you know, the field have a rough ride over the last 20 years, went up and down, lots of bad findings. And in the end it turns out that we are there where we have been 20 years ago, the mammalian heart essentially doesn't regenerate. So anything which would improve that would be of very major importance. Why is it a good paper? Because it starts from a very clear finding, one mouse, which looks like strongly regenerating after MI, another mouse line, which doesn't. And so by applying, let's say, classical genetic, very stringent methodology, Loren Field and his group identified this troponin I kinase to be the culprit. And they also proved it, because putting it back in the strain with a low, so-called, regeneration brought it back to the other level. So it's a very clear, nice methodology.

And finally, it's also a bit provocative because others in a very prominent paper, actually, have shown that this kinase... Or they concluded more or less just the opposite. The reason for the discrepancy is not quite clear and I was very happy to learn that the two groups actually discussed about it. So it's not just a bad controversy, but something which brings forward science.

And finally, I think something we didn't talk about yet today, what I particularly liked, maybe the most, on this paper is that this group didn't stop at the point of DNA synthesis. Everybody else would've probably said, "Okay, here we are, one regenerate the other doesn't." But in the very important extra finding of this paper is that this is just increased DNA synthesis and not more myocytes. And this distinction is so critical to the field because people forget that adult mammalian cardiomyocytes often have several nuclei and individual nuclei have more than one set of chromosomes, so this polyploid. And so if you see DNA synthesis like in this paper, it doesn't necessarily mean more myocytes. And actually here it was shown that it is not more myocytes but more polyploidization and making this difference so clear, I think it's a very important contribution to the field.

Dr. Greg Hundley:

Very nice. Well, listeners, we're going to turn back to each of our guests today and we'll start with you Loren. Based on your results, what do you see as the next study moving forward in this sphere of research?

Dr. Loren Field:

I think these results made me appreciate for the first time that the intrinsic level of cell cycle reentry, that's just the S phase, not the cell division, is actually much higher than I had thought previously. And this was because we just fortuitously, or I guess anti-fortuitously, we're using a strain that had low levels of S phase induction. If you calculate the turnover, if every nucleus that it synthesized DNA actually went on to have that cell divide, you could replace a 50% loss of myocytes over the course of about 550 days, give or take. And to me, that's actually telling me that if we could push those cells from just being polypoid, as Thomas was saying, to actually go through cytokinesis, there would be enough intrinsic activity to go forward. So this really tells me that what we should be focusing on is now not trying to induce cell cycle, but to allow the cells that are entering the cell cycle to actually progress through it.

Dr. Greg Hundley:

Very nice. And Sean?

Dr. Sean Reuter:

Yes, well, echoing Loren's point there, it's really not necessarily cell cycle induction, it's cell cycle completion to the cytokinetic fate. And that's the key. If we can get to that point, if we can figure out the mechanism to get to that point, then we have a wonderful discovery. However, we're not quite there yet, but we hope to be.

Dr. Greg Hundley:

And Thomas.

Dr. Thomas Eschenhagen:

Well, nothing to add really from my side, except that I would like to know what this Troponin I kinase does, because that is somehow still a missing link. How does this kinase lead to more DNA synthesis or the initiation of cell cycling? That would be an important finding and I'm sure there will be more research going on. Particularly also, to solve this discrepancy, I mean, there must be something in it and we don't quite yet know how, but I think we are in a good way. I'm sure there will be papers showing that soon. So I think that's, again, a very good start for this discussion.

Dr. Greg Hundley:

Well, listeners, we want to thank Dr. Loren Field, Dr. Sean Reuter and Dr. Thomas Eschenhagen for bringing us this really informative study in mammalian myocellular regeneration, highlighting that the level of cardiomyocyte cell cycle reentry in hearts expressing TNNI3 kinase would lead to significant regenerative growth if each cardiomyocyte exhibiting S phase activity was able to progress through cytokinesis. And this in turn suggests that identification of factors which facilitate cardiomyocyte cell cycle progression beyond S phase will be key to unlocking the intrinsic regenerative capacity of the heart.

Well, on behalf of Carolyn, Peder and myself, we want to wish you a great week and we will catch you next week on the run. This program is copyright of the American Heart Association 2023. 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, please visit ahajournals.org.