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.