Circulation September 14, 2021 Issue

This week's episode features special Guest Host Mercedes
Carnethon, as she interviews author Miriam Cortese-Krott and
Associate Editor Charles Lowenstein as they discuss the article
"Red Blood Cell and Endothelial eNOS Independently Regulate
Circulating Nitric Oxide Metabolites and Blood
Pressure."


Dr. Carolyn Lam:


Welcome to Circulation on the Run, your weekly podcast, summary,
and backstage pass to the journal and its editors. We're your
co-host I'm Dr. Carolyn Lam, associate editor from The National
Heart Center in Duke National University of Singapore.


Dr. Greg Hundley:


And I'm Dr. Greg Hundley, associate editor, director of the
Pauley Heart Center at VCU Health in Richmond, Virginia.


Dr. Carolyn Lam:


Greg, today's feature paper is one of those really, really
landmark papers that really advance our understanding of Nitric
oxide signaling. And it's about red blood cell and Endothelial
eNOS, and how they independently regulate circulating nitric
oxide, metabolites, and blood pressure. A real, real must, but
let's go on and look at the other papers in this issue first.
Greg, you want to go first?


Dr. Greg Hundley:


You bet, Carolyn. Better grab a cup of coffee. And my first paper
is from professor Nathan Mewton from Hôpital Louis Pradel
Hospices Civils de Lyon. Carolyn, these authors hypothesized that
Colchicine a potent anti-inflammatory agent may reduce infarct
size in left ventricular remodeling at the acute phase of STEMI.
And so to address this hypothesis, they performed a double-blind
multi-center trial and randomly assigned patients admitted for a
first episode of STEMI referred for primary PTCA to receive oral
Colchicine two-milligram loading dose followed by 0.5 milligrams
twice a day, or matching placebo from admission to day five and
the primary efficacy outcome was infarct size determined by
cardiovascular magnetic resonance imaging at five days. And the
relative left ventricular end-diastolic volume change at three
months and infarct size at three months was also assessed by
cardiac MRI. And these were secondary outcomes.


Dr. Carolyn Lam:


Nice. Okay. So what were the results?


Dr. Greg Hundley:


Right, Carolyn. So 192 patients were enrolled. 101 in the
Colchicine group and 91 in the controls. And as a result of this
trial, the oral administration of high dose Colchicine at the
time of Reperfusion. And for five days thereafter did not reduce
infarct size assessed by cardiac MRI. And so Carolyn, the
clinical implications of these results suggest that other studies
exploring the timing, pharma kinetics, and dose-response of
Colchicine, as well as other anti-inflammatory agents are needed
to identify an effective method to reduce infarct size and limit
remodeling in this group of patients.


Dr. Carolyn Lam:


Wow, it's just such a rich field done with all this about
Colchicine. Well, our next paper is a pre-specified sub-analysis
of the randomized EAST-AFNET 4 Trial and the sub-analysis assess
the effect of systematic early rhythm control therapy that is
using Antiarrhythmic drugs or catheter ablation compared to usual
care, which means allowing rhythm control therapy to improve
symptoms in patients with heart failure. And this was defined in
the sub-analysis as the presence of heart failure symptoms of New
York Heart Association status two to three or a left ventricular
ejection fraction of less than 50%.


Dr. Carolyn Lam:


Now, the authors led by Dr. Kirchhof at University Heart and
Vascular Center UKE in Hamburg, Germany included 798 patients in
this sub-analysis of whom 442 had HFpEF, 211 had heart failure
with mid-range ejection fraction and 132 had HF-rEF over a median
of 5.1 years of follow-up the composite primary outcome of
cardiovascular death stroke or hospitalization for worsening
heart failure, or for acute coronary syndrome occurred less often
in patients randomized to early rhythm control therapy compared
with patients randomized to usual care. And this was not altered
by heart failure status with an interaction P-value of 0.6. Left
ventricular function, symptoms, and quality of life improved
equally in both treatment strategies.


Dr. Greg Hundley:


Wow, Carolyn, a lot of information here. So what can we take away
from this?


Dr. Carolyn Lam:


Well, let's remember that this is a sub-analysis, albeit
pre-specified of that randomized trial of the EAST-AFNET 4 Trial,
but nonetheless, the data supports a treatment strategy of rhythm
control therapy with Antiarrhythmic drugs or ablation within a
year of diagnosing atrial fibrillation in patients with signs and
symptoms of heart failure to reduce cardiovascular outcomes.


Dr. Greg Hundley:


Very nice, Carolyn. So, Carolyn, my next paper pertains to
Alarmin Interleukin-1 Alpha, and it comes to us from Dr.
Thimoteus Speer at Saarland University. So, Carolyn, Alarmin
Interleukin-1 Alpha is expressed in a variety of cell types,
promoting sterile systemic inflammation. And the aim of the
present study was to examine the role of Alarmin Interleukin-1
Alpha in mediating inflammation in the setting of acute
myocardial infarction and chronic kidney disease.


Dr. Carolyn Lam:


Wow, sterile inflammation. It's a really hot topic now. So what
did these authors find?


Dr. Greg Hundley:


Right, Carolyn. So we're going to call Alarmin Interleukin-1
Alpha. Let's just call it IL-1 Alpha and so increased IL-1 Alpha
surface expression on monocytes from patients with acute
myocardial infarction in patients with chronic kidney disease was
found to be associated with cardiovascular events. Next, IL-1
Alphas itself served as an adhesion molecule, mediating
leukocyte-endothelial adhesion, and finally, abrogation of IL-1
alpha prevented inflammation after myocardial infarction and
ameliorated chronic kidney disease in Vivo.


Dr. Carolyn Lam:


Wow. So what does this mean clinically?


Dr. Greg Hundley:


Right, Carolyn, so perhaps targeted therapeutic inhibition of
IL-1 Alpha might represent a novel anti-inflammatory treatment
strategy in patients with myocardial infarction and in patients
with chronic kidney disease.


Dr. Carolyn Lam:


Amazing. Thanks, Greg. Well, in today's issue, there's also an
exchange of letters between doctors Lother and Filippatos on
Finerenone and risk of hyperkalemia in CKD and type two diabetes.
There's an On My Mind paper by Dr. Sattler on the single-cell
immunology and cardiovascular METs in, do we know yet what we
don't know?


Dr. Greg Hundley:


And then Carolyn, from the mailbag, a Research Letter from
Professor Wehrens entitled “Atrial Specific LK Beta One Knockdown
Represents a Novel Mouse Model of Atrial Cardiomyopathy with
Spontaneous Atrial Fibrillation.” Well, Carolyn, how about we
turn our attention to those red blood cells and endothelial
nitric oxide synthase.


Dr. Carolyn Lam:


Yeah. Can't wait.


Dr. Mercedes Carnethon:


Well, welcome to this episode of Circulation on the Run. Our
podcasts, where we have an opportunity to speak with authors of
important papers that are appearing in the journal of
circulation. I'm pleased to introduce myself. My name is Mercedes
Carnethon, professor and vice-chair of preventive medicine at the
Northwestern University Feinberg School of Medicine. And I'm
pleased today to invite our guest author, Miriam Cortese-Krott,
who is the faculty of the University of Duesseldorf, and a guest
professor at the Karolinska Institute in Stockholm. And we have
with us as well the other associate editor who handled the piece
for circulation, Dr. Charlie Lowenstein from Johns Hopkins
University. So welcome to each of you this morning.


Miriam Cortese-krott:


Thank you.


Dr. Charles Lowenstein :


Thanks for having me.


Dr. Mercedes Carnethon:


Well, thank you. I'm really excited to jump right into this
piece, Miriam, can you tell me a little bit about the rationale
for carrying out the study, why you pursued it?


Professor Miriam Cortese-Krott:


The reason is because when I was working as a post-doc, I had to
isolate an enzyme from red blood cells, which is a very, very
difficult. And if you know, this enzyme is endothelial nitric
oxide synthase, which produce nitric oxide, and actually, the red
blood cell is full of the worst enemy of nitric oxide, which is
hemoglobin. So actually, when I was talking about my project,
everybody was asking, "Why are you doing that?" And I was
actually able to isolate the enzyme and look at activity and be
sure that the enzyme was fine, but the function of this enzyme
was absolutely unknown.


Professor Miriam Cortese-Krott:


And the only way to study proteins in red blood cells is to make
modification in the bone marrow of the mice. So in the Erythroid
cells, because you can not, of course, if there are cells without
nucleus you don't have any chance to modify them in culture,
something like that. So the only way was to generate mice with
modification specifically in the red blood cells. And I had the
chance to create, to generate red cell-specific eNOS knockout
mice. And of course, as a control endothelial-specific eNOS
knockout mice by using the Cre-loxP technology. And with this
technology, I could really understand what's happening to the
physiology of the mouse if you remove this protein from the red
blood cells. And so this was the whole idea.


Dr. Mercedes Carnethon:


Thank you so much. It was really exciting for me to read this
piece. We are on opposite ends of the scientific inquiry spread
as I'm an epidemiologist who does things at the population level,
and you're identifying things at the basic science level. I
thought the paper was extremely well-written and that encouraged
people to dig in, even if you're unfamiliar, and in part that's
because you provided such a great explanation of how your
findings are used and how they're relevant to the process. Do you
mind sharing a little bit about your findings and how you expect
that they will be used by our scientific community?


Professor Miriam Cortese-Krott:


I think the main finding of this paper is that if you remove eNOS
from the red blood cells if the mice are hypertensive, have
hypertension, and this is completely something that you actually
will not expect, as I told you that indeed red cells are full of
the enemy of nitric oxide that remove it immediately. So you can
ask yourself how it is possible. But I think the key finding here
in this paper was that I also generated the opposite model. So I
created the model a conditional eNOS Knockout model where you can
decide in which tissue you want to have your enzyme. And of
course, I applied for red blood cells. And what you see in this
model is that you start from a global knockout mouse with
hypertension, you reintroduce the eNOS just in the red blood
cells, you have normal tension. So this means, this is the main
finding. You have a switch in the red blood cells, which is the
enzyme eNOS, which it's behaving in a completely different way
clearly as compared to the vessel wall eNOS and still regulating
blood pressure.


Dr. Mercedes Carnethon:


Well, thank you so much. I think this is the point at which I
like to turn to the associate editor who handled the piece.
Charlie, you and I don't get to talk as often given the diversity
of work that we each pursue, but Charlie, tell me a little bit
about what excited you about this piece?


Dr. Charles Lowenstein:


Thanks, Mercedes. So I love this piece. I thought Miriam, your
article is so great. So a couple of thoughts. One is nitric oxide
and nitric oxide synthase are so important in biology and
medicine, nitric oxide regulates blood pressure. It regulates
neurotransmission. It regulates inflammation. And this is true,
not only in the lab, looking at cells in mice, but also in the
human. So genetic variance in the endothelial nitric oxide
synthase gene or NOS3 are associated with risks for diseases like
coronary artery disease. So eNOS is just so important in biology
and medicine. And now some ancient history. When I was a
cardiology fellow, about a hundred years ago, I worked in the lab
that first purified nitric oxide synthase proteins, and we cloned
two of the nitric oxide synthase genes that was in the lab of Dr.
Solomon Snyder at Johns Hopkins back in the 1700s.


Dr. Charles Lowenstein:


So when we cloned the nitric oxide synthase genes, when we and
others did, we made a huge mistake. We chose the names for these
isoforms from the tissue where they were first isolated. So we
called the brain nitric oxide synthase nNOS, because it's a
neurons, macrophages MCnos we called it MCnos and in endothelial
cells, we called it the nitric oxide synthase eNOS or endothelial
NOS. But in the last 20 years, lots of investigators have found
these isoforms are in other cells, not just the original cells at
discovery. And so Miriam's question is just so important, which
cells make endothelial NOS also called NOS3. That's the history.
Now what Miriam has discovered is just so important. I was so
fascinated by her work because as she just said, she made two
amazing discoveries. One, red blood cells make endothelial nitric
oxide synthase.


Dr. Charles Lowenstein:


And that's been a controversy for a long time. Some people have
said, "Yes." Some, "No." And Miriam made the definitive answer.
Yes, red blood cells make eNOS, and secondly, she has discovered
so much about the physiology of ENO coming from red blood cells,
the nitric oxide that's made inside red blood cells regulates
blood pressure. What a magical, interesting, and important
finding. That's a little bit about the history. Nitric oxide and
NOS are important in medicine. The people who originally cloned
and purified the nitric oxide synthase isoforms named them after
the tissue in which they discovered. And Miriam has made a major
discovery that it's not only endothelial cells that make nitric
oxide but also red blood cells.


Dr. Mercedes Carnethon:


Thank you so much for that summary. And I guess, I would have
thought perhaps this was something of an Elixir of youth because
if you've been working in this area for 200 plus years and
Miriam, you started working on this as part of your dissertation
work, you both have a lot of insight and background on where
we've been and what the advances are. Miriam, can you tell me a
little bit about how you'd like to see these findings used by the
scientific community?


Professor Miriam Cortese-Krott:


I think I would like that the scientific community would use my
mice first because I think, as Charles has said, it's not only
red cells that express eNOS and it's not only endothelial cells.
There are other cells producing eNOS and the function in the
other cells is not known even in leukocytes, even when they have
iNOS of course, but also have eNOS. So you can use my mice since
it's a flux model. You can choose whatever you want, what cell
you want, and then knock it in and knock it out. So this is one
thing that I think the community could really do. I cannot do
everything. So I'm happy to give my mice away.


Professor Miriam Cortese-Krott:


And the second thing is I would like too that in particular, the
clinical community would see this link between Emathology and
cardiovascular disease. This is something that was started, of
course, there are studies looking at anemia and cardiovascular
disease, but these studies have sometimes some issues I of course
cannot speak as a basic scientist. I cannot speak about huge
clinical trials, but I think this link exists and exists at the
molecular level and it can be a target for pharmacological
therapy. So I think this is what I would like to transport with
this study to the clinical community and the basic science
community.


Dr. Mercedes Carnethon:


Yeah. I think this is the point at which Charlie, I turn it to
you because you really stand at the intersection of both of those
communities. What questions do you have for Miriam going forward,
as you think about spreading the word on this important work?


Dr. Charles Lowenstein:


So Miriam's discovery is just so important and she now has the
tools to help answer really, really important questions. How is
nitric oxide made in red blood cells? How is it stored in red
blood cells? How is it transported throughout the body in red
blood cells? What is the chemistry of nitric oxide, when it is
stored, when it combines with oxygen when it forms nitrite and
nitrate, how is it released from red blood cells? How is it
targeted from a red blood cell to the vasculature? So there're
these great basic science questions that Miriam and her
colleagues are now poised to answer. So there's the science part
of it. Then there's the medicine part of it because Miriam's mice
and her great discovery have really huge implications for
medicine. And so the question is, how can we use ENO? How can we
deliver it? How can we target ENO to human tissues?


Dr. Charles Lowenstein:


How can we turn on erythrocyte, nitric oxide synthase? How can we
turn it off? Because there are all these medical diseases where
too much nitric oxide is bad, like in sepsis or inadequate
amounts, don't protect the vasculature like atherosclerosis. Then
there are all these other interesting questions. When we
transfuse red blood cells, sometimes if you transfuse aged red
blood cells, it's not good. You can harm people. Maybe we can
load up or activate eNOS in stored red blood cells and then help
deliver more ENO to patients who need red blood cells. So there
are all these fascinating medical questions that we can look at
based on Miriam's really important discovery.


Dr. Mercedes Carnethon:


Well, thank you so much. We're coming to the end of this
wonderful and informative podcast. And I guess, I'd just ask
Miriam, do you have anything else you'd like our listeners to
know about your work and about the findings from this study?


Professor Miriam Cortese-Krott:


I would like people know that hard work help a lot, and that you
have to believe in what you are doing and the quality of your
science at the end would bring their true discoveries. So I think
it's important specifically, for the young women in science that
having this message too. So the science per se must be excellent
and to proceed, you need a lot of work, but then the work goes to
a good end.


Dr. Mercedes Carnethon:


Miriam, thank you so much for that inspirational note. The hard
work that scientists need, the persistence across one's career
and building from earlier discoveries, and bringing those forward
through one's career are always critically important. And so I
hope everyone has really enjoyed this episode and this
opportunity to hear from Dr. Cortese-Krott. Miriam, you've done
such wonderful work, and thank you as well, Charlie, for your
insights about the intersection of this work with clinical care
and basic science.


Professor Miriam Cortese-Krott:


Thank you.


Dr. Charles Lowenstein:


Thank you.


Dr. Mercedes Carnethon:


Thank you all very much for joining us today in this episode of
Circulation on the Run.


Dr. Greg Hundley:


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, visit ahajournals.org.