February 2021 Discover CircRes - a podcast by Cynthia St. Hilaire, PhD & Milka Koupenova, PhD
from 2021-02-18T19:00
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.
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