May 2021 Discover CircRes

This month on Episode 24 of Discover CircRes, host Cindy St. Hilaire highlights the topics covered in the May 14th Compendium on Heart Failure, as well as discussing two original research articles and a brief overview of the Review Series on Calcific Aortic Valve Disease from the April 30th issue of Circulation Research. This episode also features an in-depth conversation with Dr David Durgan and Huanan Shi from Baylor School of Medicine about their study Restructuring The Gut Microbiota by Intermittent Fasting Lowers Blood Pressure.   Article highlights:   Vacante, et al. CARMN Regulates Atherosclerosis via SMC Modulation   Hanna, et al. Cardiac Neuronal Control of the Sinoatrial Node   Cuevas, et al. Introduction to the Aortic Valve Disease Series   Compendium on Heart Failure   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 will be highlighting topics presented in our April 30th and May 14th issues of Circ Res. I'll also speak with Dr David Durgan and his graduate student, Huanan Shi, from Baylor School of Medicine about their study Restructuring The Gut Microbiota by Intermittent Fasting Lowers Blood Pressure.   Cindy St. Hilaire:        The first article I want to share comes from the April 30th issue of Circ Res and is titled CARMN Loss Regulates Smooth Muscle Cells and Accelerates Atherosclerosis in Mice. The first author is Francesca Vacante and the corresponding author is Andrew Baker, and they're from the University of Edinburgh. The increased proliferation and migration of local vascular smooth muscle cells is part of the complex pathology of atherosclerotic plaques. These proatherogenic changes to smooth muscle cells are regulated in part via two micro RNAs, miR-143 and miR-145. And these are located together on human chromosome five. In this very same genetic locus is also a gene encoding a long non-coding RNA called cardiac mesoderm enhancer-associated non-coding RNA or CARMN. Cindy St. Hilaire:        This team found that levels of CARMN and miR-143/145 RNAs in mouse and human atherosclerotic plaque decreased as the condition worsened. Mechanistic experiments showed that this decrease drove smooth muscle cell pathology. Knock down of all three RNAs promoted increased proliferation and migration of human artery smooth muscle cells with the loss of CARMN specifically and independently, triggering increased proliferation. The team went on to show that in mice, loss of CARMN accelerated the progression of induced atherosclerosis. Together, the work highlights the interplay between these noncoding RNAs and atherosclerotic disease progression. Cindy St. Hilaire:        The second article I want to share is titled Innervation and Neuronal Control of the Mammalian Sinoatrial Node, a Comprehensive Atlas. The first author is Peter Hanna and the corresponding author is Kalyanam Shivkumar from UCLA. The nervous system regulates cardiac physiology and influences pathophysiological adaptations to disease. Mapping the intrinsic neurocircuitry of the heart is necessary if we are to fully understand how neural circuits function in health and in diseases such as arrhythmia. Neural circuits from outside the heart meet up with those within the heart at ganglionated plexuses on the heart surface. One such plexus is the right atrial ganglionated plexus, RGAP. And RAGP is thought to regulate the signal inputs from the vagus nerve into the sinoatrial node or the SAN, which is the heart's pacemaker. Cindy St. Hilaire:        To develop a detailed description of the connections between the RAGP and the SAN, this group used the pig's heart as it is a close anatomical match to that in a human's. Performing a combination of tissue clearing, immunohistochemistry and 3D fluorescent microscopy, this group showed that approximately 99% of the neurons in RAGP and most of those innervating the SAN, are cholinergic neurons. In spite of this, single cell transcriptomic analysis revealed a great deal of phenotypic diversity among these RAGP neurons. Through electrophysiological and neural ablation studies, the team revealed the extent of RAGPs modulation of the sinoatrial node functions, which characterizes the RAGP as an integrative neural structure and not just a relay station within the intrinsic cardiac nervous system. This work now creates a very detailed reference atlas of the RAGP sinoatrial node conductivity and a framework for mapping other aspects of the intrinsic cardiac nervous system. Cindy St. Hilaire:        The April 30th issue of Circ Res also has a short review series on calcific aortic valve disease. Dr Rolando Cuevas and I write an introduction to this series. Dr Joy Lincoln covers genetic and developmental contributors to aortic stenosis, Dr Jonathan Butcher covers inflammatory and biomechanical drivers of endothelial interstitial interactions in calcific aortic valve disease. Dr Tom Gleason covers current therapeutic options in aortic stenosis and Dr  Elena Aikawa covers multi-ohmic approaches to define calcific aortic valve disease pathogenesis. Cindy St. Hilaire:        The May 14th issue of Circulation Research is the heart failure compendium. This features 10 articles written by the leading experts, who present an update on the state of the field of heart failure research and current therapeutic options. Dr Douglas Mann is the guest editor of this compendium, and in his introduction, he emphasized his vision that the authors of this series, "Focusing on linking disease pathophysiology with the mechanisms action of current therapies, with the hope that past successes would serve as a prologue for the development of future therapies." Together, these Reviews present the recent therapeutic advances in heart failure and is truly representative of the successful transition of bench top research to the bedside of patients. Cindy St. Hilaire:        In the first article in the compendium, Dr Veronique Rogers provides an update on heart failure epidemiology, including a focus on the role of healthcare disparities. Dr Michael Felker, and Dr Mann follow with an overview of the pathophysiology of heart failure with reduced ejection fraction and highlight how several successful heart failure trials fit or do not fit into the current conceptual translational models of heart failure. Dr Walter Paulus and Michael Zile discuss heart failure with preserved ejection fraction, with a focus on the role of systemic inflammation and myocardial stiffness, and relate this pathophysiology to distinct clinical phenotypes and tailored medical therapies. Cindy St. Hilaire:        Drs Joyce Njoroge and John Teerlink discuss what is currently known regarding pathophysiology of acute decompensated heart failure, and present a handful of new therapy developments. Drs Gary Lopaschuk, Qutuba Karwi, Rong Tian, Adam Wende, and Dale Abel discuss cardiac energy metabolism and heart failure and review several promising approaches to beneficially altering metabolism in the failing heart. Highly relevant to the long-term cardiovascular phenotype seen in patients who have had COVID-19, Drs Ray Hershberger, Jason Cowan, Elizabeth Jordan, and Daniel Kinnamon reviewed the genetic basis for dilated cardiomyopathy and discuss what is known regarding the interaction of genetic risk and environmental factors. Cindy St. Hilaire:        Drs Jan Griffin, Hannah Rosenblum and Matthew Maurer discussed cardiac amyloidosis due to light chain or transthyretin amyloidosis and cover current effective therapeutic strategies and active clinical trials. Drs Virginia Hahn, Kathleen Zhang, Lova Sun, Vivek Narayan, Daniel Lenihan, and Bonnie Ky covered the development of heart failure due to targeted cancer therapies and discuss the rationale and evidence supporting different cardiotherapeutic approaches. Cindy St. Hilaire:        The Compendium concludes with an article by Drs Daniel Burkhoff, Veli Topkara, Gabriel Sayer, and Nir Uriel that discusses the current state of left ventricular assist devices or LVADs and the structural, cellular and molecular aspects of LVAD associated reverse left ventricle remodeling. This comprehensive Compendium on Heart Failure is found in the May 14th issue of Circulation Research. Cindy St. Hilaire:        So today, Dr David Durgan and Huanan Shi from Baylor College of Medicine are here with me to discuss their study, Restructuring the Gut Microbiota by Intermittent Fasting Lowers Blood Pressure, which is in our April 30th issue of Circulation Research. So thank you both very much for joining me today. David Durgan              Pleasure to be here. Cindy St. Hilaire:        So this study is bringing together two hot fields, the gut microbiome and intermittent fasting, and it's in the context of high blood pressure, which obviously is a national and global crisis. But before we jump into the details of the paper, could you just define what is meant by gut microbiome and intermittent fasting for the purposes of the discussion? David Durgan:             Sure. So when were you referred to the gut microbiome or the gut microbiome, what we're really referring to there are all the microbes that are residing in the gut. So this can be the complex composition of bacteria, viruses, fungi. However, for the purposes of our studies, we really focus in just on the bacteria. Cindy St. Hilaire:        So how did you even come to this question? What was the premise that existed such that you wanted to ask this question? Microbiome, intermittent fasting, and hypertension? Dr David Durgan:       It really started in terms of understanding the connection between the biome and hypertension. And this actually all started in a separate model of hypertension that we developed here in our lab. And that was a model of obstructive sleep apnea. When we were first developing this and characterizing this, one of the strange observations that we found is that these animals did not have any change in blood pressure, which was contrary to what we see in patients and even what they see in the intermittent hypoxia models. David Durgan:             So when we started thinking about OSA and the patient, we started thinking about all these other co-morbidities, one of them being obesity and poor diet. So at this point we started adding in other morbidities, such as a high-fat diet. And we found that very quickly within one week, actually, when we had the combination of both apnea and high fat diet, that this was then leading to the increase in blood pressure. And really lucky, right place at the right time was that we were thinking about what the high-fat diet was doing. And there was a seminar here on campus about the gut microbiota, which we really had done nothing with up to that point. And after attending that, it quickly became obvious that our high-fat diet was going to be shifting the biota. So this is what really led us to making this connection between changes to the microbiome and blood pressure. Cindy St. Hilaire:        So can you tell me little bit about the design of your study, about the animal systems you use and the diet and the regime that you put them on? David Durgan:             Sure. So we went into this with two overall questions. So we had already shown previously in this model that the biota was disrupted and that was contributing at least to the hypertensive phenotype. So we came into this and wanted to address the questions of, one, what are the mechanisms through which the microbiota is influencing host blood pressure? And then two, is there some type of intervention that we could do to shift the makeup of the microbiota and see how that affected the hypertensive phenotype? David Durgan:             So to address those two components, we took the spontaneously hypertensive stroke prone rat, and it's normotensive parent strain, the WKY, and we put them either on a normal ad libitum food access or every other day fasting, which is just as it sounds, it was a full 24 hours of ad-lib access followed by 24 hours of no food access at all. David Durgan:             And this went on for 10 weeks with constant assessment of food intake, body weight, blood pressure. And then at the end, we isolated fecal content in order to look at the effects on the biome. And that was done with whole genome shotgun sequencing, but we also did a on-targeted metabolomics approach of both the fecal content and the plasma in order to get a real understanding of what are some of the microbial metabolites that could be influencing hosts. Cindy St. Hilaire:        Such an interesting question. And it's such a complex idea, but I thought you did a really great job winnowing it down as your paper progressed. And you did find that the every other day feeding reduced blood pressure in the hypertensive stroke prone rats, and interestingly or maybe not interestingly to you, but I thought it was interesting, is that those every other day fed animals, they certainly ate more on the days when they were allowed to eat. And obviously on the days they weren't, they were eating less. And so their overall food intake was less. And ultimately at the end of your trial period, their weight was less. So are these effects that you see on blood pressure more directly related to the weight loss or to the actual microbiome? And how did you confirm that? Huanan Shi:                So that's actually a very good question. A lot of the intermittent fasting related studies definitely can separate the effects of the fasting itself and the effects of the weight loss as intermittent fasting has been used very frequently as a method for obesity and reduce body weight. So to confirm that the effects is through intermittent fasting, to restructuring the microbiome, a sort of indirect route from the weight loss, so we collected fecal sample of these animals that have been fed either on the intermittent fasting protocol or ad libitum with food access every day. We then transferred the fecal content through our garage into germ-free rats which they do not have an established gut microbiota. Huanan Shi:                So these germ-free animals who'll receive the hypertensive HSR mode] biota with just regular feeding pattern also developed high blood pressure compared to those who received the normotensive microbiota. So interestingly is that the animals that received the microbiota from the hypertensive animal that also was fed on the fasting protocol did not develop a high blood pressure. So this study actually tells us that maybe the weight loss have some effects, but through the microbiota transplant study, we show that majority or the conjoined factors mostly from the changes in microbiota instead of the weight loss. Cindy St. Hilaire:        Yeah. It also makes me wonder how much of the microbiota changes actually influence the weight loss as well. I wonder that's probably a whole another black box to open. You did find that in the feeding regime differences, there was a difference in the actual communities of bacteria. Can you talk maybe about the implications of what that means, and also does that mean this is perhaps something that we could recapitulate with a pill, like with a probiotic pill of some sort? David Durgan:             Yeah. So some of the overall changes that we see, we do see pretty drastic changes in the beta diversity of the community. This being things like richness, evenness, the number of species that are actually present and really pretty interestingly, the way that we saw those shift was that the SHR that were undergoing the fasting protocol, their community structure overall seemed to shift more closely to resemble that of the normotensive WKY. So there were some pretty large shifts. And then when we get down to some of the genera and species levels, we again see that many of these are being normalized to look much more like the communities of the WKY. David Durgan:             In terms of taking a pill or something along that source, somewhat surprisingly, actually we found that in the hypertensive animal, a number of species that are commonly thought of as probiotics. So for instance, bifidobacteria and lactobacillus, which are two of the only FDA approved genera for probiotics, they were actually higher in our SHR control fed animals. So I think there's still a lot of work to be done to understand exactly the contribution of individual species. Maybe what's more important is understanding the functional output from the community as a whole. So what are some of the metabolites that are actually influencing the host? Cindy St. Hilaire:        So it may not be the bacteria itself, but perhaps the products that create. David Durgan:             Right. And the thing that's frustrating, but also exciting about this is that there's so much functional redundancy between different species, meaning that while you could have loss of one species, it may look very significant on paper, but it could be that other species in the community are making up for that. So they're able to make the same metabolites and thereby overcome that deficiency. Cindy St. Hilaire:        Got it. Got it. So it may not really be that big of a shift per se. David Durgan:             Yeah. We can't always go off of just what is the species change and that's why we really moved and thought it was important to move on to looking at the metabolites themselves. Cindy St. Hilaire:        So you did see that these hypertensive rats had more an inflammatory profile in certain sections of the gut. And I was trying to think about this in terms of humans. And I don't know if it's known, but do patients with IBS or with chronic diseases like Crohn's disease or some other gut inflammation phenotype, do they actually have more hypertension or develop it earlier? I guess I'm thinking of this in terms of cause and consequence, the hypertension influence the gut microbiome, or do you think the microbiome perhaps is driving the hypertension? David Durgan:             That's a great question. I've tried to look and see if there's any real conclusive evidence for inflammatory GI disorders and a concrete connection to elevated blood pressure. Personally, I've not found convincing evidence of that at this point in time. Cindy St. Hilaire:        So in terms of the metabolites, I thought it was really interesting that you found, I think it was a reduction in bile acids, and specifically you then explored further choline, that that was at play in this hypertensive state. So can you discuss what it is you exactly found and then what this might mean in terms of hypertension pathogenesis? David Durgan:             Yeah. So from our un-targeted metabolomics data, we performed random forest analysis to try and understand some of the broad pathways that were altered. First of all, just differences between our hypertensive and normotensive control animals, but then also how the fasting affected those metabolites. And there were a number of pathways of interests, which need to be followed up on, but the one that really stood out to us was primary and secondary bile acid metabolism. Now, we followed up on this by doing a targeted approach to look at a specific panel of primary and secondary bile acids. And we were really very surprised at just how different they were. We measured, I believe, 17 different bile acids. And we found that in the plasma, that 12 of these were significantly lower in a hypertensive model. So this was really exciting. David Durgan:             And the more we looked into this, it really all make sense in terms of that if you look at where bile acid receptors are located, they're present in the endothelium and smooth muscle, in brain, on inflammatory cells. So we've really, I think, just started to see the tip of the iceberg in terms of their effects systemically. In a final figure of our paper, we look at their effects on vascular function and show that by giving a TGR5 agonist, which is one of the bile acid receptors that we could improve vascular function in this hypertensive model. I mean, bile acids classically have really been looked at in regards of strictly in the liver and the GI and in the inner hepatic circulation. But just the fact that we see these receptors so widespread systemically really tells us that even though the concentrations may seem low and plasma relative to in the GI tract, that they're very likely having pretty profound effects on overall physiology. Cindy St. Hilaire:        . So do either of you follow an intermittent fasting diet and also, I guess more specifically about IF is these rats, the study, you did every other day feeding. So for humans, obviously I think right now it's Ramadan so a lot of people are almost doing that now. But for humans, that seems like a stretch. I don't think I would really want to do that regularly. So do you think any of these findings could also be similar for different forms of intermittent fasting? I know like that 8/16 hour breakdown is the popular one. What do you think about that? David Durgan:             I personally have not tried it. There have been some grad students that have come through the lab and actually one of the investigators on this paper who worked with me to develop this idea, he was very into ... I think he did the 16/8 that you're referring to. Cindy St. Hilaire:        Okay. Yeah. David Durgan:             But yeah, I mean, that's really kind of been a hindrance almost in the field is that you go to understand clinical studies on intermittent fasting, and there's just so many different protocols out there. Whether it be looking at outcomes or blood pressure or whatever effect on physiology during Ramadan, during a 16/8, during every other day. And it's really muddied the waters in terms of understanding the overall effects, but looking through all of that, it does appear that even in the small clinical studies that are out there, that there does appear to be some benefit. There is a every other day fasting. So very similar to exactly the same as our protocol in a small randomized controlled trial. And it should be said that these were healthy individuals, but even after just four weeks of EODF or every other day fasting, there was about a 5 mm decrease in blood pressure in individuals. David Durgan:             The followup papers from that group really should be very interesting. So these individuals have now gone back on a normal feeding regimen, but they plan, I believe, to look at intervals out to two years to see how long lasting these effects are. Cindy St. Hilaire:        Interesting. The other thing with humans, I mean, obviously your rats, they're eating one meal, the same meal, essentially. Humans eat a variety of things at every meal at every different day. Sometimes they have a bag of candy because it's Easter or whatever, so that doesn't help either. So what was the most challenging part of the study? David Durgan:             This required a lot of legwork by Fred in terms of some of the multi-omics analysis. Huanan Shi:                For me, two part. One part is definitely analyze these data using the machine learning protocol. A lot of things I had to learn from scratch. And eventually, it's a lot of time-consuming troubleshooting, but I'm glad everything went through pretty well. I guess something else will be since these rats are eating and fasting at the same time every day and so you have to come in every day at the same time to change cages,  like food. So, yeah. Cindy St. Hilaire:        Collect poop. Well, it was a beautiful, really well done study. I thought it was super interesting. We talk about what's the next podcast going to be at all of our editorial meetings and this paper, everyone thought it was a great topic. It's just really timely with the intermittent fasting. It was really wonderful. What do you think is next? What are you going to do next on this? David Durgan:             So I think there's a lot to do next. I think that one of the most interesting ideas really I alluded to earlier, and that is the widespread distribution of these bile acid receptors. So while we've taken an initial look at vascular function, I think that there's a lot to do elsewhere. We're really interested in how this could be affecting the neurological component of hypertension. Many of these bile acids signaling pathways have been shown to be anti-inflammatory. So do we see changes in neuro inflammation, in sympathetic output? One we're capable of elevating bile acids, which are capable of passing the blood-brain barrier should be noted. So that's definitely one. And then also just beginning to look at how translational this might be. So do we see changes in bile acids in hypertensive patients as well? Cindy St. Hilaire:        You're talking about the receptors and that last figure paper figure seven where you use, I forget if it was an agonist or antagonist, but you modulated that receptor activity. Do resistance arteries, which have a bigger role in hypertension, do they have higher or different levels of expression than other vascular beds in the body? Or do we not know that yet? David Durgan:             I can think of studies that have shown similar results in terms of bile acids on vascular function, both in aorta and in mesenteric arteries. But whether the distribution is different on these receptors, I'm not really sure that's known. Cindy St. Hilaire:        Well, there's lots of super interesting questions. I mean, I came up with bunches more that I wanted to know based on the study. So I'm sure that will pan out for you, hopefully with lots more great papers like this one and funding and congrats on an excellent graduate student paper. It was a real great story. And thank you both for joining me today. David Durgan:             Thank you very much. Cindy St. Hilaire:        That's it for the highlights from the April 30th and May 14th issues of Circulation Research. Thank you for listening. Please check out the Circ Res Facebook page and follow us on Twitter and on Instagram with the handle @circres and hashtag discovercircres. Thank you to our guests, Dr David Durgan and Huanan Shi. 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 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 up-to-date and exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association, 2021. The opinions expressed by the speakers in this podcast are their own and not necessarily those of the editors or the American Heart Association. For more information, visit ahajournals.org.