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This month on Episode 65 of Discover CircRes, host Cindy St. Hilaire highlights articles featured in the September 27th and October 11th issues of Circulation Research. This Episode also includes a discussion with Dr Ken Walsh and Dr Ariel Polizio about their study, Experimental TET2 Clonal Hematopoiesis Predisposes to Renal Hypertension Through an Inflammasome-Mediated Mechanism.
Article highlights:
Ju, et al. NAE1 Crotonylation Regulates Cardiac Hypertrophy
Pirri, et al. EPAS1 Atheroprotection via Fatty Acid Metabolism
Saleem, et al. Myeloid CD11c+ Cells and JAK2/STAT3/SMAD3 in SSBP
Pietsch, et al. Chronic Activation of Tubulin Tyrosination
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This month on Episode 64 of Discover CircRes, host Cindy St. Hilaire highlights articles featured in the August 30th and September 13th issues of Circulation Research. This Episode also includes a discussion with Drs Stephanie Chung, Ahmed Gharib, and Khalid Abd-Elmoniem from NIDDK about their study, Endothelial Dysfunction in Youth-Onset Diabetes Type 2, A Clinical Translational Study.
Article highlights:
Zhao, et al. AMPK Phosphorylation of β-Arrestin-1 Blocking β-AR
Bashore, et al. Monocytes Profiling and Cardiovascular Disease
Chu, et al. Oxysterol-GPR183 Axis and Endothelial Senescence
Sigle, et al. Targeting Secreted Cyclophilin A in Failing Hearts
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This month on Episode 63 of Discover CircRes, host Cindy St. Hilaire highlights articles featured in the August 2nd and August 16th issues of Circulation Research. This Episode also includes a discussion with Drs Chen Gao and Yibin Wang about their study, Glucagon Receptor Antagonist for Heart Failure with Preserved Ejection Fraction
Article highlights:
Douvdevany, et al. Imaging the Turnover of the Sarcomere
Quelquejay, et al. Wnk1 Deletion in Smooth Muscle Cells Induces Aortitis
Paulke, et al. The Role of Dysferlin in Cardiac Hypertrophy
Morais, et al. Predictors of Outcome in SPAN
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This month on Episode 62 of Discover CircRes, host Cindy St. Hilaire highlights articles featured in the July 5th and July 19th issues of Circulation Research. This Episode also includes a discussion with the four finalists for the Basic Cardiovascular Sciences Outstanding Early Career Investigator Award.
Article highlights:
Mallaredy, et al. Extracellular Vesicle Reduction Prevents Heart Failure
Mori, et al. CD163 Macrophages and EndMT in Plaque Progression
Wang, et al. Rbx2 Regulates Mitophagy
Nakayama, et al. ARRDC4 Limits Cardiac Reserve in Diabetes
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This month on Episode 61 of Discover CircRes, host Cindy St. Hilaire highlights articles featured in the June 7th and June 21st issues of Circulation Research. This Episode also includes a discussion with Dr Chris O'Callaghan and Jiahao Jiang from the University of Oxford about their study, A Novel Macrophage Subpopulation Conveys Increased Genetic Risk of Coronary Artery Disease.
Article highlights:
Compendium on Interface Between Cardioimmunology, Myocardial Health, and Disease
Zafeiropoulo, et al. Splenic Ultrasound Improves Pulmonary Hypertension
Roman, et al. MICU3 Enhances Mitochondrial Ca2+ Uptake
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This month on Episode 60 of Discover CircRes, host Cindy St. Hilaire highlights original research articles featured in the May 10 and May 24th issues of Circulation Research. This Episode also includes a discussion with Dr Sophie Astrof and Dr AnnJosette Ramirez from Rutgers University about their study, Buffering Mechanism in Aortic Arch Artery Formation and Congenital Heart Disease.
Article highlights:
Tamiato, et al. Pericyte RGS5 in Cardiac Aging
Zifkos, et al. PTP1B and Venous Thromboinflammation
Ma, et al. NR4A3 in Vascular Calcification
Sultan, et al. VEGF-B Induced Coronary Endothelial Cell Lineage
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This month on Episode 59 of Discover CircRes, host Cindy St. Hilaire highlights original research articles featured in the April 12 and April 26th issues of Circulation Research. This Episode also includes a discussion with Dr Craig Morrell and Chen Li from University of Rochester about their study, Thrombocytopenia Independently Leads to Changes in Monocyte Immune Function.
Article highlights:
Arkelius, et al. LOX-1 and MMP-9 Inhibition Improves Stroke Outcomes
Cruz, et al. C122Y Disrupts Kir2.1-PIP2 Interaction in ATS1
Blaustein, et al. Environmental Impacts on Cardiovascular Health and Biology: An Overview
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This month on Episode 58 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the March 1 and March 15th issues of Circulation Research. This Episode also includes a discussion with Drs Frank Faraci, Tami Martino, and Martin Young about their contributions to the Compendium on Circadian Mechanisms in Cardiovascular and Cerebrovascular Disease.
Article highlights:
Yan, et al. GCN2 in Ponatinib-Induced Cardiotoxicity
Wang, et al. Activating v-ATPase Ameliorates Cardiac Cardiomyopathy
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This month on Episode 57 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the February 2nd and February 19th issues of Circulation Research. This Episode also includes a discussion with Dr Kathryn Howe and Dr Sneha Raju from University of Toronto, about their manuscript titled Directional Endothelial Communication by Polarized Extracellular Vesicle Release.
Article highlights:
Ren, et al. ZBTB20 Regulates Cardiac Contractility
Faleeva, et al. Sox9 Regulates Vascular Extracellular Matrix Aging
Bai, et al. PKA Is Critical for Cardiac Growth
Wang, et al. Indole-3-Propionic Acid Protects Against HFpEF
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This month on Episode 56 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the January 5th and January 19th issues of Circulation Research. This Episode also includes a discussion with Dr Julie Freed and Gopika Senthilkumar from the Medical College of Wisconsin about their study, Necessary Role of Ceramides in the Human Microvascular Endothelium During Health and Disease.
Article highlights:
He, et al. T Cell LGMN Deficiency Prevents Hypertension
Salyer, et al. TnI-Y26 Phosphorylation Improves Relaxation
Jacob, et al. MFN2 in Megakaryocyte and Platelet Function
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This month on Episode 55 of Discover CircRes, host Cynthia St. Hilaireaire highlights two original research articles featured in the December 8th issue of Circulation Research. This Episode also includes a discussion with Dr José Luis de la Pompa and Dr Luis Luna-Zurita from the National Center for Cardiovascular Research in Spain about their study, Cooperative Response to Endocardial NOTCH Reveals Interaction With Hippo Pathway.
Article highlights:
Shi, et al. Nat10 Mediated ac4C in Cardiac Remodeling
Knight, et al. CDK4 Oxidation Attenuates Cell Proliferation
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This month on Episode 54 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the October 27th and November 10th issues of Circulation Research. This Episode also includes a discussion with Dr Sophie Susen and Dr Caterina Casari about their study, Shear Forces Induced Platelet Clearance Is a New Mechanism of Thrombocytopenia, published in the October 27th issue.
Article highlights:
Pass, et al. Single Nuclei Transcriptome of PAD Muscle
Liu, et al. Myocardial Recovery in DCM: CDCP1 and Fibrosis
Grego-Bessa, et al. Neuregulin-1 Regulates Chamber Morphogenesis
Agrawal, et al. A New Model of PH due to HFpEF
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This month on Episode 53 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the September 29th and October 13th issues of Circulation Research. This Episode also includes a discussion with Dr Margaret Schwarz and Dr Dushani Ranasinghe about their study, Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension, published in the September 29 issue.
Article highlights:
Serio, et al. p300/CBP-Upregulated Glycolysis and Cardiac Aging
Sharifi, et al. ADAMTS-7 and TIMP-1 in Atherosclerosis
Zhang, et al. TMEM215 Represses Endothelial Apoptosis
Perike, et al. PPP1R12C Promotes Atrial Hypocontractility in AF
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This month on Episode 52 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the September 1 and September 15th issues of Circulation Research. This Episode also includes a discussion with Dr Manuel Mayr about the study, Proteomic Atlas of Atherosclerosis, the Contribution of Proteoglycans to Sex Differences, Plaque Phenotypes and Outcomes, published in the September 15 issue.
Article highlights:
Sun, et al. CCND2 modRNA Remuscularization Hearts with AMI
Ho, et al. Lymphatic Genes Prevent Cardiac Valve Disease
Shanks, et al. Cardiac Vagal Activity Increases During Exercise
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This month on Episode 51 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the August 4th and August 18th issues of Circulation Research. This Episode also includes a discussion with Dr Eric Small and Dr Xiaoyi Liu from the University of Rochester Medical Center about their article p53 Regulates the Extent of Fibroblast Proliferation and Fibrosis in Left Ventricular Pressure Overload, published in the July 21st issue of the journal.
Article highlights:
Régnier, et al. CTLA-4 Pathway Is Pivotal in Giant Cell Arteritis
Zarkada, et al. Chylomicrons Regulate Lacteal Permeability
Schuermans, et al. Age at Menopause, Telomere Length, and CAD
Bayer, et al. T-cell MyD88 Regulates Fibrosis in Heart Failure
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This month on Episode 50 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the June 23, July 7, and July 21 issues of Circulation Research. This Episode also includes a discussion with BCVS Outstanding Early Career Investigator Award Qiongxin Wang from University of Washington St. Louis, Haobo Li from Massachusetts General Hospital, and Asma Boukhalfa from Tufts Medical Center.
Article highlights:
Tong, et al. The Role of DRP1 in Mitophagy
Abe, et al. ERK5-NRF2 Axis and Senescence-Associated Stemness
Dai, et al. Therapeutic Targeting of Endocytosis Defects in DCM
Weng, et al. PDCD5 Suppresses Cardiac Fibrosis
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This month on Episode 49 of Discover CircRes, host Cynthia St. Hilaire highlights two original research articles featured in the May 26th issue and provides an overview of the June 9th Compendium on Early Cardiovascular Disease of Circulation Research. This Episode also includes a discussion with Dr Tejasvi Dudiki and Dr Tatiana Byzova about their study, Mechanism of Tumor Platelet Communications in Cancer.
Article highlights:
Nichtová, et al. Mitochondria-SR Tethering and Cardiac Remodeling
Ferrucci, et al. Muscle Transcriptomic and Proteomic in PAD
Compendium on Early Cardiovascular Disease.
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This month on Episode 48 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the April 28th issue of Circulation Research. This Episode also includes a discussion between Dr Mina Chung, Dr DeLisa Fairweather and Dr Milka Koupenova, who all contributed to manuscripts to the May 12th Compendium on Covid-19 and the Cardiovascular System.
Article highlights:
Heijman, et al. Mechanisms of Enhanced SK-Channel Current in AF
Chen, et al. IL-37 Attenuates Platelet Activation
Enzan, et al. ZBP1 Protects Against Myocardial Inflammation
Compendium on Covid-19 and the Cardiovascular System.
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. Today, I'm going to be highlighting articles from our April 28th and May 12th issues of Circulation Research. I'm also going to have a chat with Dr Mina Chung, Dr DeLisa Fairweather and Dr Milka Koupenova, who all contributed to articles in the May 12th COVID Compendium. But before we have that interview, let's first talk about some highlights.
The first article I want to present is titled Enhanced Calcium-Dependent SK-Channel Gating and Membrane Trafficking in Human Atrial Fibrillation. This article is coming from the University of Essen by Heijman and Zhou, et al. Atrial fibrillation is one of the most common forms of heart arrhythmia in humans and is characterized by irregular, often rapid heartbeats that can cause palpitations, dizziness and extreme fatigue.
Atrial fibrillation can increase a person's risk of heart failure, and though treatments exist such as beta blockers, blood thinners and antiarrhythmia medications, they can have limited efficacy and side effects. A new family of drugs in development are those blocking small-conductance calcium-activated potassium channels called SK channels, which exhibit increased activity in animal models of AF and suppression of which attenuates the arrhythmia. In humans however, the relationship between SK channels and atrial fibrillation is less clear, at least in terms of SK channel mRNA levels. Because mRNA might not reflect actual channel activity, this group looked at just that and they found indeed that channel activity was increased in cardiomyocytes from atrial fibrillation patients compared to those from controls even though the mRNA and protein levels themselves were similar. The altered currents were instead due to changes in SK channel trafficking and membrane targeting.
By confirming that SK channels play a role in human atrial fibrillation, this work supports the pursuit of SK channel inhibitors as possible new atrial fibrillation treatments.
The next article I want to present is titled IL-37 Attenuates Platelet Activation and Thrombosis Through IL-1R8 Pathway. This article comes from Fudan University by Chen and Hong, et al. Thrombus formation followed by the rupture of a coronary plaque is a major pathophysiological step in the development of a myocardial infarction. Understanding the endogenous antithrombotic factors at play could provide insights and opportunities for developing treatments. With this in mind, Chen and Hong, et al. investigated the role of interleukin-1 receptor 8, or IL-1R8, which suppresses platelet aggregation in mice, and of IL-37, a newly discovered human interleukin that forms a complex with IL-1R8 and is found at increased levels in the blood of patients with myocardial infarction.
Indeed, the amount of IL-37 in myocardial infarction patients negatively correlates with platelet aggregation. They also show that treatment of human platelets in vitro with IL-37 suppresses the cell's aggregation and does so in a concentration-dependent manner. Moreover, injection of the protein into the veins of mice inhibits thrombus development and better preserves heart function even after myocardial infarction. Such effects were not seen in mice lacking IL-1R8. This suggests IL-37's antithrombotic action depends on its interaction with the receptor. Together, the results suggest IL-37 could be developed as a antithrombotic agent for use in MI patients or indeed perhaps other thrombotic conditions.
The last article I want to present before our interview is titled ZBP1 Protects Against Mitochondrial DNA-Induced Myocardial Inflammation in Failing Hearts. This article is coming from Kyushu University and is by Enzan, et al. Myocardial inflammation is a key factor in the pathological progression of heart failure and occurs when damaged mitochondria within the stricken cardiomyocyte release their DNA, triggering an innate inflammatory reaction. In a variety of cells, DNA sensors such as Z-DNA-binding protein 1 or ZBP1 are responsible for such mitochondrial DNA-induced inflammation. In theory then, it's conceivable that therapeutic suppression of ZBP1 might reduce myocardial inflammation in heart failure and preserve function. But as Enzan and colleagues have now discovered to their surprise, mice lacking ZBP1 exhibited worse, not better heart inflammation and more failure after induced myocardial infarction.
Indeed, the test animals' hearts had increased infiltration of immune cells, production of inflammatory cytokines and fibrosis together with decreased function compared with the hearts of mice with normal ZBP1 levels. Experiments in rodent cardiomyocytes further confirmed that loss of ZBP1 exacerbated mitochondrial DNA-induced inflammatory cytokine production while overexpression of ZBP1 had the opposite effect. While the reason behind ZBP1's opposing roles in different cells is not yet clear, the finding suggests that boosting ZBP1 activity in the heart might be a strategy for mitigating heart inflammation after infarction.
Cindy St. Hilaire: The May 12th issue of Circulation Research is our COVID compendium, which consists of a series of 10 reviews on all angles of COVID-19 as it relates to cardiovascular health and disease. Today, three of the authors of the articles in this series are here with me. Dr Mina Chung is a professor of medicine at the Cleveland Clinic. She and Dr Tamanna Singh and their colleagues wrote the article, A Post Pandemic Enigma: The Cardiovascular Impact of Post-Acute Sequelae of SARS-CoV-2. Dr DeLisa Fairweather, professor of medicine, immunology and clinical and translational science at the Mayo Clinic, and she and her colleagues penned the article, COVID-19 Myocarditis and Pericarditis. Dr Milka Koupenova is an assistant professor of medicine at the UMass Chan School of Medical and she led the group writing the article, Platelets and SARS-CoV-2 During COVID-19: Immunity, Thrombosis, and Beyond. Thank you all for joining me today.
DeLisa Fairweather: Thank you so much for having us.
Mina Chung: Thank you.
Milka Koupenova: Thank you for having us, Cindy.
Cindy St. Hilaire: In addition to these three articles, we have another seven that are on all different aspects of COVID. Dr Messinger's group wrote the article, Interaction of COVID-19 With Common Cardiovascular Disorders. Emily Tsai covered cell-specific mechanisms in the heart of COVID-19 patients. Mark Chappell and colleagues wrote about the renin-angiotensin system and sex differences in COVID-19. Michael Bristow covered vaccination-associated myocarditis and myocardial injury. Jow Loacalzo and colleagues covered repurposing drugs for the treatment of COVID-19 and its cardiovascular manifestations. Dr Stephen Holby covered multimodality cardiac imaging in COVID, and Arun Sharma covered microfluidic organ chips in stem cell models in the fight against COVID-19.
Cindy St. Hilaire As of today, worldwide, there have been over six hundred million individuals infected with the virus and more than six and a half million have died from COVID-19. In the US, we are about a sixth of all of those deaths.
Obviously now we're in 2023, the numbers of individuals getting infected and dying are much, much lower. As my husband read to me this morning, one doctor in Boston was quoted saying, "People are still getting wicked sick." In 75% of deaths, people have had underlying conditions and cardiovascular disease is found in about 60% of all those deaths. In the introduction to the compendium, you mentioned that the remarkable COVID-19 rapid response initiative released by the AHA, which again is the parent organization of Circ Research and this podcast, if I were to guess when that rapid response initiative started, I would've guessed well into the pandemic, but it was actually March 26th, 2020. I know in Pittsburgh, our labs have barely shut down. So how soon after we knew of SARS-CoV-2 and COVID, how soon after that did we know that there were cardiovascular complications?
Mina Chung: I think we saw cardiovascular complications happening pretty early. We saw troponin increases very early. It was really amazing what AHA did in terms of this rapid response grant mechanism. You mentioned that the RFA was announced, first of all, putting it together by March 26th when we were just shutting down in March was pretty incredible to get even the RFA out. Then the grants were supposed to be submitted by April 6th and there were 750 grants that were put together and submitted. They were all reviewed within 10 days from 150 volunteer reviewers. The notices were distributed April 23rd, less than a month out.
Cindy St. Hilaire: Amazing.
Mina Chung: So this is an amazing, you're right, paradigm for grant requests and submissions and reviews.
DeLisa Fairweather: For myocarditis, reports of that occurred almost immediately coming out of China, so it was incredibly rapid.
Cindy St. Hilaire: Yeah, and that was a perfect lead up to my next question. Was myocarditis, I guess, the first link or the first clue that this was not just going to be a respiratory infection?
DeLisa Fairweather: I think myocarditis appearing very early, especially it has a history both of being induced by viruses, but being strongly an autoimmune disease, the combination of both of those, I think, started to hint that something different was going to happen, although a lot of people probably didn't realize the significance of that right away.
Cindy St. Hilaire: What other disease states, I guess I'm thinking viruses, but anything, what causes myocarditis and pericarditis normally and how unique is it that we are seeing this as a sequelae of COVID?
DeLisa Fairweather: I think it's not surprising that we find it. Viruses around the world are the primary cause of myocarditis, although in South America, it's the parasite Trypanosoma cruzi. Really, many viruses that also we think target mitochondria, including SARS-CoV-2, have an important role in driving myocarditis. Also, we know that SARS-CoV-1 and MERS also reported myocarditis in those previous infections. We knew about it beforehand that they could cause myocarditis.
Cindy St. Hilaire: Is it presenting differently in a COVID patient than say those South American patients with the... I forget the name of the organism you said, but does it come quickly or get worse quickly or is it all once you get it, it's the same progression?
DeLisa Fairweather: Yeah. That's a good question. Basically, what we find is that no matter what the viral infection is, that myocarditis really appears for signs and symptoms and how we treat it identically and we see that with COVID-19. So that really isn't any different.
Cindy St. Hilaire: Another huge observation that we noticed in COVID-19 patients, which was the increased risk of thrombic outcomes in the patients. Dr Koupenova, Milka, you are a world expert in platelets and viruses and so you and your team were leading the writing of that article. My guess is knowing what you know about platelets and viruses, this wasn't so surprising to you, but could you at least tell us the state of the field in terms of what we knew about viruses and platelets before COVID, before Feb 2020?
Milka Koupenova: Before Feb 2020, we actually knew that influenza gets inside in platelets. It leads to not directly prothrombotic events, but it would lead to release of complement 3 from them. That complement 3 would actually increase the immunothrombosis by pushing neutrophils to release their DNA, forming aggregates. In cases when you have compromised endothelium and people with underlying conditions, you would expect certain thrombotic outcomes. That, we actually published 2019 and then 2020 hit. The difference between influenza and SARS-CoV-2, they're different viruses. They carry their genome in a different RNA strand. I remember thinking perhaps viruses are getting inside in platelets, but perhaps they do not. So we went through surprising discoveries that it seemed like it is another RNA virus. It also got into platelets. It was a bit hard to tweak things surrounding BSL-3 to tell you if the response was the same.
It is still not very clear how much SARS or rather what receptor, particularly when it gets inside would induce an immune response. There are some literature showing the MDA5, but not for sure, may be responsible. But what we found is that once it gets in platelets, it just induces this profound activation of programmed cell death pathways and release of extracellular vesicles and all these prothrombotic, procoagulant form of content that can induce damage around, because platelets are everywhere. So that how it started in 2019 and surprisingly progressed to 2021 or 2020 without the plan of really studying this virus.
Cindy St. Hilaire: How similar and how different is what you observe in platelets infected, obviously in the lab, so I know it's not exactly the same, but how similar and how different is it between the flu? Do you know all the differences yet?
Milka Koupenova: No offense here, they don't get infected.
Cindy St. Hilaire: Okay.
Milka Koupenova: Done the proper research. The virus does not impact platelets, but induces the response.
Cindy St. Hilaire: Okay.
Milka Koupenova: That goes back to sensing mechanism. Thank goodness platelets don't get infected because we would be in a particularly bad situation, but they remove the infectious virus from the plasma from what we can see with function.
Cindy St. Hilaire: Got it. So they're helping the cleanup process and in that cleaning up is where the virus within them activates. That is a really complicated mechanism.
Milka Koupenova: Oh, they're sensing it in some form to alert the environment. It's hard to say how similar and how different they are unless you study them hint by hint next to each other. All I can tell is that particularly with SARS-C, you definitely see a lot more various kinds of extracellular vesicles coming out of them that you don't see the same way or rather through the same proportion with influenza. But what that means in how platelet activates the immune system with one versus the other, and that goes back to the prothrombotic mechanisms. That is exactly what needs to be studied and that was the call for this COVID compendium is to point out how much we have done as a team. As scientists who put heads together, as Mina said, superfast response, it's an amazing going back and looking at what happened to think of what we achieved.
There is so much more, so much more that we do not understand how one contributes to all of these profound responses in the organs themselves, such as myocarditis. We see it's important and that will be the problem that we're dealing from here on trying to figure it out and then long COVID, right?
Cindy St. Hilaire: Yeah. Related to what you just said about the mechanism, this cleanup by the platelets or the act of cleaning up helps trigger their activation, is that partly why the antiplatelet and anticoagulant therapies failed in patients? Can you speculate on that? I know the jury's still out and there's a lot of work to be done, but is that part of why those therapies weren't beneficial?
Milka Koupenova: The answer to that in my personally biased opinion is yes. Clearly, the antiplatelet therapies couldn't really control the classical activation of a platelet. So what I think we need to do from here on is to look at things that we don't understand that non-classically contribute to the thrombotic response downstream. If we manage to control the immune response in some way or the inflammation of the infection or how a platelet responds to a virus, then perhaps we can ameliorate a little bit of the downstream prothrombotic effect. So it's a lot more for us to trickle down and to understand in my personal opinion.
DeLisa Fairweather: There is one thing that was really remarkable to me in hearing your experience, Milka, is that I had developed an autoimmune viral model of myocarditis in mice during my postdoc. So I've been studying that for the last 20 years. What is unique about that model is rather than using an adjuvant, we use a mild viral infection so it doesn't take very much virus at all going to the heart to induce it. I also, more recently, started studying extracellular vesicles really as a therapy, and in doing that, inadvertently found out that actually, the model that I'd created where we passage the virus through the heart to induce this autoimmune model, we were actually injecting extracellular vesicles into the mice and that's what was really driving the disease. This is really brought out. So from early days, I did my postdoc with Dr Noel Rose. If you've heard of him, he came up with the idea of autoimmune disease in the '50s. We had always, in that environment, really believed that viruses were triggering autoimmune disease and yet it took COVID before we could really prove that because no one could identify them.
Here we have an example and I think the incidence rates with COVID were so high for myocarditis because for the first time, we had distinguished symptoms of patients going to the doctor right at the beginning of their infection having an actual test to examine the virus, knowing whether it's present or not, whether PCR or antibody test, and then being able to see when myocarditis happened.
Cindy St. Hilaire: Yeah. I think one thing we can all appreciate now is just some of the basic biology we've learned on the backend of this. Actually, those last comments really led well to the article that your team led, Dr Chung, about what we call long COVID, which I guess I didn't realize has an actual name, post-acute sequelae of SARS-CoV-2 or PASC is the now more formal name for long COVID. But what is it? We hinted at it that there's these bits about autoimmune and things like that. What counts as long COVID?
Mina Chung: Yeah. Our article was led by Tamanna Singh. She did a fantastic job of putting this together. We've had, and others, theorized that the huge palette of symptoms that you can experience post-COVID, they can affect all these organ systems with brain fog, these atypical chest pains, postural orthostatic tachycardia, a lot of palpitations, atrial fibrillation, many weakness and fatigue. To us, really, you can get GI symptoms. We've been very interested in, is this an autoimmune phenomenon directed against nerves and all those things. It's also very interesting because many of the non-COVID syndromes that existed pre-COVID like POTS and chronic fatigue syndrome and a lot of other syndromes are associated with autoantibodies. So that is a very interesting area to explore. Is there a persistence of viral fragments. Is there autoimmunity? Is it also a component of persistence of the damage from the initial infection? So it's an area that still needs a lot of work and a lot of work is going into it, but this is like a post or inter pandemic of itself, so hopefully we'll get more insights into that.
Cindy St. Hilaire: Yeah, it's really interesting. I have a friend who has very debilitating long COVID and one of her doctors had said, "If I didn't know any better, I would just describe this as a autoimmune type X." What do we know, I guess, about the current hypothesis of the pathogenesis of PASC? Are there any prevailing theories right now as to why it's occurring? Is the virus still active or is it these domino effects that are leading to multi-organ collapse of some sort?
Mina Chung: Yeah. In some people, persistent viral particles can be identified for months, but whether or not that's what's triggering it, it's hard to know. We see more autoimmune disease that's been reported and various antibodies being reported. So those are clearly processes to be investigated. The microthrombosis is still up there in terms of potentially playing a role in long COVID.
Milka Koupenova: Mina, you probably know better because you see patients, but to all I have been exposed to, long COVID does not really have a homogeneous symptom presentation and then a few theories as to what may be going on in these patients. Not everybody has a microthrombosis. Not everybody have a D-dimer elevated, but some people do. Some people have, as you pointed out, these spectacularly profound brain fog. People can't function. It's probably your friend, Cindy, right?
Cindy St. Hilaire: Yeah.
Milka Koupenova: So one of the theories that I have been, from a viral perspective, very interested in is that a lot of the symptoms in certain individuals such as fatigue, brain fog, sensitivity to light and skin can very well be explained by a flare-up of Epstein-Barr virus that may be what SARS-CoV-2 somehow is inducing. I don't know, DeLisa, what your experience with long COVID is as a scientist. I hope only. But I would like to hear your perspective too because it's so heterogeneous and it is amazing what happens.
DeLisa Fairweather: I have a very interesting perspective from a number of different directions. One, as I mentioned before, my long history with Dr Rose and I've written many articles theorizing how viruses could cause autoimmune disease. This has grown and really, I think this has been extremely revealing during COVID for many of those theories. One thing that I write about in the review for this article is that mast cells, from all the research I've done with myocarditis in our model, mast cells are central to what is driving everything. We show they're the first innate immune cell acting as an antigen-presenting cell, completely driving the response in a susceptible pattern. One of the things that's very important in autoimmune disease is both sex and race. I'd say one of the big weaknesses we have in myocarditis pre-COVID and post-COVID has been ignoring what's going on with race.
In the United States, myocarditis is 90%, 95% white men that are under 50 years of age and most of the cases are under 40 or some of the ones really associated with sudden cardiac death are under 30. So it's very specific. I've been studying sex and race differences and we see those exact differences in our animal models. In animal models, whether you're susceptible or not depends on how many mast cells you have.
Well, I've proposed from the beginning, looking, I've written a lot of different sex difference reviews looking at viruses and autoimmune disease with different autoimmune diseases and hypothesizing and really seeing that mast cells do a lot of the things we're talking about. They have all of the receptors, the whole group of them that have been related to SARS-CoV-2 so they can be activated or stimulated by the virus itself. They act as a antigen-presenting cell. They're critical in the complement pathway as well as macrophages. We see the dominant immune phenotype really being macrophages. Mast cells just are usually not counted anywhere. And of course, these receptors, a lot of them have to do with enzymes and things that are all related to mast cells pathways. Then how they activate the immune response and lead it towards the pathway that leads to chronic autoimmune disease with increased autoantibodies in females, mast cells are very different by sex.
This has to do also when we talked in the Review about myocarditis and pericarditis. It's both those appearing. Although clinically, we have really boxed them as separate things, because there is some definite clinical pericarditis phenotypes that are different, myocarditis in animal models is always myopericarditis. It always then, in that outer pericardial areas where mast cells sit, they sit around the vascular area in most concentrated. So when they degranulate, we see inflammation coming in the vessel, but really concentrated with fibrosis there and along the pericardium. So that's very typical of what's going on. When we shift anything that shifts that, it changes whether you have more pericarditis or less pericarditis and the vascular inflammation by altering anything that affects the mast cells. I talk a little bit about in the review, I think there's only been a few recent things looking at it in COVID, but I think mast cells and certain susceptibility to autoimmune diseases that occur more often in women can really predispose.We need to pay more attention to mast cells and what they might indicate for all these pathways.
Milka Koupenova: I think we should study the platelet mast cell access at this point.
DeLisa Fairweather: Yes.
Milka Koupenova: Because as you're talking about these sex differences, which is spectacular, these things to me are so mind-boggling how one, the infection itself would be more prevalent in men, but then long COVID is more prevalent in women. All of these things and why we understand so very little, what we found about a few years ago in the Framingham Heart Study in the platelets from those people is that all toll-like receptors are expressed at the higher level in women and they associate with different things between men and female. For instance, toll-like receptors in women will associate more with a prothrombotic response while in male with pro-inflammatory response. I think they grossly underestimate the amount of our sex differences from cell to cell.
DeLisa Fairweather: It is, yeah.
Mina Chung: One other thing that I learned about the sex differences from this compendium is Mark Chappell also notes, you mentioned TLR and TLR7 and ACE2 are X chromosome in an area that he says escapes X-linked inactivation. So it could very well be involved in further.
DeLisa Fairweather:
Further, yeah. And ACE2 is expressed more highly in male cells for what's been researched because of the sex difference in COVID, both the COVID infection
Cindy St. Hilaire: So a variety of organ systems are impacted in patients with PASC, also referred to as long COVID, the lungs, the heart, the pancreas, the GI system, pretty much any system, the brain, nervous system. We've just been talking about the mast cell impact. I was really thinking in my head, well, the one thing that connects all of it is the vasculature. I'm a vascular biologist, so I have certain biases, I'm sure, but how much of the sequelae that we see is a function of vascular phenotypes?
Milka Koupenova: I do think the vasculature is super important. It's clear that not all endothelial cells, for instance, will pick up the virus and respond to it. That's why you have this patchy breakage when you look at autopsies. Hence, platelets will respond according to what's local. That's why you find these micro thrombotic events at certain places. Why does it happen in each organ? How does the virus get to each organ to respond? Or is it just inflammation, but why is it in specific places? That's what we don't understand. That's where we need to go. Perhaps, as DeLisa points out, perhaps it's a lot more complicated than how we traditionally think of thrombosis. Actually, my personal bias, again 100% sure that it is a lot more complicated than the traditional mechanisms that we have understood, and that's where the immune system comes and autoimmunity perhaps stems from and they probably speak to each other, right? It's not just one thing.
DeLisa Fairweather: Yeah. I think really, EVs are bringing lots of understanding. A lot of things we used to just think were maybe free-floating and the serum are inside EVs. I think that the immune response is perhaps even more specific than we ever thought and more regulated than we ever understood.
When an EV comes through a cardiomyocyte, whether it's from the mitochondria or through a lysosome, is part of what goes into its outer membrane, something that tells the immune system that that came from the heart, so it knows to go. This will solve a lot of our questions with autoimmune disease if it's very specific like that. It doesn't just have to be the release of free-floating cardiac myosin. We know cardiac myosin is the driver of the autoimmune response in myocarditis, but they're probably much more fine-tuned.
Cindy St. Hilaire: Yeah. I just would love to end with hearing from each of you. You each have your own domain of specialty. If I gave you a massive pot of money, what would be the question you would want to tackle? What's the gap you would love to answer?
Milka Koupenova: We still don't understand specifically what kind of vesicles are coming out, what are their contents in addition to those vesicles. We don't understand. When it comes to platelets, what comes from their granules? We see these breakages of the membrane. Those are non-granule proteins, and non-granule proteins, they serve as dangerous associated molecular pattern signals and can be profoundly inflammatory to the surrounding environment, can be procoagulant. What are those? How are they affecting the surrounding environment? Ultimately, why is there a microthrombi? Why is there not a profound thrombosis everywhere? Thank goodness there isn't, but why isn't? That's what I would do with my money.
DeLisa Fairweather: I think I would do something very similar. All of our research in our animal model, on the one side, we are looking in this viral myocarditis animal model and finding the EVs that come from that are driving myocarditis. On the other hand, we're using EVs that come from healthy human plasma or fat, and we're seeing a profound downregulation of everything if you give it early and we're trying to see how late you can give it and still get an effect. So looking at those and really understanding the components in the context of COVID and COVID vaccines to understand those components, I really think that's the future of where we're going to find what's causing disease and also how we can find therapies. They may be able to reverse this.
Mina Chung: Yeah, I'm interested very much in the autoimmunity and the autoantibodies that are and how they may react with those microthrombi. Perhaps there's autoantibodies within a lot of that material. We're looking at using human and pluripotent stem cell-derived cell models to study the effects of those. That is what I would use our money for.
Cindy St. Hilaire: Well, Dr Mina Chung, Dr DeLisa Fairweather, Dr Milka Koupenova, thank you all so much for joining me today and talking about not only the articles that you wrote and with your colleagues, but also other articles in this amazing compendium. I do think this is one of the first all-encompassing compendiums or group of articles that focus specifically on COVID and cardiovascular disease. So thank you all so much.
Mina Chung: Thank you.
DeLisa Fairweather: Thank you.
Milka Koupenova: You're welcome.
Cindy St. Hilaire: That's it for highlights from the April 28th and May 12th 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. Thank you to our guests, Dr Mina Chung, Dr DeLisa Fairweather and Dr Milka Koupenova. This podcast is produced by Ishara 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. 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 information, visit ahajournals.org.
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This month on Episode 47 of Discover CircRes, host Cynthia St. Hilaire highlights three original research articles featured in the March 31 issue of Circulation Research. We’ll also provide an overview of the Compendium on Increased Risk of Cardiovascular Complications in Chronic Kidney Disease published in the April 14 issue. Finally, this episode features an interview with Dr Elizabeth Tarling and Dr Bethan Clifford from UCLA regarding their study, RNF130 Regulates LDLR Availability and Plasma LDL Cholesterol Levels.
Article highlights:
Shi, et al. LncRNAs Regulate SMC Phenotypic Transition
Chen, et al. Bilirubin Stabilizes Atherosclerotic Plaque
Subramaniam, et al. Mapping Non-Obvious cAMP Nanodomains by Proteomics
Compendium on Increased Risk of Cardiovascular Complications in Chronic Kidney Disease
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'm going to share three articles selected from our March 31st issue of Circulation Research and give you a quick summary of our April 14th Compendium. I'm also excited to speak with Dr Elizabeth Tarling and Dr Bethan Clifford from UCLA regarding their study, RNF130 Regulates LDLR Availability and Plasma LDL Cholesterol Levels.
So first the highlights. The first article we're going to discuss is Discovery of Transacting Long Noncoding RNAs that Regulates Smooth Muscle Cell Phenotype. This article's coming from Stanford University and the laboratory of Dr Thomas Quertermous. Smooth muscle cells are the major cell type contributing to atherosclerotic plaques. And in plaque pathogenesis, the cells can undergo a phenotypic transition whereby a contractile smooth muscle cell can trans differentiate into other cell types found within the plaque, such as macrophage-like cells, osteoblast-like cells and fibroblast-like cells. These transitions are regulated by a network of genetic and epigenetic mechanisms, and these mechanisms govern the risk of disease.
The involvement of long non-coding RNAs, or Lnc RNAs as they're called, has been increasingly identified in cardiovascular disease. However, smooth muscle cell Lnc RNAs have not been comprehensively characterized and the regulatory role in the smooth muscle cell state transition is not thoroughly understood. To address this gap, Shi and colleagues created a discovery pipeline and applied it to deeply strand-specific RNA sequencing from human coronary artery smooth muscle cells that were stressed with different disease related stimuli. Subsequently, the functional relevancy of a few novel Lnc RNAs was verified in vitro.
From this pipeline, they identified over 4,500 known and over 13,000 unknown or previously unknown Lnc RNAs in human coronary artery smooth muscle cells. The genomic location of these long noncoding RNAs was enriched near coronary artery disease related transcription factor and genetic loci. They were also found to be gene regulators of smooth muscle cell identity. Two novel Lnc RNAs, ZEB-interacting suppressor or ZIPPOR and TNS1-antisense or TNS1-AS2, were identified by the screen, and this group discovered that the coronary artery disease gene, ZEB2, which is a transcription factor in the TGF beta signaling pathway, is a target for these Lnc RNAs. These data suggest a critical role for long noncoding RNAs in smooth muscle cell phenotypic transition and in human atherosclerotic disease.
Cindy St. Hilaire: The second article I want to share is titled Destabilization of Atherosclerotic Plaque by Bilirubin Deficiency. This article is coming from the Heart Research Institute and the corresponding author is Roland Stocker. The rupture of atherosclerotic plaque contributes significantly to cardiovascular disease. Plasma concentrations of bilirubin, a byproduct of heme catabolism, is inversely associated with risk of cardiovascular disease, but the link between bilirubin and atherosclerosis is unknown.
Chen et el addressed this gap by crossing a bilirubin knockout mice to a atherosclerosis prone APOe knockout mouse. Chen et el addressed this gap by crossing the bilirubin knockout mouse to the atherosclerosis-prone APOE knockout mouse, and used the tandem stenosis model of plaque instability to address this question. Compared with their litter mate controls, bilirubin-APOE double knockouts showed signs of increased systemic oxidative stress, endothelial dysfunction, as well as hyperlipidemia. And they had higher atherosclerotic plaque burden.
Hemeatabolism was increased in unstable plaques compared with stable plaques in both of these groups as well as in human coronary arteries. In mice, the bilirubin deletion selectively destabilized unstable plaques and this was characterized by positive arterial remodeling and increased cap thinning, intra plaque hemorrhage, infiltration of neutrophils and MPO activity. Subsequent proteomics analysis confirmed bilirubin deletion enhanced extracellular matrix degradation, recruitment and activation of neutrophils and associated oxidative stress in the unstable plaque. Thus, bilirubin deficiency generates a pro atherogenic phenotype and selectively enhances neutrophil-mediated inflammation and destabilization of unstable plaques, thereby providing a link between bilirubin and cardiovascular disease risk.
Cindy St. Hilaire: The third article I want to share is titled Integrated Proteomics Unveils Regulation of Cardiac Monocyte Hypertrophic Growth by a Nuclear Cyclic AMP Nano Domain under the Control of PDE3A. This study is coming from the University of Oxford in the lab of Manuela Zaccolo. Cyclic AMP is a critically important secondary messenger downstream from a myriad of signaling receptors on the cell surface. Signaling by cyclic AMP is organized in multiple distinct subcellular nano domains, regulated by cyclic AMP hydrolyzing phosphodiesterases or PDEs.
The cardiac beta adrenergic signaling has served as the prototypical system to elucidate this very complex cyclic AMP compartmentalization. Although studies in cardiac monocytes have provided an understanding of the location and the properties of a handful of these subcellular domains, an overview of the cellular landscape of the cyclic AMP nano domains is missing.
To understand the nanodynamics, Subramanian et al combined an integrated phospho proteomics approach that took advantage of the unique role that individual phosphodiesterases play in the control of local cyclic AMP. They combined this with network analysis to identify previously unrecognized cyclic AMP nano domains associated with beta adrenergic stimulation. They found that indeed this integrated phospho proteomics approach could successfully pinpoint the location of these signaling domains and it provided crucial cues to determine the function of previously unknown cyclic AMP nano domains.
The group characterized one such cellular compartment in detail and they showed that the phosphodiesterase PDE3A2 isoform operates in a nuclear nano domain that involves SMAD4 and HDAC1. Inhibition of PDE3 resulted in an increased HDAC1 phosphorylation, which led to an inhibition of its deacetylase activity, and thus derepression of gene transcription and cardiac monocyte hypertrophic growth. These findings reveal a very unique mechanism that explains the negative long-term consequences observed in patients with heart failure treated with PDE3 inhibitors.
Cindy St. Hilaire: The April 14th issue is our compendium on Increased Risk of Cardiovascular Complications in Chronic Kidney Disease. Dr Heidi Noels from the University of Aachen is our guest editor of the 11 articles in this issue. Chronic kidney disease is defined by kidney damage or a reduced kidney filtration function. Chronic kidney disease is a highly prevalent condition affecting over 13% of the population worldwide and its progressive nature has devastating effects on patient health. At the end stage of kidney disease, patients depend on dialysis or kidney transplantation for survival. However, less than 1% of CKD patients will reach this end stage of chronic kidney disease. Instead, most of them with moderate to advanced chronic kidney disease will prematurely die and most often they die from cardiovascular disease. And this highlights the extreme cardiovascular burden patients with CKD have.
The titles of the articles in this compendium are the Cardio Kidney Patient Epidemiology, Clinical Characteristics, and Therapy by Nicholas Marx, the Innate Immunity System in Patients with Cardiovascular and Kidney Disease by Carmine Zoccali et al. NETs Induced Thrombosis Impacts on Cardiovascular and Chronic Kidney disease by Yvonne Doering et al. Accelerated Vascular Aging and Chronic Kidney Disease, The Potential for Novel Therapies by Peter Stenvinkel et al. Endothelial Cell Dysfunction and Increased Cardiovascular Risk in Patients with Chronic Kidney Disease by Heidi Noels et al. Cardiovascular Calcification Heterogeneity in Chronic Kidney Disease by Claudia Goettsch et al. Fibrosis in Pathobiology of Heart and Kidney From Deep RNA Sequencing to Novel Molecular Targets by Raphael Kramann et al. Cardiac Metabolism and Heart Failure and Implications for Uremic Cardiomyopathy by P. Christian Schulze et al. Hypertension as Cardiovascular Risk Factor in Chronic Kidney Disease by Michael Burnier et al. Role of the Microbiome in Gut, Heart, Kidney crosstalk by Griet Glorieux et al, and Use of Computation Ecosystems to Analyze the Kidney Heart Crosstalk by Joachim Jankowski et al.
These reviews were written by leading investigators in the field, and the editors of Circulation Research hope that this comprehensive undertaking stimulates further research into the path flow of physiological kidney-heart crosstalk, and on comorbidities and intra organ crosstalk in general.
Cindy St. Hilaire: So for our interview portion of the episode I have with me Dr Elizabeth Tarling and Dr Bethan Clifford. And Dr Tarling is an associate professor in the Department of Medicine in cardiology at UCLA, and Dr Clifford is a postdoctoral fellow with the Tarling lab. And today we're going to be discussing their manuscript that's titled, RNF130 Regulates LDLR Availability and Plasma LDL Cholesterol Levels. So thank you both so much for joining me today.
Elizabeth Tarling: Thank you for having us.
Bethan Clifford: Yeah, thanks for having us. This is exciting.
Cindy St. Hilaire: I guess first, Liz, how did you get into this line of research? I guess, before we get into that, I should disclose. Liz, we are friends and we've worked together in the ATVB Women's Leadership Committee. So full disclosure here, that being said, the editorial board votes on these articles, so it's not just me picking my friends. But it is great to have you here. So how did you enter this field, I guess, briefly?
Elizabeth Tarling: Yeah, well briefly, I mean my training right from doing my PhD in the United Kingdom in the University of Nottingham has always been on lipid metabolism, lipoprotein biology with an interest in liver and cardiovascular disease. So broadly we've always been interested in this area and this line of research. And my postdoctoral research was on atherosclerosis and lipoprotein metabolism. And this project came about through a number of different unique avenues, but really because we were looking for regulators of LDL biology and plasma LDL cholesterol, that's sort of where the interest of the lab lies.
Cindy St. Hilaire: Excellent. And Bethan, you came to UCLA from the UK. Was this a topic you were kind of dabbling in before or was it all new for you?
Bethan Clifford: It was actually all completely new for me. So yeah, I did my PhD at the same university as Liz and when I started looking for postdocs, I was honestly pretty adamant that I wanted to stay clear away from lipids and lipid strategy. And then it wasn't until I started interviewing and meeting people and I spoke to Liz and she really sort of convinced me of the excitement and that the interest and all the possibilities of working with lipids and well now I won't go back, to be honest.
Cindy St. Hilaire: And now here you are. Well-
Bethan Clifford: Exactly.
Cindy St. Hilaire: ... congrats on a wonderful study. So LDLR, so low density lipoprotein receptor, it's a major determinant of plasmid LDL cholesterol levels. And hopefully most of us know and appreciate that that is really a major contributor and a major risk for the development of atherosclerosis and coronary artery disease. And I think one thing people may not really appreciate, which your study kind of introduces and talks about nicely, is the role of the liver, right? And the role of receptor mediated endocytosis in regulating plasma cholesterol levels. And so before we kind of chat about the nitty-gritty of your study, could you just give us a brief summary of these key parts between plasma LDL, the LDL receptor and where it goes in your body?
Elizabeth Tarling: Yeah. So the liver expresses 70% to 80% of the body's LDL receptor. So it's the major determinant of plasma lipoprotein plasma LDL cholesterol levels. And through groundbreaking work by Mike Brown and Joe Goldstein at the University of Texas, they really define this receptor mediated endocytosis by the liver and the LDL receptor by looking at patients with familial hypercholesterolemia. So those patients have mutations in the LDL receptor and they either express one functional copy or no functional copies of the LDL receptor and they have very, very large changes in plasma LDL cholesterol. And they have severe increases in cardiovascular disease risk and occurrence and diseases associated with elevated levels of cholesterol within the blood and within different tissues. And so that's sort of how the liver really controls plasma LDL cholesterol is through this receptor mediated endocytosis of the lipoprotein particle.
Cindy St. Hilaire: There's several drugs now that can help regulate our cholesterol levels. So there's statins which block that rate limiting step of cholesterol biosynthesis, but there's this new generation of therapies, the PCSK9 inhibitors. And can you just give us a summary or a quick rundown of what are those key differences really? What is the key mechanism of action that these therapies are going after and is there room for more improvement?
Bethan Clifford: Yeah, sure. So I mean I think you've touched on something that's really key about the LDR receptor is that it's regulated at so many different levels. So we have medications available that target the production of cholesterol and then as you mentioned this newer generation of things like PCSK9 inhibitors that sort of try and target LDL at the point of clearance from the plasma.
And in response to your question of is there room for more regulation, I would say that given the sort of continual rate of increased cholesterol in the general population and the huge risks associated with elevated cholesterol, there's always capacity for more to improve that and sort of generally improve the health of the population. And what we sort of found particularly exciting about RNF130 is that it's a distinct pathway from any of these regulatory mechanisms. So it doesn't regulate the level of transcription, it doesn't regulate PCSK9. Or in response to PCSK9, it's a completely independent pathway that could sort of improve or add to changes in cholesterol.
Cindy St. Hilaire: So your study, it's focusing on the E3 ligase, RNF130. What is an E3 ligase, and why was this particular one of interest to you? How did you come across it?
Elizabeth Tarling: is predTates Bethan joining the lab. This is, I think, again for the listeners and those people in training, I think it's really important to note this project has been going in the lab for a number of years and has really... Bethan was the one who came in and really took charge and helped us round it out. But it wasn't a quick find or a quick story. It had a lot of nuances to it. But we were interested in looking for new regulators of LDL cholesterol and actually through completely independent pathways we had found the RNF130 locus as being associated with LDL cholesterol in animals. And then it came out in a very specific genome-wide association study in the African American care study, the NHLBI care study. And so really what we started looking at, we didn't even know what it was.
Elizabeth Tarling: So we asked ourselves, well what is this gene? What is this protein? And it's RNF, so that's ring finger containing protein 130 and ring stands for really interesting new gene. Somebody came up with the glorious name. But proteins that contain this ring domain are very characteristic and they are E3 ubiquitin ligases. And so they conjugate the addition of ubiquitin to a target protein and that signals for that protein to either be internalized and/or degraded through different decorative pathways within the cell. And so we didn't land on it because we were looking at E3 ligases, we really came at it from an LDL cholesterol perspective. And it was something that we hadn't worked on before and the study sort of blossomed from there.
Cindy St. Hilaire: That's amazing and a beautiful, but also, I'm sure, heartbreaking story because these long projects are just... They're bears. So what does this RNF130 do to LDLR? What'd you guys find?
Bethan Clifford: As Liz said, this is a long process, but one of the key factors of RNF130 is it's structurally characteristically looked like E3 ligase. So the first thing that Liz did and then I followed up with in the lab is to see is this E3 ligase ubiquitinating in vitro. And if it is going to ubiquitinate, what's it likely to regulate that might cause changes in plasma cholesterol that would explain these human genetic links that we saw published at the same time.
And so because the LDL cholesterol is predominantly regulated by the LDL receptor and the levels of it at the surface of the parasites in the liver, the first question we wanted to see is does RNF130 interact in any way with that pathway? And I'm giving you the brief view here of the LDL receptor. We obviously tested lots of different receptors. We tested lots of different endocytose receptors and lipid regulators, but the LDL receptor is the one that we saw could be ubiquitinated by RNF130 in vitro. And so then we wanted to sort of go on from there and establish, okay, if this E3 ubiquitin ligase, is it regulating LDL receptor? What does that mean in an animal context in terms of regulating LDL cholesterol?
Cindy St. Hilaire: Yeah, and I guess we should also explain, ubiquitination, in terms of this receptor, and I guess related to Goldstein and Brown and receptor mediated endocytosis, like what does that actually mean for the liver cell and the cholesterol in the LDLR that is binding the receptor?
Bethan Clifford: \So yes, ubiquitination is a really common regulatory mechanism actually across all sorts of different cells, all sorts of different receptors and proteins. And basically what it does is it signals for degradation of a protein. So a ubiquitin molecule is conjugated to its target such as in our case the LDL receptor and that ubiquitin tells the cell that this protein is ready for proteasomal degradation. And that's just one of the many things ubiquitination can do. It can also signal for a trafficking event, it can signal for a protein to protein interaction, but it's most commonly associated with the proteasomal degradation.
Cindy St. Hilaire: So in terms of... I guess I'm thinking in terms of PCSK9, right? So those drugs are stemming from observations in humans, right? There were humans with gain and loss of function mutations, which caused either more or less of this LDLR receptor internalization. How is this RNF130 pathway different from the PCSK9 activities?
Elizabeth Tarling: Yeah, so PCSK9 is a secreted protein, so it's made by hepatocyte and actually other cells in the body and it's secreted and it binds to the LDL particle, LDL receptor complex, and signals for its internalization and degradation in the proteasome. So this is not ubiquitination event, this is a completely different trafficking event. And so the RNF130, actually what Bethan showed, is it directly ubiquitinates the LDL receptor itself, signaling for an internalization event and then ultimately degradation of the LDR receptor through a decorative pathway, which we also define in the study.
So these are two unique mechanisms and actually some key studies that we did in the paper were to modulate RNF130 in animals that do not have PCSK9. And so in that system where in the absence of PCSK9 you have a lot of LDR receptor in the liver that's internalizing cholesterol. What happens when you overexpress RNF130? Do you still regulate at the LDL receptor? And you absolutely do. And so that again suggests that they're two distinct mechanisms and two distinct pathways.
Cindy St. Hilaire: That was one thing I really loved about your paper is every kind of figure or section, the question that would pop up in my head, even ones that didn't pop in my head were beautifully answered with some of these really nice animal models, which is never an easy thing, right? And so one of the things that you brought up was difficulty in making one of the animal models. And so I'm wondering if you could share a little bit for that challenge. I think one thing that we always tend to hide is just science is hard and a lot of what we do doesn't work. And I really think especially for the trainees and really everyone out there, if we kind of share these things more, it's better. So what was one of the most challenging things in this study? And I guess I'm thinking about that floxed animal.
Elizabeth Tarling: Yeah, so I'll speak a bit about that and then I'll let Bethan address because she was really the one on the ground doing a lot of the struggles. But again, we actually weren't going to include this information in the paper. And upon discussion and actually prompted by the reviewers of the paper and some of the questions that they asked us, we realized, you know what? It's actually really important to show this and show that this happens and that there are ways around it.
And so the first story is before Bethan even arrived in the lab, we had purchased embryonic stem cells that were knockout first condition already. And so this is a knockout strategy in which the exon of interest is flanked with lots of P sites so that you can create a flox animal, but also so you can create a whole body knockout just by the insertion of this knockout first cassette.
Elizabeth Tarling: And so we got those mice actually in the first year of Bethan joining the lab. We finally got the chimeric mice and we were able to stop reading those mice. And at the same time we tried to generate our flox animals so that we could move on to do tissue-specific studies. And Bethan can talk about the pain associated with this. But over two years of breeding, we never got the right genotypes from the different crosses that you need to do to generate the flox animal.
And it was actually in discussions with Bethan where we decided we need to go back. We need to go back to those ESLs that we purchased five years ago and we need to figure out if all of the elements that the quality control step had told us were in place are actually present. And so Bethan went back and sequenced the whole locus and the cassette to figure out what pieces were present and we found that one of the essential locks P sites that's required for every single cross from the initial animal was absent and therefore we could actually never make the mouse we wanted to make.
And so that's sort of just a lesson for people going down that route and making these tools that we need in the lab to answer these questions is that despite paying extra money and getting all of the sort of QCs that you can get before you receive the ESLs, we should have gone back and done our own housekeeping and sort of a long journey told us when we went back that we didn't have what we thought we had at the beginning. And that was a real sticking point as Bethan can-
Cindy St. Hilaire: Yeah. And so you know you're not alone. My very first postdoc that I did, I went with a mouse that they had also bought and were guaranteed that it was a knockout and it was not. And it is a painful lesson, but it is critical to... You get over it.
So Bethan, maybe you can also tell us a little bit about what are the other kind of next things you tried? You pivoted and you pivoted beautifully because all the models you used I thought were quite elegant in terms of exactly asking the question you wanted to ask in the right cells. So can you maybe explain some of the in vivo models you used for this study?
Bethan Clifford: Sure, there are definitely a lot. So I mean I think Liz sort of encapsulated the trouble we have with the knockout really succinctly, but actually I want to just take this moment to sort of shout out to another postdoc in the Tarling lab, Kelsey Jarrett, who was really instrumental in the pivoting to a different model. So for the knockouts when we sort of established we didn't have exactly what we thought we did and then to compound that we also weren't getting the DeLiAn ratios breeding this whole body knockout.
We wanted to sort of look at a more transient knockout model. And that's where Kelsey really stepped in and sort of led the way and she generated AAV-CRISPR for us to target RNF130 specifically in the liver. And that had the added beauty of, one, not requiring breeding to get over this hurdle of the knockout being somewhat detrimental to breeding. But it also allowed us to ask the question of what RNF130 is doing specifically in the liver where the liver regulates LDL receptor and LDL cholesterol.
And so that was one of the key models that really, really helped get this paper over the finish line. But we did a whole barrage of experiments, as you've seen. We wanted to make sure... One of the key facets of the Tarling lab is whenever you do anything, no matter what you show Liz, it will always be, "Okay, you showed it to me one way, now show it to me a different way." Can you get the same result coming at it from different ways? And if you can't, why is that? What is the regulation behind that? And so that's really what the paper is doing is asking the same question in as many ways as we can accurately and appropriately probe what RNF130 does to the LDR receptor.
So we tried gain of function studies without adenovirus overexpression. We tried transient knockdown with antisense oligonucleotides, and then we did, as I said, the AAV-CRISPR knockdown with the help of Kelsey and our whole body knockout. And then we also repeated some of these studies such as the adenovirus and the ASO in specific genetic backgrounds. So in the absence of PCSK9, can we still regulate the LDL receptor? And then we also, just to really confirm this, in the absence of the LDL receptor, do we see a difference? And the answer is no, because this effect was really dependent on that LDL receptor being present. So there was a big combination.
Cindy St. Hilaire: It was really nice, really a beautiful step-wise progression of how to solidly answer this question. But a lot of, I think, almost all you did was in mice. And so what is the genetic evidence for relevancy in humans? Can you discuss a little bit about those databases that you then went to to investigate, is this relevant in humans?
Bethan Clifford: I think Liz might be better off answering that question.
Elizabeth Tarling: And I think this sort of pivots on what Bethan was saying. So when we had struggles in the lab, it was a team environment and a collaboration between people in the lab that allowed us to make that leap and make those next experiments possible to then really answer that question. And to be able to include the antisense oligonucleotides required a collaboration with industry. We were very lucky to have a longstanding collaboration with Ionis, who provided the antisense oligonucleotides.
And for the human genetics side of things, that also was a collaboration with Marcus Seldin, who was a former postdoc with Jake Lusis and is now our PI at UC Irvine. And what he helped us do is dive into those summary level databases and ask from that initial study in the NHLBI care population, do we see associations of RNF130 expression in humans with LDL cholesterol with cardiovascular outcomes. And so one database which I would recommend everybody use, it's publicly available, is the StarNet database. And it's in the paper and the website is there. And that allowed us to search for RNF130.
Elizabeth Tarling: And what it does is it asks how RNF130 expression in different tissues is associated with cardiometabolic outcomes and actual in CAD cases and controls, so people with and without heart disease. And we found that expression of RNF130 in the liver was extremely strongly correlated with the occurrence of cardiovascular disease in people with CAD. So in cases versus controls. And then we were also able to find many other polymorphisms in the RNF130 locus that were associated with LDL cholesterol in multiple different studies.
And I think the other message from this paper is this, unlike PCSK9 and unlike LDR receptor itself, which are single gene mutations that cause cardiovascular disease, there are many sub genome-wide significant loci that contribute to this multifactorial disease, which is extremely complex. And I think RNF130 falls within that bracket that those sort of just on the borderline of being genome-wide significant still play significant biological roles in regulating these processes. And they don't come up as a single gene hit for a disease, but combinatorialy they are associated with increased risk of disease and they have a molecular mechanism that's associated with the disease. And so that's what Marcus helped us do in terms of the human genetics is really understand that and get down to that level of data.
Cindy St. Hilaire: Yeah. Yeah, it really makes you want to go back and look at those. Everyone always focuses on that really high peak and those analyses, but what are all those other ones above the noise, right? So it's really important.
Elizabeth Tarling: I think it's really hard to do that. I think that's one where people... Again, it comes down to team science and the group of people that we brought together allowed us to ask that molecular question about how that signal was associated with the phenotype. I think by ourselves we wouldn't have been able to do it.
Cindy St. Hilaire: Yeah. So your antisense oligonucleotide experiments, they were really nice. They showed, I think it was a four-week therapy, they showed that when you injected them expression of RNF130 went down by 90%. I think cholesterol in the animals was lowered by 50 points or so. Is this kind of a next viable option? And I guess related to that, cholesterol's extremely important for everything, right? Cell membrane integrity, our neurons, all sorts of things. Is it possible with something that is perhaps really as powerful as this to make cholesterol too low?
Elizabeth Tarling: I think that what we know from PCSK9 gain and loss of function mutations is that you can drop your plasma cholesterol to very low levels and still be okay because there are people walking around with mutations that do that. I think RNF130 is a little different in that it's clearly regulatory in a homeostatic function in that it's ubiquitously expressed and it has this role in the liver to regulate LDL receptor availability, but there are no homozygous loss of function mutants people walking around, which tells us something else about how important it is in potentially other tissues and in other pathways. And we've only just begun to uncover what those roles might be.
So I think that as a therapy, it has great potential. We need to do a lot more studies to sort of move from rodent models into more preclinical models. But I do think that the human data tell us that it's really important in other places too. And so yeah, we need to think about how best it might work as a therapy. If it's combinatorial, if it's dosed. Those are the types of things that we need to think about.
Cindy St. Hilaire: Yeah, it's really exciting. Do you know, are there other protein targets of RNF130? Is that related to my next question of what is next?
Elizabeth Tarling: I mean, so I should point out, so Bethan unfortunately left the lab last year for a position at Amgen where she's working on obesity and metabolic disease. But before she left, she did two very, very cool experiments searching for new targets or additional targets of RNF130. Starting in the liver, but hopefully we'll move those into other tissues. And so she did gain of function RNF130 versus what loss of function we have of RNF130, and she did specific mass spec analysis of proteins that are ubiquitinated in those different conditions.
And by overlaying those data sets, we're hoping to carve out new additional targets of RNF130. And there are some, and they're in interesting pathways, which we have yet to completely test, but definitely there are additional pathways, at least when you overexpress and reduce expression. Now, whether they turn out to be, again, bonafide in vivo, actual targets that are biologically meaningful is sort of the next step.
Cindy St. Hilaire: Yeah. Well, I'm sure with your very rigorous approach, you are going to find out and hopefully we'll see it here in the future. Dr Elizabeth Tarling and Dr Bethan Clifford, thank you so much for joining me today. I really enjoyed this paper. It's a beautiful study. I think it's a beautiful example, especially for trainees about kind of thoroughly and rigorously going through and trying to test your hypothesis. So thanks again.
Elizabeth Tarling: Thank you.
Bethan Clifford: Thank you very much.
Cindy St. Hilaire: That's it for the highlights from the March 31st and April 14th issues of Circulation Research. Thank you for listening. Please check out the Circulation Research Facebook page and follow us on Twitter and Instagram with the handle @CircRes, and #DiscoverCircRes. Thank you to our guests, Dr Liz Tarling and Dr Bethan Clifford.
This podcast is produced by Ishara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, you're on-the-go source for the most exciting discoveries in basic cardiovascular research.
This program is copyright of the American Heart Association 2022. 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 information, visit ahajournals.org.
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This month on Episode 46 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the March 3 and March 17th issues of Circulation Research. This episode also features an interview with Dr Andrew Hughes and Dr Jessilyn Dunn about their review, Wearable Devices in Cardiovascular Medicine.
Article highlights:
Delgobo, et al. Deep Phenotyping Heart-Specific Tregs
Sun, et al. Inhibition of Fap Promotes Cardiac Repair After MI
Sun, et al. Endosomal PI3Kγ Regulates Hypoxia Sensing
Johnson, et al. Hypoxemia Induces Minimal Cardiomyocyte Division
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'm going to share four articles selected from the March 3rd and March 17th issues of CircRes. I'm also going to have a discussion with Dr Andrew Hughes and Dr Jessilyn Dunn about their review, Wearable Devices in Cardiovascular Medicine. And the Review is also featured in our March 3rd issue.
Cindy St. Hilaire: First, the highlights. The first article I'm going to present is Myocardial Milieu Favors Local Differentiation of Regulatory T-Cells. The first author is Murilo Delgobo and the corresponding author is Gustavo Campos Ramos. After myocardial infarction, the release of autoantigens from the damaged heart cells activates local and infiltrating immune cells such as the T-cell. Studies in mice have shown that fragments of the muscle protein myosin can act as autoantigens, and these myosin fragments are the dominant driver of the T-cell response.
But how do these myosin specific T-cells behave in the damaged heart to drive inflammation and repair is unknown. To find out, Delgobo and colleagues studied endogenous myosin specific T-cells, as well as those transferred into recipient mice. They found, whether exogenously supplied or endogenously created, the myosin specific T-cells that accumulated in the animals' infarcted hearts tended to adopt an immunosuppressive T-regulatory phenotype.
Strikingly, even if the exogenous cells were differentiated into inflammatory TH-17 cells prior to transfer, a significant proportion of them were still reprogrammed into T-regs within the heart. Although cells pre-differentiated into an inflammatory TH-17 phenotype were less inclined to change after the transfer, the results nevertheless indicate that, by and large, the infarcted heart promotes T-cell reprogramming to quell inflammation and drive repair. Yet exactly how the heart does this is a question for future studies.
Cindy St. Hilaire: The next article I'm going to present is titled Inhibition of FAP Promotes Cardiac Repair by Stabilizing BNP. The first authors of the study are Yuxi Sun and Mengqiu Ma, and the corresponding author is Rui Yue, and they are from Tongji University. After myocardial infarction, there needs to be a balance of recovery processes to protect the tissue. Fibrosis, for example, acts like an immediate bandaid to hold the damaged heart muscle together, but fibrosis can limit contractile function.
Similarly, angiogenesis and sufficient revascularization is required to promote survival of cardiomyocytes within the ischemic tissue and protect heart function. To better understand the balance between fibrotic and angiogenic responses, Sun and colleagues examined the role of fibroblasts activated protein, or FAP, which is dramatically upregulated in damaged hearts, and brain natriuretic peptide, or BNP, which promotes angiogenesis in the heart.
In this study, they found that genetic deletion or pharmacological inhibition of FAP in mice reduces cardiac fibrosis and improves angiogenesis and heart function after MI. Such benefits are not seen if BNP or its receptor, NRP-1, are lacking. The in vitro experiments revealed that FAP's protease activity degrades BNP, thus inhibiting the latter's angiogenic activity. Interestingly, while FAP is upregulated in the heart, its levels drop in the blood, showing that BNP inhibition is localized. Together, these results suggest that blocking FAP's activity in the heart after MI could be a possible strategy for protecting the muscle's function.
Cindy St. Hilaire: The next article I want to present is Hypoxia Sensing of Beta-Adrenergic Receptor is Regulated by Endosomal PI-3 Kinase Gamma. The first author of this study is Yu Sun, and the corresponding author is Sathyamangla Naga Prasad. Hypoxia is the most proximate acute stress encountered by the heart during an ischemic event. Hypoxia triggers dysfunction of the beta-adrenergic receptors, beta-1AR and beta-2AR, which are critical regulators of cardiac function.
Under normoxic conditions, activation of PI3K-gamma by beta-adrenergic receptors leads to feedback regulation of the receptor by hindering its dephosphorylation through inhibition of protein phosphatase 2A or PP2A. Although it is known that ischemia reduces beta-adrenergic receptor function, the impact of hypoxia on interfering with this PI3K feedback loop was unknown.
Using in vitro and in vivo techniques, this group found that activation of PI3K-gamma underlies hypoxia sensing mechanisms in the heart. Exposing PI3K-gamma knockout mice to acute hypoxia resulted in preserved cardiac function and reduced beta-adrenergic receptor phosphorylation. And this was due to a normalized beta-2AR associated PP2A activity, thus uncovering a unique role for PI3K-gamma in hypoxia sensing and cardiac function.
Similarly, challenging wild-type mice post hypoxia with dobutamine resulted in an impaired cardiac response that was normalized in the PI3K-gamma knockout mice. These data suggests that preserving beta-adrenergic resensitization by targeting the PI3K-gamma pathway would maintain beta-adrenergic signaling and cardiac function, thereby permitting the heart to meet the metabolic demands of the body following ischemia.
Cindy St. Hilaire: The last article I want to highlight is Systemic Hypoxia Induces Cardiomyocyte Hypertrophy and Right Ventricle Specific Induction of Proliferation. First author of this study is Jaslyn Johnson, and the corresponding author is Steven Houser, and they're at Temple University.
The cardiac hypoxia created by myocardial infarction leads to the death of the heart tissue, including the cardiomyocytes. While some procedures such as reperfusion therapy prevent some cardiomyocyte death, true repair of the infarcted heart requires that dead cells be replaced. There have been many studies that have attempted new approaches to repopulate the heart with new myocytes. However, these approaches have had only marginal success.
A recent study suggested that systemic hypoxemia in adult male mice could induce cardiac monocytes to proliferate. Building on this observation, Johnson and colleagues wanted to identify the mechanisms that induced adult cardiomyocyte cell cycle reentry and wanted to determine whether this hypoxemia could also induce cardiomyocyte proliferation in female mice.
Mice were kept in hypoxic conditions for two weeks, and using methods to trace cell proliferation in-vivo, the group found that hypoxia induced cardiac hypertrophy in both the left ventricle and the right ventricle in the myocytes of the left ventricle and of the right ventricle. However, the left ventricle monocytes lengthened while the RV monocytes widened and lengthened.
Hypoxia induced an increase in the number of right ventricular cardiomyocytes, but did not affect left ventricular monocyte proliferation in male or in female mice. RNA sequencing showed upregulation of cell cycle genes which promote the G1 to S phase transition in hypoxic mice, as well as a downregulation of cullen genes, which are the scaffold proteins related to the ubiquitin ligase complexes. There was significant proliferation of non monocytes in mild cardiac fibrosis in the hypoxic mice that did not disrupt cardiac function.
Male and female mice exhibited similar gene expression patterns following hypoxia. Thus, systemic hypoxia induced a global hypertrophic stress response that was associated with increased RV proliferation, while LV monocytes did not show increased proliferation. These results confirm previous reports that hypoxia can induce cardiomyocyte cell cycle activity in-vivo, and also show that this hypoxia induced proliferation also occurs in the female mice.
Cindy St. Hilaire: With me today for our interview, I have Dr Andrew Hughes and Dr Jessilyn Dunn, and they're from Vanderbilt University Medical Center. And they're here to discuss the review article that they helped co-author called Wearable Devices in Cardiovascular Medicine. And just as a side note, the corresponding author, Evan Brittain, unfortunately just wasn't able to join us due to clinical service, but they're going to help dissect and discuss this Review with us. Thank you both so much for joining me today. Andy, can you just tell us a little bit about yourself?
Andy Hughes: Yeah, thank you, Cindy. I'm Andy Hughes. I'm a third year medicine resident at Vanderbilt University who is currently on an NIH supported research year this year. And then will be applying to cardiology fellowships coming up in the upcoming cycle.
Cindy St. Hilaire: Great, thank you. And Jessilyn, I said you are from Vanderbilt. I know you're from Duke. It was Evan and Andy at Vanderbilt. Jessilyn, tell us about yourself.
Jessilyn Dunn: Thanks. I am an Assistant Professor at Duke. I have a joint appointment between biomedical engineering and biostatistics and bioinformatics. The work that my lab does is mainly centered on digital health technologies in developing what we call digital biomarkers, using data from often consumer wearables to try to detect early signs of health abnormalities and ultimately try to develop interventions.
Cindy St. Hilaire: Thank you. We're talking about wearable devices today, and obviously the first thing I think most of us think about are the watch-like ones, the ones you wear on your wrists. But there's really a whole lot more out there. It's not just Apple Watches and Fitbits and the like. Can you just give us a quick summary of all these different types of devices and how they're classified?
Jessilyn Dunn: Yeah, absolutely. We have a wide variety of different sensors that can be useful. A lot of times, we like to think about them in terms of the types of properties that they measure. So mechanical properties like movement, electrical properties like electrical activity of the heart. We have optical sensors. And so, a lot of the common consumer wearables that we think about contain these different types of sensors.
A good example that we can think about is your consumer smartwatch, like an Apple Watch or a Fitbit or a Garmin device where it has something called an accelerometer that can measure movement. And oftentimes, that gets converted into step counts. And then it may also have an optical sensor that can be used to measure heart rate in a particular method called PPG, or photoplethysmography. And then some of the newer devices also have the ability to take an ECG, so you can actually measure electrical activity as well as the optical based PPG heart rate measurement. These are some of the simpler components that make up the more complex devices that we call wearables.
Cindy St. Hilaire: And how accurate are the measurements? You did mention three of the companies, and I know there's probably even more, and there's also the clinical grade at-home ECG machines versus the one in the smartwatch. How accurate are the measurements between companies? And we also hear recent stories about somebody's Apple Watch calling 911 because they think they're dead, things like that. Obviously, there's proprietary information involved, but how accurate are these devices and how accurate are they between each other?
Jessilyn Dunn: This is a really interesting question and we've done quite a bit of work in my lab on this very topic, all the way from what does it mean for something to be accurate? Because we might say, "Well, the more accurate, the better," but then we can start to think about, "Well, how accurate do we need something to be in order to make a clinical decision based off of that?" And if it costs significantly more to make a device super, super accurate, but we don't need it to be that accurate to make useful decisions, then it actually might not be serving people well to try to get it to that extreme level of accuracy.
So there are a lot of trade-offs, and I think that's a tough thing to think about in the circumstances, is these trade-offs between the accuracy and, I don't know, the generalizability or being able to apply this to a lot of people. That being said, it also depends on the circumstances of use. When we think about something like step counts, for example, if you're off by a hundred step counts and you're just trying to get a general view of your step counts, it's not that much of a problem.
But if we're talking about trying to detect an irregular heart rhythm, it can be very bad to either miss something that's abnormal or to call something abnormal that's not and have people worried. We've been working with the Digital Medicine Society to develop this framework that we call V3, which is verification, analytic validation and clinical validation. And these are the different levels of analysis or evaluation that you can do on these devices to determine how fit for purpose are they.
Given the population we're trying to measure in and given what the goal of the measurement is, does the device do the job? And what's also interesting about this topic is that the FDA has been evolving how they think about these types of devices because there's, in the past, been this very clear distinction between wellness devices and medical devices. But the problem is that a lot of these devices blur that line. And so, I think we're going to see more changes in the way that the FDA is overseeing and potentially regulating things like this as well.
Cindy St. Hilaire: These consumer-based devices have started early on as the step counters. When did they start to bridge into the medical sphere? When did that start to peak the interest of clinicians and researchers?
Jessilyn Dunn: Yeah, sure. What's interesting is if we think back to accelerometers, these have been used prior to the existence of mobile phones. These really are mechanical sensors that could be used to count steps. And when we think about the smartwatch in the form that we most commonly think of today, probably looking back to about 2014 is when ... maybe between 2012, 2014 is when we saw these devices really hitting the market more ... Timing for when the devices that we know as our typical consumer smartwatch today was around 2012 to 2014.
And those were things that were counting steps and then the next generation of that
added in the PPG or photoplethysmography sensor. That's that green light when we look on the back of our watch that measures heart rate. And so, thinking back to the early days, probably Jawbone, there was a watch called Basis, the Intel Basis watch. Well, it was Basis and then got acquired by Intel. Fitbit was also an early joining the market, but that was really the timing.
Cindy St. Hilaire: How good are these devices at actually changing behavior? We know we're really good at tracking our steps now and maybe monitoring our heartbeat or our oxygen levels. How good are they at changing behavior though? Do we know yet?
Andy Hughes: Yeah, that's a great question and certainly a significant area of ongoing research right now with physical activity interventions. Things that we've seen right now is that simple interventions that use the wearable devices alone may not be as effective as multifaceted interventions. And what I mean by that is interventions that use the smartwatch but may be coupled with another component, whether that is health education or counseling or more complex interventions that use gamification or just in time adaptive interventions.
And gamification really takes things to another level because that integrates components, competition or support or collaboration and really helps to build upon features of behaviors that we know have an increased likelihood of sustaining activity. With that being said, that is one of the challenges of physical activity interventions, is the sustainability of their improvements over the course of months to years.
And something that we have seen is the effects do typically decrease over time, but there is work on how do we integrate all of these features to develop interventions that can help to sustain the results more effectively. So we have seen some improvement, but finding ways to sustain the effects of physical activity is certainly an area of ongoing research.
Cindy St. Hilaire: I know it's funny that even as adults we love getting those gold stars or the circle completions. All of these devices, whether it's smartwatches like we're just talking about, or the other things for cardiac rehabilitation, they're generating a ton of data. What is happening with all this data? Who's actually analyzing it? How is it stored and what's that flow through from getting from the patient's body to the room where their physician is looking at it?
Andy Hughes: And that is certainly a challenge right now that is limiting the widespread adoption of these devices into routine clinical care is, as Jessilyn mentioned. The wearables generate a vast amount of data, and right now, we need to identify and develop a way as clinicians to sort through all of the noise in order to be able to identify the information that is clinically meaningful and worthy of action without significantly increasing the workload.
And a few of the barriers that will be necessary in order to reach that point is, one, finding ways to integrate the wearables' data into the electronic health record and also developing some machine learning algorithms or ways with which we can use the computational power of those technologies to be able to identify when there is meaningful data within all of the vast data that comes from wearables. So it's somewhere that certainly we need to get to for these devices to reach their full clinical potential, but we are limited right now by a few of those challenges.
Jessilyn Dunn: I was just going to say, I will add on to what Andy was saying about this idea behind digital biomarkers because this fits really nicely with this idea that giving people this huge data deluge is not helpful, but if we had a single metric where we can say, "Here's the digital biomarker of step count, and if you're above some threshold, you're good to go. And if you're below some threshold, some intervention is needed." That's a lot of the work that we've been doing, is trying to develop what are these digital biomarkers and how can they be ingested in a really digestible way?
Cindy St. Hilaire: Yeah, that's great. Regarding the clinical and the research grade devices, I know a Fitbit or Apple Watch can sometimes be used for those, but I guess I'm talking also about the other kind of more clinically oriented devices, how good is compliance and how trustable is that data? Everybody's on probably their best behavior when they're in the office with the physician or if they're on the treadmill in the cardiac lab, but home is a different story. And what don't we know about compliance when people are out of the office and the reliability of that data that's generated in that space?
Andy Hughes: I think you touched on a really important point right here, and one of the potential advantages of these wearable devices is that they provide continuous long-term monitoring over the course of weeks to months to years as opposed to those erratic measurements that we get from the traditional office visits or hospitalizations where, for example, the measurements we're taking are either in a supervised environment with a six-minute walk distance, for example, or self-reported or questionnaires.
So we build upon that information, but then additionally, we go beyond the observer effect where many individuals, the first week or two that you're wearing this new device, you may be more prone to increase your activity because you know that you're being monitored or you have this novel technology, but as you wear it for months to years, you outgrow those potential biases and you really can garner more comprehensive information.
In terms of compliance, we can speak to some of the research studies that have either really struggled with compliance and that limits the interpretability of their results and something we'll need to address in the future, but I think that's something that can be addressed with future studies keeping in mind all of the advantages that these devices offer compared to some of the traditional measures that we have used in the past.
Cindy St. Hilaire: With all this data we're collecting, whether it be biological data or even just behavioral data, have we actually learned anything new? And I mean that in terms of All Of Us study this, I don't know, it was like 5,000 patients I think, and lo and behold, it found out that higher step count correlated with lower risk for a ton of diseases, which is not exactly groundbreaking. So are we, at this point in time, learning anything new from the use of these at-home devices, or are they really just able to help us enforce what we thought we knew regarding behavior?
Andy Hughes: I think these devices have certainly provided some novel insights that build upon our understanding of physical activity. Many of us can hypothesize that decreased activity would have poor outcomes on health, which the studies have demonstrated in many facets. But in reference to All Of Us study that you mentioned, I think it's interesting to look as well at some of the diagnoses or conditions that were associated with decreased activity.
For example, reflux disease was also highlighted in that study, which may not have been identified if we didn't have the vast data and ability to really look for associations with diseases that have not been previously studied or thought to be related to physical activity. So I think that's one of the strong features of that database, is the wealth of knowledge that really will be hypothesis generating and help to inform future studies as we look even beyond cardiovascular conditions.
Cindy St. Hilaire: One question, and you did bring it up in a bit of the discussion in your piece, is the bias that is in these devices. We know from COVID at-home pulse oximeters do not work as efficiently on darker skin. We actually know that going into bathrooms with the hand sensors that spit out the paper towels. So what kind of disparities or biases do these devices create or reinforce in the population?
Jessilyn Dunn: This is such a critical topic because a lot of these issues had been discovered retrospectively because the people who were developing the technologies were not the representative of the people who were using the technologies. I think that's something that across the board we've been looking at from device development to AI implementation, which is having people who are going to be using the devices in the process of developing the technology and having voices heard from across the board.
We did a detailed look when we were evaluating devices for their accuracy at this exact question of where the heart rate sensors in smartwatches use optical based technology. And there was some evidence that was also an issue for people with varying skin tones, for people with wrist tattoos or more hair or freckles. And so, we did a deep dive and the generation of devices that we looked at which would meet this study was probably about three years ago.
We didn't see any discrepancies. And so, that's just one study and there are many more to be done, but I think prior to the technology development as well as once the technology comes out, keeping an eye on how that technology is doing, whether there are continued reports of failure of the technologies is really important. And there are a lot of ways that we can be vigilant about that.
Cindy St. Hilaire: Yeah, that's great. And so, Andy, regarding patient populations, I can also see perhaps socioeconomic implications of this because smartwatches are not cheap. So how do we see that in terms of helping our patients? Are we going to be able to get a smartwatch through our insurance company?
Andy Hughes: I think that's one of the really important next steps, is finding ways to make sure that as we advance the field of wearable devices in clinical care, that we recognize some of the existing inequities in terms of access to care, access to digital technologies that currently exist, and find ways by partnering with health insurance companies and the industry and providers and members of that community, finding ways to not only advance wearables, but use it in a way that we can decrease health disparities by really helping to increase access for these digital technologies to the underserved communities.
Jessilyn Dunn: Yeah, the beauty of these technologies is that truthfully, at their core, they're very cheap. They're not difficult to develop, they're not difficult to build and disseminate. So a lot of what we think about is the infrastructure that goes around these devices. Does it require a smartphone to transfer data? Does it require internet access? What are the other pieces that need to be in place for these devices to work within an ecosystem? So this starts to get to questions beyond the devices themselves, but there's certainly a lot to think about and be done in the area of equity and ensuring that these devices can help everyone.
Cindy St. Hilaire: And there's also the, I guess, ethical considerations of who owns this data. Obviously, if it's a consumable that you went and bought at Target, that's probably different than the one you're getting from your cardiologist. But who owns the data? Who has access to it? And are there any cases in the literature where an individual who's had certain measurements taken, have those measurements come back to bite them?
And I guess I'm thinking of something like cardiac rehab. If a patient doesn't get up and move enough or doesn't follow their physical therapy enough or lose weight quick enough, could their insurance coverage get cut? Could their premiums go up? What safeguards are in place for these very tricky situations? Are there safeguards in place?
Andy Hughes: And on the clinical side, I think it will be important to treat this information just like any other protected health information that we have as part of the electronic health record. And so, there will be inherently safeguards around that in a similar manner for how we treat other protected health information.
But I think another important component of that will be a very clear consent policy when we reach the point that patients are consenting to include this information and their electronic health record, in terms of what the proposed benefits are and the potential risks associated with it, because it really is a vast amount of unique data that needs to be protected and safeguarded. And part of that comes by treating it as protected health information, but we will also need to make sure that there's a very clear consent policy that goes with it.
Cindy St. Hilaire: Yeah. What do we see as the next steps in wearable devices? What do you guys see as the next big thing? I know one's coming from the actual AI and device side of things, and the other one is coming from the clinical side of things. What do each of you see as the next thing in this field?
Jessilyn Dunn: I think on the device and AI side of things, I think we're thinking toward improving battery life, increasing the suite of sensors that are being added to these devices so we have a wider variety of measurements that are more representative of physiology, and then better algorithms to have better detection of sleep or activity or certain types of activity or certain types of arrhythmias. This combination of hardware and software and algorithms, I think coming together as all of these different pieces evolve will show us some really cool technology in the years to come.
Andy Hughes: And I think from a clinical side, it's really twofold moving forward. I think as Jessilyn mentioned, there's a lot of novel sensor technologies that have a lot of exciting and evolving potential that we can hopefully integrate into the clinical space, but on the other hand, it's how can we use these wearable devices to enhance traditional therapies that we're already using?
For example, if we take the heart failure population, is there a way that we can use the wearable devices and the existing measurements with heart rate and physical activity and blood pressure to find a way to improve remote management and safely up-titrate guideline directed medical therapy, which are medications that we know have clinical benefit. But can we augment their clinical benefit and their utility by using some of the existing technologies that we already have?
And then lastly, building upon the initial studies with larger trials in more diverse generalizable populations to really enhance our understanding of the benefits that these devices may have for different cardiovascular conditions.
Cindy St. Hilaire: Well, this was wonderful. Dr Andrew Hughes and Dr Jessilyn Dunn, thank you so much for joining me. The review, Wearable Devices in Cardiovascular Medicine, will be out in our March 3rd issue of Circulation Research. I forget which one, so I'll have to edit that out. Thank you so much for joining us, and I learned a ton. This was great.
Jessilyn Dunn: Thank you.
Andy Hughes: Thank you.
Cindy St. Hilaire: That's it for our highlights from the March 3rd and March 17th issues of Circulation Research. Thank you for listening. Please check out the Circulation Research Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Dr Andrew Hughes and Dr Jessilyn Dunn.
This podcast is produced by Ishara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy texts for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, you're on-the-go Source for the most exciting discoveries in basic cardiovascular research.
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 information, visit ahajournals.org.
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