Podcasts about cardiomyocytes

Play Episode Listen Later Jul 24, 2023

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.22.550175v1?rss=1 Authors: Trewin, A. J., Weeks, K. L., Wadley, G. D., Lamon, S. Abstract: Cardiomyocyte calcium homeostasis is a tightly regulated process. The mitochondrial calcium uniporter (MCU) complex can buffer elevated cytosolic Ca2+ levels and consists of pore-forming proteins including MCU, and various regulatory proteins such as mitochondrial calcium uptake proteins 1 and 2 (MICU1/2). The stoichiometry of these proteins influences the sensitivity to Ca2+ and activity of the complex. However, the factors that regulate their gene expression remain incompletely understood. Long non-coding RNAs (lncRNAs) regulate gene expression through various mechanisms, and we recently found that the lncRNA Tug1 increased the expression of Mcu and associated genes. To further explore this, we performed antisense LNA knockdown of Tug1 (Tug1 KD) in H9c2 rat cardiomyocytes. Tug1 KD increased MCU protein expression, yet pyruvate dehydrogenase dephosphorylation, which is indicative of mitochondrial Ca2+ uptake was not enhanced. However, RNA-seq revealed that Tug1 KD increased Mcu along with differential expression of greater than 1000 genes including many related to Ca2+ regulation pathways in the heart. To understand the effect of this on Ca2+ signalling, we measured phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and its downstream target cAMP Response Element-Binding protein (CREB), a transcription factor known to drive Mcu gene expression. In response a Ca2+ stimulus, the increase in CaMKII and CREB phosphorylation was attenuated by Tug1 KD. Inhibition of CaMKII, but not CREB, partially prevented the Tug1 KD-mediated increase in Mcu. Together, these data suggest that Tug1 modulates MCU expression via a mechanism involving CaMKII and regulates cardiomyocyte Ca2+ signalling which could have important implications for cardiac function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

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Cardiomyocyte mechanical memory is regulated through the talin interactome and DLC1 dependent regulation of RhoA

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Play Episode Listen Later Jul 19, 2023

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.19.549635v1?rss=1 Authors: Marhuenda, E., Xanthis, I., Pandey, P., Azad, A., Richter, M., Pavlovic, D., Gehmlich, K., Faggian, G., Ehler, E., Levitt, J., Ameer-Beg, S., Iskratsch, T. Abstract: Mechanical properties are cues for many biological processes in health or disease. Likewise, in the heart it is becoming clearer that mechanical signals are critically involved in the disease progression. Cardiomyocytes sense the mechanical properties of their environment at costameres through integrins and associated proteins, including the mechanosensitive protein talin as an integral component. Our previous work indicated different modes of talin tension, depending on the extracellular matrix stiffness. Here, we wanted to study how this leads to downstream mechanotransduction changes, further influencing the cardiomyocyte phenotype. Combining immunoprecipitations and Fluorescence Recovery after Photobleaching (FRAP) experiments, we identify that the talin interacting proteins DLC1, RIAM and paxillin each preferentially bind to talin at specific extracellular matrix stiffness and this interaction is preserved even in absence of tension. This demonstrates a mechanical memory, which we confirm further in vivo in mouse hearts. The mechanical memory is regulated through adhesion related kinase pathways. Optogenetic experiments using the LOVTRAP systems confirm direct competition between the individual proteins, which again is altered through phosphorylation. DLC1 regulates RhoA activity in a stiffness dependent way and both loss and overexpression of DLC1 results in myofibrillar disarray. Together the study demonstrates a mechanism of imprinting mechanical information into the talin-interactome to finetune RhoA activity, with impacts on cardiac health and disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Non-cell autonomous cardiomyocyte regulation complicates gene supplementation therapy for LMNA cardiomyopathy

Play Episode Listen Later Jul 18, 2023

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.18.549413v1?rss=1 Authors: Sun, Y., Guo, C., Chen, Z., Lin, J., Yang, L., Zhang, Y., Wu, C., Zhao, D., Jardin, B., Pu, W., Zhao, M., Dong, E., Hu, X., Zhang, S., Guo, Y. Abstract: Aims: Recombinant adeno-associated viruses (rAAVs) are federally approved gene delivery vectors for in vivo gene supplementation therapy. Loss-of-function truncating variants of LMNA, the coding gene for Lamin-A/C, are one of the primary causes of inherited dilate cardiomyopathy (DCM). Here we aim to study whether AAV-based LMNA supplementation could treat LMNA deficiency-triggered cardiac defects. Methods and Results: We compared whole-body, cardiomyocyte-specific and genetic-mosaic mouse models that carry Lmna truncating variants at the same genetic loci and uncovered primarily a non-cell autonomous impact of Lmna on cardiomyocyte maturation. Whole-body lamin-A supplementation by rAAVs moderately rescued the cardiac defects in Lmna germline mutants. By contrast, cardiomyocyte-specific lamin-A addback failed to restore the cardiomyocyte growth defects. A Cre-loxP-based AAV vector that expresses lamin-A throughout the body but excluding the heart was able to restore cardiomyocyte growth in Lmna germline mutants. Conclusions: Lmna regulates cardiomyocyte growth non-cell autonomously. Non-myocytes are the key cell targets for a successful gene therapy for LMNA-associated cardiac defects. Translational perspective: LMNA truncating mutations are among the major causes of inherited DCM. AAV gene supplementation therapy is emerging as a promising strategy to treat genetic cardiomyopathy, but whether this strategy is suitable for LMNA cardiomyopathy remained unclear. Our study counterintuitively showed that the cardiomyocytes are not necessarily the correct therapeutic cell targets for AAV-based treatment of LMNA cardiomyopathy. By contrast, careful elucidation of cell-autonomous versus non-cell-autonomous gene functions is essential for the proper design of a gene supplementation therapy for cardiomyopathy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Cardiomyocyte-specific adenylyl cyclase type-8 overexpression induces cell-autonomous activation of RelA and non-cell-autonomous myocardial and systemic inflammation

Play Episode Listen Later Jul 16, 2023

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.15.549173v1?rss=1 Authors: Kumar, V., Bermea, K. C., Kumar, D., Singh, A., Verma, A., Kaileh, M., Sen, R., Lakatta, E. G., Adamo, L. Abstract: Mice with cardiac-specific overexpression of adenylyl cyclase (AC) type 8 (TGAC8) are under a constant state of severe myocardial stress and have been shown to have a remarkable ability to adapt to this stress. However, they eventually develop accelerated cardiac aging and cardiac fibrosis, and experience reduced longevity. Here we show that young (3-month-old) TGAC8 animals are characterized by a broad and extensive inflammatory state, that precedes the development of cardiac fibrosis. We demonstrate that activation of ACVIII in the cardiomyocytes results in cell-autonomous RelA-mediated NF-{kappa}B signaling. This is associated with non-cell-autonomous activation of proinflammatory and age-associated signaling in myocardial endothelial cells, increases in serum levels of inflammatory cytokines, changes in myocardial immune cells, and changes in the size or composition of lymphoid organs. Finally, we provide evidence suggesting that ACVIII-driven RelA activation in cardiomyocytes might be mediated by calcium-Protein Kinase A (PKA) signaling. Our findings highlight a novel mechanistic connection between cardiomyocyte stress, myocardial para-inflammation, systemic inflammation, and aging, and therefore point to novel potential therapeutic targets to reduce age-associated myocardial deterioration. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

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Complement C3 reduces apoptosis in human cardiomyocytes

Play Episode Listen Later May 2, 2023

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.01.538962v1?rss=1 Authors: Zhang, M., Fang, Z., Li, X., Yang, F., Xiaoli, A. M. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

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The Effect of E-Cigarettes on the Human Heart Studied using Cardiomyocytes Generated from Induced Pluripotent Stem Cells

Play Episode Listen Later Apr 6, 2023

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.06.535946v1?rss=1 Authors: Bhowmik, A. T., Zhao, S. R., Wu, J. C. Abstract: Electronic cigarettes (e-cigarettes) have become increasingly popular with adolescents in recent years as a result of aggressive marketing schemes, false safety claims, and appealing flavors targeted towards teens from e-cigarette companies. In the past 8 years alone, e-cigarette use amongst youth has increased by 18 times. Although many dangerous effects of smoking e-cigarettes on lungs have come to light, limited and qualitative effort has been made to analyze the impact of smoking e-cigarettes on the human heart directly. In this study, we determined e-liquid cardiotoxicity in both healthy cells and cells with long QT syndrome by treating healthy and diseased human cardiomyocytes with e-liquids with varying nicotine concentrations. These cardiomyocytes were generated from human induced pluripotent stem cells. The cardiomyocytes were divided into 5 groups, a control group and 4 test groups, each treated with e-liquid containing varying amounts of nicotine between 0% and 70%. The cells' biological indicators such as heart rate, pulse pressure, essential protein concentration, and metabolic activity, were measured, characterized using three different functional assays: contractility, Western blot, and viability, and tracked closely over 2 weeks. The results demonstrated that acute exposure to e-liquid led to tachycardia, hypertension, decreased protein levels, and cell death. The rate of cardiotoxicity increases with higher nicotine concentrations. The basal fluid also showed non-negligible toxicity. Under identical conditions, the functionality of the diseased heart cells declined at a faster rate compared to healthy cells. Overall, this work systematically establishes the harmful physiological effects of e-cigarettes on the human heart quantitatively. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

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A Novel Transcription Factor Combination for Direct Reprogramming to a Spontaneously Contracting Human Cardiomyocyte-like State

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Play Episode Listen Later Mar 15, 2023

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.14.532629v1?rss=1 Authors: Tejeda, M. R., Fonoudi, H., Weddle, C. J., DeKeyser, J.-M., Lenny, B., Fetterman, A. K., Magdy, T., Sapkota, Y., Epting, C., Burridge, P. W. Abstract: The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts. However, this process has been less successful in human cells, thus limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross- species concordance between the required transcription factor combinations for mouse and human cells. To address this issue, we identified novel transcription factor candidates to induce cell conversion between human fibroblasts and cardiomyocytes, using the network-based algorithm Mogrify. We developed an automated, high-throughput method for screening transcription factor, small molecule, and growth factor combinations, utilizing acoustic liquid handling and high- content kinetic imaging cytometry. Using this high-throughput platform, we screened the effect of 4,960 unique transcription factor combinations on direct conversion of 24 patient-specific primary human cardiac fibroblast samples to cardiomyocytes. Our screen revealed the combination of MYOCD, SMAD6, and TBX20 (MST) as the most successful direct reprogramming combination, which consistently produced up to 40% TNNT2+ cells in just 25 days. Addition of FGF2 and XAV939 to the MST cocktail resulted in reprogrammed cells with spontaneous contraction and cardiomyocyte-like calcium transients. Gene expression profiling of the reprogrammed cells also revealed the expression of cardiomyocyte associated genes. Together, these findings indicate that cardiac direct reprogramming in human cells can be achieved at similar levels to those attained in mouse fibroblasts. This progress represents a step forward towards the clinical application of the cardiac direct reprogramming approach. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

The ion channel Trpc6a regulates the cardiomyocyte regenerative response to mechanical stretch.

Play Episode Listen Later Mar 14, 2023

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.14.532536v1?rss=1 Authors: Rolland, L., Faucherre, A., Mancilla Abaroa, J., Drouard, A., Jopling, C. Abstract: Myocardial damage caused for example by cardiac ischemia leads to ventricular volume overload resulting in increased stretch of the remaining myocardium. In adult mammals, these changes trigger an adaptive cardiomyocyte hypertrophic response which, if the damage is extensive, will ultimately lead to pathological hypertrophy and heart failure. Conversely, in response to extensive myocardial damage, cardiomyocytes in the adult zebrafish heart and neonatal mice proliferate and completely regenerate the damaged myocardium. We therefore hypothesized that in adult zebrafish, changes in mechanical loading due to myocardial damage may act as a trigger to induce cardiac regeneration. Based, on this notion we sought to identify mechanosensors which could be involved in detecting changes in mechanical loading and triggering regeneration. Here we show using a combination of knockout animals, RNAseq and in vitro assays that the mechanosensitive ion channel Trpc6a is required by cardiomyocytes for successful cardiac regeneration in adult zebrafish. Furthermore, using a cyclic cell stretch assay, we have determined that Trpc6a induces the expression of components of the AP1 transcription complex in response to mechanical stretch. Our data highlights how changes in mechanical forces due to myocardial damage can be detected by mechanosensors which in turn can trigger cardiac regeneration. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

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Cardiomyocyte transcriptomic signatures in response to Trypanosoma cruzi infection underpin Chagas cardiomyopathy progression.

Play Episode Listen Later Mar 3, 2023

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.27.530371v1?rss=1 Authors: Candray, K., Nakagama, Y., Masamichi, I., Nakagama, S., Tshibangu-Kabamba, E., Takeda, N., Sugiura, Y., Nitahara, Y., Michimuko-Nagahara, Y., Kaku, N., Onizuka, Y., Arias, C.-E., Mejia, M., Alas, K., Pena, S., Maejima, Y., Komuro, I., Nakajima-Shimada, J., Kido, Y. Abstract: Chagas disease can lead to life-threatening cardiac manifestations that occur more frequently in geographic areas more prevalent with the TcI/II circulating genetic strains. To elucidate the differential transcriptomic signatures of the cardiomyocyte resulting from infection with TcI/II or TcVI T. cruzi strains and explore their relationships with pathogenesis, HL-1 rodent cardiomyocytes were infected with TcI/II or TcVI T. cruzi trypomastigotes. RNA was isolated serially post-infection for microarray analysis. Enrichment analyses of differentially expressed genes (fold-change greater than or equal to 2 or less than or equal to 0.5) highlighted the over-represented biological pathways. We found that Oxidative stress-related GO terms, 'Hypertrophy model', 'Apoptosis', and 'MAPK signaling' pathways (all with p less than 0.01) were upregulated. 'Glutathione and one-carbon metabolism' pathway, and 'Cellular nitrogen compound metabolic process' GO term (all with p less than 0.001) were upregulated exclusively in the cardiomyocytes infected with the TcI/II strains. Upregulation in the oxidative stress-related and hypertrophic responses are shared hallmarks with viral myocarditis, another inflammatory cardiac pathology. Nitrogen metabolism upregulation and Glutathione metabolism imbalance may implicate the relation of nitrosative stress and poor oxygen radicals scavenging in the unique pathophysiology of chagasic cardiomyopathy development. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Cardiomyocyte-fibroblast interaction regulates ferroptosis and fibrosis after myocardial injury

Play Episode Listen Later Feb 8, 2023

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.07.527364v1?rss=1 Authors: Mohr, M. E., Li, S., Trouten, A. M., Stairley, R. A., Roddy, P. L., Liu, C., Zhang, M., Sucov, H., TAO, G. Abstract: Neonatal mouse hearts have transient renewal capacity which is lost in juvenile and adult hearts. After myocardial infarction (MI) in neonatal hearts, an initial loss of cardiomyocytes occurs but it is unclear through which type of regulated cell death (RCD). In the current studies, we induced MI in neonatal and juvenile mouse hearts, and show that ischemic cardiomyocytes primarily undergo ferroptosis, a non-apoptotic and iron-dependent form of RCD. We demonstrate that cardiac fibroblasts (CFs) protect cardiomyocytes from ferroptosis through paracrine factors and direct cell-cell interaction. CFs show strong resistance to ferroptosis due to high ferritin expression. Meanwhile, the fibrogenic role of CFs, typically considered detrimental to heart function, is negatively regulated by paired-like homeodomain 2 (Pitx2) signaling from cardiomyocytes. In addition, Pitx2 prevents ferroptosis in cardiomyocytes by regulating ferroptotic genes. Understanding the regulatory mechanisms of cardiomyocyte survival and death can identify potentially translatable therapeutic strategies for MI. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

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Catalytic antibodies in arrhythmogenic cardiomyopathy patients cleave desmoglein 2 and N-cadherin and impair cardiomyocyte cohesion

Play Episode Listen Later Feb 8, 2023

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.08.527624v1?rss=1 Authors: Yeruva, S., Stangner, K., Jungwirth, A., Hiermaier, M., Shoykhet, M., Kugelmann, D., Hertl, M., Egami, S., Ishii, N., Koga, H., Hashimoto, T., Weis, M., Beckmann, B. M., Biller, R., Schuettler, D., Kaab, S., Waschke, J. Abstract: Aims: Arrhythmogenic cardiomyopathy (AC) is a severe heart disease predisposing to ventricular arrhythmias and sudden cardiac death caused by mutations affecting intercalated disc (ICD) proteins and aggravated by physical exercise. Recently, autoantibodies targeting ICD proteins, including the desmosomal cadherin desmoglein 2 (DSG2), were reported in AC patients and were considered relevant for disease development and progression, particularly in patients without underlying pathogenic mutations. However, it is unclear at present whether these autoantibodies are pathogenic and by which mechanisms show specificity for DSG2 and thus can be used as a diagnostic tool. Methods and Results IgG fractions were purified from 15 AC patients and 4 healthy controls. Immunostainings dissociation assays, atomic force microscopy (AFM), western blot analysis and Triton-X-100 assays were performed utilizing human heart left ventricle tissue, HL-1 cells, and murine cardiac slices. Immunostainings revealed that autoantibodies against ICD proteins are prevalent in AC and most autoantibody fractions have catalytic properties and cleave the ICD adhesion molecules DSG2 and N-cadherin, thereby reducing cadherin interactions as revealed by AFM. Furthermore, most of the AC-IgG fractions causing loss of cardiomyocyte cohesion activated p38MAPK, which is known to contribute to a loss of desmosomal adhesion in different cell types, including cardiomyocytes. In addition, p38MAPK inhibition rescued the loss of cardiomyocyte cohesion induced by AC-IgGs. Conclusion Our study demonstrates that catalytic autoantibodies play a pathogenic role by cleaving ICD cadherins and thereby reducing cardiomyocyte cohesion by a mechanism involving p38MAPK activation. Finally, we conclude that DSG2 cleavage by autoantibodies could be used as a diagnostic tool for AC Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

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Remote-refocusing light-sheet fluorescence microscopy enables 3D imaging of electromechanical coupling of hiPSC-derived and adult cardiomyocytes in co-culture

Play Episode Listen Later Jan 29, 2023

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.28.526043v1?rss=1 Authors: Dvinskikh, L., Sparks, H., Brito, L., MacLeod, K. T., Harding, S. E., Dunsby, C. Abstract: Improving cardiac function through stem-cell regenerative therapy requires functional and structural integration of the transplanted cells with the host tissue. Visualizing the electromechanical interaction between native and graft cells necessitates 3D imaging with high spatio-temporal resolution and low photo-toxicity. A custom light-sheet fluorescence microscope was used for volumetric imaging of calcium dynamics in co-cultures of adult rat left ventricle cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes. Aberration-free remote refocus of the detection plane synchronously to the scanning of the light sheet along the detection axis enabled fast dual-channel 3D imaging at subcellular resolution without mechanical sample disturbance at up to 8 Hz over a {approx}300 m x 40 m x 50 m volume. The two cell types were found to undergo electrically stimulated and spontaneous synchronized calcium transients and contraction. Electromechanical coupling was found to improve with co-culture duration, with 50% of adult-CM coupled after 24 hours of co-culture, compared to 19% after 4 hours (p = 0.0305). Immobilization with para nitroblebbistatin did not prevent calcium transient synchronization, with 35% and 36% adult-CM coupled in control and treated samples respectively (p = 0.91), indicating that electrical coupling can be maintained independently of mechanotransduction. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Circulation January 10, 2023 Issue

Play Episode Listen Later Jan 9, 2023 24:41

Please join authors Loren Field and Sean Reuter, as well as Associate Editor Thomas Eschenhagen as they discuss the article "Cardiac Troponin I-Interacting Kinase Affects Cardiomyocyte S-Phase Activity But Not Cardiomyocyte Proliferation." Dr. Greg Hundley: Welcome listeners, to this January 10th issue of Circulation on the Run, and I am Dr. Greg Hundley, associate editor, director of the Pauley Heart Center at VCU Health in Richmond, Virginia. Dr. Peder Myhre: I am Dr. Peder Myhre from Akershus University Hospital and University of Oslo in Norway. Dr. Greg Hundley: Well, listeners, this week's feature discussion delves into the world of preclinical science and evaluates cardiac troponin I and its impact on S phase activity in cardiomyocytes, and does that relate to cardiomyocyte proliferation. But before we get to that, how about we grab a cup of coffee and Peder and I will work through some of the other articles in the issue. Peder, how about this week I go first? Dr. Peder Myhre: Go ahead, Greg. Dr. Greg Hundley: Right. So Peder, this first study evaluated whether the burden of positive coronary artery calcification on cardiovascular disease differed by multidimensional individual characteristics, and so the investigators led by Dr. Kosuke Inoue from Kyoto University sought to investigate the heterogeneity in the association between positive coronary artery calcium and incident cardiovascular disease. And so Peder, to examine this question, the authors implemented a cohort study design that included adults aged greater than 45 years, free of cardiovascular disease, from the Multi-Ethnic Study of Atherosclerosis, or MESA, and after propensity score matching in a one-to-one ratio, they applied a machine learning causal forest model to, first, evaluate the heterogeneity in the association between positive coronary artery calcium and incident cardiovascular disease and then, second, to predict the increase in cardiovascular disease risk at 10 years when the coronary artery calcium score was greater than zero, so versus is it zero at all at the individual level? Dr. Peder Myhre: Oh, Greg, that is so cool, so using machine learning for coronary artery calcium and risk prediction, I'm very excited. What did they find? Dr. Greg Hundley: Right, Peder, so the expected increases in cardiovascular disease risk when the coronary artery calcium score was greater than zero were heterogeneous across individuals. Moreover, nearly 70% of people with low atherosclerotic cardiovascular disease risk showed a large increase in cardiovascular disease risk when the coronary calcium score was greater than zero, highlighting the need for coronary artery calcium screening among such low-risk individuals. And Peder, future studies are really needed to assess whether targeting individuals for coronary artery calcium measurements based on not only the absolute ASCVD risk, but also the expected increase in CVD risk when a CAC score is greater than zero and whether that improves overall assessment of cardiovascular outcomes. Dr. Peder Myhre: Wow, that is so clinically relevant and very interesting. And we're actually going to stay clinically relevant with the next paper which is about anti-platelet therapy after PCI. And this paper describes the long-term results of the HOST-EXAM trial. To remind you, Greg, the HOST-EXAM trial was an investigator-initiated prospective, randomized, open label, multicenter trial done at 37 sites in Korea. They enrolled patients who had undergone PCI with DES and maintained dual anti-platelet therapy without any clinical event for a mean 12 months and then they were randomized one to-one to either clopidogrel, 75 milligrams once daily, or aspirin, 100 milligram once daily. The primary results of this trial was published in Lancet in 2021 and showed superiority of clopidogrel over aspirin in prevention of the composite of MACE and major bleeding during 24 months of followup. And then, through the current paper, this describes the results of the post trial extended followup of about five years. Dr. Greg Hundley: Very nice, Peder, so aspirin versus clopidogrel and looking at the maintenance of that monotherapy and cardiovascular outcomes. Wow, so what did they find? Dr. Peder Myhre: Yeah, Greg. They, in this extended followup study, had a total of 5.8 years median followup, and the primary endpoint occurred in 12.8% in the clopidogrel group versus 16.9% in the aspirin group, and that has a range of 0.74 with a 95% conference interval ranging from 0.63 to 0.86. So also the clopidogrel group had lower risk of the secondary thrombotic endpoint and the secondary bleeding endpoint while there was no significant difference in the incident on all caused death. So Greg, to conclude, these very interesting results from the primary analysis of the HOST-EXAM trial was consistent through the longer followup, and this support the use of clopidogrel over aspirin monotherapy from 12 months onwards after PCI. Dr. Greg Hundley: Very nice Peder, beautiful description and sounds like long-term clopidogrel use over aspirin was quite beneficial. Well, the next study comes to us from the world of preclinical science, and it is from the investigative group led by Dr. Yunzeng Zou from Shanghai Institute of Cardiovascular Diseases and the Zhongshan Hospital and Fudan University. Peder, the study pertains to diabetes. So diabetic heart dysfunction is a common complication of diabetes mellitus and cell death is a core event that leads to diabetic heart dysfunction. However, the time sequence of cell death pathways and the precise intervening time of particular cell death type remained largely unknown in diabetic hearts. And so, Peder, this study aimed to identify the particular cell death type that is responsible for diabetic heart dysfunction and propose a promising therapeutic strategy by intervening in this cell death pathway. Dr. Peder Myhre: Wow, Greg, that is really interesting. Heart dysfunction in diabetes is something that we really have to learn more about and I'm so excited to hear what these authors found, Greg. Dr. Greg Hundley: Right. So first, Peder, the authors identified necroptosis as the predominant cell death type at later stages in the diabetic heart. And then second, Peder, the CB2 receptor, and we'll call that CB2-R, recruits transcription factor Bach2 to repress necroptosis and protects against diabetic heart injury while hyperglycemia and MLKL in turn phosphorylates CB2-R to promote ubiquitous dependent degradation of CB2-R, thus forming a CB2-R centric feedback loop of necroptosis. And finally, Peder, cardiac CB2-R or Bach2 expression negatively correlates with both MLKL 10 expression and the extent of diabetic heart injuries in humans. And so the clinical implications of these findings, Peder, are that the CB2-R centric necrotic loop represents a promising target for the clinical treatment of diabetic heart injuries. Dr. Peder Myhre: So Greg, this paper that comes to us from corresponding author Amanda Paluch from University of Massachusetts Amherst, is a meta-analysis of eight prospective studies with device measured steps including more than 20,000 adults who were followed for CVD events. And the mean age of participants in this study was 63 years and 52% were women. And the participants were followed for a median of 6.2 years and 1,523 cardiovascular events occurred. So first, Greg, there was a significant difference in the association of steps per day in cardiovascular disease between older, that is greater or equal to 60 years, and younger, that is less than 60 years adults. So for older adults that has the ratio for cardiovascular disease using Q1 as reference was 0.80 for Q2, 0.62 for Q3, and 0.51 for Q4. And for younger adults that has ratio for cardiovascular disease using Q1 as reference was 0.79 for Q2, 0.90 for Q3, and 0.95 for Q4. And in the paper, Greg, there are some beautiful, restricted cubic lines that really illustrate the association between daily steps and the risk of cardiovascular disease among older adults and in younger adults. So the authors conclude that for older adults taking more daily steps is associated with a progressively lower risk of cardiovascular disease. And monitoring and promoting steps per day is a simple metric for clinician patient communication and population health to reduce the risk of cardiovascular disease. Dr. Greg Hundley: Well, Peder, we've got some other very interesting articles in this issue and how about we dive into that mail bag and discuss a few of those. So I'll go first. The first is a Perspective piece by Professor Powell-Wiley entitled “Centering Patient Voices through Community Engagement in Cardiovascular Research.” A very important topic where can those in the community actually help us design meaningful outcomes for our research initiatives? And next Peder, there is a Research Letter from Professor Evans entitled “Increasing Mononuclear deployed Cardiomyocytes by Loss of E2F7/8, and does that fail to improve cardiac regeneration post myocardial infarction?” Dr. Peder Myhre: Thanks, Greg. We also have an ECG Challenge by Dr. Li entitled, “What Is The Truth Behind Abnormal ECG Changes?” And this is describing a very rare and interesting cause of ST segment elevation. I recommend everyone to read that case. We also have our own Nick Murphy who gives us the Highlights from the Circulation Family of Journals where he summarizes five papers from the Circulation subspecialty journals. First, the experience with a novel visually assisted ablation catheter is reported in circulation A and E. The impact of various exercise training approaches on skeletal muscle in heart failure with preserved the F is presented in circulation heart failure. Gaps in heart failure treatment over a decade are reported in circulation cardiovascular quality and outcomes, and the associations of machine learning approaches to plaque morphology from coronary CTA with ischemia are reported in circulation cardiovascular imaging. And finally, Greg, an observational study of left main PCI at sites with and without surgical backup is reported in circulation cardiovascular interventions. Let's go on to the feature paper today describing the cardiac troponin I interacting kinase and the impact on cardiomyocyte S phase activity. Dr. Greg Hundley: Great, let's go. Welcome listeners to this January 10th feature discussion. Very interesting today as we are going to delve into the world of preclinical science. And we have with us today Dr. Loren Field and Dr. Sean Reuter from University of Indiana in Indianapolis, Indiana. And our own associate editor, Dr. Thomas Eschenhagen from University Medical Center of Hamburg in Hamburg, Germany. Welcome gentlemen. Well, Loren, we're going to start with you. Can you describe for us some of the background information that went into the preparation of your study, and what was the hypothesis that you wanted to address? Dr. Loren Field: Sure. This study actually came about in a rather roundabout fashion. We were doing a study with Kai Wollert in Hanover, Germany, where we were looking at the impact of a CXCR4 antagonist, which is used to mobilize stem cells from the bone marrow. And we had sent our mice over to Kai's lab and we have a mouse model that allows us to track S phase activity in cardiac myocytes, so these are cells are starting to replicate. And Kai crossed them into a different genetic background. And when he sent the mice back to us to analyze the hearts, we observed that we saw things that we never saw before in our experiments here. His injury model was different than ours and now the mouse also had a genetic background, so we had to spend about a year to figure out if it was the injury model or the background. It turned out to be the genetic background, and the phenotype was these mice had about a 15-fold elevated level of cell cycle reentry. So then it became a relatively simple genetics game where we took the progenitor mice, made F1 animals, looked for the phenotype, did backcross animals, and basically identified the gene responsible for the phenotype. Dr. Greg Hundley: Very nice. And so in this study moving forward, what hypothesis did you want to address? Dr. Loren Field: Well, the main hypothesis was to figure out what the gene was and then secondarily to figure out the degree of cell cycle progression. When the cell is proliferating, the first task is to replicate its genome, which is S phase activity that's followed by the nuclei dividing and then finally by the cell itself becoming two cells. So our task was to identify, first, the gene and secondly, how far through the cell cycles the cells progressed. Dr. Greg Hundley: Very nice. And how did you construct your experiment? Dr. Loren Field: It was, again, very straightforward. It was simply setting up the appropriate genetic crosses to produce the animals. For the past 10, 15 years, we've been developing a computer assisted assay that allows us to identify the anatomical position of S phase positive cardiac myocytes in sections of the heart. And basically, we apply that program to the different genetic backgrounds and after that it's a ball of mapping studies, QTL mapping. Dr. Greg Hundley: So really mechanistic understanding. Well listeners, we're next going to turn to Sean, and Sean, can you describe for us your study results? Dr. Sean Reuter: Yes, as Loren stated, we saw a 15-fold increase in the S phase activity within the remote zone. Now we partition the heart in three different zones after injury, so the scar, the border zone, and then the remote zone or injury. And as Loren stated, we saw a 15-fold increase in the S phase activity, cell cycle activity, in the remote zone. And it's only because we have this system in hand that we can anatomically map the S phase activity within the heart that we were able to detect and also quantify this. And I think that's the reason we discovered this particular phenotype. But in addition to that, we performed RNA-seq or Exome sequencing and discovered that TNNI3K was the responsible gene for elevated S phase activity within the remote zone and border zone, but interestingly not in the scar. Dr. Greg Hundley: Very interesting, Sean, and so describe for us the importance of the TNNI3K and its relationship to this S phase. Dr. Sean Reuter: Sure. This particular gene was first discovered around 2000, and it's been studied for a while now, but the targets of this kinase specifically expressed in the heart, and it does get elevated after injury, but the actual targets are not well described or well known. It's believed that it phosphorylates some mild filament fibers and structural proteins, but the actual mechanism and the consequence of this is not known. So when we saw this in the remote zone, the elevated S phase, our current theory is that we believe that it's probably increasing oxidative stress that would basically further out from the at-risk zone or the border zone and then it now is in the remote zone. So we think it's just causing the heart, a pathological area of the heart, basically to expand. And so that's our current theory. Other groups have published on the oxidative stress in over expression of TNNI3K as well. Dr. Greg Hundley: Very nice. Well listeners, next we are going to turn to our associate editor, Thomas many articles come your way and come across your desk. What attracted you to this particular article, and how do we put its results really in the context of cardiac regeneration? Dr. Thomas Eschenhagen: Indeed, there were several arguments. It's a cool paper and the whole field is still very important. As probably most of you know, the field have a rough ride over the last 20 years, went up and down, lots of bad findings. And in the end it turns out that we are there where we have been 20 years ago, the mammalian heart essentially doesn't regenerate. So anything which would improve that would be of very major importance. Why is it a good paper? Because it starts from a very clear finding, one mouse, which looks like strongly regenerating after MI, another mouse line, which doesn't. And so by applying, let's say, classical genetic, very stringent methodology, Loren Field and his group identified this troponin I kinase to be the culprit. And they also proved it, because putting it back in the strain with a low, so-called, regeneration brought it back to the other level. So it's a very clear, nice methodology. And finally, it's also a bit provocative because others in a very prominent paper, actually, have shown that this kinase... Or they concluded more or less just the opposite. The reason for the discrepancy is not quite clear and I was very happy to learn that the two groups actually discussed about it. So it's not just a bad controversy, but something which brings forward science. And finally, I think something we didn't talk about yet today, what I particularly liked, maybe the most, on this paper is that this group didn't stop at the point of DNA synthesis. Everybody else would've probably said, "Okay, here we are, one regenerate the other doesn't." But in the very important extra finding of this paper is that this is just increased DNA synthesis and not more myocytes. And this distinction is so critical to the field because people forget that adult mammalian cardiomyocytes often have several nuclei and individual nuclei have more than one set of chromosomes, so this polyploid. And so if you see DNA synthesis like in this paper, it doesn't necessarily mean more myocytes. And actually here it was shown that it is not more myocytes but more polyploidization and making this difference so clear, I think it's a very important contribution to the field. Dr. Greg Hundley: Very nice. Well, listeners, we're going to turn back to each of our guests today and we'll start with you Loren. Based on your results, what do you see as the next study moving forward in this sphere of research? Dr. Loren Field: I think these results made me appreciate for the first time that the intrinsic level of cell cycle reentry, that's just the S phase, not the cell division, is actually much higher than I had thought previously. And this was because we just fortuitously, or I guess anti-fortuitously, we're using a strain that had low levels of S phase induction. If you calculate the turnover, if every nucleus that it synthesized DNA actually went on to have that cell divide, you could replace a 50% loss of myocytes over the course of about 550 days, give or take. And to me, that's actually telling me that if we could push those cells from just being polypoid, as Thomas was saying, to actually go through cytokinesis, there would be enough intrinsic activity to go forward. So this really tells me that what we should be focusing on is now not trying to induce cell cycle, but to allow the cells that are entering the cell cycle to actually progress through it. Dr. Greg Hundley: Very nice. And Sean? Dr. Sean Reuter: Yes, well, echoing Loren's point there, it's really not necessarily cell cycle induction, it's cell cycle completion to the cytokinetic fate. And that's the key. If we can get to that point, if we can figure out the mechanism to get to that point, then we have a wonderful discovery. However, we're not quite there yet, but we hope to be. Dr. Greg Hundley: And Thomas. Dr. Thomas Eschenhagen: Well, nothing to add really from my side, except that I would like to know what this Troponin I kinase does, because that is somehow still a missing link. How does this kinase lead to more DNA synthesis or the initiation of cell cycling? That would be an important finding and I'm sure there will be more research going on. Particularly also, to solve this discrepancy, I mean, there must be something in it and we don't quite yet know how, but I think we are in a good way. I'm sure there will be papers showing that soon. So I think that's, again, a very good start for this discussion. Dr. Greg Hundley: Well, listeners, we want to thank Dr. Loren Field, Dr. Sean Reuter and Dr. Thomas Eschenhagen for bringing us this really informative study in mammalian myocellular regeneration, highlighting that the level of cardiomyocyte cell cycle reentry in hearts expressing TNNI3 kinase would lead to significant regenerative growth if each cardiomyocyte exhibiting S phase activity was able to progress through cytokinesis. And this in turn suggests that identification of factors which facilitate cardiomyocyte cell cycle progression beyond S phase will be key to unlocking the intrinsic regenerative capacity of the heart. Well, on behalf of Carolyn, Peder and myself, we want to wish you a great week and we will catch you next week on the run. This program is copyright of the American Heart Association 2023. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more, please visit ahajournals.org.

Integrated transcriptome and lineage analyses reveal novel catecholaminergic cardiomyocytes contributing to the cardiac conduction system in murine heart

Play Episode Listen Later Nov 4, 2022

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.04.515095v1?rss=1 Authors: Sun, T., Grassam-Rowe, A., Pu, Z., Ren, H., An, Y., Guo, X., Hu, W., Liu, Y., Li, Y., Liu, Z., Kou, K., Ou, X., Chen, T., Fan, X., Liu, Y., Tu, S., He, Y., Ren, Y., Chen, A., Shang, Z., Xia, Z., Miquerol, L., Smart, N., Zhang, H., Tan, X., Shou, W., Lei, M. Abstract: Cardiac conduction system (CCS) morphogenesis is essential for correct heart function yet is incompletely understood. Here we established the transcriptional landscape of cell types populating the developing heart by integrating single-cell RNA sequencing and spatial enhanced resolution omics-sequencing (Stereo-seq). Stereo-seq provided a spatiotemporal transcriptomic cell fate map of the murine heart with a panoramic field of view and in situ cellular resolution of the CCS. This led to the identification of a previously unrecognized cardiomyocyte population expressing dopamine beta-hydroxylase (Dbh+-CMs), which is closely associated with the CCS in transcriptomic analyses. To confirm this finding, genetic fate mapping by using DbhCre/Rosa26-tdTomato reporter mouse line was performed with Stereo-seq, RNAscope, and immunohistology. We revealed that Dbh+-derived CMs first emerged in the sinus venosus at E12.5, then populated the atrial and ventricular CCS components at E14.5, with increasing abundance towards perinatal stages. Further tracing by using DbhCFP reporter and DbhCreERT/Rosa26-tdTomato inducible reporter, we confirmed that Dbh+-CMs are mostly abundant in the AVN and ventricular CCS and this persists in the adult heart. By using DbhCre/Rosa26-tdTomato/Cx40-eGFP compound reporter line, we validated a clear co-localization of tdTomato and eGFP signals in both left and right ventricular Purkinje fibre networks. Finally, electrophysiological optogenetic study using cell-type specific Channelrhodopsin2 (ChR2) expression further elucidated that Dbh+-derived CMs form a functional part of the ventricular CCS and display similar photostimulation-induced electrophysiological characteristics to Cx40CreERT/ChR2- tdTomato CCS components. Thus, by utilizing advanced transcriptomic, mouse genetic, and optogenetic functional analyses, our study provides new insights into mammalian CCS development and heterogeneity by revealing novel Dbh+-CMs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

The IgCAM CAR regulates gap junction mediated coupling on embryonic cardiomyocytes and affects their beating frequency

PaperPlayer biorxiv neuroscience

Play Episode Listen Later Nov 2, 2022

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.02.514878v1?rss=1 Authors: Matthaeus, C., Juettner, R., Gotthardt, M., Rathjen, F. G. Abstract: The IgCAM coxsackie-adenovirus receptor (CAR) is essential for embryonic heart development and for electrical conduction in the mature heart. However, it is not well-understood how CAR exerts these effects at the cellular level. To address this question, we analysed the spontaneous beating of cultured embryonic hearts and cardiomyocytes from wildtype and CAR knockout (KO) embryos. Surprisingly, in the absence of CAR, cultured cardiomyocytes showed an increased frequency of beating and calcium cycling. Increased beating of heart organ cultures was also induced by application of reagents that bind to the extracellular region of CAR, such as the adenovirus fiber knob. However, the calcium cycling machinery, including calcium extrusion via SERCA2 and NCX, was not disrupted in CAR KO cells. In contrast, CAR KO cardiomyocytes displayed an increase in the size, but decrease in total number, of membrane-localized Cx43 clusters. This was accompanied by improved cell-cell coupling between CAR KO cells, as demonstrated by increased intercellular dye diffusion. Our data indicate that CAR may modulate the localization and oligomerization of Cx43 at the plasma membrane, which could in turn influence electrical propagation between cardiomyocytes via gap junctions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

llc car beating copy ko increased frequency junction coupling mediated regulates embryonic biorxiv cardiomyocytes ncx cx43

Optogenetic manipulation of second messengers in neurons and cardiomyocytes with microbial rhodopsins and adenylyl cyclase

Play Episode Listen Later Oct 27, 2022

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.25.513731v1?rss=1 Authors: Hagio, H., Hosaka, S., Koyama, W., Song, A. D., Narantsatsral, J., Matsuda, K., Shimizu, T., Hososhima, S., Tsunoda, S. P., Kandori, H., Hibi, M. Abstract: Even though microbial photosensitive proteins have been used for optogenetics, their use should be optimized to precisely control second messengers in vivo. We exploited GtCCR4 and KnChR, cation channelrhodopsins from algae, BeGC1, a guanylyl cyclase rhodopsin from a fungus, and photoactivated adenylyl cyclases (PACs) from cyanobacteria (OaPAC) or bacteria (bPAC), to control cell functions in zebrafish. Optical activation of GtCCR4 and KnChR in the hindbrain reticulospinal V2a neurons, which are involved in locomotion, immediately induced swimming behavior, whereas activation of BeGC1 or PACs was achieved at a short latency. KnChR had the highest locomotion-inducing activity of all the channelrhodopsins examined. Activation of GtCCR4 and KnChR in cardiomyocytes induced cardiac arrest, whereas activation of bPAC gradually induced bradycardia. KnChR activation led to an increase in intracellular Ca2+ in the heart, suggesting that depolarization caused cardiac arrest. These data suggest that these optogenetic tools can be used to reveal the roles of second messengers in various cell types in vertebrates. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

song llc copy manipulation activation messengers optical neurons pacs microbial shimizu matsuda biorxiv ca2 koyama cardiomyocytes bpac

September 2022 Discover Circ Res

#ExpertAnswers - an InsideScientific Podcast

Play Episode Listen Later Sep 15, 2022 28:48

This month on Episode 40 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the September 2 and September 16 issues of the journal. This episode also features an interview with Dr Jun Yoshioka, and Dr Yoshinobu Nakayama, from the City University of New York, about their study, Interaction of ARRDC-4 with GLUT1 Mediates Metabolic Stress in the Ischemic Heart. Article highlights: Jin, et al. Gut Dysbiosis Promotes Preeclampsia Mengozzi, et al. SIRT1 in Human Microvascular Dysfunction Hu, et al. Racial Differences in Metabolomic Profiles and CHD Garcia-Gonzales, et al. IRF7 Mediates Autoinflammation in Absence of ADAR1 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 be highlighting some articles from September 2nd, and September 16th issues of CircRes. And I'm also going to have a conversation with Dr Jun Yoshioka, and Dr Yoshinobu Nakayama, from the City University of New York, about their study, Interaction of ARRDC-4 with GLUT1 Mediates Metabolic Stress in the Ischemic Heart. But, before I get to the interview, I'm going to highlight a few articles. The first article is from our September 2nd issue, and it's titled, Gut Dysbiosis Promotes Preeclampsia by Regulating Macrophages, and Trophoblasts. The first author is Jiajia Jin, and the corresponding author is Qunye Zhang from the Chinese National Health Commission. Preeclampsia is a late-stage pregnancy complication that can be fatal to the mother, and the baby. It's characterized by high blood pressure, and protein in the urine. The cause is unknown, but evidence suggests the involvement of inflammation, and impaired placental blood supply. Because gut dysbiosis can influence blood pressure, and inflammation has been observed in preeclamptic patients, Jin and colleagues examined this link more closely. They found that women with preeclampsia had altered gut microbiome. Specifically, a reduction in a species of bacteria that produced short-chain fatty acids, and lower short-chain fatty acid levels in their feces, in their serum, and in their placentas. And preeclamptic women had lower short-chain fatty acid levels in their feces, in their serum, and in their placentas compared with women without preeclampsia. They found that fecal transfers from the preeclampsia women to rats with a form of the condition exacerbated the animals' preeclampsia symptoms, while fecal transfers from control humans alleviated the symptoms. Furthermore, giving rats an oral dose of short-chain fatty acids or short-chain fatty acid producing bacteria decreased the animals' blood pressure, reduced placental inflammation, and improved placental function. This work suggests that short-chain fatty acids, and gut microbiomes could be a diagnostic marker for preeclampsia. And microbial manipulations may even alleviate the condition. The second article I want to share is also from our September 2nd issue, and it's titled, Targeting SIRT1 Rescues Age and Obesity-Induced Microvascular Dysfunction in Ex Vivo Human Vessels. And this study was led by Alessandro Mengozzi from University of Pisa. With age, the endothelial lining of blood vessels can lose its ability to control vasodilation, causing the vessel to narrow and reduce blood flow. This decline in endothelial function has been associated with age related decrease in the levels of the enzyme, SIRT1. And artificially elevating SIRT1 in old mice improves animals' endothelial function. Obesity, which accelerates endothelial dysfunction, is also linked to low SIRT1 levels. In light of these SIRT1 findings, Mengozzi, and colleagues examined whether increasing the enzyme's activity could improve the function of human blood vessels. The team collected subcutaneous microvessels from 27 young, and 28 old donors. And both age groups included obese, and non-obese individuals. SIRT1 levels in the tissue were, as expected, negatively correlated with age and obesity, and positively correlated with baseline endothelium dependent vasodilatory function. Importantly, incubating tissue samples from older, and obese individuals with a SIRT1 agonist, restored the vessel's vasodilatory functions. This restoration involved a SIRT1 induced boost to mitochondrial function, suggesting that maintaining SIRT1 or its metabolic effect might be a strategy for preserving vascular health in aging, and in obesity. The third article I want to share is from our September 16th issue. And this one is titled, Differences in Metabolomic Profiles Between Black And White Women and Risk of Coronary Heart Disease. The first author is Jie Hu, and the corresponding author is Kathryn Rexrode, and they're from Brigham and Women's Hospital, and Harvard University. In the US, coronary heart disease, and coronary heart disease-related morbidity, and mortality is more prevalent among black women than white women. While racial differences in coronary heart disease risk factors, and socioeconomic status have been blamed, this group argues that these differences alone cannot fully explain the disparity. Metabolomic variation, independent of race, has been linked to coronary heart disease risk. Furthermore, because a person's metabolome is influenced by genetics, diet, lifestyle, environment and more, the authors say that it reflects accumulation of many cultural, and biological factors that may differ by race. This group posited that if racial metabolomic differences are found to exist, then they might partially account for differences in coronary heart disease risk. This study utilized plasma samples from nearly 2000 black women, and more than 4500 white women from several different cohorts. The team identified a racial difference metabolomic pattern, or RDMP, consisting of 52 metabolites that were significantly different between black, and white women. This RDMP was strongly linked to coronary heart disease risk, independent of race, and known coronary heart disease risk factors. Thus, in addition to socioeconomic factors, such as access to healthcare, this study shows that racial metabolomic differences may underlie the coronary heart disease risk disparity. The last article I want to share is also from our September 16th issue, and it is titled, ADAR1 Prevents Autoinflammatory Processes in The Heart Mediated by IRF7. The first author is Claudia Garcia-Gonzalez, and the corresponding author is Thomas Braun, and they are from Max Planck University. It's essential for a cell to distinguish their own RNA from the RNA of an invading virus to avoid triggering immune responses inappropriately. To that end, each cell makes modifications, and edits its own RNA to mark it as self. One type of edit made to certain RNAs is the conversion of adenosines to inosines. And this is carried out by adenosine deaminase acting on RNA1 or ADAR1 protein. Complete loss of this enzyme causes strong innate immune auto reactivity, and is lethal to mice before birth. Interestingly, the effects of ADAR1 loss in specific tissues is thought to vary. And the effect in heart cells in particular has not been examined. This study, which focused on the heart, discovered that mice lacking ADAR1 activity specifically in cardiomyocytes, exhibit autoinflammatory myocarditis that led to cardiomyopathy. However, the immune reaction was not as potent as in other cells lacking ADAR1. Cardiomyocytes did not exhibit the sort of upsurge in inflammatory cytokines, and apoptotic factors seen in other cells lacking ADAR1. And the animals themselves did not succumb to heart failure until 30 weeks of age. The author suggests that this milder reaction may ensure the heart resists apoptosis, and inflammatory damage because, unlike some other organs, it cannot readily replace cells. Cindy St. Hilaire: Today I have with me, Dr Jun Yoshioka, and Dr Yoshinobu Nakayama, and they're from City University of New York. And today we're going to talk about their paper, Interaction of ARRDC4 With GLUT1 Mediates Metabolic Stress in The Ischemic Heart. And this is in our September 2nd issue of Circulation Research. So, thank you both so much for joining me today. Jun Yoshioka: Thank you for having us. We are very excited to be here. Cindy St. Hilaire: It's a great publication, and also had some really great pictures in it. So, I'm really excited to discuss it. So, this paper really kind of focuses on ischemia, and the remodeling in the heart that happens after an ischemic event. And for anyone who's not familiar, ischemia is a condition where blood flow, and thus oxygen, is restricted to a particular part of the body. And in the heart, this restriction often occurs after myocardial infarctions, also called heart attacks. And so, cardiomyocytes, they require a lot of energy for contraction, and kind of their basic functions. And in response to this lack of oxygen, cardiomyocytes switch their energy production substrate. And so, I'm wondering if before we start talking about your paper, you can just talk about the metabolic switch that happens in a cardiac myocyte in the healthy state versus in the ischemic state. Jun Yoshioka: Sure. As you just said, that the heart never stops beating throughout the life. And it's one of the most energy demanding organs in the body. So, under normal conditions, cardiac ATP is mainly derived from fatty acid oxidation, and glucose metabolism contributes a little bit less in adult cardiomyocytes. However, under stress conditions such as ischemia, glucose uptake will become more critical when oxidative metabolism is interrupted by a lack of oxygen. That is because glycolysis is a primary anaerobic source of energy. We believe this metabolic adaptation is essential to preserve high energy phosphates and protect cardiomyocytes from lethal injuries. The concept of shifting the energy type of stress preference toward glucose, as you just said, has been actually long proposed as an effective therapy against MI. For example, GIK glucose insulin petition is classic. Now, let me explain how glucose uptake is regulated. Glucose uptake is facilitated by multiple isophones of glucose transporters in cardiomyocytes. Mainly group one and group four, and the minor, with a minor contribution of more recently characterized STLT1. In this study, we were particularly interested in group one because group one is a basal glucose transporter. Dr Ronglih Liao, and Dr Rong Tian's groups reported nearly two decades ago that the cardiac over-expression of group one prevents development of heart failure, and ischemic damage in mice. Since they are remarkable discoveries, the precise mechanism has not yet been investigated enough, at least to me. Especially how acute ischemic stress regulates group one function in cardiomyocytes. We felt that this mechanism is important because there is a potential to identify new strategies around group one, to reduce myocardiac ischemic damage. That is why we started this project hoping to review a new mechanism by which a protein family, called alpha-arrestins, controls cardiac metabolism under both normal, and diseased conditions. Cindy St. Hilaire: That is a perfect segue for my next question, actually, which is, you were focusing on this arrestin-fold protein, arrestin domain-containing protein four or ARRDC4. So, what is this family of proteins? What are arrestin-fold proteins? And before your study, what was known about a ARCCD4, and its relationship to metabolism, and I guess specifically cardiomyocyte metabolism? Jun Yoshioka: So, the arrestin mediated regulation of steroid signaling is actually common in cardiomyocytes. Especially beta, not the alpha, beta-arrestins have been well characterized as an adapter protein for beta-adrenergic receptors. Beta-arrestins combine to activate beta-adrenergic receptors on the plasma membrane, promote their endosomal recycling, and cause desensitization of beta-adrenergic signaling. Over the past decade, however, this family, the arrestin family, has been extended to include a new class of alpha-arrestins. But unlike beta-arrestins, the physiological functions of alpha-arrestins remain largely unclear based in mammalian cells. Humans, and mice have six members of alpha-arrestins including Txnip, thioredoxin interacting protein called Txnip, and five others named alpha domain-containing protein ARRDC1 2, 3, 4 and 5. Among them Txnip is the best studied alpha-arrestin. And Txnip is pretty much the only one shown to play a role in cardiac physiology. Txnip was initially thought to connect alternative stress and metabolism. However, it is now known that the Txnip serves as an adapter protein for the endocytosis of group one, and group four to mediate acute suppression of glucose influx to cells. In fact, our group has previously shown that the Txnip knockout mice have an enhanced glucose uptake into the peripheral tissues, as well as into the heart. Now, in this study, our leading player is ARRDC4. The arrestin-domains of ARRDC4 have 42% amino acid sequence similarities to Txnip. This means that the structurally speaking ARRDC4 is a brother to Txnip. So, usually the functions of arrestins are expected to be related to their conserved arrestin-domains. So, we were wondering whether two brothers, Txnip, and ARRDC4, may share the same ability to inhibit the glucose transport. That was a starting point where we initiated this project. Cindy St. Hilaire: That's great. And so, this link between ARRDC4, and the cardiac expression of gluten one and gluten four, I guess, mostly gluten one related to your paper, that really wasn't known. You went about this question kind of based on protein homology. Is that correct? Jun Yoshioka: That is right. Cindy St. Hilaire: And so, ARRDC4 can modulate glucose levels in the cell by binding, and if I understand it right, kind of helping that internalization process of glute one. Which makes sense. You know, when you have glucose come into the cell, you don't want too much. So, the kind of endogenous mechanism is to shut it off, and this ARRDC4 helps do that. But you also found that this adapter protein impacts cellular stress, and the cellular stress response. So, I was wondering if you could share a little bit more about that because I thought that was quite interesting. It's not just the metabolic impact of regulating glucose. There's also this cellular stress response. Jun Yoshioka: Right? So, Txnip is known to induce oxidative stress. But about the ARRDC4, we found that ARRDC4 actually does not induce oxidative stress. Instead, we found that it reproducibly causes ER, stress rather than oxidative stress. So, let Yoshinobu talk about the ER stress part. Yoshinobu, can you talk about how you found the ER stress story? Yoshinobu Nakayama: So, then let's talk about the, yeah, ER stress caused by ARRDC4. The ER stress caused by ARRDC4, year one was the biggest challenge in this study, because it's a little bit difficult to how we found a link of the glucose metabolism to the effect of the ARRDC4, only our stress. And at the other point of the project, we noticed that a ARRDC4 causes ER stress reproducibly, but we did not know how. So, both group one, and ARRDC4 are membrane proteins mainly localized near the plasma membrane. Then how does ARRDC4 regulate the biological process inside in the plasma radical? So, we then hypothesize that ARRDC4 induces intercellular glucose depravation by blocking cellular glucose uptake, and then interferes with protein glycosylation, thereby disturbing the ER apparatus. That makes sense because inhibition of group one trafficking by ARRDC4 was involved in the unfolded protein response in ischemic cardiomyocytes. Cindy St. Hilaire: So how difficult was that to figure out? How long did that take you? Yoshinobu Nakayama: How long? Yeah. Is this the question? Cindy St. Hilaire: It's always a hard question. Yoshinobu Nakayama: I think it's not several weeks. Maybe the monthly, months project. Yeah. Cindy St. Hilaire: Okay. It's always fun when, you know, you're focusing on one angle, and then all of a sudden you realize, oh, there's this whole other thing going on. So, I thought it was a really elegant tie-in between the metabolism, but also just the cellular stress levels. It was really nice. So, you created a full body knockout of ARRDC4 in the mouse, and you did all the proper kind of phenotyping. And at baseline everything's normal, except there's a little bit of changes in the blood glucose levels. But I also noticed when you looked at the expression of ARRDC4 in different tissues, it was very high in the lungs, and also in the intestines. And so, I know your study didn't focus on those tissues, but I was wondering if you could possibly speculate what ARRDC4 is doing in those tissues? Is it something similar? Do those cells under stress have any particular metabolic switching that's similar? Jun Yoshioka: Well, actually we don't have any complete answer for that question, because like you said, we didn't focus on lung, and other tissues. But I could say that actually the brother of ARRDC4, Txnip, is also highly expressed in lung, and bronchus, and in those organs. So, it's interesting because, which means that, the molecule is very oxygen sensitive, I will say. Both brothers. But that's all we know for now. But that's a very great point. And then we are excited to, you know. Cindy St. Hilaire. Yeah. Jun Yoshioka: Move on to the other tissues. Cindy St. Hilaire: I was thinking about it just because I've actually recently reviewed some papers on pulmonary hypertension. So, when I saw that expression, that was the first thing I thought of was, oh, they should put these mice in a sugen/hypoxia model, and see what happens. Jun Yoshioka: Right? Cindy St. Hilaire: So, there's an idea for you, Yoshinobu. A K-99 grant or something. And also, because it's a full body knockout, even when you're looking at the heart, obviously the cardiomyocytes are really the most metabolically active cell, but cardiac fibroblasts are also a major component of the heart tissue. And so, do you know, is the, I guess, effects or the protectiveness of the ARRDC4 knockout heart, is it mostly because of the role in the cardiomyocytes or is there a role for it also in the fibroblast? Yoshinobu Nakayama: Yeah, that's a very great question. Yeah. So, although we use the systemic knockout mice in the study, we believe that the beneficial effect of ARRDC4 deficiency is cardiac, autonomous. But this is because cardioprotection was demonstrated in the isolated heart experiments. But, you know, root is still uniformly expressing all cell types within the heart. To address this, we have tested the specific effects of ARRDC4 on cardiac fibroblasts, and inflammatory cells. ARRDC4 knockout hearts had a twofold increase in myocardial glucose uptake over wild-type hearts during insulin-free perfusion. However, an increase in glucose uptake in isolated cardiac fibroblast or inflammatory cells was relatively mild, with about 1.2 fold increase over wild-type cells. Thus we conclude that cardiomyocytes are the measure contributed to the cardiac metabolic shift. And then the mechanism within cardiomyocytes should play the major role in cardioprotection. Jun Yoshioka: I might, at one point, because, you know, the fibroblasts, they don't need to beat, right? Cindy St. Hilaire: Right. Jun Yoshioka: The inflammasome cells. They don't need to beat neither. So, they don't need that much energy. So, the cardiomyocytes energy metabolism is very important. So, that's why this mechanism is kind of more important in cardiomyocytes than other cell types. Cindy St. Hilaire: Yeah. And I think, you know, your phenotyping of the mice at baseline show that there's really no effect in a cell that's not under stress. So, it's really, really nice finding. Yeah. This article, I should say, is featured on the cover of the September 2nd Circulation Research issue. And it's got this really nice 3D modeling of the binding of ARRDC4 to glute one. And I was reading the paper, and the methods said, you use some AI for that. So, I'm sure other people have heard, too, AI in protein modeling is important. But AI in art, right? There's that new DALL-E 2 program. So how are you able to do this? How did that work? Jun Yoshioka: So, our study used is called AlphaFold, which applies the artificial intelligence-based deep learning method. AlphaFold, nowadays, everybody really is interested in AlphaFold. AlphaFold uses structural, and genetic data to come up with a model of what the protein of interest should look like. So, that is also how we got the protein structure, ARRDC4. We think that the ability of AlphaFold to precisely predict the protein structure from amino acid sequence would be a huge benefit to life sciences, including of course, cardiovascular science research, because of high cost, and technical difficulties in experimental methods. It's very useful if you can computationally predict the complex from individual structures of ARRDC4. And group one, which is actually structure of group one, is available in a protein data bank. But ARRDC4, it was not available. That's why we used AlphaFold. And then we use the docking algorithm called Hdoc. So, based on these AI analysis, we could successfully identify specific residues in a C terminal arrestin domain as an international interface, that regulates group one function. So, we believe this AI method will pretty much accelerate efforts to understand the protein, protein interactions. And we believe that will enable more advanced drug discovery, for example, in very near future. Cindy St. Hilaire: Yeah, it's really great. I started thinking about it in terms of some of the things I'm studying. So yeah, it was really nice. Jun Yoshioka: Try next time. Cindy St. Hilaire: Yeah, I will, I will. Actually, I went to the website, and was playing with it before I got on the call with you. So, how do you think your findings can be leveraged towards informing clinical decision making or even developing therapeutics? Jun Yoshioka: So, let me talk about what needs to be done. There are more things we must do. Cindy St. Hilaire: Always. Yeah. Jun Yoshioka: One of the most clinically relevant questions is whether ARRDC4 inhibition actually can mitigate development of post MI heart failure, and reduce mortality in the chronic phase, not the acute phase. Because in this paper we just did the seven day post MI, which is kind of like acute to subacute phase. But you never know what's going to happen in the chronic phase, right? And that is actually not so simple to answer because there are so many issues that you should consider. For example, Dr E. Dale Abel's lab has reported previously that cardiomyacites, specific group one, knockout in mice does not really accelerate the transition from compensated hypotrophy to heart failure. Also, the same group has shown that the overexpression group one does not actually prevent LV dysfunction in the mouse model of pressure overload. So, it is possible that ARRDC knockout can be, do much, or even harmful to LV remodeling in a chronic phase because chronic phase, it's not, it's getting hypoxy conditions, right? Cindy St. Hilaire: Yeah. So, it really might be something, I guess, personalized medicine is not the phrase I'm looking for. But I guess temporarily modulated, it would be something maybe we can figure out in an acute phase versus. Jun Yoshioka: Chronic phase. Cindy St. Hilaire: Yeah. Yeah. Jun Yoshioka: This makes sense. Because, you know, high capacity of ATP synthesis, by oxidating metabolism, could be important for chronic heart failure. So, it's selecting substrates. Energy substrates is no longer, you know, that issue. So, I'm not sure I'm answering your question, but this is the point that we consider to move on to the next. Cindy St. Hilaire: Well, that's great. And I think that was my next question, really. What is next? Are you really going to try to pinpoint where you could possibly target? Jun Yoshioka: Right. So, the first point we have to figure out about chronic phase, and another point we are interested in, is what's going on at the level of mitochondria. Does ARRDC4 knockout hearts have a different activity of electron transport chain or glycolytic enzymes within mitochondria? Cindy St. Hilaire: Or even mitochondrial fission infusion because it's, you know, it's a machinery. Jun Yoshioka: Yeah. And how about the other essential pathways in glucose metabolism such as mTOR, AMPK and HEF1, and so on. So, all these must be determined to help understand the more precise role of ARRDC4 in cardiac metabolism, we believe. Cindy St. Hilaire: It's a wonderful study, and now we have even more questions to ask using your great model. Congratulations again. Yoshinobu Nakayama: Thank you so much. Cindy St. Hilaire: Dr Yoshioka, and Dr Nakayama. Jun Yoshioka: Thank you. Cindy St. Hilaire: A wonderful paper, and congrats on getting the cover, and thank you so much for joining me today. Jun Yoshioka: Thanks well so much for having us. Yoshinobu Nakayama: Thank you. Cindy St. Hilaire: That's it for the highlights from our September 2nd, and our September 16th issues of Circulation Research. Thank you so much for listening. Please check out our CircRes Facebook page, and follow us on Twitter, and Instagram with the handle @circres, and hashtag discovercircres. Thank you to our guests, Dr Jun Yoshioka, and Dr Yoshinobu Nakayama. This podcast is produced by Ishara Ratnayaka, edited by Melissa Stonerm, and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on the go source for the most 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, please visit ahajournals.org.

#ExpertAnswers: Joe Soughayer and Adam Veteto on Excitation-Contraction Coupling in Cardiomyocytes

Play Episode Listen Later May 4, 2022 9:26

Joe Soughayer and Adam Veteto discuss excitation-contraction coupling in cardiomyocytes and the tools used to characterize this.

contraction coupling excitation cardiomyocytes

#ExpertAnswers: Joe Soughayer and Adam Veteto on Excitation-Contraction Coupling in Cardiomyocytes

#ExpertAnswers - an InsideScientific Podcast

Play Episode Listen Later May 4, 2022 9:26

Joe Soughayer and Adam Veteto discuss excitation-contraction coupling in cardiomyocytes and the tools used to characterize this.

contraction coupling excitation cardiomyocytes

Circulation March 8, 2022 Issue

Play Episode Listen Later Mar 7, 2022 17:33

This week, join author Marco Valgimigli and Associate Editor Mark Link as they discuss the original research article "Amulet or Watchman Device for Percutaneous Left Atrial Appendage Closure: Primary Results of the SWISS-APERO Randomized Clinical Trial." Dr. Carolyn Lam: Welcome to Circulation on the Run, your weekly podcast summary and backstage fast as a journal and editors. We're your co-host, I'm Dr. Carolyn Lam, Associate Editor from the National Heart Center and Duke National University of Singapore. Dr. Greg Hundley: I'm Dr. Greg Hundley, Associate Editor, Director of Poly Heart Center at VCU Health in Richmond, Virginia. Dr. Carolyn Lam: Greg, I've got a personal interest in this feature paper that's coming up. I've always been very intrigued with the left atrial appendant closure. Well guess what? This is the results of the Swiss-Apero Randomized Clinical Trial, comparing the Amulet with the Watchman device for percutaneous left atrial appendage closure, really interesting stuff coming right up, but let's hold everyone in suspense. As we go through some of the other favorite papers in today's issue. Would you like to go first? Dr. Greg Hundley: You bet Carolyn, this first paper really pertains to driving restrictions and earlier arrhythmia is in patients receiving a secondary prevention, implantable cardioverter defibrillator, and it comes to us from Dr. Christian Steinberg. So Carolyn, regulatory authorities of most industrialized countries recommend six months of private driving restriction after implantation of a secondary prevention ICD and these driving restrictions result in significant inconvenience in social implications. And so Carolyn, the purpose of this study was to assess the instance rate of appropriate device therapies in contemporary recipients of a secondary prevention ICD using a retrospective across three Canadian tertiary care centers enrolling 721 consecutive patients with new secondary prevention ICD implants between the years of 2016 and 2020. And they were followed for a median of 760 days. Dr. Carolyn Lam: Nice. An important question. So what did they find Greg? Dr. Greg Hundley: Right Carolyn. So they found that the cumulative incidents of arrhythmic syncope resulting in sudden cardiac incapacitation was 1.8% within the first 90 days, and subsequently dropped to 0.4% between 91 and 180 days after ICD insertion. So Carolyn the incidence rate of appropriate therapies resulting in sudden cardiac incapacitation in contemporary recipients of a secondary prevention ICD is much lower than previously reported and significantly declines after the first three months, lowering driving restrictions to months after the index cardiac event seems safe and revision of the existing guidelines should be considered in countries still adhering to a six month period. Dr. Carolyn Lam: Oh, love it Greg. Elegant study with clinically impactful results as with the next paper, I'm going to talk about a post talk analysis of the Danish trial in which authors led by Dr. Boas from Denmark and Dr. Bower from Austria and their colleagues. And what they did is they tested whether periodic repolarization dynamics or P R D, which is a marker of repolarization instability associated with increased sympathetic activity. Could indeed identify patients with non-ischemic cardiomyopathy that may benefit from prophylactic ICD implantation. So, 748 patients were included in this P R D sub-study. And they were included if they had a 24 hour whole term monitor recording at baseline with technically acceptable ECG signals during the night hours. P R D as a reminder of periodic repolarization dynamics was assessed using wavelet analysis according to previously validated models. Dr. Greg Hundley: Very interesting, Carolyn. So what did they find? Dr. Carolyn Lam: Periodic repolarization dynamics was independently associated with mortality. More over P R D was significantly associated with mortality in the control group, but not in the ICD group in this Danish trial. There was a significant interaction between P R D and effect of ICD implantation on mortality, such that patients with higher P R D had greater benefit in terms of mortality reduction with the ICD. Based on P R D the investigators could identify a new group of patients where prophylactic ICD implantation was associated with a significant absolute mortality reduction of 17.5% after eight years corresponding to a number needed to treat of only six. So this is the first sub-study of Danish to identify a marker on top of age, that can predict the treatment effect of prophylactic ICD implantation in patients with nonischemic cardiomyopathy. Dr. Greg Hundley: Very nice Carolyn. Well, my next paper comes to us from Dr. Kory Levine from the Washington University School of Medicine. And Carolyn recent studies have established that cc chemokine receptor type two are CCR2 marks the pro inflammatory subsets of monocytes, macrophages and dendritic cells that contribute to adverse left ventricular remodeling and heart failure progression. Now elucidation of the effector mechanisms that mediate adverse effects of CCR2 plus monocytes, macrophages and dendritic cells could yield important insights into therapeutic strategies to suppress myocardial inflammation. Dr. Carolyn Lam: Hmm, indeed. And so what did these author determine regarding the suppression of myocardial inflammation? Dr. Greg Hundley: So Carolyn this team utilized mouse models of re perfused myocardial infarction, and angiotensin two and phenylephrine infusion, and diphtheria toxin cardiomyocyte ablation to investigate cc chemokine ligand 17. They found that cc chemokine ligand 17 serves as a pro-inflammatory mediator of CCR2 macrophages and dendritic cells, and their results suggest that inhibition of cc chemokine ligand 17 may serve as an effective strategy to promote T-cell regulation recruitment, and thereby suppress myocardial inflammation. Dr. Carolyn Lam: Wow, thanks, Greg. That was beautifully summarized great stuff. Well, there are other exciting articles in today's issue. There's an exchange of letters between Doctors Nagareddy and Spear on “Interleukin One Alpha as a Central Regulator of Leukocyte Endothelial Adhesion in Myocardial Infarction and Chronic Kidney Disease. There's an ECG challenge by Dr. Yang entitled, “A Fatal Case of Y QRS tachycardia following Sintilimab treatment for lung cancer. It can happen.” There's an On My Mind paper by Dr. Faed on “CYP2C19 Genotyping in Anticoagulated Patients Post PCI: Should it be Routine?” Dr. Greg Hundley: Great, Carolyn and I've got a Research Letter from Professor Poo entitled “Efficient in Vivo Hemology- Directed Repair within Cardiomyocytes.” Well, how about we get onto that feature, discuss and learn more about Watchman devices and left atrial appendage closure. Dr. Carolyn Lam: Ooh, let's go. Dr. Greg Hundley: Well, listeners. Now we are onto the feature discussion today, and we're so privileged to have with us, Dr. Marco Valgimigli from Bern, Switzerland and our own Associate Editor, Dr. Mark Link from UT Southwestern. Welcome gentlemen. Well, Marco, we will start with you. Can you describe for us a little bit of the background material as to why you wanted to perform this study and what was the hypothesis that you wanted to address? Dr. Marco Valgimigli: But thank you so much for having me left atrial appendage closure is a therapeutic option for a patient who have formal indication to oral anti-population because of atrial fibrillation yet have some relative or absolutely contraindication to the treatment because mainly of high bleeding risk features, mainly because of previous bleeding complications. The two most frequently use device to accomplish that procedure are from one side Watchman, which is FDA approved. And on the other hand, the Amulet, which has been historically the device, which has been most frequently used in Europe, these two devices have been formally compared in one head to head study, which is Amulet IDE and also reported in the journal some month ago. However, it was important also to compare the new Watchman iteration, which the Watchman Flex, which was not part of the original Amulet IED, which only compared Amulet with Watchman 2.5. Dr. Marco Valgimigli: Before we set up this multicenter study, which recruited patients across eight centers and four European countries, we roughly screened 450 patients. And we ended up randomizing 222 to either of these two device being Amulet or Watchman. We set up this study to answer a superiority hypothesis, actually, of Amulet versus Watchman and we assume that Amulet would be superior compared to Watchman with the respect to the need for crossover to a known randomly allocated device or LA Patency at 45 day SCCTA. Dr. Greg Hundley: Very nice Marco. And so now describe for us your study design. And then what was your study population? Dr. Marco Valgimigli: That was an investigate initiated and conducted multicenter study, which took place across eight centers in four European countries. We screened roughly 450 patients to be able to randomize. Finally, 222 were evenly allocated to either of these two device. We selected patients with either fibrillation and form lead for oral anti-regulation to with relatively high chat box score on average four and very high has bled at least three or greater. In fact, 95% of our patient had prior bleeding before being considered for the study. Dr. Greg Hundley: Very good. And describe for us your results. Dr. Marco Valgimigli: So the superiority that we assumed was actually not met. The primary point, which was again, crossover to a known allocated device or patency rate at 45 day SCCTA was pretty much similar with rates being 68% in the Watchman group and 70% in the Amulet group. So basically no real difference. We did see though some interesting differences with respect to some key secondary endpoints. Dr. Greg Hundley: And describe those. Dr. Marco Valgimigli: So for example, the type of leaks that was recorded was actually different with respect to the two devices with intra device leaks, being much more frequent with Amulet and peri device leak, being much more frequent with Watchman. Also the rate of any or major procedure complications were slightly yet significantly higher in the Amulet group. And interestingly as well, we had some minor differences with respect to the device related thrombosis, which was numerically slightly higher in Watchman as compared to Amulet also, we performed at 45 day TEE, Transesophageal Echo and with that respect, we did see a slightly, yet numerically and significantly higher rate of peri device leak, detected at Transesophageal Echo with Watchman as compared to Amulet, which is in a way reflecting our SCCTA findings. Dr. Greg Hundley: Very nice. And so lots of results comparing these two devices for a left atrial appendage closure. So Mark, you see many papers come across your desk. What attracted you to this particular paper? Dr. Mark Link: Yeah, this is a very important clinical paper. The left atrial occlusion devices are going to continue to be used and with increasing frequency and there can be very beneficial. The Amulet is not approved in the United States. And so comparison between Watchmen and the Amulet are very important, both for the US and for Europe and the rest of the world. And that's why we were attracted to this paper, it's a nice randomized paper comparing the two most commonly use devices. Dr. Greg Hundley: Help us put the results of this study into the context in really how we might manage patients and how we're considering these devices, both from the European perspective and then also here in the US. Dr. Mark Link: I think what this study shows is that they're more or less equivalent and there may be theoretical reasons why one may be better than the other, but in this trial it didn't really come out. So I think the Amulet used more in Europe. The Watchman is used exclusively here, but I think this opens a window for the Amulet to come to America also and be approved by the FDA. Dr. Greg Hundley: Very good, well, coming back to you, Marco, where do you see is the next study that needs to be performed in this arena of research? Dr. Marco Valgimigli: I think now we have relatively effective device in accomplishing what we would like to accomplish. Namely sitting the LAA for preventing subsequent thromboembolic events. However, the procedure despite operators have been growing in terms of interest and experience is still associated with some degree of complications. Some of them are minor, but some of them are not. I think the next future, we should try to minimize those complication to the lowest possible event so that this procedure can potentially be also offered to a broader patient population, perhaps not just as a replacement of oral anticoagulation, but perhaps in association with oral anticoagulation to further minimize the stroke and other thrombotic events. Dr. Greg Hundley: And Mark, do you have anything to add here? Dr. Mark Link: Yes. I think what we need more information on is two things. One is procedural complications, procedural complications were higher with the Amulet device. And is that because it was a newer device and people are just learning how to use it, or is there something there that's really important? So that's one and then the really important one is long term clinical outcomes. This was a relatively short term trial when you're talking about strokes and anticoagulation. And so what I'd like to see is longer term follow up with both devices in the future. Dr. Greg Hundley: Well, thank you Mark. So now Marco, do you have any additional studies that you're performing really on this same dataset? Dr. Marco Valgimigli: Well, I actually fully agree with Mark, very long term follow up is absolutely mandatory in this patient population. And I'm very glad to announce that this Swiss Apero study that we have been discussing will actually continue. Continuing patients follow up five years so that we will have much more clinical information, at least comparative effectiveness, clinical information between two devices. And also we'll be performing at 13 months, a second SCCTA to better understand the dynamic nature of these peri device or inter device list, whether they actually do see over time and which of them may or not play a clinical role. Dr. Greg Hundley: Very nice well listeners, we want to thank Dr. Marco Valgimigli from Bern, Switzerland and our own associate editor, Dr. Mark Link from UT Southwestern for really bringing us this very important paper that compared the results of the Watchman, which is for primarily used in the US versus the Amulet, primarily used in Europe, a left atrial appendage closure device. Well, on behalf of Carolyn and myself, we want to wish you a great week and we will catch you next week on the run. Speaker 5: This program is copied right 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, please visit ahajournals.org.

Circulation January 18, 2022 Issue

Play Episode Listen Later Jan 18, 2022 20:23

Please join author Mohamed Abdel-Wahab and Associate Editor Stefan James as they discuss the article "Comparison of a Pure Plug-Based Versus a Primary Suture-Based Vascular Closure Device Strategy for Transfemoral Transcatheter Aortic Valve Replacement: The CHOICE-CLOSURE Randomized Clinical Trial." Dr. Carolyn Lam: Welcome to Circulation on the Run. Your weekly podcast summary and backstage pass to the Journal and its editors. We're your co-hosts. I'm Dr. Carolyn Lam, associate editor from the National Heart Center and Duke National University of Singapore. Dr. Greg Hundley: And I'm Dr. Greg Hundley, associate editor, director of the Pauley Heart Center at VCU Health in Richmond, Virginia. Well, Carolyn, this week's feature, a very interesting topic, looking at closure devices at the sites of access for patients that are undergoing TAVR procedures. But before we get to that, how about if we grab a cup of coffee and start with some of the other articles in the issue. Would you like to go first? Dr. Carolyn Lam: I would love to and I would like to describe not just one, but two articles from recent SGLT2 inhibitor trials. So, the first paper is an analysis of the DAPA-HF trial. Now we know that circulating high sensitivity, cardiac troponin T predominantly reflects myocardial injury. And higher levels are associated with a higher risk of worsening heart failure and death in patients with heart failure with reduced ejection fraction or HFrEF. But what about the prognostic significance of changes in high sensitivity troponin T over time and the effects of Dapagliflozin and on clinical outcomes in relation to baseline levels, as well as the effect of dapagliflozin on the high sensitivity troponin T levels? Well, this is what this study answers. It's a biomarker substudy of the DAPA-HF trial from Dr. Berg of the TIMI study group at Brigham women's hospital and colleagues. Dr. Greg Hundley: Wow. Carolyn, very interesting. So remind us about the DAPA heart failure trial. What was it about? Dr. Carolyn Lam: Ah, well, DAPA-HF was a randomized double blind placebo control trial of dapagliflozin in patients with symptomatic HFrEF defined by injection fraction 40% or less wherein dapagliflozin significantly reduced the primary endpoint of cardiovascular death or worsening heart failure events. And in today's biomarker substudy increases in high sensitivity, cardiac troponin T over a one year interval of time were highly predictive of subsequent risk of worsening heart failure and cardiovascular death. The effect of dapagliflozin on the primary endpoint was consistent irrespective of baseline troponin T concentration with no evidence of attenuated treatment benefit in those with very high troponin T concentrations. Dr. Greg Hundley: Very interesting Carolyn. Now you've got another study. Is this one on EMPA? Dr. Carolyn Lam: You are right. Thank you. The next paper is and analysis of the Emperor-Preserved trial. As a reminder, Emperor-preserved study the SGLT2 inhibitor empagliflozin in patients with HFpEF this time, which is a left ventricular ejection fraction above 40, and showed a significant reduction in the risk of cardiovascular death or heart failure hospitalization. The current paper evaluated the efficacy of empagliflozin on health related quality of life in patients with HFpEF and whether the clinical benefit observed with empagliflozin varied according to baseline health status. Dr. Greg Hundley: Very nice, super review Carolyn. So what were the results of this study? Dr. Carolyn Lam: In Emperor-Preserved, baseline health status and quality of life did not influence the magnitude of effect of empagliflozin on the risk of cardiovascular death or hospitalization for heart failure. Empagliflozin improved health status and quality of life as assessed by the Kansas city cardiomyopathy questionnaire across all domains and at all measured time points. Thus an effect that appeared early and was sustained for at least one year. Dr. Greg Hundley: Very nice. So two really informative papers on SGLT2 inhibitors. Well Carolyn, I'm going to turn the conversation to the world of preclinical science and talk about Titin. So Carolyn, titin truncation variants are the most common inheritable risk factor for dilated cardiomyopathy and their pathogenicity has been associated with structural localization. The A-band variants with overlapping myosin heavy chain binding domains appear more pathogenic than the I-band variants and the mechanisms for this are not well understood. So these investigators led by Dr. Hinson at the Jackson Laboratory for genomic medicine, performed a study demonstrating why A-Band variants are highly pathogenic for dilated cardiomyopathy and how they could reveal new insights into dilated cardiomyopathy pathogenesis. Titin functions and therapeutic targets were assessed. Dr. Carolyn Lam: Wow, interesting. So what did they enroll in? How did they do this? what did they find? Dr. Greg Hundley: Great Carolyn, so human Cardiomyocytes and cardiac micro tissue functional assays revealed that highly pathogenic A-Band Titin truncation mutations generate four shortened titin poisoned peptides and diminish full length, titin protein levels. While less pathogenic I-band titin mutations only diminish titin protein levels. And so Carolyn, the authors developed a one and done, genome editing therapeutic approach using CRISPR technology to repair the reading frame of Titin truncation mutations in cardiomyocytes. And therefore these genome editing therapeutics could correct the underlying genetic lesion responsible for dilated cardiomyopathy due to these Titin mutations. Dr. Carolyn Lam: Wow. Interesting. One and done genome editing. You learn something new every day with circulation. You've got another paper? Dr. Greg Hundley: Yes, Carolyn. Thank you. And so this paper comes to us from Dr. Beiyan Zhou From the Yukon health, school of medicine and again, from the world of preclinical science. So Carolyn, while several interventions can effectively lower lipid levels and people at risk for atherosclerotic cardiovascular disease, cardiovascular event risks remain, suggesting an unmet medical need to identify factors contributing to this cardiovascular event risk. Now monocytes and macrophages play central roles in atherosclerosis, but previous work has yet to provide a detailed view of macrophage populations involved in increased atherosclerotic cardiovascular disease risk. Dr. Carolyn Lam: Huh? Okay. Well, I'm super excited to hear what these investigators did Greg. Dr. Greg Hundley: Right, Carolyn. Well these authors developed a novel computational program. They call AtheroSpectrum, which identified a specific gene expression profile associated with inflammatory macrophage foam cells. And additionally, a subset of 30 genes expressed in circulating monocytes jointly contributed to the prediction of symptomatic atherosclerotic vascular disease. So therefore Carolyn, in the future, perhaps incorporating this new pathogenic foaming gene set with known risk factors may significantly strengthen the power to predict atherosclerotic cardiovascular disease risk. Dr. Carolyn Lam: Wow. Super interesting and well summarized. Thank you, Greg. Well also in today's issue, there's a Perspective by Dr. Kirtane on “The Long-Awaited Revascularization Guidelines are Out. What's In Them?” A Research Letter by Dr. Laffin on rise in blood pressure observed among us adults during the COVID 19 pandemic. Dr. Greg Hundley: Very Nice Carolyn. Well in our Cardiovascular News Segment, there's a piece on metabolic risk factors and how they drive the burden of Ischemic heart disease. Well, what a great issue here and now, how about we get onto that feature discussion? Dr. Carolyn Lam: Very Cool. Closure devices after TAVR. Here we go. Dr. Greg Hundley: Well, listeners welcome today to our feature discussion and we have with us Dr. Mohamed Abdel Wahab from Leipzig Germany. And we are going to discuss some issues pertaining to transcatheter aortic valve replacement, in terms of access to the arteries in the lower extremity. Welcome Mohamed. And can you start with, what was some of the background that led you to perform your study and what was the hypothesis that you wanted to address? Dr. Mohamed Abdel-Wahab: Thank you, Greg. And thank you for having me here. So as you mentioned, there are several cardiovascular procedures that currently require large-bore arterial access. The most common of these procedures is transcatheter aortic valve replacement. But there are other procedures as well, like endovascular aortic repair, mechanical circulatory devices. All of these require large-bore arterial access and of course, closure afterwards. And what we were interested in looking at was whether different types of vascular access site closure devices or strategies behave differently in the setting. Particularly in the setting of transcatheter aortic valve replacement. The reason behind this is that for many years, we only had one technique, to percutaneously close arterial access sites after these procedures. And these were mainly based on suture based devices or suture based techniques. Very recently, alternative techniques based on collagen plugs have been introduced. Dr. Mohamed Abdel-Wahab: And we know these types of devices or closure techniques from usual coronary intervention procedures for smaller access sites or for smaller sheath size. But they have been developed a step further for these large-bore procedures. These newer devices, particularly what we call the MANTA device, which is based on the collagen plug has been shown in initial visibility studies and also in registry based analysis to be very safe and effective. It leads to a very rapid hemostasis. And data from observational studies have suggested that it may be even superior to the suture based techniques, largely based on what we call the ProGlide device or the [inaudible 00:10:56]. And this is actually what we were aiming to look at. To compare these two different strategies based on two different devices. The suture based, the classical suture based technique using two ProGlides compared to the newer plug based technique using the MANTA in a population treated with TAVR. Dr. Greg Hundley: Very nice. And describe for us, your study design. And then also maybe explain a little bit more about the study population. Who did you include in this study? Dr. Mohamed Abdel-Wahab: So the design was more or less, very inclusive. So we designed the trial to more or less represent real word population. More or less [inaudible 00:11:40] population receiving transcatheter aortic valve replacement. So we included patients, of course where the procedure is being thought to be indicated and feasible by a multidisciplinary heart team. And also where the heart team thought that the transfemoral access route, which is the main route for the majority of patients, is obtainable and use of a percutaneous closure device is also possible. Dr. Mohamed Abdel-Wahab: Of course we had some exclusions. For example, patients where the use of a surgical access technique was necessary. They couldn't be naturally included in the trial. Patient that already had complications related, for example, to previous coronary angiogram PCI at the access site, they couldn't be included. But we were more or less, very inclusive in this trial. The trial population reflects the patients that are currently being treated with TAVR, so more or less an elderly population. More or less equally split-by males and females, which is very particular, again to the TAVR population. So this is a little bit different than the population that receives PCI, where we usually have a predominantly male population. This is not the case here. So these are the broad lines. Also reflecting current practice, the population that has been included in the trial is more or less overall, an intermediate risk population, when you look at the surgical scores. Dr. Greg Hundley: Very nice. So this was multicenter and then also patients were randomized to each of the two therapies, I believe. And was that a one to one randomization? Dr. Mohamed Abdel-Wahab: Exactly. So it was a multicenter trial. Patients were randomized between these two techniques. We mentioned the ProGlide based and the MANTA based in 1:1 fashion. And steering committee of course was more or less dominated by interventional cardiologists. Of course, in the context of this particular trial setting, the trial was only performed in Germany and it was an investigative initiated trial, not sponsored by the industry. Dr. Greg Hundley: Very nice. And can you describe for us, Mohamed, your results? Dr. Mohamed Abdel-Wahab: Yes. We actually hypothesized based on the observational data we have, that we will have less vascular complications with the MANTA based technique or the collagen based technique. At the end of the day, what we observed is completely the opposite. So the primary endpoint of the trial, which was what we call major and minor vascular complications defined according to the standardized criteria provided by the valve academic research consortium. These events occurred significantly more common in patients that were randomized to the MANTA based technique, as opposed to the ProGlide based technique, which was statistically significant. Dr. Greg Hundley: And did you observe those results across both the men and the women? And also, were there any differences in the results related to participants' age? Dr. Mohamed Abdel-Wahab: Yeah. So there were no interactions with various subgroups, both the predefined ones, including age and sex, as you mentioned. But also we looked at some post hoc subgroups, including for example, whether this is being affected by the size of the access vessels or by the presence and location of calcification, for example. But there were no interactions in all subgroups we looked at, with one exception which was chronic renal insufficiency. But all other subgroups showed actually no significant interaction, favoring the suture based, ProGlide based technique in all subgroups. Dr. Greg Hundley: Very good. And so can you describe in terms of, for individuals performing TAVR procedures and obtaining access, how do we use the results of your study to inform how we might move forward with closure of the artery in the future? Dr. Mohamed Abdel-Wahab: I mean, the first thing I would like to stress is the importance of doing randomized trials in general. Because I think this is not the first time we see opposite results when we are comparing randomized evidence with the evidence from observation studies, with the known limitations of observational comparative analysis. The second thing I think is really reassuring that the suture based technique that we know and that we have been using for many years now is safe and appears to be even more effective than the newly developed plug based technique. So this is one important information I think from this trial. The third piece of information is that the recently developed plug based technique, although being inferior in the study, it still may have some advantages in selected patients. And this is what we probably need to look at in a little bit more details in the future. Dr. Mohamed Abdel-Wahab: For example, what we realized from the study is that it could be a good option as a bailout device. So in some cases where the suture based technique has failed in the study, the crossover to the MANTA device was successful in the majority of cases. And may lead or help avoid complex endovascular interventions and implanting for example, stents or covered stents or even doing surgery. So this is something that is a nice observation from the dataset we have, but of course needs validation in larger studies. Dr. Greg Hundley: Very nice. And so really you've answered, kind of one of our key questions is, your thoughts on the next study that you see needs to be performed really in this area of research? Dr. Mohamed Abdel-Wahab: Yeah, so I think there are several things. One thing is, again, to look at potential patient subgroups that may benefit from the plug based device from the beginning. So probably it's not something that we should be using as a default strategy based on the results of this trial. But there could be certain subgroups we need maybe to dig a little bit more into the details or subgroups, if you wish to say so. Look a little bit more granularly at some patient groups that could benefit. But as mentioned, I think that the bailout indication is a very interesting one and needs to be looked at. Dr. Mohamed Abdel-Wahab: Not only in the TAVR setting, but also in the setting of other procedures. Such as for example, the use of mechanical circulatory assist device or ECMOs, where it may be difficult to apply these sutures post hoc. So the sutures that we apply during a TAVR procedure and what we use in this trial, this is the so-called preclosure technique. So you apply the sutures after gaining access. Then you insert your large-bore sheaths through the procedure. And then the sutures are already there and you can close the access site, usually without problems. Which is difficult, if you obtain access, for example, with an ECMO or an Impella. And then after a couple of days, you need to close it. So the sutures are not yet in place. In this particular scenario, it may be beneficial to use a plug afterwards. Or as a bailout device as previously Mentioned. Dr. Greg Hundley: Very nice well listeners. We want to thank Dr. Mohamed Abdel Wahab from Leipzig Germany for bringing us this study indicating that among patients treated with transfemoral TAVR, this pure plug based vascular closure technique using the MANTA VCD was associated with a higher rate of access site or access related vascular complications. Well, on behalf of Carolyn and myself, we want to wish you a great week and we will catch you next week on the run. Dr. Greg Hundley: 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, please visit AHA journals dot org.

covid-19 american germany band journal patients run kansas comparison singapore richmond perspective berg emperor closure aha crispr yukon brigham circulation pci manta ecmo hinson sglt2 ischemic tavr empa hfpef dapa hfref laffin empagliflozin vcu health duke national university dapagliflozin cardiomyocytes impella leipzig germany carolyn lam titin emperor preserved

Circulation November 30, 2021 Issue

covid-19 cytokine ipscs cytokine storm cardiomyocytes

Play Episode Listen Later Nov 29, 2021 22:09

Please join first author Cecilia Bahit and Associate Editor Graeme Hankey as they discuss the article "Predictors of Development of Atrial Fibrillation in Patients With Embolic Stroke Of Undetermined Source: An Analysis of the RE-SPECT ESUS Trial." Dr. Carolyn Lam: Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to the journal and its editors. We're your co-hosts. I'm Dr. Carolyn Lam, Associate Editor from the National Heart Center, and Duke; National University of Singapore. Dr. Greg Hundley: And I'm Dr. Greg Hundley, Associate Editor and Director of the Pauley Heart Center at VCU Health in Richmond, Virginia. Well, Carolyn, this week's feature, we're going to analyze the RE-SPECT ESUS trial. What does that pertain to? Well, you're going to have to wait and find out, but it relates to atrial fibrillation and embolic stroke. But before we get to that, how about we grab a cup of coffee and go through some of the other articles in the issue? Would you like to go first? Dr. Carolyn Lam: I sure would. Greg, we know that Chronic kidney disease is associated with adverse outcomes among patients with established cardiovascular disease or diabetes. The question is: What are the effects of Icosapent Ethyl across the range of kidney function in patients with established cardiovascular disease or diabetes from the REDUCE-IT trial? Dr. Greg Hundley: Ah, Carolyn, can you remind us what was the REDUCE-IT trial? What did it encompass there? Dr. Carolyn Lam: The REDUCE-IT trial was a multicenter double-blind, placebo-controlled trial that randomized statin treated patients with elevated triglycerides, who had cardiovascular disease or diabetes, and one additional risk factor, two treatment with icosapent ethyl at 4g daily versus placebo. After a median follow up period of 4.9 years, the study drug demonstrated a 25% relative risk reduction in the primary composite endpoint of cardiovascular death, myocardial infarction, stroke, coronary revascularization, or unstable angina. Dr. Greg Hundley: Ah, great summary of the original paper, but now this is sort of a follow-up paper. What did this paper research? Dr. Carolyn Lam: Well first, remember they focused on renal function and the median baseline GFR was 75 ml/min with a range of 17 to 123 mL/min/1.73 m2. Treatment with Icosapent Ethyl led to consistent reduction in both primary and secondary composite endpoints across the baseline GFR categories. Patients with the GFR >60 treated with Icosapent Ethyl had the largest absolute, but similar relative risk reduction for the primary composite endpoint. And while patients with GFR >60 treated with Icosapent Ethyl had the highest numerical rates of atrial fibrillation of flutter and serious bleeding. The hazard ratios for atrial fibrillation flutter and serious bleeding were similar across GFR categories. In summary Icosapent Ethyl reduced cardiovascular events among patients with elevated triglycerides in a well-controlled LDL on statin therapy across a wide range of baseline renal function. Dr. Greg Hundley: Oh, Carolyn. Beautiful presentation. That presentation was so good that I know you are ready for a quiz. We haven't had Carolyn's quiz in a week, so we've got to get right back to that. Dr. Carolyn Lam: No, we don't (laughs). Dr. Greg Hundley: Can you describe the primary sequelae of Hutchinson-Gilford progeria syndrome? Dr. Carolyn Lam: Oh wow. Okay. So this is the syndrome where there's premature aging, there's a lot of vascular stiffening, calcification. I'm going to guess some sort of atherosclerotic consequence (laughs). Dr. Greg Hundley: Very nicely done Carolyn. Oh my goodness. I need to get you to take my ABIM recertification- Dr. Carolyn Lam: (laughing) Dr. Greg Hundley: Beautifully done. So Carolyn, this paper comes to us from Dr. Vicente Andrés from Centro Nacional De Investigaciones Cardiovasculares Carlos III, and Hutchinson-Gilford progeria syndrome is a rare disorder characterized, just like you said, Carolyn by premature aging and death, mainly due to myocardial infarction, stroke or heart failure. The disease is provoked by progerin, a variant of lamin A expressed in most differentiated cells. Carolyn, these patients look healthy at birth and symptoms typically emerge in the first or second year of life. In assessing the reversibility of progerin induced damage, and the relative contribution of specific cell types is critical to determining the potential benefits of late treatment and to developing new therapies. Dr. Carolyn Lam: Wow, you've really, really piqued my interest. So what did these investigators do and what did they find? Dr. Greg Hundley: Oh Carolyn, very clever design. So the authors use CRISPR-Cas9 technology to generate mice engineers to ubiquitously express progerin while lacking lain A and allowing progestin suppression in lain A restoration in a time and cell type specific manner upon CRE recombinase activation. They characterize the phenotype of these engineered mice and cross them with CRE transgenic lines to assess the effects of suppressing progestin and restoring lain A ubiquitously at different disease stages, as well as specifically in vascular smooth muscle cells and cardiomyocytes. So Carolyn, what did they find? Well, number one, like Hutchinson-Milford progenia syndrome patients, their engineered mice appeared healthy at birth, and progressively developed Hutchinson-Milford progenia syndrome symptoms, including failure to thrive, Lipodystrophy, vascular smooth muscle cell loss, vascular fibrosis, electric cardiographic anomalies and early death. Their median lifespan was 15 months versus 26 months in the wild types. Dr. Greg Hundley: Second, ubiquitous progestin suppression in lain A restoration significantly extended lifespan, when induced in six month old, mildly symptomatic mice, and even in severely ill animals aged 13 months, although the benefit was much more pronounced upon the early intervention. And then finally, Carolyn remarkably major vascular alterations were prevented and lifespan normalized in engineered Hutchinson-Milford progenia syndrome mice when progestin suppression and lain A restoration were restricted to: just Vascular smooth muscle cells and Cardiomyocytes. Dr. Carolyn Lam: Wow, just fascinating, but, okay. What is the clinical take home message? Dr. Greg Hundley: Right, Carolyn. So these authors findings suggest that it is never too late to treat Hutchinson-Milford progenia syndrome, although the benefit is much more pronounced when progestin is targeted early in mice with mild symptoms. Also, restricting its suppression to Vascular smooth muscle cells in Cardiomyocytes is sufficient to prevent Vascular disease and normalize lifespan in mice, and therefore these data suggest that strategies to treat Hutchinson-Milford progenia syndrome through gene therapy or RNA therapy should consider targeting Vascular smooth muscle cells and Cardiomyocytes. Dr. Carolyn Lam: Oh wow. Very, very cool. Well, my next paper is a basic science paper that's significant for both its methods and its results. Dr. Greg Hundley: Oh wow, Carolyn, I can't wait. So tell us about this novel methodology. Dr. Carolyn Lam: Well, this paper is from Dr. Chang from Westlake University in Hangzhou, China, and colleagues who use a gene editing approach to efficiently institute Exon Skipping without introducing DNA double-strand breaks. So harnessing a fusion of a nuclease defective Case protein, and a cytidine deaminase, which is, we're going to abbreviate it as Targeted AID-induced mutagenesis (TAM) or base editor three (BE3), their approach precisely edited conserved guanines at splice sites, thus abrogating Exon recognition resulting in a programmable skipping of the targeted Exons. Isn't that neat? Dr. Greg Hundley: Yeah, it really is sophisticated Carolyn, wow. So what did they do using these methods? Dr. Carolyn Lam: A novel mirroring model of Duchenne muscular dystrophy was generated, which recapitulated many cardiac defects observed in the human form of the disease, including dilated cardiomyopathy, reduced left ventricular function and extensive cardiac fibrosis. Using this model, they examined the feasibility of using a cytidine base editor to install Exon Skipping and rescue the dystrophic cardiomyopathy in vivo. A single dose administration of an Adenovirus 9EtAm, instituted over 50% targeted Exon Skipping in the Chengdu muscular dystrophy transcripts and restored up to 90% dystrophin in the heart. And as a result, early ventricular remodeling was prevented and cardiac and skeletal muscle function were improved, leading to an increased lifespan of the mice. Despite gradual decline of the Adenovirus vector and base editor expression, the dystrophin restoration and pathophysiological rescue of muscular dystrophy lasted for at least a year. And so this technique really has the potential to be applied to monogenic human diseases, to modulate Exon Skipping or inclusion. Isn't that cool? Dr. Greg Hundley: Absolutely, Carolyn. Beautifully explained. Dr. Carolyn Lam: Well, let me end by sharing what else is in today's issue. There's a Perspective piece by Dr. Alexander on “Chest Pain Redux: Updated and Patient Centered.” There is an In Depth paper by Dr. Kroemer on NAD plus metabolism in cardiac health, aging and disease. And there's a Research Letter by Dr. Shepherd on sudden death in female athletes, with insights from a large regional registry in the United Kingdom. Dr. Greg Hundley: Very good, Carolyn. What a great issue. Now, how about we get to that feature discussion? Dr. Carolyn Lam: Let's go, Greg. Dr. Greg Hundley: Welcome listeners to our feature discussion today on this November 30th. And we have with us Dr. Cecilia Bahit from Rosario, Argentina and our own associate editor, Dr. Graeme Hankey from Perth, Australia to talk to us about a paper pertaining to Atrial Fibrillation. Welcome to you both, and Cecilia, we'll start with you. Could you describe for us a little bit of the background information that went into formulating your study, and then what hypothesis did you want to address? Dr. Cecilia Bahit: Thank you for the invitation. So we all know that embolic stroke of undetermined source, which is called ESUS isn't just a subset of cryptogenic stroke, and is associated with stroke recurrence about 3-6% per year. And on the other hand, we know that continuous cardiac monitoring in this patient population shows that atrial fibrillation can be detected between 10% at six months or 30% at three years. So the underlying atrial fibrillation may be a mechanism for the recurrent thromboembolic stroke in this patient population. So we know that prior studies have identified some predictors of atrial fibrillation in these patients. And if we are able to identify which patients could benefit from cardiac monitoring and have a higher yield to detect atrial fibrillation, we could do a better job at treating them. So, that was our idea behind the paper. So using the RE-SPECT ESUS trial, which was a trial that included patient with ESUS stroke and were randomized to the bigger trend versus Aspirin, we look at predictors of atrial fibrillation unassociated regarding stroke. Dr. Greg Hundley: Very nice. And so, now was this a sub-study here and maybe define for us a little bit, your study design and specific study population. Dr. Cecilia Bahit: So this was a secondary analysis of a randomized clinical trial that as mentioned it was not a sub-study, it was a secondary analysis. We thought all along to do it because of the interest of the clinical question. We look at the total patient population was 5,390 patients. And we looked at those patients who developed atrial fibrillation during the 19 months of follow-up. And it was 7.5%, 403 patients developed atrial fibrillation. Dr. Greg Hundley: Very good. And what were your results? Dr. Cecilia Bahit: So, as I mentioned, we saw that 7.5% of our patient population developed atrial fibrillation during the follow-up. And we know those patients were older, were like, have higher morbidities, and we assessed, we did an one variable analysis and then a multi-variable analysis, trying to identify predictors for atrial fibrillation. And for our model, we identified different predictors, older age, hypertension, lack of diabetes, and higher body mass index, were independent predictors of atrial fibrillation. So the patients who have atrial fibrillation have a higher recurrence of stroke, it was 7.2 versus four, compared to those that did not have atrial fibrillation. Dr. Cecilia Bahit: So I think there's an important part, that 20% of the patient population of the overall trial, this is a little more than a thousand patients, had NT-prob measure at baseline. And when we included this biomarker into the model, only older age and NT-prob were independent predictors of atrial fibrillation. In addition, even though this was not the objective of this analysis, we look at the treatment effect of the bigger trend. And even though we saw that there was a statistical benefit of the bigger trend versus Aspirin in the higher group of these in our score, the overall treatment effect was not there. So we couldn't assess the fact that the bigger trend was better compared to Aspirin in patient with atrial fibrillation, but of course the numbers were very small. Dr. Greg Hundley: Very good. Thank you so much for that wonderful description. And Graeae, now we'll turn to you as associate editor for us at Circulation, and also the editorialist on this particular paper. What caught your attention about this particular study and the results from the many papers that really come across your desk. Dr. Graeme Hankey: Thank you, Greg. And congratulations to Cecilia and her RE-SPECT ESUS colleagues. I mean, this is a landmark study, the RE-SPECT ESUS study, and just to go back, embolic stroke of undetermined source is really common. About one in four ischemic strokes, we don't know the cause of, and it's one of the major subtypes of cryptogenic stroke is an embolic ischemic stroke in which the source could have come from the heart or the aortic arch or the carotids. And we're not really sure. And we think that some of these patients have occult atrial fibrillation, but we can't pick it up at the time. So one way is to try and monitor them with prolonged ECG monitoring. And another way is to actually treat them with anticoagulation because we know that, that's more effective in people with cardio embolic stroke. And so RE-SPECT ESUS and NAVIGATE ESUS used the latter strategy and said, let's see if treating people with ESUS with anticoagulation is more effective than antiplatelet therapy. Dr. Graeme Hankey: And both studies were not significant in terms of showing that Dabigatran or Parovarian for NAVIGATE ESUS was more effective than antiplatelet therapy. So we're left now with this default that all patients with ESUS just get Aspirin, but we have a hunch that some of them actually have cardiogenic embolism and are being undertreated with Aspirin and need anticoagulation. So it's a heterogeneous entity, but we're treating it homogeneously with a sort of weak antiplatelet. So we want to try and find out who's going to get AF or who's already got it that is occult. And this study is a really great and prospective study with 5,000 patients as Cecilia said, who of whom 7% did develop AF just through annual ECG reporting and just with symptom reporting. And that's probably an under report. You know, if they'd had monitoring, they probably would've found about 20 or 30% would've developed AF during that time of 19 months follow up. Dr. Graeme Hankey: And it's the first study to really then show that not just the AF people had a higher stroke rate, but in that group who they predicted to be at high risk of AF with older age and the NT-prob, that the high risk group had a significant reduction with Dabigatran versus Aspirin in that high risk group. It's just, when you look for hetero homogeneity or heterogeneity across the risk groups, it wasn't quite significant. And that might be because it's not significant or it might be that study was underpowered to look at those three, across those three risk subgroups. And also it might be a bit confounded because of it, the patients weren't randomized according to their risk status for AF, they were just randomized, whether they had ESUS, so it's further excited us that there might be a subgroup who needs anticoagulation. And that's why the ARCADIA trial is ongoing now, looking at where the people with ESUS who have high risk of AF benefit from a apixaban versus aspirin. Dr. Greg Hundley: Very nice. And so, with these results that we have here, maybe come back to Cecilia, what do you think would be the next series of studies that needs to be performed in this area of research? Dr. Cecilia Bahit: Well, there's one side that's ongoing as Dr. Hankie mentioned, but I think we should be able to identify which patients have a higher risk of atrial fibrillation and those patients who use cardiac monitoring for long term to identify atrial fibrillation and to treat properly. So I think that would be key in this area. Dr. Greg Hundley: Very nice. And Graeae, what are your thoughts? Dr. Graeme Hankey: Yes. Well, one way is to have our ESUS patients have prolonged ECG monitoring by implantable loop recorders, for example, and then those who develop AF randomizing them to anticoagulation versus antiplatelet therapy. Although if they declare themselves with AF they're usually just go straight onto anticoagulation therapy. So the burning question is, in these people with ESUS who haven't declared themselves as AF, but have predictors of AF like those shown in RESPECT ESUS, like older age, high blood pressure, high BMI ,prob, and perhaps echo features, like left atrial size or ECG features like lots of premature atrial contractions or P wave of abnormalities. Dr. Graeme Hankey: Are these, the subgroups or even LV dysfunction, are these subgroups who need to be more specifically targeted in a randomized trial rather than the whole group of ESUS. And also with longer follow up. NAVIGATE ESUS stopped after 11 months. The bigger RESPECT ESUS stopped after a median follow up of 16 months and the curves were diverging. Maybe with five years follow up, a lot of these people would've developed AF and would've benefited from longer term anticoagulation, but the trials were stopped early, because there wasn't a signal of benefit and there was an early risk of bleeding with anticoagulation. Dr. Greg Hundley: Very good. Well listeners, this has been a really interesting study and we want to thank Cecilia and Graeme for sharing results of the RESPECT ESUS study, highlighting that, in patients with embolic stroke of undetermined source, atrial fibrillation occurs and is a possible source of this stroke, and then also older age, and elevation of NT-prob can be associated with development of atrial fibrillation, subsequent to that stroke event. Dr. Greg Hundley: Well listeners, we want to wish you a great week. And on behalf of Carolyn and myself, look forward to catching you next week on The Run. This program is copyright of the American Heart Association, 2021. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more visit ahajournals.org.

COVID and the Heart

Steministas

Play Episode Listen Later Nov 23, 2021 18:32

In this episode, we talk about COVID-19 and the heart. How does COVID affect the heart? Listen to find out why underlying cardiovascular issues are a risk factor for severe COVID and to hear what damage COVID can to heart function. Previous COVID episode: https://open.spotify.com/episode/6JeH5o3NVi9da1cajvJ3vL?si=15f11115272040b8 Previous RNA vaccine episode: https://open.spotify.com/episode/5xDPH8WoMTJFLumyqDyfS5?si=390f4dee74e240e1 //Sources// COVID and heart what we know: https://www.health.harvard.edu/blog/covid-19-and-the-heart-what-have-we-learned-2021010621603 ACE-2 receptor: https://theconversation.com/what-is-the-ace2-receptor-how-is-it-connected-to-coronavirus-and-why-might-it-be-key-to-treating-covid-19-the-experts-explain-136928 ACE-2 ANG II: https://www.frontiersin.org/articles/10.3389/fcimb.2020.00317/full Cardiovascular disease: https://www.who.int/news-room/fact-sheets/detail/cardiovascular-diseases-(cvds) SARS ACE-2 receptor: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1287568/ SAR-COV-2 ACE-2 receptor: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7778857/ COVID and heart damage: https://pubmed.ncbi.nlm.nih.gov/33092737/ Cardiomyocyte injury: Cytokine storm: https://www.news-medical.net/health/What-is-Cytokine-Storm.aspx#:~:text=During%20a%20cytokine%20storm%2C%20various,can%20lead%20to%20organ%20damage. Direct and indirect mechanisms: https://jamanetwork.com/journals/jama/fullarticle/2776538 COVID autopsy review: https://www.clinicalkey.com/#!/content/playContent/1-s2.0-S1054880720301046?returnurl=https:%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1054880720301046%3Fshowall%3Dtrue&referrer=https:%2F%2Fjamanetwork.com%2F NEJM COVID Vaccine Myocarditis: https://www.nejm.org/doi/full/10.1056/NEJMoa2110737 SARS-COV-2 and cardiomyocyte iPSCs: https://www.science.org/doi/10.1126/scitranslmed.abf7872

November 2021 Discover CircRes

waves hyper ecg hyperkalemia cardiomyocytes

Play Episode Listen Later Nov 18, 2021 27:17

This month on Episode 30 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the October 29 and November 12 issues of Circulation Research. This episode also features a conversation with Dr Elisa Klein from the University of Maryland about her study, Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity. Article highlights: Subramani, et al. CMA of eNOS in Ischemia-Reperfusion Liu, et al. Macrophage MST1 Regulates Cardiac Repair Van Beusecum, et al. GAS6/Axl Signaling in Hypertension Pati, et al. Exosomes Promote Efferocytosis and Cardiac Repair Cindy St. Hilaire: Hi and welcome to Discover CircRes, the podcast of the American Heart Association's Journal Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh and today I'll be highlighting articles presented in our October 29th and November 12th issues of Circulation Research. I also will speak with Dr Elisa Klein from the University of Maryland about her study, Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity. Cindy St. Hilaire: The first article I want to share is titled, Chaperone-Mediated Autophagy of eNOS in Myocardial Ischemia Reperfusion Injury. The first author is Jaganathan Subramani and the corresponding author is Kumuda Das from Texas Tech University Health Sciences Center. Reestablishing blood flow to ischemic heart muscle after myocardial infarction is critical for restoring muscle function but the return of flow itself can cause damage, a so-called reperfusion injury. The generation of reactive oxygen species or ROS and loss of nitric oxide or NO both contribute to reperfusion injury. Reperfusion injury is exacerbated when the NO producing enzyme, endothelial nitric oxide synthase or eNOS, produces damaging super oxide anions instead of NO. This switch in eNOS function is caused by glutathionylation of the enzyme, termed SG-eNOS. But how long this modification lasts and how it is fixed is unclear. This group used an in vitro model of ischemia reperfusion where human endothelial cells are exposed to several hours of hypoxia followed by reoxygenation. In this model, they found the level of SG-eNOS steadily increases for 16 hours and then sharply decreases. By blocking several different cellular degradation pathways, they discovered that this decrease in S-G eNOS was due to chaperone mediated autophagy with the chaperone protein, HSC70, being responsible for SG-eNOS destruction. Importantly, this team went on to show that pharmacological D-glutathionylation of eNOS in mice promoted NO production and reduced reperfusion injury, suggesting this approach may be of clinical benefit after myocardial infarction. Cindy St. Hilaire: The second article I want to share is titled Macrophage MST1/2 Disruption Impairs Post-Infarction Cardiac Repair via LTB4. The first author is Mingming Liu and the corresponding author is Ding Ai and they're from Tianjin Medical University. Myocardial infarction injures the heart muscle. These cells are unable to regenerate and instead a non-contractile scar forms and that fibrotic scar can lead to heart failure. Cardiomyocytes specific inhibition of the kinase MST1 can prevent infarction induced death of the cells and preserve the heart function, suggesting that it may have clinical utility. However, MST1 also has anti-inflammatory properties in macrophages. So inhibition of MST1 in macrophages may delay inflammation resolution after MI and impair proper healing. Thus, targeting this enzyme for therapy is not a straightforward process. This study examined mice lacking MST1 in macrophages and found that after myocardial infarction, the inflammatory mediator leukotriene B4 was upregulated in macrophages and the animal's heart function was reduced compared to that of wild type controls. Blocking the action of leukotriene B4 in mice reduced infarction injuries in the hearts of MST1-lacking animals and enhanced repair in the injured hearts of wild type animals given an MST1 inhibitor. The results suggest that if MST1 inhibition is used as a future post infarction regenerative therapy, then leukotriene B4 blockade may prevent its inflammatory side effects. Cindy St. Hilaire: The next article I want to share is titled Growth Arrest Specific-6 and Axl Coordinate Inflammation and Hypertension. The first author is Justin Beusecum and the corresponding author is David Harrison and they're from Vanderbilt University. Inflammation contributes to hypertension pathology but the links of this relationship are unclear. It's thought one trigger of inflammation may be the hypertension-induced mechanical stretch of vascular endothelial cells. Mechanical stretch causes endothelial cells to release factors that convert circulating monocytes into inflammatory cells. And one such factor is the recently identified Axl and Siglec-6 positive dendritic cells, also called AS DCs. AS DCs produce a large amount of inflammatory cytokines but little is known about the role of AS DCs or their cytokines in hypertension. This group found elevated levels of AS DCs in hypertensive people compared to normal tensive individuals. Mechanical stretch of human endothelial cells promoted the release of GAS6, which is an activator of the AS DC cell surface kinase, Axl. This stretch induced GAS6 release also promoted conversion of co-cultured monocytes to AS DCs. Inhibition of GAS6 or Axl in the co-cultured system prevented conversion of monocytes to AS DCs. This team went on to show that hypertensive humans and mice have elevated levels of plasma GAS6 and that blocking Axl activity in mice attenuated experimentally induced hypertension and the associated inflammation. This work highlights a new signaling pathway, driving hypertension associated inflammation and identifies possible targets to treat it. Cindy St. Hilaire: The last article I want to share is titled Novel Mechanisms of Exosome- Mediated Phagocytosis of Dead Cells in Injured Heart. The first author is Mallikarjun Patil and Sherin Saheera and the corresponding author is Prasanna Krishnamurthy from the University of Alabama, Birmingham. After myocardial infarction inflammation must quickly be attenuated to avoid excessive scarring and loss of muscle function. Macrophage mediated efferocytosis of dead cells is a critical part of this so-called inflammation resolution process. And resolution depends in part on the protein. MFGE8. MFGE8 helps macrophages engage with eat me signals on the dead cells and loss of macrophage MFGE8 delays inflammation resolution in mice. Because stem cell-derived exosomes promote cardiac repair after infarction and are anti-inflammatory and express MFGE8, this group hypothesized that perhaps part of a stem-cell derived exosomes proresolven activity may be due to boosting macrophage efferocytosis. They showed that stem cell derived exosomes did indeed boost efferocytosis of apoptotic cardiomyocytes in vitro and in vivo. An in vitro experiments showed that if exosomes lacked MFGE8 then efferocytosis by macrophages was reduced. Furthermore, after myocardial infarction in mice, treatment with MFGE8 deficient exosomes did not reduce infarct size and did not improve heart function compared to control exosomes. These results suggest MFGE8 is important for the cardioprotective effects of stem cell-derived exosomes. And that this protein may be of interest for boosting efferocytosis after myocardial infarction and in other pathologies where inflammation is not readily resolved. Cindy St. Hilaire So today, Dr Elisa Klein from the Department of Biomedical Engineering at the University of Maryland is with me to discuss her study Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity and this article is in our November 12th issue of Circulation Research. So Dr Klein, thank you so much for joining me today. Elisa Klein: Thank you for having me. Cindy St. Hilaire: Yeah. So broadly your study is investigating how blood flow patterns specifically, kind of, laminar and oscillatory flow, how those blood flow patterns impact protein modifications and activity. So before we, kind of, get to the details of the paper, I was wondering if you could just introduce for us the concept of blood flow patterns, how they change in the body naturally but then how they might influence or contribute to disease pathogenesis in the vessels? Elisa Klein: Sure. So obviously we have blood flow through all of our vessels and since we are complex human beings, we have complex vascular beds that turn and that split or bifurcate. And so every place we get one of these bifurcations or a turn in a vessel, the blood flow can't quite make that turn or split perfectly. So you get a little area where the flow is a oscillatory or what we call disturbed. There's lots of different kinds of disturbed flow. And the reason why that's important is because you tend to develop atherosclerotic plaques at locations where the blood flow is disturbed. So in my lab, we look a lot at what it is about that disturbed flow that makes the endothelial cells there dysfunctional and that leads to the atherosclerotic plaque development. Cindy St. Hilaire: That is so interesting. So I can picture how this is happening in a mouse at the bifurcation of different arteries but how are you able to model this in vitro? Can you describe the setup and then also how that setup can mirror the physiological parameters? Elisa Klein: Sure. So we have a couple of different systems we can use to model this and they all have their advantages and disadvantages, right? So a few years ago we made a system that's a parallel plate flow chamber. So you basically have your cells that you see that on a microscope slide and you use a gasket that's a given shape and that either drives the flow… Usually it drives the flow straight across the cells. So that's a nice laminar steady flow. And we see that the cells align and they produce nitric oxide in that type of flow which are measures that they are responding to the flow in vitro. So, a few years ago we made a device that actually makes the flow zigzag as it goes across the endothelial cells. And that creates these little pockets of disturbed flow and we did that in our parallel plate flow chamber. And that parallel plate flow chamber is really good for visualizing the cells. So you can stick it on a microscope. You can see what's happening, we can label for specific markers but it's not good for doing the things that we did in this Circ Research paper, where we want it to measure metabolism, because you need a lot more cells to measure metabolism and we needed a better media to cell ratio, so less media and more cells. So for this one, we designed and built a cone-and-plate device. So what it is, it's a cone and you spin that cone on top of a dish of endothelial cells and that cone produces flow. So it's going around in a circle. And if we just make it go around in a circle, it'll produce a steady laminar flow but if we oscillated it, so basically we kind of turn it back and forth, it'll make this oscillating disturbed flow. And then we have our dish of cells. We do this in a 60-millimeter dish and then we have a small amount of media in there and a lot of cells. And we can culture the cells in there for a while. Cindy St. Hilaire: That is so neat. And so I'm assuming that then your cone system is very tuneable. You could either speed it up, slow it down or change that oscillatory rate with different, I guess, shifts of it? Elisa Klein: Yeah, that's exactly right. So we can do all those things. It's programmable with a motor and so we can run whatever type of flow we want. Cindy St. Hilaire: That's great. So before your study, what was known regarding this link between hemodynamics and endothelial cell dysfunction and also endothelial cell metabolism? Because I feel like that's a really interesting space that a lot of people look at, kind of, metabolism and EC dysfunction or they just look at shear stress and EC dysfunction and you're, kind of, combining the three. So what was kind of the knowledge gap that you were hoping to investigate? Elisa Klein: Yeah, so we're really interested in macrovascular endothelial cell dysfunction. So this pro atherosclerotic phenotype that you can get in endothelial cells. And most of the work on endothelial cell metabolism had actually been done in the context of angiogenesis. So how much energy and how do cells get their energy to make new blood vessels? And that's more of a microvascular thing. So there was a study that came out before ours, actually, before we started this study, that was looking at how steady laminar flow could decrease endothelial cell glycolysis. And so that was after 72 hours of flow and they showed some gene expression changes at that time. Our study is shorter than that and we were still able to see a decrease in glycolysis in our cells in laminar flow. Before we started this study, no one had really looked at disturbed flow. So in the meantime, there are a few other papers that came out showing that the cells don't decrease glycolysis when they're in disturbed flow but not so much connecting them back to this function of making nitric oxide. Cindy St. Hilaire: So we were kind of dancing to the topic of O linked N acetylglucosamine or how do you say it? Elisa Klein: GlcNAC. Cindy St. Hilaire: GlcNAC? O- GlcNAC. So, O- GlcNAC is a sugar drive modification and I think it's added to Syrian and three Indian residues and proteins. Elisa Klein: Yup, that's right. Cindy St. Hilaire: Okay, good. And that modification, it does help dictate a protein's function. And you were investigating the role of this moiety on endothelial nitric oxide synthase or eNOS and so what exactly does this GlcNAC do for eNOS' function and under what conditions or disease states is this modification operative? Elisa Klein: Yeah. So there's some really important studies from a little bit ago that showed that eNOS gets GlcNAcylated in animals with diabetes, right? So if you have constantly high sugar levels, you get this modification of eNOS. The thought was that eNOS gets GlcNAcylated at the same site where it gets phosphorylated. But a more recent study came out and said, well, maybe that's not the case but it definitely gets GlcNAcylated somewhere where it affects this phosphorylation site. So it may be near it and prevent the folding or prevent the phosphorylation site availability. So if the eNOS gets GlcNAcylated, the thought is that it can't get phosphorylated and therefore it can't make nitric oxide. Cindy St. Hilaire: And so an interesting thing about this GlcNAcylation, which is probably the hardest thing I've ever said on this podcast, is that it's integrated with lots of different things. Obviously you need glycolysis and the substrates from the breakdown of sugars to make that substrate but also the enzymes that make that substrate are required. And so what's known about that balance in endothelial cells? Is there much known regarding the metabolic rate of the cells and this N-Glcynation? Elisa Klein: Yeah. So endothelial cells are thought to be highly glycolytic in terms of how they use glucose but they definitely take up glutamine to fuel the tricarboxylic acid or TCA cycle. And another paper came out a few years ago showing that quiescent and endothelial cells metabolize a lot of fatty acids. So they're fueling their energy needs that way. So there wasn't a lot known about GlcNAcylation in endothelial cells. A lot of this work has been done in cancer cells, which are also highly glycolytic but their metabolism actually seems like it's maybe more diverse than people have thought for a long time. So the weird thing about GlcNAcylation, which if you're used to working with phosphorylation there's a thousand different enzymes that can phosphorolate right. But with GlcNAcylation there's one enzyme that's known to put the GlcNAC on and one enzyme that's known to take it off. And so they're global, right? So in our studies, if we say, okay, we're going to knock down that enzyme, you're effecting every single protein in the cell that's GlcNAcylated. And obviously ourselves in particular, we're not a big fan of that. Especially once you put them in flow, they were, like, nope, we're not going to make it. Cindy St. Hilaire: Well, and that's a perfect segue to my next question because your results show that this flow really did not alter the expression of these enzymes that either add or subtract to the moiety. And rather it was the Hexosamine Biosynthetic Pathway that was decreased itself. So can you maybe give us a quick primer on what that is exactly and how that pathway feeds into the glycosylation... I think you wrote in the paper of over 4,000 proteins? So how would that fit in and why eNOS then? Elisa Klein: Yeah, so the Hexosamine Biosynthetic Pathway is one of these branch pathways that comes off glycolysis and there are these numbers sometimes there are these pathways out there and people say for the HBP in particular, 2% to 5% of the glucose that's going down through glycolysis gets shunted off into the HBP. We've done a lot of looking to try and figure out exactly where that 2% to 5%- Cindy St. Hilaire: Yeah, what exact percentage? Elisa Klein: Yeah, but some percentage of it comes down and we really thought there were going to be changes in these enzymes that do the GlcNacylation, we thought there might be changes in the localization of the proteins and it's possible that those things do occur. We just couldn't detect them in our cells. And in the end, what we showed was the main thing was that when you have cells and steady laminar flow, you just decreased glycolysis. And therefore, that 2% to 5% goes down. So you seem to make less of this UDP- GlcNAC, which is the substrate that gets put on to eNOS in this case. The really strange thing that we could not explain despite a lot of work and obviously we don't get to put all of our experiments that didn't work in the paper- Cindy St. Hilaire: The blood, sweat and tears gets left out. So- Elisa Klein: Exactly. So we tried really hard to figure out why it was eNOS specifically, right? Because in steady laminar flow, you see a lot of these like GlcNAcylated proteins and a lot of them didn't change but eNOS changed hugely, essentially this GlcNAcylation just went away for the cells and steady laminar flow. So we couldn't quite answer that. We're still working on that part of the question and looking at some of the other proteins that maybe get GlcNAcylated more in this case and trying to figure out what they are. Cindy St. Hilaire: I thought one of the cool results in your paper was one of the last ones. It was the one in healthy mice. In that you looked at healthy mice, just normal C57 black 6 mice that were 10 weeks old. So they just, kind of, reached maturity but you looked at their kind of these bifurcations and you looked at the inner aortic arch where there is more disturbed flow and you saw, similar to your in vitro studies, that there was this higher level of O-GlcNAcylation compared to the outer arch in the descending order. So my question is, these are healthy mice that are relatively young, they're not even full adults yet. That takes a couple more months. And so what are your thoughts about the role of this O-GlcNAcylation specifically on eNOS in driving atherogenesis. Where do you think this is happening in the disease process? It appears if it's in these wild type mice, it's already happening early. So where do you think this is most operative in the disease pathogenesis? Elisa Klein: I mean, I think it's very early, the effects of disturbed flow on endothelial cells. I can't imagine that there's a time when it's not having an effect on the cells. So I teach college students and I tell them all the time you think you're invincible now but these choices you're making today are going to affect your cardiovascular future in 50 years, which is very hard to accept. So I think it's very early in the process and I think it's only made worse by the things that we eat, in particular, that changed our blood sugar and our blood fatty acids and things like that. And our lab is looking into this more to try and see how when you change your blood metabolites then how does that then also affect this GlcNAcylation and the endothelial cell metabolism and then how does that affect endothelial cell function? Cindy St. Hilaire: Yeah. And it's funny, it's really making me think of those, kinds of, extreme diets like keto diets and things like that where you're just like depleting sugar. And obviously there's lots of controversy in that field, but if you just think about the sugar aspect what is that doing to those EC cells? Why do you think endothelial cells have this response? Meaning why do you think it is that they've adapted to induce a metabolic shift in response to disturbed flow? Because, obviously it's not going to be perfect laminar flow everywhere. So what do you think it is that provides some sort of advantage in the shift? Elisa Klein: That's a really good question. I haven't thought about the advantage that it might provide. There are a lot of things that are going on in this area of disturbed flow. So there is the shear stress, the differential shear stress that the cells are experiencing. There's also transport issues, right? So if you have this area of disturbed flow, you have blood and the contents of the blood, including the white blood cells and the red blood cells, everything else that's, kind of, sitting around in that area and not getting washed downstream as quickly. So it is possible that maintaining glycolysis provides energy for repair or for protecting the endothelial cell from some sort of inflammatory insult or something like that, that's happening in the area of disturbed flow. And I feel like I just read something recently, it was in a different genre but... if they stopped the increased glycolysis or stop the metabolic shifts, it actually was worse. Right? So I also believe that we treat humans for a single metabolic change, right? So if you have diabetes, I'm going to give you this drug and if you have high triglycerides, I'm going to give you this drug. But it's possible that if you have this metabolic abnormality, your body shifts the rest of your metabolism to protect the cells because of that metabolic abnormality. And so part of what we do as engineers is try and build computational models or we can take into account some of this complexity. So that's a really interesting question and my guess is that there are some protective aspects of this maintenance of high glycolysis and disturbed flow. Cindy St. Hilaire: Yeah, maybe it would be perfectly fine until we get athero and then it all goes awry. So in terms of... obviously it's early days and I know you're a bioengineer but in terms of translational potential, what do you think your findings suggest about future potential therapies or future targets for which we can use to develop therapies? Is modulating this O-GlcNAcylation itself, a viable option? Elisa Klein: I don't think that modulating it is a super viable option, right? Because as I said, when we tried to change those enzymes ourselves did not enjoy going through flow or anything else. So it's very hard to change it overall. What I think is these things that are coming out about how metabolism may shift for endothelial cells when they're activated versus when they're quiescent, right? So when laminar flow or cells are quiescent, they decrease glycolysis, they increase fatty acid oxidation. Those things are important to take into consideration when you are treating a person who has a metabolic disorder. So that's the biggest translational piece that I think is, how do we give therapies that modify the metabolism of a cell holistically instead of trying to hit one pathway in particular. We have done some studies where we tried to give endothelial cells something to inhibit a specific metabolic pathway and you see the cell shifts its entire metabolism to account for that. So we're starting to look at some of these other drugs like statins or metformin that do change endothelial cell metabolism, possibly even the SGLT2 inhibitors and trying to see not just how they change glycolysis but how they change metabolism as a whole and how that then affects endothelial cell function. Cindy St. Hilaire: So what are you going to do next on this project? Elisa Klein: So on this project, so we have some stuff in the works like I said on statins and how statins work together. And one of our big goals is to sort of build a comprehensive metabolic model of the endothelial cell. So this study really focused on glucose but there are other things that endothelial cells metabolize, glutamine, and fatty acids, and trying to look at some of those and then seeing how changes in the glycolytic pathway may affect some of those other pathways. We also have some really nice mass spec data part of which is in this paper but part of which is going to go into our next work, which is looking at how laminar flow impacts some of the other side branch pathways that are in metabolism and coming off of glycolysis as well as the TCA cycle, right? So we don't think of endothelial cells as being big mitochondrial energy producers but they do use their mitochondria. And so we think it's really interesting and part of our goal of building an endothelial cell model and then hopefully a model of the complexity of the whole vascular wall. Cindy St. Hilaire: Wow. That would be amazing. Well, Dr Elisa Klein from the University of Maryland, thank you so much for joining me today. This is an amazing study and I'm looking forward to seeing hopefully more of your future work. Elisa Klein: Thank you so much. It was a pleasure. Cindy St. Hilaire: That's it for the highlights the from October 29th and November 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 or #DiscoverCircRes. Thank you to our guest, Dr Elisa Klein. This podcast is produced by Asahara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy texts for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on-the-go source for the most exciting discoveries and basic cardiovascular research. This program is copyright of the American Heart Association, 2021. The opinions expressed by speakers on this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, visit AHAjournals.org.

Ep.40 - Let's Get Hyper about T Waves

Current ECG Podcast

Play Episode Listen Later Nov 3, 2021 12:55

On this episode Dave shares about acute coronary syndrome and some of the important ECG shapes and morphologies that can occur in the very beginnings of the injury to the Cardiomyocytes of your heart. Also In This Episode Abnormally wide T waves ECG Tracing examples Don't get confused with Hyperkalemia Subscribe to the video version of this podcast to have access to the visuals that accompany the audio as well as additional tools and resources to help improve your understanding. Subscribe now at CurrentECG.com And Stay Current!

October 2021 Discover CircRes

rich trial patients rescue injury commentary seneca mitochondria extracellular jacc vesicles doxorubicin cardiomyocytes

Play Episode Listen Later Oct 21, 2021 30:54

This month on Episode 29 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the September 17th and October 1st issues of Circulation Research. This episode also features conversations with BCVS Outstanding Early Career Investigator Award finalists, Dr Jiangbin Wu from the University of Rochester, Dr Chen Gao from UCLA, and Dr Chris Toepfer from Oxford University. Article highlights: Raftrey, et al. Dach1 Extends Arteries and Is Cardioprotective Zhang, et al. Blood Inflammatory Exosomes and Stroke Outcome Joyce, et al. Cardiovascular Health and Epigenetic Age Liu, et al. Wls Suppresses Fibrosis in Heart Regeneration Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh. And today, I'll be highlighting articles presented in our September 17th and October 1st issues of Circulation Research. I also am going to speak with the BCVS Outstanding Early Career Investigator Award finalists, Dr Jiangbin Wu from the University of Rochester, Dr Chen Gao from UCLA, and Dr Chris Toepfer from Oxford University. Cindy St. Hilaire: The first article I want to share is titled, Dach1 Extends Artery Networks and Protects Against Cardiac Injury. The first author is Brian Raftrey, and the corresponding author is Kristy Red-Horse from Stanford University. Coronary artery disease occurs when blood vessels supplying the heart develop atherosclerotic plaques that limit blood flow, which prevents oxygen and nutrients from reaching the cardiac tissue and often leads to a heart attack or cardiac arrest. The suggested strategy for treating coronary artery disease is to promote the growth of new blood vessels to compensate for the dysfunctional ones. Several factors are known to control coronary blood vessel development, including the transcription factor, DACH1. In mice lacking DACH1, embryonic coronary artery development is stunted. But whether increasing DACH1 protein levels boosts heart vessel development, and whether this would work in mirroring coronary arteries, were unanswered questions. Cindy St. Hilaire: This group engineered inducible gain-of-function DACH1 mice and found that DACH1 over expression in the embryo boosted coronary artery development. The team then used the same model to induce DACH1 in adult mice for six weeks. While there was no apparent differences in the artery growth between the animals and the controls under normal conditions, after myocardial infarction, the mice over expressing DACH1 had better recovery and survival with increased artery growth and heart function. The results paved the way for studying the mechanisms of DACH1-mediated protection, and how they might be leveraged as potential coronary artery disease treatments. Cindy St. Hilaire: The second article I want to share is titled Circulating Pro-Inflammatory Exosomes Worsen Stroke Outcomes in Aging. The first author is Hongxia Zhang, and the corresponding author is Kunlin Jin from University of North Texas Health Science Center. Aging is associated with declining tissue function and an assortment of health issues. But in rodents at least, certain factors, including the plasma of youthful animals and the exosomes of stem cells, can have rejuvenating effects on old animals. Exosomes are small membrane-bound particles containing cellular contents that circulate in the blood after they're released from cells. This group has shown that as rats age, the animals' serum exosomes accumulate pro-inflammatory mediators, such as C3a and C3b. Cindy St. Hilaire: When these aged rats were subjected to stroke, and then injected with serum exosomes isolated from either old or young rats, those receiving youthful exosomes fared much better in terms of infarct size and sensory motor deficits, while those receiving aged exosomes fared worse. The team went on to show that injected exosomes accumulate at the site of stroke injury, but those from old donors caused more neuronal damage, as seen by reduced synaptic function. Preventing C3a activity on microglia reversed the effects of the old exosomes and improved stroke outcome, suggesting that such modulation of inflammatory molecules might be a treatment strategy for stroke. Cindy St. Hilaire: The next article I want to share is titled Epigenetic Age Acceleration Reflects Long-Term Cardiovascular Health. The first author is Brian Joyce, and the corresponding author is Donald Lloyd-Jones. And they're from Northwestern University. DNA methylation is an epigenetic modification that regulates gene transcription. Studies of young and old individuals have shown that at certain locations in the genome, methylation status is highly correlated with age. These methylation patterns are also linked to measures of cardiovascular health, including blood pressure, cholesterol level and body mass index. This suggests that if a person has particularly good or particularly poor cardiovascular health, their DNA may appear younger or older than the individual's actual age. Cindy St. Hilaire: This group tested the hypothesis that people with poor cardiovascular health exhibit methylation changes more commonly found in elderly individuals than those with good cardiovascular health. And if so, DNA methylation patterns might be useful for predicting future cardiovascular risk. Cindy St. Hilaire: The team examined DNA methylation of over a thousand individuals enrolled in a prospective heart health cohort, testing them around age 40 and then again at around age 45. Changes in methylation status were then compared to individuals' cardiovascular health scores over a longer period. Sure enough, faster epigenetic changes did correlate with poor cardiovascular health later in life. Data from the second cohort of individuals supported the initial findings. This study indicates that DNA methylation status may be an early biomarker that signals cardiovascular issues, and may therefore allow for prompt implementation of treatment and prevention strategies. Cindy St. Hilaire: The last article I want to share is titled, Yap Promotes Noncanonical Wnt Signaling from Cardiomyocytes for Heart Regeneration. The first author is Shijie Liu, and the corresponding author is James Martin. And they're from Baylor College of Medicine. After a heart attack, cardiomyocytes are destroyed and replaced with a fibrotic scar that interferes with the contractile function of the heart. While adult mouse and human hearts are similar in this regard, the hearts of newborn mice possess greater regenerative capacity, and this regeneration capacity persists for approximately one week. The transcription factor YAP is known to regulate regenerative processes in neonatal hearts of mice. And its deletion eliminates regeneration, and its over-activation in adult cardiomyocytes reduces fibrosis. Cindy St. Hilaire: These experiments suggest cardiomyocytes transmit signals to cardiac fibroblasts. Wntless protein regulates the release of Wnt signaling molecules and also is a target of YAP. Mice that lack Wntless in their cardiomyocytes appear to have normal heart development and function. However, their neonatal regenerative capacity was impaired. In the weeks after heart injury, the mice that lack Wntless had reduced heart function, increased scar size and increased numbers of activated cardiac fibroblasts compared with that seen in controls. The study indicates that Wntless is critical to the regeneration of cardiac tissue, and may perhaps be leveraged to minimize scarring after heart attacks. Cindy St. Hilaire: I'm really excited to have with me today the three finalists of the BCVS Outstanding Early Career Investigator Award. The first person I'm going to be speaking with is Jiangbin Wu, who is a research assistant professor at the Aab Cardiovascular Research Institute at the University of Rochester. Thank you so much for joining me today. Jiangbin Wu: Thank you. Cindy St. Hilaire: And congratulations, actually. I know this is a highly competitive award that gets a lot of applications, so congrats on becoming a finalist. Before we get to your abstract, which is related to mitochondria and calcium influx in cardiomyocytes, I was wondering if you could share a bit about yourself. Maybe what your research path was, and what brought you to study cardiomyocytes and the mitochondria that are within them? Jiangbin Wu: Yeah. Right now, I'm an assitant professor at Cardiovascular Research Institute of University of Rochester. Previous, I was actually studying in the cancer field and also some kind of mitochondria work in some cancer cells. Although when I came to the University of Rochester and I switched to cardiovascular and then we are working on a kind of microRNA[at the initial. The way we screen for these is just by doing the RNA-Seq is target the microRNA. and then we start to study the function of these genes, and found that it's a mitochondria calcium channel regulator. Cindy St. Hilaire: The title of your abstract is FAM210A Maintains Cardiac Mitochondrial Homeostasis Through Regulating LETM1-Dependent Calcium Efflux. So before we unpack what all those words in the abstract title mean, could you tell me how you ended up focusing on FAM210A? What does this protein do, and why'd you focus on it? Jiangbin Wu: Yeah. As I mentioned that we just gathered this protein actually is by some kind of chance as a microRNA target. And this protein full name is family with similarity 210 A, actually is a family of proteins. This is just one of them. And the way discover is localized in mitochondria in the membrane. And also, there is some other people's report is in mitochondria. And we want to sort out its function inside the mitochondria and in the cardiac background. So we do some kind of omics or mass spec to get its interlocking interacting proteins. And then we found LETM1. It's a calcium channel inside the mitochondria in the membrane. So we figured out is, this FAM210 protein regulate LETM1 function in calcium, pump calcium is part of the mitochondria matrix. And I think this is a very important, because calcium overload is always happening in the very heart of the cardiomyocytes. Cindy St. Hilaire: That's a perfect segue, because my next question was really what is the gap in knowledge that your study was trying to address? Were you really focused on just the function of this one protein, or what was the greater goal of this study? Jiangbin Wu: Actually, the function this protein is the initial step. Our final aim is to use this protein, to over expression this protein in the heart failure patient or in some kind of heart failure models to do the, sort of do the work in some heart failure patients. Cindy St. Hilaire: Maybe a gene therapy approach, or if there's a pharmacological way to up regulate this protein? Jiangbin Wu: Yeah, because we've proposed that the self expression of this proteins will reduce the calcium overloading cardiomyocytes, which is a major cause for the cardiomyocytes death in heart failure process. So over expression will reduce this kind of process. And then it will make the cardiomyocytes survival in the failure heart. Cindy St. Hilaire: That is interesting. I mean, obviously you were using a mouse knockout model, so you know what's driving the expression down in that case. But in humans, what do we know about the regulation of this protein? Is anything known, or any known causes that cause its reduction in expression? Jiangbin Wu: Actually, we do. Its expression in heart failure is slightly increased in heart failure. So we feel it's a kind of some kind of compensating effect to try to save the heart from failing. Cindy St. Hilaire: Interesting. It's just not turned on early enough, in that case then. Jiangbin Wu: Yeah. And for the regulating protein for this one, I think we find microRNA can suppress its expression, but not too many other influences on these regulator proteins. Cindy St. Hilaire: That is so interesting. So what's next? What are you going to do next on this project? Jiangbin Wu: Yeah. I think currently, we are just at the start to do some kind of therapeutic effect that use to these proteins. I think we will do more deep in the therapeutic effects for over expression of these genes in... Currently, we are working on mouse models. Maybe in different heart failure models to prove that it's very benefiting to the heart failure patients. Cindy St. Hilaire: Wonderful. Well, congratulations on an excellent study. Really looking forward to your presentation, which is coming up shortly, and really looking forward to your future research in this field. Jiangbin Wu: Okay, thank you. Cindy St. Hilaire: So I also have with me, Dr Chris Toepfer, who's another finalist for the BCVBS outstanding early career investigator award. He's a principal investigator from the University of Oxford, and his abstract is titled, Defining Diverse Disease Pathway Mechanisms Across Thick And Thin Filament, Hypertrophic Cardiomyopathy Variance. So congratulations, Chris, and thank you for joining me today. Chris Toepfer: Thank you very much. It's great to be here. Cindy St. Hilaire: Before we start to discuss your abstract, I was wondering if you could just share a little bit about yourself. Maybe your career path, and how you came to study hypertrophic cardiomyopathy? Chris Toepfer: Yeah, sure. I guess this story gets longer and longer every time somebody asks it,right, in your career? Cindy St. Hilaire: That's a good thing. Chris Toepfer: Yeah. I started out as an undergraduate in London, and actually during the second year of my undergraduate degree, I fell into a lab kind of out of interest. It was starting to study cardiac muscle mechanics. And that was the lab of Professor Michael Ferenczy. And ended up, after I finished my undergraduate degree, I joined him for a PhD. I had a PhD program that also took me overseas to the NIH to work with Dr James Sellers, who was a muscle motor protein biochemist. And we really, I sort of really fell in love, with the idea of studying disease of multiple levels, and understanding how the heart would function from the basic molecule up to the entire organ and looking at different systems in between. Chris Toepfer: And that's what led me to then, so my postdoctoral position to seek out a completely different direction in some ways, but something that could also extend how we could look at the heart. And that's where I moved to Boston to work with Christine and Jonathan Seidman. I'm looking at more of the genetic basis then of hypertrophic cardiomyopathy rather than just, sort of more diffusely the mechanisms underlying cardiac muscle contraction. And then two years ago, I moved back to the UK to Oxford to sets up my own group, which has been fun during the pandemic as you can imagine. Cindy St. Hilaire: It's hard enough starting up a lab under normal times. I can't imagine doing it during a pandemic. Chris Toepfer: And we are now completely focused on stem cell models and CRISPR CAS engineering, and trying to understand hypertrophic cardiomyopathy in a dish. Cindy St. Hilaire: That's wonderful. And actually I looked at your CV. We actually overlapped a little bit. I was doing my postdoc at NIH in the NHLBI while you were there for your graduate school. So I too fell in love with kind of the starting with the human as the model path of research. So maybe you can kind of fill in all the listeners in who aren't cardiomyopathy experts. So what is, I guess, in a nutshell, hypertrophic cardiomyopathy, and what gap in knowledge was your study specifically addressing? Chris Toepfer: So in general, about one in 500 people have hypertrophic cardiomyopathy. And for those that are genetically linked, a lot of them are in the key contractile proteins of the heart, the drive muscle contraction. And what you often see in those people is they have thickened hearts. And what happens is actually the heart begins to be too hard, and it actually relaxes very poorly in between beats. Chris Toepfer: So what we are really trying to understand in this disease and with this abstract was how are different forms of hypertrophic cardiomyopathy created? Because it can be a couple of different forms. There are different proteins involved that have very vastly different functional mechanisms within the cell. So would this, we went away, we generated some stem cell models where we could then differentiate into cardiomyocytes. Model the disease in a dish. And we made kind of a group of good methods to go and look at what was happening inside the cells. And then we could screen drugs against what's happening inside those cells, so that was kind of the idea of what we were looking at, at the time. And what's fallen out of all of that is a drug now called Melacamptin that's starting to get to the clinic, which addresses some of these underlying mechanisms we were beginning to study. So that's what I'll talk about a bit later on in our session today. Cindy St. Hilaire: It's great. One of the things you focused on in the abstract is comparing these thick and thin filament variants. What are the implications of those, I guess, in the human disease state, but also in how you could design or use your stem cells as a model, and were any of the results that you found surprising? Chris Toepfer: So I think what was the really key finding that we saw was that the thick filament variants seemed to be switching myosin, which is a molecular motor that drives cardiac muscle contraction very much to arm”ON”. And my sort of analogy to that is they're all very sort of bodybuilder like. Myosin switched on, ready to go to work causing way too much contraction. And the compound that we were using at the time Myocamptin, we could turn those off and resolve the disease. Whereas with the thin filament variants, they were operating through a completely different mechanism. And when we tried to treat them with the same compound, they wouldn't always salvage disease. So though the face of it, they look the same in the dish, in that they contracted too much, relaxed very poorly. You're clearly doing it via complete different mechanism. And that's what we're starting to dig into now. And that's what we'll be talking about. Cindy St. Hilaire: Yeah. And that's actually kind of the question I was going to finish up with you. What are the, I guess translational implications? No, yes. You're using this drug. Is that only good for thick filament-like variants? And are you going to be able to screen patients to tell which variant they have, and therefore if this or that drug might be useful? Chris Toepfer: So we're in a real golden age now for genomics where I guess patients can come into the clinic and they can be sequenced and you could maybe tell them now what might be the underlying cause of their disease. I am not a clinician, but what we, as a basic scientist can say is, well, we can go away and try and understand whether this variant you may have in your genome is causative of disease. And if it is what mechanism that may fall under, what may be causing them to have this phenotype? Chris Toepfer: And I think what we can do is we can try and then bin the subpopulations of variants, and try and find novel drugs or novel pathways that we could try and find drugs for to treat the disease, and to differentiate them from each other. So I think it's too early to say whether Mylocamptin will be able to sort this for everybody, I guess we will find out in the next years. But I think already we can start thinking about, well, what would be the next step after this? We can bring precision medicine even further. And that's, I think the goal where we're heading towards. Cindy St. Hilaire: Well, that's wonderful, and this is a wonderful abstract. I'm really looking forward to seeing the full study and your presentation later on. And thank you so much for joining. Chris Toepfer: No. Yeah. Thank you for having me. I'm really looking forward to it later on. Cindy St. Hilaire: Great. Dr Chen Gaol is the third finalist for the BCBS Outstanding Early Career Investigator Award. She's an assistant researcher at UCLA, and her abstract is titled, Functional Impact of RBFox1C in Cardiac, Pathological Remodeling through Targeted MRNA Stability Regulation. So congratulations, and thank you so much for joining me today. Chen Gal: Absolutely, thank you for having me. Cindy St. Hilaire: Before we jump into your abstract, could you share with us a little bit about your career path, and how you came to study the role of RNA binding proteins, I guess specifically in pathological cardiac remodeling? Chen Gal: Yes, I think my research over the years has been into the very basic questions, which is I'm interested in looking at how the RNA is being regulated. For example, how the RNA is being spliced, is being ideated, and how the RNA is being degraded if it's ever been translated into protein. And the second half of my research is of course, physiological driven, because I'm interested in different type of cardiac disease, starting from the traditional heart attack to the now more emerging medical need, which is the cardiometabolic disease. So I was trained as a molecular biologist. I started in molecular biology Institute at UCLA. My PhD supervisor is Dr Yibin Wang, who first introduced me to understand there is actually a whole new world of R regulation at a post-transcription level. Chen Gal: So at that time we basically utilized the R sequencing. Just look for the easiest to heart, and try to understand how these RNA are differentially spliced in the heart. And I was so interested in understanding more about a cardiology. So I decided, even if I move out to my postdoc research I still want to continue working in the heart, although at a totally different angle. And that is when I started to really try to understand different aspects of RNA regulation. So now I am starting to be a junior faculty, establishing my own lab. And I really wanted to understand more how different steps of our metabolism is regulated. Cindy St. Hilaire: Really timely research. And I really like how you are doing a great job combining extremely basic biochemical processes with advanced disease states. An extra, that's why this abstract made it as a finalist. So congrats on that. So your study was focused on the RNA binding protein, RB Fox one, which has several isoforms. And so can you tell us which isoform you were looking at, and why you were interested in that particular isoform? Chen Gal: Yes, actually I've studied about ISO form of RPFox1. It itself, is actually subject to alternative splicing, while generating one nuclear, and another simosolic isoform. Where I was a PhD student, I was very simple minded, just trying to screen for the R binding protein that actually is expressed in the diseased heart. So RBFox1 is at least at a transcriptional level, the only one that we identify to be to decreased in the fatal heart. The nuclear function, the nucelo ISO form of RPFox1 is mainly regulating alternative splicing. But it is when I was studying this nuclear function of the RBFox1, I identified there is actually another isoform where she is in the set ourselves based on the different of c terminal domains of the RFox1. So I was just wondering, apparently you shouldn't be regulating and splicing anymore. I just move on to another layer of RA regulation. And then what I found most interesting is these RBFox1 is regulating the R stability, which is something that we'll talking about later today. Cindy St. Hilaire: That's great. So to do this study, you actually created a new knockout mouse model where you specifically deleted this one C isoform. What was kind of the baseline and maybe the disease state phenotypes that you saw in that mouse? Chen Gal: The result and phenotype so far is very striking. We utilize the CAS nine CRISPR technology simply because for, we were lucky the settle the Fox warehouse, one extra axon. So that does allow us to coach the lox P side, just blanking in that particular AXA. And in theory we could across it with different CRE, and to generate either cardiac or different tissue, specifically knock out. Even at a baseline we see a decreased cardiac function when we inactivate this isoform in the adult heart. And when we look at the gene expression profile is, I call mind-blowing type of experience, because turns out this gene not only is regulating some of the inflammatory genes, but also is helping involve protein translation and delivery metabolism, which I hope in the future will set us on the path to really understand the role of this RP Fox1. Not only into HFpEF, but also in the cardiometabolic disorder. Cindy St. Hilaire: Yeah, that's great. It's so rewarding when you do this one really big kind of risky experiment, and it turns into not just one interesting path to study, but multiple. One of the things that you mentioned in the abstract is clip seek. I was wondering if you could tell us a little bit about this technology, and how you used it in your study? Chen Gal: Yeah. I think one of the rewarding parts for me focusing on the R metabolism is really driving different accounting and sequencing tools, and utilize that in the heart. So cardiomyocyte has been traditionally viewed now to be very easy to work with type of model comparing helo cells, right? And I think in the field, we are still so short of knowledge, what type of the cutting-edge tools that we can use in the heart. My research involved clip seek, which is to use UV crosslinking the RNA with the R binding protein. So that will allow us to understand which are the RNA targets that are directly interacting with the RNA binding protein. I'm also using great seek, which is to find dynamically label the recency size to RNA. And that will allow us to look forward to RA degradation profile at a global level in the baseline or under disease. So I thought those are really cool technologies, and that's something that makes me excited about my work on a daily basis. Cindy St. Hilaire: Yeah, that's wonderful. So what's next? What are you going to do after this initial study? What's the next question you're going to go after? Chen Gal: Yeah, like I mentioned, I'm interested in, honestly, different type of heart disease, not just the stress induced heart failure, but also the recent years, I started to branch out a little bit to understand more of the biology of HFpEF. For example, how the R binding protein that we are studying right now is playing a role in the development of HFpEF. Or we actually understand very little about them, the micromechanism for HFpEF development, right. What are the RNA splicing profile in the cardio metabolic disorder on account? We also find differential regulation of R stability in the HfPEF compared to the HFpEF compared to the HFrEF. So I thought those are really interesting questions that I would like to pursue in the future. Cindy St. Hilaire: That's great and best of luck in those future studies. Chen Gal: Thank you. Cindy St. Hilaire: Before we leave, I was wondering if you could share with us any advice that you would give to a trainee, maybe something that you wish you knew ahead of time in this kind of early career stage. Chen Gal: I consider myself a really, really lucky person. And if I have one word to give to the younger people, younger than me, is to find great mentors for your career. And luckily our field has a lot of good mentors who are ready to help us every single step of our career. For example, my PhD supervisor, Dr Wang. And I have met a lot of good mentors inside and outside of UCLA. I'm pretty sure this is the same thing for Chris, who is trained by Dr Seidman, and everybody know how great a mentor she is. So I think having a great mentor will help you every step of your career development to making sure you're always on the right track. And that, that is also something that you will do when we have our own lab, because we want to be great mentors for our trainees as well. Cindy St. Hilaire: I know. That's something I strive for too, is to emulate my amazing mentors that I've had. What do you think is a good quality for a good mentor? Like what's one of the, I guess key features that you look for in someone that you would like to be your mentor? Chen Gal: For me, I think my mentors are all cheerleaders. They never try to push me to move out one career path versus the other. They are good listeners, and they are also my role models. Cindy St. Hilaire: That's wonderful. Chris, what's a piece of advice that you would like to share with trainees that your former self wish you knew of? Chris Toepfer: I think it's very important to echo the message of a good mentorship, and a good lab environment that allows you to flourish and really helps you to grow yourself to the future. And also helps you understand the bits of you that you could actually grow as well, a little bit better. So you become a more rounded scientist. I think something that's really important or something that I've always found very infectious is to find mentorship and mentors that are also incredibly enthusiastic about you as an individual, as well as the science. I think that that can really drive you. And I think that's also an important thing to have in yourself, to have, to find that question for yourself that really drives you and you can be really enthusiastic about. Cindy St. Hilaire: I totally agree. Well, thank you again for joining me today. Congratulations on being a finalist, and I wish everyone the best of luck in their presentations later on at BCBS. Chen Gal: Thank you so much. Jiangbin Wu: Thank you. Chris Toepfer: Thank you very much. Cindy St. Hilaire: That's it for the highlights from the September 17th and October 1st 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 #Discover CircRes. Thank you to our guests, BCBS Outstanding Early Career Investigator Award Finalists, Dr Jaobing Wu, Dr Chen Gal, and Dr Chris Toepfer. And a special congratulations to Dr Toepfer who won this year's competition. This podcast is produced by Asahara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of circulation research. Some of the copy texts for 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, 2021. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American heart association. For more information, please visit AHAjournals.org

JACC: CardioOncology - Mitochondria Rich Extracellular Vesicles Rescue Patient-Specific Cardiomyocytes From Doxorubicin Injury: Insights to the SENECA Trial

JACC Speciality Journals

Play Episode Listen Later Sep 21, 2021 3:19

Commentary by Dr. Nathan Wang

August 2021 Discover CircRes

Play Episode Listen Later Aug 19, 2021 26:50

This month on Episode 27 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the July 23rd and August 6th issues of Circulation Research. This episode also features an in-depth conversation with Drs Ana Gomez and John Pierre Benitah, from INSERM and the Paris-Saclay University, about their study, Impaired Binding to Junctophilin 2 and Nanostructural Alterations in CPVT Mutation. Article highlights: Glasenap, et al. Imaging Inflammation and Fibrosis in Heart Failure Shi, et al. Cardiomyocyte Pyroptosis Aggravates MI/R Injury Koenis, et al. SPM Temper Phagocyte Responses in COVID-19 Zhang, et al. Common Origin of Heart and Extraembryonic Lineages Cynthia St. Hilaire: Hi, and welcome to Discover CircRes, the podcast to the American Heart Association's journal, Circulation Research. I'm your host, Dr Cynthia St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'll be highlighting articles presented in our July 23rd and August 6th issues of Circulation Research. I also will speak with Drs Ana Gomez and John Pierre Benitah, from Inserm and the Paris-Saclay University, about their study, Impaired Binding to Junctophilin 2 and Nano-structural Alterations in CPVT Mutation. Cynthia St. Hilaire: The first article I want to share comes from the July 23rd issue of Circ Res, and it's titled Molecular Imaging and Inflammation and Fibrosis in Pressure Overload Heart Failure. The first author is Aylina Glasenapp and the corresponding author is James Thackeray, and they're from Hanover Medical School in Germany. After a heart attack, inflammation and fibrosis of the heart alter cardiac contraction and can lead to its failure. Currently, for ischemic heart failure, doctors use imaging techniques such as positron emission tomography, and cardiac magnetic resonance imaging, to measure the inflammation and fibrosis to provide a prognosis. Cynthia St. Hilaire: However, whether these imaging techniques are useful for non-ischemic heart failure was unknown. To find out, this group performed transverse aortic constriction on mice, which is a commonly used method to model non-ischemic heart failure, and then they analyzed the animal's hearts with positron emission tomography to assess the inflammation and cardiac magnetic resonance imaging to quantify scar tissue. Compared with Sham-operated animals, those that underwent TAC exhibited increased heart inflammation for at least three weeks and significant fibrosis for at least six weeks. The degree of scarring and inflammation was inversely correlated with heart function. The team also found that reversal of TAC led to reduced inflammation and fibrosis over time. Together, the results confirm that these imaging modalities are valuable for monitoring fibrosis and inflammation in non-ischemic heart failure, and they could potentially be useful for assessing the effectiveness of interventions. Cynthia St. Hilaire: The second article I want to share is titled GSDMD Mediated Cardiomyocyte Pyroptosis Promotes Myocardial Ischemia Reperfusion Injury. The first author is Huairui Shi and the corresponding author is Junbo Ge, and they're from Fudan University in China. After myocardial infarction, restoring blood flow is essential to saving muscle function. However, restoration of flow itself causes damage by inducing inflammation and cell death. This study found that the cell death aspect of a reperfusion injury occurs via a process called pyroptosis, which is a controlled form of necrosis that is due to excessive inflammation. Cynthia St. Hilaire: The team developed an in vitro model of reperfusion injury, where cultured cardiomyocytes are starved and then resupplied with oxygen. Using this model, they found that cells exhibited features of pyroptosis, including the release of inflammatory factors, increased production of the pyroptotic factor gasdermin D and cell death. Cardiomyocytes lacking gasdermin D did not display signs of pyroptosis under these same conditions. The team went on to show that gasdermin D was significantly increased in the hearts of mice following ischemia reperfusion. And compared with control animals, mice whose cardiomyocytes were engineered to lack gasdermin D, suffered less necrosis and smaller reperfusion injuries in their hearts. Together, these findings provide insights into the mechanisms that should be targeted to minimize pyroptosis and subsequent ischemia reperfusion injury, following myocardial infarctions. Cynthia St. Hilaire: The next article I want to share is titled Disruptive Resolution Mechanisms Favor Altered Phagocyte Responses in COVID-19. The first authors are Duco Steven Koenis, Issa Beegun and Charlotte Camille Jouvene, and the corresponding author is Jesmond Dalli. And they're from Queen Mary University of London. Inflammation is essential in the early stages of battling and invading pathogen, but at the same time, inflammation can become damaging to the host if it is not resolved in a timely manner. Prolonged and unresolved inflammation is responsible for the hospitalizations and deaths of many COVID-19 patients. An excess of circulating pro-inflammatory cytokines is one of the key features of severe COVID-19. And now, Koenis and colleagues show that certain pro-resolving factors are out of balance in these severe patients. Cynthia St. Hilaire: Blood samples from patients with mild COVID-19 showed an increase in specialized pro-resolving lipid mediators. However, blood from patients with severe COVID-19 had lower levels of these pro-resolving lipid factors. Expression of specialized pro-resolving lipid mediator receptors on phagocytes was also higher in patients with mild disease than those with severe COVID-19. And, in line with this, the proportion of activated pro-inflammatory phagocytes was higher in patients with severe disease. Cynthia St. Hilaire: When patients were treated with the steroid dexamethasone, they subsequently inhibited the increased levels of the specialized pro-resolving lipid mediators in the blood. Together, these results reveal specialized pro-resolving lipid mediators are dysregulated in severe cases of COVID-19, and the findings suggest increasing these pro-resolving lipid mediators could promote resolution of out-of-control inflammation. Cynthia St. Hilaire: The last article I want to share is titled Unveiling Complexity and Multi Potentiality of Early Heart Fields. The first authors are Qinqguan Zhang and Daniel Carlin, and the corresponding authors are Sylvia Evans, Joshua Bloomekatz, and Neil Chi, and they're from UC, San Diego. The developing heart is thought to originate from two populations of cells; the first and the second heart fields. And these are first identifiable at stages E 7.5 in the mouse, or on day 15 in the human embryo. Genes controlling the development of these fields have been linked to congenital heart defects, but interestingly, congenital heart defects are also sometimes linked to placental abnormalities. However, the mechanisms underlying this link have been unclear. Now this study has gone on to discover an unexpected link between the first heart field and extra embryonic tissues, which give rise to the yolk sack and the placenta. Cynthia St. Hilaire: Through lineage tracing experiments and single cell transcriptomics, the team discovered that the first heart field consists of two sources of mesoderm progenitor cells, one source that is embryonic in nature and the other source arises from the interface between the extra embryonic and the embryonic tissue of the early gastrula. This latter population of progenitor cells, which is defined by the expression of the transcription factor hand one, gives rise to extra embryonic mesoderm cells in addition to the two Hartfield cell populations. The discovery of this shared source of mesodermal progenitors not only blurs the lines between the embryo and its supporting tissue but may also explain the link between placental abnormalities and congenital heart defects. Cynthia St. Hilaire: Today I have with me Drs Ana Gomez and Jean-Pierre Benitah, and they're from Inserm and the Paris-Saclay University. And today we'll discuss their study Impaired Binding of Junctophilin 2 and Nano-structural Alterations in CPVT Mutation. And this article is in our July 23rd issue of Circulation Research. So thank you both very much for joining me today. Jean-Pierre Benitah: Thank you. Ana Gomez: Thank you. Cynthia St. Hilaire: You're in Paris, so we're trying to match it so we're all meeting our normal workday on a Friday. So I very much appreciate you taking the time to meet with me. So this study is investigating a rare disease called Catecholaminergic Polymorphic Centricular Tachycardia, or CPVT. So can you describe to us what is CPVT and how does this disease present in patients? Ana Gomez: Okay, so CPVT stands for Catecholaminergic Polymorphic Centricular Tachycardia. So it is a genetic disease that appears mainly in childhood and youth with sudden death. So the patients don't have any remarkable problem, either in the electrocardiogram or arteries, or in the cardiac structure by echocardiography, and they seem healthy. But when they have stress, it can be emotional or it can be physical, so during exercise, it presents with syncope or sudden cardiac arrest. So the problem is that, many of the times, the first symptom is the death of a child playing soccer or doing exercise and then the only treatment that they, so far, it's beta blockers, to avoid this stress, and also flecainide and propafenol. But these treatments are still not completely efficacious, or sometimes the people need to get implant defibrillator. It's a big cost and it's also stressful because if the patient feels that they have to recharge, that supposes stress, and this stress is bad for them. Cynthia St. Hilaire: Right, so it's like if they feel a flutter, it makes them more stressful, which can exacerbate. That is terrifying. And so the goal, I guess, regarding gaps in knowledge that are leading to your investigation, what was known about this disease before you started your study? And where did you leap off from that? Jean-Pierre Benitah: Up to now, what we know about the disease is an alteration of the calcium homeostasis in cardiac myocyte. That could induce trivial activity, and then arrhythmia and cardiac sudden death. So mainly the mutation related to an intracellular calcium channel called Ryanodine receptor. So it's up to 60% of the patient with this mutation, but also you have a mutation related also to proteins that are in-buried in the control of the Ryanodine receptor activity, priadine, calmodulin. Cynthia St. Hilaire: Yeah, that was actually going to be my next question. So I know this cardiac Ryanodine receptor 2, or RYR2, it's obviously the channel component that helps to release that calcium signal, but it's part of a larger complex. I believe it's called the Calcium Release Unit. Can you talk about what is in that unit in terms of proteins and then where those other genetic mutations fit into that? Ana Gomez: Yeah, so the Calcium Release Unit is formed by a cluster of Ryanodine receptors. So in the reticular cardiomyocytes, these are mostly in the junction of sarcoplasmic reticulum that is very close to the sarcoplasmic reticulum membrane inside the cardiomyocyte, inside the cell. So the channel is internal. But it's very close to the sarcolemma in the T-tubule invaginations where the L-type calcium channels are located. So this is... The channels are very important to activate contraction, so it's heartbeat. The calcium entry through the attached calcium channel on the surface makes some calcium get into these very restricted spaces, like 20 nanometers, and in this space this calcium activates the Ryanodine receptor. So the Ryanodine receptor is activated by calcium and these release much more calcium than is needed for the contraction. So the problem of the CPVT is that the channels may release calcium during diastole, so when calcium should be low because they had to relax. Ana Gomez: For your new question, which proteins? So the main proteins are the Ryanodine receptor. But Ryanodine receptors are a very big macro complex. They are the biggest channels that are known and they have a big cytoplasmic portion with proteins that can bind to them, and most of them just keep the channel quiet. So this may be calmodulin, FKPB 12.6, or 12, sorcin. And then there are also some other proteins that scaffold kinases, like PKA and CaM kinase. And also they have some proteins that moderate the channel from the luminal side. So, calsequestrin, triadin and junctin. And this agents to fill in that we will speak later. It's important because it binds to the L-type calcium channel and to the ryanodine receptor. So it's important to keep the dyad structure. It's not only a structural role. Cynthia St. Hilaire: Yeah, that is so interesting. So your study focused on a very specific mutation. It's the RYR2 arginine in the 420 spot to glutamine mutation. So I guess my first question is based on the patient population, how common is this specific mutation? How common is that? Ana Gomez: Yeah. So in fact, I'm going to say that it's very common, because normally CPVT is one mutation, one family. Cynthia St. Hilaire: I see. Ana Gomez: Even if they are located in hotspots, but these particular mutations, we were approached by a cardiologist working in Spain who had this family with a child that died at the age 14, playing soccer game. And so Dr Zorio in Valencia, she found this RyR2 420Q mutation. And at this time this was the first mutation in this site. I mean, not really in the site, there was already RyR2 420W that was already, so it was the same spot, but different. Cynthia St. Hilaire: That was my next follow up question to that. My PhD was biochemistry, so this brought back having to memorize the amino acid structure. So arginine is large and positively charged to glutamine is neutral. So what were the experiments that you designed to help determine the functional causes of this mutation? You know, in addition to just, okay, obviously there's a charge change, so there's probably a structural or a binding change, but how did you determine the functional consequences of this mutation? Ana Gomez: The structure, as you say, this has been shown. In fact, they was the first family, but then also in this region, there was another family and in Israel also there is another family. So there are three, but the structural limitations that these arginine is neutral. It has been shown by a laboratory, who works in Vancouver, in a structural and the end terminal has like three logs and these are 420. It's important to hold a chloride that in the middle and, and to hold the position. So, but this is not the functional, the functional is what we were going to analyze. So the first thing that we did is to analyze calcium sparks because calcium sparks is the functional, let's say elementary event, of calcium release to RyR2 receptors. So we start analyzing calcium sparks in the cells and we found strange things, like very long calcium sparks that was not so clear in other CPVT models, even one that we studied earlier. And so then we started to continue to know why we have longer calcium sparks and different kind of analysis. So we also collaborate with some other laboratories to do the ultrastructure of the dyad by electromicroscopy. Ana Gomez: And then we found that the sarcoplasmic reticulum, junctional sarcoplasmic reticulum, was enlarged. So we thought, well, maybe the channel, the calcium spark is longer because locally they delayed depletion. So we did another kind of experiment changing the volume of the SR and it was not so concluded so we found that it may contribute to longer calcium sparks, but it doesn't explain for it. So then we start with to analyze different proteins candidates, also the phosphorylation of course. And then we didn't find in most of these proteins, like FKVP. Cynthia St. Hilaire: Kind of a standard go-tos. None of them were involved. Yeah. Ana Gomez: Yeah. And then, because there is this ultrastructural alteration, we thought of junctophilin and that is how we found that junctophilin binding was impaired. Cynthia St. Hilaire: That's a perfect segue. You're hitting all of my next questions. So can you tell us a little bit about, what did you find regarding junctophilin and the RyR2 channel? Jean-Pierre Benitah: So mainly, junctophilin act to us the good structural design between the ryanodine receptor and the trigger L-type calcium channel. And people say that junctophilin binds to both proteins to keep them close to each other. So mainly what we found is that we don't have activation of the expression of junctophilin, but it seems that with this mutation the junctophilin is less in contact with ryanodine receptor. But it's not the case for the L-type calcium channel. It seems that coimmunoprecipitation experiments that we've done show that junctophilin stayed still with the L-type calcium channel, but have a lower affinity to the ryanodine receptor when you have this mutation. What was really important is that we saw that not only in the mouse model where we induce this mutation, but also in cardiomyocytes derived from induced pluripotent stem cells from patients that have this mutation. Cynthia St. Hilaire: I think that's one of the great strengths of your study. You know, I like how you took a multi-faceted approach, you know, using these IPS cells from the patients and also created a knock in model. Previous studies had used more global or whole exon deletions. So how is your knock-in able to identify additional information that built upon those former studies? Ana Gomez: Maybe this is not an exact answer to your question, but what I think is that the strength of our study or one of the strengths of our study is that we have the patients with electrocardiograms working, we have the cells from the patients. So we have...Our IPS cell is from one of the persons that have been patient, and the control line is from his brother. So we have the two brothers. They are still living, and we have the mice and everything is in the same point mutation. So in this thing, because there is a lot of, let's say, critics to the IPS cells studies because they are not mature and they don't look like an adult cardiomyocyte. And I think that besides CPVT, we can also show that of course cardiomyocytes derive from IPS cells. They are not adult, but they are still a good model because we recapitulate the same thing. Ana Gomez: So we can mix the human context to really have what happened in patients, because that is the important thing, but we also need to manipulate the in vivo animals and there are some things that we cannot do. We cannot get adult cardiomyocytes from patients, so for that, we have the mice and we can also analyze from in vivo to the molecular level. So I think that it's a big strong point from our study that you take compared to others, that they are only in mice or only in IPS, cannot do this correlation. Then, each mutation, we think that it may, or at least each region of the mutation, may have different mechanisms. So if we find these longer calcium sparks in these R420Q mutation, it doesn't mean that because we also have other studies in C-terminal mutation, and we don't find longer calcium sparks, we just find more. So this is not because of the design of the study, but because the mechanism of the mutation is different. Cynthia St. Hilaire: In terms of translational potential, what do your findings suggest about either the ability to screen patients potentially for the development of CPVT or actually more importantly, you know, therapies to help treat these patients when they're identified? Jean-Pierre Benitah: Yeah. It's one of the big problems with the CPVT, especially since when you look at the different mutations, those are different mutations that have been reported on the ryanodine receptor located on different hotspots on the ryanodine receptor. And it's seems that each hotspot could have a different type of mechanism behind that. So, for example, we show, there you see, you know, different mutations in collaboration with CPVT or 420Q mutation. So the mechanism was related to an alliteration of the sensitivity of the ryanodine receptor to the calcium. So the group of branching show that in other mutations, in other spots, hot spots, it was related in fact, to a modification of this. Also the sensitivity of calcium of the ryanodine receptor calcium, but from the luminal side. Ana Gomez: Regarding your first question was diagnosis. I think that after our work, we may also include junctophilin, because so far there has not been any link to junctophilin for sensitivity. So when a patient has CPVT, they start screening for mutations in the ryanodine receptor, since it was found that this child was involved and then in other proteins. So I think now if they don't find in a patient, because there are still like 40% of CPVT patients that the mutation has not been found. Ana Gomez: For therapeutic side maybe find a molecule that stimulates the binding of junctophilin to ryanodine receptor, but also maybe some smaller molecule that may interact between the N-terminal and the core solenoid because we found that in the interim molecular structure, they show tighter association between the N-terminal and the core solenoid. So maybe it's more of a tide or something that can be in between too. I mean, I don't know, but it's first line there. Cynthia St. Hilaire: Potential, but still far off. That's wonderful. So are some of these mechanisms, I assume, they would also be relevant in non-genetic forms of tachycardia? Is that the case? Could some of your findings also perhaps be applied to the tachycardia related to heart failure or other types of disease states? Ana Gomez: I think it's actually, for example, junctophilin binding to ryanodine receptor in heart failure. It has not been yet studied, but we want to do it. It's something because as you say heart failure, it's a very common disease. So it's also very relevant to the public health. This is something that we need to know. Jean-Pierre Benitah: One of the things that happens in heart failure is that it seems also that you are a dissociation between the calcium channels and the ryanodine receptor because you have less tissue formation. So perhaps this is difficult to try to figure out whether it would be the same, but perhaps this activation between the communication between the two channels is one of the main points that we have in CPVT and in heart failure related to tachycardia. Ana Gomez: Yeah. In fact, many years ago we showed that. We showed that in heart failure there is a defect in calcium channel and ryanodine receptor. So in this study it was only functional. We didn't do the structure, but of course it is something that we have to keep in mind, continue investigating. Cynthia St. Hilaire: Yeah. Well that sounds like a great future project. Well, I want to thank you so much for joining me today and helping to discuss your paper. I love it when we take rare diseases and figure out the mechanism with hopefully applying it to more common disease states. That's what I do in my lab with vascular calcification, and so thank you so much for joining me and for this great publication. And we look forward to your future work that is hopefully in Circ Res. Jean-Pierre Benitah: Thank you for the invitation. Ana Gomez: Yeah, thank you very much for your time. Cynthia St. Hilaire: That's it for the highlights from the July 23rd and August 6th issues of Circulation Research. Thank you for listening. Cynthia St. Hilaire: Please check out the Circ Res Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, doctors Ana Gomez and John-Pierre Benitah. Cynthia St. Hilaire: This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles was provided by Ruth Williams. I'm your host, Dr Cynthia 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, 2021. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.

March 2021 Discover CircRes