Morrbid is a new identified leukocyte-specific long noncoding RNA (lncRNA). However, the expression and biological functions of Morrbid in cardiomyocytes and in heart disease are currently unclear. The study is to determine the roles of cardiac Morrbid in acute myocardial infarction (AMI) and to identify the potential cellular and molecular mechanisms involved. We found that both human and mouse cardiomyocytes could express a significant amount of Morrbid and its expression was increased in cardiomyocytes with hypoxia or oxidative stress, and in mouse hearts with AMI. Overexpression of Morrbid reduced the myocardial infarct size and cardiac dysfunction, whereas the infarct size and cardiac dysfunction were deteriorated in cardiomyocyte-specific Morrbid knockout (Morrbidfl/fl/Myh6-Cre) mice. We identified that Morrbid had a protective effect against hypoxia or H2O2-induce apoptosis, which was also confirmed in vivo in mouse hearts after AMI. We further discovered that serpine1 was a direct target gene of Morrbid, which was involved in Morrbid-mediated protective effect on cardiomyocytes. In summary, we have found for the first time that the cardiac Morrbid is a stress-enhanced lncRNA, which protects hearts from AMI via anti-apoptosis through its target gene serpine1. Morrbid may be a novel promising therapeutic target for ischemic heart diseases such as AMI.
Yang Yu, Haiqiong Yang, Qiuting Li, Nianhui Ding, Jiali Gao, Gan Qiao, Jianguo Feng, Xin Zhang, Jianming Wu, Yajun Yu, Xiangyu Zhou, Xiaobin Wang, Chunxiang Zhang
Almost half of patients recovering from open chest surgery experience atrial fibrillation (AF) that results principally from inflammation in the pericardial space surrounding the heart. Given that post-operative AF is associated with increased mortality, effective measures to prevent AF after open-chest surgery are highly desirable. In this study, we tested the concept that extracellular vesicles (EVs) isolated from human atrial explant-derived cells can prevent post-operative AF. Middle-aged female and male rats were randomized to undergo sham operation or induction of sterile pericarditis followed by trans-epicardial injection of human EVs or vehicle into the atrial tissue. Pericarditis increased the probability of inducing AF while EV treatment abrogated this effect in a sex independent manner. EV treatment reduced infiltration of inflammatory cells and production of pro-inflammatory cytokines. Atrial fibrosis and hypertrophy seen after pericarditis was markedly attenuated by EV pre-treatment; an effect attributable to suppression of fibroblast proliferation by EVs. Our study demonstrates that injection of extracellular vesicles at the time of open-chest surgery shows prominent anti-inflammatory effects and prevents AF due to sterile pericarditis. Translation of this finding to patients might provide an effective new strategy to prevent post-operative AF by reducing atrial inflammation and fibrosis.
Sandrine Parent, Ramana Vaka, Yousef Risha, Clarissa Ngo, Pushpinder Kanda, Stanley Nattel, Saad Khan, David Courtman, Duncan J. Stewart, Darryl R. Davis
Previous studies implicate extracellular adenosine signaling in attenuating myocardial ischemia and reperfusion injury (IRI). This extracellular adenosine signaling is terminated by its uptake into cells by equilibrative nucleoside transporters (ENTs). Thus, we hypothesized that targeting ENTs would function to increase cardiac adenosine signaling and concomitant cardioprotection against IRI. Mice were exposed to myocardial ischemia and reperfusion injury. Myocardial injury was attenuated in mice treated with the nonspecific ENT inhibitor dipyridamole. A comparison of mice with global Ent1 or Ent2 deletion showed cardioprotection only in Ent1–/– mice. Moreover, studies with tissue-specific Ent deletion revealed that mice with myocyte-specific Ent1 deletion (Ent1loxP/loxP Myosin Cre+ mice) experienced smaller infarct sizes. Measurements of cardiac adenosine levels demonstrated that postischemic elevations of adenosine persisted during reperfusion after targeting ENTs. Finally, studies in mice with global or myeloid-specific deletion of the Adora2b adenosine receptor (Adora2bloxP/loxP LysM Cre+ mice) implied that Adora2b signaling on myeloid-inflammatory cells in cardioprotection provided by ENT inhibition. These studies reveal a previously unrecognized role for myocyte-specific ENT1 in cardioprotection by enhancing myeloid-dependent Adora2b signaling during reperfusion. Extension of these findings implicates adenosine transporter inhibitors in cardioprotection against ischemia and reperfusion injury.
Wei Ruan, Jiwen Li, Seungwon Choi, Xinxin Ma, Yafen Liang, Ragini Nair, Xiaoyi Yuan, Tingting W. Mills, Holger K. Eltzschig
Excessive activation of cardiac fibroblasts (CFs) in response to injury provokes cardiac fibrosis, stiffness, and failure. The local mediators counter-regulating this response remain unclear. Exogenous C-type natriuretic peptide (CNP) exerted antifibrotic effects in preclinical models. To unravel the role of the endogenous hormone, we generated mice with fibroblast-restricted deletion (KO) of guanylyl cyclase-B (GC-B), the cGMP-synthesizing CNP receptor.CNP activated GC-B/cGMP signaling in human and murine CFs, preventing proliferative and promigratory effects of AngiotensinII (AngII) and TGF-β. Fibroblast-specific GC-B-KO mice showed enhanced fibrosis in response to AngII infusions. Moreover, after two weeks of mild pressure-overload induced by transverse aortic constriction (TAC), such KO mice had augmented cardiac fibrosis and hypertrophy, together with systolic and diastolic contractile dysfunction. This was associated with increased expression of the profibrotic genes collagen I, III and periostin. Notably, such responses to AngII and TAC were greater in female as compared to male KO mice. Enhanced AngII-induced CNP expression in female hearts and augmented GC-B expression and activity in female CFs may contribute to this sex disparity. The results show that paracrine CNP signaling in CFs has antifibrotic and antihypertrophic effects. The CNP/GC-B/cGMP pathway might be a target for therapies combating pathological cardiac remodeling.
Franziska Werner, Estefania Prentki Santos, Konstanze Michel, Hanna Schrader, Katharina Völker, Tamara Potapenko, Lisa Krebes, Marco Abesser, Dorothe Möllmann, Martin Schlattjan, Hannes Schmidt, Boris V. Skryabin, Katarina Špiranec Spes, Kai Schuh, Christopher P. Denton, Hideo A. Baba, Michaela Kuhn
Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop a clinically aggressive dilated cardiomyopathy (DCM). Genetic mutation knock-in (KI) animal models imply that altered function of the arginine-serine-rich (RS) domain is crucial for severe DCM. To test this hypothesis, we generated an RS domain deletion mouse model (Rbm20ΔRS). We show that Rbm20ΔRS mice manifest DCM with mis-splicing of RBM20 target transcripts. We found that RBM20 is mis-localized to the sarcoplasm in Rbm20ΔRS mice, which led to the formation of RBM20 granules similar to those detected in mutation KI animals. In contrast, mice lacking the RNA recognition motif (RRM) show similar mis-splicing of RBM20 target genes, but do not develop DCM or exhibit RBM20 granule formation. Using in vitro studies with immunocytochemical staining, we demonstrate that only DCM-associated mutations in the RS domain facilitate RBM20 nucleocytoplasmic transport and promote granule assembly. Further, we defined the core nuclear localization signal (NLS) within the RS domain. Mutation analysis of phosphorylation sites in the RS domain indicate that this modification is dispensable for RBM20 nucleocytoplasmic transport. Collectively, our findings revealed that disruption of RS domain-mediated nuclear localization is crucial for severe DCM caused by NLS mutations.
Yanghai Zhang, Zachery R. Gregorich, Yujuan Wang, Camila U. Braz, Jibin Zhang, Yang Liu, Peiheng Liu, Jiaxi Shen, Nanyumuzi Aori, Timothy A. Hacker, Henk Granzier, Wei Guo
Myocardial fibrosis and calcification associate with adverse outcomes in nonischemic heart failure. Cardiac fibroblasts (CF) transition into myofibroblasts (MF) and osteogenic fibroblasts (OF) to promote myocardial fibrosis and calcification. However, common upstream mechanisms regulating both CF-to-MF transition and CF-to-OF transition remain unknown. microRNAs are promising targets to modulate CF plasticity. Our bioinformatics revealed downregulation of miR–129-5p and upregulation of its targets small leucine–rich proteoglycan Asporin (ASPN) and transcription factor SOX9 as common in mouse and human heart failure (HF). We experimentally confirmed decreased miR–129-5p and enhanced SOX9 and ASPN expression in CF in human hearts with myocardial fibrosis and calcification. miR–129-5p repressed both CF-to-MF and CF-to-OF transition in primary CF, as did knockdown of SOX9 and ASPN. Sox9 and Aspn are direct targets of miR–129-5p that inhibit downstream β-catenin expression. Chronic Angiotensin II infusion downregulated miR–129-5p in CF in WT and TCF21-lineage CF reporter mice, and it was restored by miR–129-5p mimic. Importantly, miR–129-5p mimic not only attenuated progression of myocardial fibrosis, calcification marker expression, and SOX9 and ASPN expression in CF but also restored diastolic and systolic function. Together, we demonstrate miR–129-5p/ASPN and miR–129-5p/SOX9 as potentially novel dysregulated axes in CF-to-MF and CF-to-OF transition in myocardial fibrosis and calcification and the therapeutic relevance of miR–129-5p.
Lejla Medzikovic, Laila Aryan, Grégoire Ruffenach, Min Li, Nicoletta Savalli, Wasila Sun, Shervin Sarji, Jason Hong, Salil Sharma, Riccardo Olcese, Gregory Fishbein, Mansoureh Eghbali
Central conducting lymphatic anomaly (CCLA) due to congenital maldevelopment of the lymphatics can result in debilitating and life-threatening disease with limited treatment options. We identified 4 individuals with CCLA, lymphedema, and microcystic lymphatic malformation due to pathogenic, mosaic variants in KRAS. To determine the functional impact of these variants and identify a targeted therapy for these individuals, we used primary human dermal lymphatic endothelial cells (HDLECs) and zebrafish larvae to model the lymphatic dysplasia. Expression of the p.Gly12Asp and p.Gly13Asp variants in HDLECs in a 2‑dimensional (2D) model and 3D organoid model led to increased ERK phosphorylation, demonstrating these variants activate the RAS/MAPK pathway. Expression of activating KRAS variants in the venous and lymphatic endothelium in zebrafish resulted in lymphatic dysplasia and edema similar to the individuals in the study. Treatment with MEK inhibition significantly reduced the phenotypes in both the organoid and the zebrafish model systems. In conclusion, we present the molecular characterization of the observed lymphatic anomalies due to pathogenic, somatic, activating KRAS variants in humans. Our preclinical studies suggest that MEK inhibition should be studied in future clinical trials for CCLA due to activating KRAS pathogenic variants.
Sarah E. Sheppard, Michael E. March, Christoph Seiler, Leticia S. Matsuoka, Sophia E. Kim, Charlly Kao, Adam I. Rubin, Mark R. Battig, Nahla Khalek, Erica Schindewolf, Nora O’Connor, Erin Pinto, Jessica R.C. Priestley, Victoria R. Sanders, Rojeen Niazi, Arupa Ganguly, Cuiping Hou, Diana Slater, Ilona J. Frieden, Thy Huynh, Joseph T. Shieh, Ian D. Krantz, Jessenia C. Guerrero, Lea F. Surrey, David M. Biko, Pablo Laje, Leslie Castelo-Soccio, Taizo A. Nakano, Kristen Snyder, Christopher L. Smith, Dong Li, Yoav Dori, Hakon Hakonarson
Regular exercise leads to widespread salutary effects, and there is increasing recognition that exercise-stimulated circulating proteins can impart health benefits. Despite this, limited data exist regarding the plasma proteomic changes that occur in response to regular exercise. Here, we perform large-scale plasma proteomic profiling in 654 healthy human study participants before and after a supervised, 20-week endurance exercise training intervention. We identify hundreds of circulating proteins that are modulated, many of which are known to be secreted. We highlight proteins involved in angiogenesis, iron homeostasis, and the extracellular matrix, many of which are novel, including training-induced increases in fibroblast activation protein (FAP), a membrane-bound and circulating protein relevant in body-composition homeostasis. We relate protein changes to training-induced maximal oxygen uptake adaptations and validate our top findings in an external exercise cohort. Furthermore, we show that FAP is positively associated with survival in 3 separate, population-based cohorts.
Jeremy M. Robbins, Prashant Rao, Shuliang Deng, Michelle J. Keyes, Usman A. Tahir, Daniel H. Katz, Pierre M. Jean Beltran, François Marchildon, Jacob L. Barber, Bennet Peterson, Yan Gao, Adolfo Correa, James G. Wilson, J. Gustav Smith, Paul Cohen, Robert Ross, Claude Bouchard, Mark A. Sarzynski, Robert E. Gerszten
The adult mammalian heart has limited regenerative capacity, while the neonatal heart fully regenerates during the first week of life. Postnatal regeneration is mainly driven by proliferation of preexisting cardiomyocytes and supported by proregenerative macrophages and angiogenesis. Although the process of regeneration has been well studied in the neonatal mouse, the molecular mechanisms that define the switch between regenerative and nonregenerative cardiomyocytes are not well understood. Here, using in vivo and in vitro approaches, we identified the lncRNA Malat1 as a key player in postnatal cardiac regeneration. Malat1 deletion prevented heart regeneration in mice after myocardial infarction on postnatal day 3 associated with a decline in cardiomyocyte proliferation and reparative angiogenesis. Interestingly, Malat1 deficiency increased cardiomyocyte binucleation even in the absence of cardiac injury. Cardiomyocyte-specific deletion of Malat1 was sufficient to block regeneration, supporting a critical role of Malat1 in regulating cardiomyocyte proliferation and binucleation, a landmark of mature nonregenerative cardiomyocytes. In vitro, Malat1 deficiency induced binucleation and the expression of a maturation gene program. Finally, the loss of hnRNP U, an interaction partner of Malat1, induced similar features in vitro, suggesting that Malat1 regulates cardiomyocyte proliferation and binucleation by hnRNP U to control the regenerative window in the heart.
Galip S. Aslan, Nicolas Jaé, Yosif Manavski, Youssef Fouani, Mariana Shumliakivska, Lisa Kettenhausen, Luisa Kirchhof, Stefan Günther, Ariane Fischer, Guillermo Luxán, Stefanie Dimmeler
Arrhythmogenic cardiomyopathy (AC) is a familial heart disease partly caused by impaired desmosome turnover. Thus, stabilization of desmosome integrity may provide potential new treatment options. Desmosomes, apart from cellular cohesion, provide the structural framework of a signaling hub. Here, we investigated the role of the epidermal growth factor receptor (EGFR) in cardiomyocyte cohesion. We inhibited EGFR under physiological and pathophysiological conditions using the murine plakoglobin knockout AC model, in which EGFR was upregulated. EGFR inhibition enhanced cardiomyocyte cohesion. Immunoprecipitation showed an interaction of EGFR and desmoglein 2 (DSG2). Immunostaining and AFM revealed enhanced DSG2 localization and binding at cell borders upon EGFR inhibition. Enhanced area composita length and desmosome assembly were observed upon EGFR inhibition, confirmed by enhanced DSG2 and desmoplakin (DP) recruitment to cell borders. PamGene Kinase assay performed in HL-1 cells treated with Erlotinib, an EGFR inhibitor, revealed upregulation of RhoA associated protein kinase (ROCK). Erlotinib-mediated desmosome assembly and cardiomyocyte cohesion were abolished upon ROCK inhibition. Thus, inhibiting EGFR, thereby stabilizing desmosome integrity via ROCK, might provide new treatment options for AC.
Maria Shoykhet, Orsela Dervishi, Philipp Menauer, Matthias Hiermaier, Sina Moztarzadeh, Colin Osterloh, Ralf J. Ludwig, Tatjana Williams, Brenda Gerull, Stefan Kaab, Sebastian Clauss, Dominik Schüttler, Jens Waschke, Sunil Yeruva
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