Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
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.
Authors
Lejla Medzikovic, Laila Aryan, Grégoire Ruffenach, Min Li, Nicoletta Savalli, Wasila Sun, Shervin Sarji, Jason Hong, Salil Sharma, Riccardo Olcese, Gregory Fishbein, Mansoureh Eghbali
Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
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.
Authors
Galip S. Aslan, Nicolas Jaé, Yosif Manavski, Youssef Fouani, Mariana Shumliakivska, Lisa Kettenhausen, Luisa Kirchhof, Stefan Günther, Ariane Fischer, Guillermo Luxán, Stefanie Dimmeler
Abstract
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.
Authors
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
Abstract
Cardiac fibrosis is associated with an adverse prognosis in cardiovascular disease, which results in a decreased cardiac compliance and ultimately heart failure. Recent studies have identified the role of long non-coding RNA (lncRNA) in cardiac fibrosis. However, the functions of many lncRNAs in cardiac fibrosis remain to be characterized. Through a whole-transcriptome sequencing and bioinformatics analysis on a mouse model of pressure overload-induced cardiac fibrosis, we screened a key lncRNA termed thrombospondin 1 antisense 1 (THBS1-AS1), which was positively associated with cardiac fibrosis. In vitro functional studies demonstrated that the silencing of THBS1-AS1 ameliorated TGF-β1 effects on cardiac fibroblasts (CFs) activation and the overexpression of THBS1-AS1 displayed the opposite effect. A mechanistic study revealed that THBS1-AS1 could sponge miR-221/222 to regulate the expression of TGFBR1. Moreover, under TGF‐β1 stimulation, the forced expression of miR-221/222 or the knockdown TGFBR1 significantly reversed the THBS1-AS1 overexpression induced further cardiac fibroblast activation. In vivo, specific knockdown of THBS1-AS1 in activated cardiac fibroblasts significantly alleviated transverse aorta constriction (TAC)-induced cardiac fibrosis in mice. Finally, we demonstrated that the human THBS1-AS1 can also affect the activation of cardiac fibroblasts by regulating TGFBR1. In conclusion, this study reveals that lncRNA THBS1-AS1 is a novel regulator of cardiac fibrosis and may serve as a potential target for the treatment of cardiac fibrosis.
Authors
Junteng Zhou, Geer Tian, Yue Quan, Qihang Kong, Fangyang Huang, Junli Li, Wenchao Wu, Yong Tang, Zhichao Zhou, Xiaojing Liu
Abstract
Heart failure (HF) is characterized by global alterations in myocardial DNA methylation, yet little is known about epigenetic regulation of the non-coding genome and potential reversibility of DNA methylation with left ventricular assist device (LVAD) therapy. Genome-wide mapping of myocardial DNA methylation in 36 HF patients at LVAD implantation, 8 patients at LVAD explantation, and 7 non-failing donors using a high-density bead array platform identified 2079 differentially methylated positions (DMPs) in ischemic cardiomyopathy and 261 DMPs in non-ischemic cardiomyopathy. LVAD support resulted in normalization of only 3.2% of HF-associated DMPs. Methylation-expression correlation analysis yielded several protein-coding genes that are hypomethylated and upregulated (HTRA1, FBXO16, EFCAB13, AKAP13) or hypermethylated and downregulated (TBX3) in HF. A novel cardiac-specific super-enhancer lncRNA (LINC00881) is hypermethylated and downregulated in human HF. LINC00881 is an upstream regulator of sarcomere and calcium channel gene expression including MYH6, CACNA1C, and RYR2. LINC00881 knockdown reduces peak calcium amplitude in the beating human iPS cell derived cardiomyocytes. Collectively, these data suggest that HF-associated changes in myocardial DNA methylation within coding and non-coding genome are minimally reversible with mechanical unloading. Epigenetic reprogramming strategies may be necessary to achieve sustained clinical recovery from heart failure.
Authors
Xianghai Liao, Peter J. Kennel, Bohao Liu, Trevor R. Nash, Richard Z. Zhuang, Amandine F. Godier-Furnemont, Chenyi Xue, Rong Lu, Paolo C. Colombo, Nir Uriel, Muredach P. Reilly, Steven O. Marx, Gordana Vunjak-Novakovic, Veli K. Topkara
Abstract
In pulmonary arterial hypertension (PAH), inflammation promotes a fibroproliferative pulmonary vasculopathy. Reductionist studies emphasizing single biochemical reactions suggest a shift toward glycolytic metabolism in PAH; however, key questions remain regarding the metabolic profile of specific cell types within PAH vascular lesions in vivo. We used RNA-seq to profile the transcriptome of pulmonary artery endothelial cells (PAECs) freshly isolated from an inflammatory vascular injury model of PAH ex vivo, and these data were integrated with information from human gene ontology pathways. Network medicine was then used to map all amino acid and glucose pathways to the consolidated human interactome, which includes data on 233,957 physical protein-protein interactions. Glucose and proline pathways were significantly close to the human PAH disease module, suggesting that these pathways are functionally relevant to PAH pathobiology. To test this observation in vivo, we used multi-isotope imaging mass spectrometry (MIMS) to map and quantify utilization of glucose and proline in the PAH pulmonary vasculature at subcellular resolution. Our findings suggest suggest that elevated glucose and proline avidity underlies increased biomass in PAECs and the media of fibrosed PAH pulmonary arterioles. Overall, these data show that anabolic utilization of glucose and proline are fundamental to the vascular pathology of PAH.
Authors
Bradley M. Wertheim, Rui-Sheng Wang, Christelle Guillermier, Christiane V.R. Hütter, William M. Oldham, Jörg Menche, Matthew L. Steinhauser, Bradley A. Maron
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