Background: Conversion of cardiac stromal cells into myofibroblasts is typically associated with hypoxia conditions, metabolic insults, and/or inflammation, all of which are predisposing factors to cardiac fibrosis and heart failure. We hypothesized that this conversion could be also mediated by response of these cells to mechanical cues through activation of the Hippo transcriptional pathway. The objective of the present study was to assess the role of cellular/nuclear straining forces acting in myofibroblast differentiation of cardiac stromal cells under the control of YAP (yes-associated protein) transcription factor and to validate this finding using a pharmacological agent that interferes with the interactions of the YAP/TAZ (transcriptional coactivator with PDZ-binding motif) complex with their cognate transcription factors TEADs (TEA domain transcription factors), under high-strain and profibrotic stimulation. Methods: We employed high content imaging, 2-dimensional/3-dimensional culture, atomic force microscopy mapping, and molecular methods to prove the role of cell/nuclear straining in YAP-dependent fibrotic programming in a mouse model of ischemia-dependent cardiac fibrosis and in human-derived primitive cardiac stromal cells. We also tested treatment of cells with Verteporfin, a drug known to prevent the association of the YAP/TAZ complex with their cognate transcription factors TEADs. Results: Our experiments suggested that pharmacologically targeting the YAP-dependent pathway overrides the profibrotic activation of cardiac stromal cells by mechanical cues in vitro, and that this occurs even in the presence of profibrotic signaling mediated by TGF-β1 (transforming growth factor beta-1). In vivo administration of Verteporfin in mice with permanent cardiac ischemia reduced significantly fibrosis and morphometric remodeling but did not improve cardiac performance. Conclusions: Our study indicates that preventing molecular translation of mechanical cues in cardiac stromal cells reduces the impact of cardiac maladaptive remodeling with a positive effect on fibrosis.

Patients with severe respiratory syndrome caused by SARS-CoV-2 undergo cardiac complications due to hyper-inflammatory conditions. Although the presence of the virus has been detected in the myocardium of infected patients, and infection of induced pluripotent cell-derived cardiomyocytes has been demonstrated, the reported expression of Angiotensin-Converting Enzyme-2 (ACE2) in cardiac stromal cells suggests that SARS-CoV-2 may determine cardiac injury by sustaining productive infection and increasing inflammation. We analysed expression of ACE2 receptor in primary human cardiac stromal cells derived from cardiospheres, using proteomics and transcriptomics before exposing them to SARS-CoV-2 in vitro. Using conventional and high sensitivity PCR methods, we measured virus release in the cellular supernatants and monitored the intracellular viral bioprocessing. We performed high-resolution imaging to show the sites of intracellular viral production and demonstrated the presence of viral particles in the cells with electron microscopy. We finally used RT–qPCR assays to detect genes linked to innate immunity and fibrotic pathways coherently regulated in cells after exposure to the virus. Our findings indicate that cardiac stromal cells are susceptible to SARS-CoV-2 infection and produce variable viral yields depending on the extent of cellular ACE2 receptor expression. Interestingly, these cells also evolved towards hyper-inflammatory/pro-fibrotic phenotypes independently of ACE2 levels. Thus, SARS-CoV-2 infection of myocardial stromal cells could be involved in cardiac injury and explain the high number of complications observed in severe cases of COVID-19.

Background: The left ventricular remodeling (LVR) process has limited the effectiveness of therapies after myocardial infarction. The relationship between autoantibodies activating AT1R‐AAs (angiotensin II receptor type 1‐AAs) and ETAR‐AAs (autoantibodies activating endothelin‐1 receptor type A) with myocardial infarction has been described. Among patients with ST‐segment–elevation myocardial infarction, we investigated the relationship between these autoantibodies with LVR and subsequent major adverse cardiac events. Methods and Results: In this prospective observational study, we included 131 patients with ST‐segment–elevation myocardial infarction (61±11 years of age, 112 men) treated with primary percutaneous coronary intervention. Within 48 hours of admission, 2‐dimensional transthoracic echocardiography was performed, and blood samples were obtained. The seropositive threshold for AT1R‐AAs and ETAR‐AAs was >10 U/mL. Patients were followed up at 6 months, when repeat transthoracic echocardiography was performed. The primary end points were LVR, defined as a 20% increase in left ventricular end‐diastolic volume index, and major adverse cardiac event occurrence at follow‐up, defined as cardiac death, nonfatal re‐myocardial infarction, and hospitalization for heart failure. Forty‐one (31%) patients experienced LVR. The prevalence of AT1R‐AAs and ETAR‐AAs seropositivity was higher in patients with versus without LVR (39% versus 11%, P <0.001 and 37% versus 12%, P =0.001, respectively). In multivariable analysis, AT1R‐AAs seropositivity was significantly associated with LVR (odds ratio [OR], 4.66; P =0.002) and represented a risk factor for subsequent major adverse cardiac events (OR, 19.6; P =0.002). Conclusions: AT1R‐AAs and ETAR‐AAs are associated with LVR in patients with ST‐segment–elevation myocardial infarction. AT1R‐AAs are also significantly associated with recurrent major adverse cardiac events. These initial observations may set the stage for a better pathophysiological understanding of the mechanisms contributing to LVR and ST‐segment–elevation myocardial infarction prognosis.

Cardiovascular diseases remain the leading cause of mortality worldwide, underscoring the urgent need for reliable in vitro models that recapitulate the complexity of the native myocardium. Conventional two-dimensional (2D) cultures lack structural and biochemical complexity, whereas in vivo models are costly, raise ethical concerns, and have poor translational potential. In this study, we developed a novel hydrogel scaffold derived from decellularized porcine ventricular myocardium (dECM). A newly optimized decellularization strategy effectively removed cellular and nuclear components while preserving essential extracellular matrix proteins. The dECM-based hydrogel exhibited reproducible self-crosslinking, gelation kinetics, and stability. Cytocompatibility assays using human bone marrow-derived mesenchymal stem cells demonstrated excellent viability and proliferation upon contact with the biomaterial. Multidimensional hydrogel applications (2.5D and 3D) in vitro revealed higher cell densities than those observed under 2D conditions. Moreover, using human umbilical vein endothelial cells, the dECM-based hydrogel proved to be a valid tool for fabricating cardiovascular in vitro models. As such, this cardiac dECM-based hydrogel is a structurally preserved, biocompatible platform that supports both short- and long-term cell culture. The scaffold has the potential to serve promising applications in cardiac tissue engineering, disease modeling, and cardiotoxicity screening by offering a closer mimicry of the native myocardial environment.

Cardiac fibrosis is characterized by a maladaptive remodeling of the myocardium, which is controlled by various inflammatory pathways and cytokines. This remodeling is accompanied by a significant stiffening of the matrix, which may contribute to further activate collagen synthesis and scar formation. Evidence suggests that TGF-β1 signaling, the main pro-fibrotic pathway in cardiac fibrosis, might cooperates with the Hippo transcriptional pathway by activating YAP/TAZ. To directly test the cooperation of mechanical cues and paracrine signaling in cardiac fibrosis, we developed a 3D model of cardiac extracellular matrix remodeling by generating tissue blocks with Gelatin Methacrylate, a bioink with tunable stiffness, and human cardiosphere-derived stromal cells. Using this strategy, we assessed the cooperation of TGF-β1 and YAP/TAZ transcriptional complex to matrix compaction. Using mechanical compression tests, Masson’s trichrome staining, immunofluorescence, and RT-qPCR, we demonstrate that pharmacological inhibition of YAP/TAZ complex reverts almost completely the pro-compaction phenotype and the matrix-remodeling activity of cells treated with TGF-β1. Our data show a direct connection between the classical pro-fibrotic signaling driven by TGF-β1 and the mechanically activated pathways under the control of YAP/TAZ in cardiac remodeling. Treatment with the elective drug targeting YAP/TAZ is sufficient to override this cooperation with potential benefits for anti-fibrotic therapeutic applications. Funding Information: This work was supported by Institutional grants (Ricerca Corrente, 5 per 1000) issued by the Italian Ministry of Health to M.P. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The use of human cardiac stromal cells was performed after authorization by the local ethical committee at Centro Cardiologico Monzino (approval date: 19 May 2012 and subsequent renewal on 16 May 2016) and have been performed in compliance with the Declaration of Helsinki. Patients signed an informed consent authorizing the donation of small fragments of cardiac tissue for research scopes.