(A-34) Modulation of matrix mechanosensing potentiates the reversal of cardiac fibroblast activation and alleviates contractile dysfunction in fibrotic hearts

Introduction:: Cardiac fibrosis is largely considered irreversible with limited available therapies, in part because the mechanisms governing the long-term persistence and plasticity of cardiac myofibroblasts (MyoFb) – the key mediators of the fibrotic heart – are incompletely understood. Here, we investigate the hypothesis that extracellular matrix (ECM)-presented biomechanical cues play pivotal roles in directing dynamic MyoFb state transitions in the fibrotic heart, and that modulating these mechanosensing pathways may hold the key to developing next generation antifibrotic therapies.


Materials and Methods:: We combine: i) dynamically softening photo-responsive hydrogels, ii) human iPSC-based 2D and 3D models of cardiac fibrosis, iii) single-cell RNA sequencing (scRNA-seq), and iv) mouse models of heart failure to investigate MyoFb responses to changing ECM mechanics. We leverage fluorescent reporter iPSC-derived cardiac fibroblasts (e.g., iPSC-CFs expressing CFP-SM22α) to enable real-time examination of cellular responses to ECM softening either with or without TGFβ inhibitors, and use scRNA-seq based trajectory inference algorithms to analyze dynamic MyoFb state transitions under well-defined chemo-mechanical perturbations. We validate these findings by a series of in vitro functional, morphological, and metabolic assays. We then perform meta-analyses of public scRNA-seq datasets to identify druggable targets along the cell’s mechanosensory apparatus, and we test the efficacy of candidate drugs in fibrotic engineered heart tissues (EHTs) and in a mouse transverse aortic constriction (TAC) model of heart failure.


Results, Conclusions, and Discussions:: We observe that ECM softening, combined with acute TGFβ inhibition, potentiates a dramatic transcriptomic shift toward quiescence in persistently activated iPSC-MyoFbs. This shift is accompanied by reversal of the functional, morphological, and metabolic phenotypes of MyoFbs. Importantly, these cell state transitions are achieved only in the presence of both mechanical and biochemical cues, suggesting synergy between the two is required for partial de-differentiation. Based on our meta-analysis of 6 public scRNA-seq datasets, we identify SRC as a CF/stroma-enriched mechanosensor that is downstream of matrix stiffness and upstream of multiple pro-fibrotic gene programs. We demonstrate that inhibition of SRC, combined with TGFβ suppression, mimics the effects of matrix softening in vivo and results in acute inhibition of mechanoresponsive pathways (e.g., YAP/TAZ and SRF/MRTF) concomitant with alleviation of contractile dysfunction in fibrotic EHTs and in pressure-overloaded mouse TAC hearts. Our findings thus point to SRC-mediated mechano-signaling as a potential stroma-enriched therapeutic target in cardiovascular fibrosis.


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