Profibrotic activation of fibro-adipogenic progenitors impairs myogenesis through extracellular matrix synthesis and soluble factors

Abstract

The primary source of fibrosis in skeletal muscle is fibro-adipogenic progenitors (FAPs) which deposit extensive extracellular matrix (ECM). FAPs are key in supporting regeneration and myogenesis in healthy muscle but become pathogenic in fibrosis. The goal of this study was to determine the effect of fibrotic FAPs on muscle stem cells (MuSC) based myogenesis while distinguishing between signaling through fibrotic FAP ECM deposition or FAP soluble factors. Primary MuSCs and FAPs were isolated from wildtype and D2.mdx mice for in vitro assays. FAPs pro-fibrotic activation was modulated in vitro with transforming growth factor beta (TGF-β) and verteporfin for activation and inhibition of activation respectively. FAP activation state was also modulated by substrate stiffness. Transwell co-culture systems of FAPs and MuSCs were used to interrogate soluble factor signaling. Cell-derived matrices were synthesized by FAPs and used as a substrate to query MuSC myogenesis. Imaging collagen deposition was via second harmonic generation to measure ECM architecture of FAP-derived matrices. Profibrotic activation of FAPs by TGF-β impaired myogenesis in transwell co-cultures. FAPs induced with TGF-β produced ECM with increased collagen deposition, whereas FAPs derived from D2.mdx mice produced ECM with increased collagen fiber diameter. Verteporfin, a suppressor of the YAP-TEAD complex, decreased collagen deposition and impaired collagen fibrillogenesis in FAPs. Across conditions, there was a negative relationship between FAP activation state and myogenic differentiation of MuSCs on the cell-derived matrices (CDMs) synthesized. Further, ECM synthesized by FAPs on underlying substrates matching patho-fibrotic stiffness significantly impaired myogenic differentiation. Overall, profibrotic FAPs produce soluble factors and altered ECM that impairs myogenic differentiation of MuSCs.