Project Summary/Abstract Fibrosis is a consequence of a myriad of skeletal muscle diseases including Duchenne muscular dystrophy. Fibrosis is the pathological accumulation of extracellular matrix (ECM) and impairs muscle function resulting in a loss of mobility and significant reduction in strength. Muscle resident fibro-adipogenic progenitors (FAPs) are the key source of ECM deposition in skeletal muscle. Pro-regenerative FAPs support regeneration by activating in response to injury, depositing ECM to replace the damaged matrix, and releasing pro-myogenic signals. However, in the context of fibrosis, there is a significant upregulation of a more pro-fibrotic FAP subpopulation. FAPs activate into myofibroblasts and remain at chronically high levels, leading to excess ECM deposition. What drives the development of a fibrotic versus regenerative FAP phenotype is not well understood. The mechanics and architecture of the ECM is altered in fibrotic muscle compared to healthy muscle, providing mechanical cues to surrounding cells. FAPs are known to be sensitive to these changes in mechanics and architecture, however, what drives this signaling pathway is not understood. Yes-associated protein (YAP) is strongly correlated with FAP activation into myofibroblasts on stiff substrates. Blocking YAP activity is a potential method to manipulate FAP-ECM signaling and reduce myofibroblast activation in the context of fibrosis. ECM signaling to FAPs may also influence the heterogeneity of FAPs, with the levels of pro- regenerative and pro-fibrotic FAPs changing during injury and fibrosis. How the subpopulations of FAPs may be contributing to ECM deposition in fibrosis and regeneration is not well understood. Elucidating the differential role of FAP subpopulations provides specific targets for anti-fibrotic and pro-myogenic therapies. Our central hypothesis is that blocking FAPs’ sensitivity to stiff substrates through inhibition of YAP will reduce the number of pro-fibrotic FAPs resulting in ECM deposition that promotes myogenesis. We will test our central hypothesis via two aims. In Aim 1, we will inhibit YAP activity in order to determine its role in FAPs’ sensitivity to engineered matrix substrates mimicking the mechanical and architectural features of healthy and fibrotic ECM. In Aim 2, we will determine the differences in ECM deposition from pro-fibrotic and pro- regenerative FAPs and its role on myogenesis to assess how the heterogeneity with the FAP population affects the development of fibrosis and regeneration. Success in these aims will identify the interactions between FAPs and the ECM in the context of fibrosis, which can be used as targets for anti-fibrotic therapies.