PROJECT SUMMARY Fibrosis is the accumulation of extracellular matrix components that disrupt tissue function and is prevalent across many muscle diseases. Muscle functions compromised in fibrosis include muscles primary function to contract as well as its ability to be compliantly stretched when not active. This results in weak and stiff muscle decreasing mobility and producing joint contractures. Skeletal muscle regenerates following injury from resident muscle stem cells, however those cells are sensitive to the organization and mechanics of fibrotic extracellular matrix. Another muscle resident stem cell, fibro-adipogenic progenitors, support myogenesis following injury, but in the context of fibrosis contribute to the pathologic buildup of extracellular matrix. However, the sensitivity of fibro-adipogenic progenitors to their mechanical environment is unknown. Nor is it known how fibro-adipogenic progenitors production of extracellular matrix signals to muscle stem cells to support or impair myogenesis. In order to target effective anti-fbrotic therapies the mechanisms that of communication between fibro-adipogenic progenitors and the extracellular matrix that defines fibrosis must be revealed. Further, the fibrotic environment can act as a barrier to restorative gene therapies for muscular dystrophy, but how fibrosis may influence the efficacy of promising gene therapies is unknown. Fibrosis is particularly common in Duchenne muscular dystrophy, with associated joint contractures. Yet, even removal of functional dystrophin from more fibrotic mouse strains yields a less severe fibrosis, motivating a conjunction of studies in both mice and humans. Fibro-adipogenic progenitors can be activated into pro- fibrogenic cells to resist apoptotic signals and produce excessive extracellular matrix components. This fibrotic extracellular matrix is mainly made of fibrillar collagen, which is the dominant load-bearing structure within healthy and fibrotic extracellular matrix. However, the organization of collagen fibers in the extracellular matrix can alter both the mechanics and adherent cell phenotypes. Fibro-adipogenic progenitors are similar to mesenchymal stromal cells, yet how extracellular matrix organization and mechanical signals drive conversion to the pro-fibrotic state are not known. Nor is it known how one of the primary functions of fibro-adipogenic progenitors, to secrete extracellular matrix, impacts the muscle stem cells responsible for myogenesis. This the potential to create a positive feedback cycle between fibro-adipogenic protenitors and the extracellular matrix. Promising gene therapy using micro-dystrophin is able to largely restore the integrity of myofibers. However, it isn’t known if once the pro-fibrotic cycle is in place if restoring the myofiber integrity and the initiating signals of fibrosis will be sufficient to reverse prominent fibrosis and the associated decline in function. Thus, our objective is to reveal fibro-adipo...