Project Summary The loss of skeletal muscle mass with age is of clinical importance because it is associated with increased morbidity and mortality, as well as a marked deterioration in the quality of life. There is also a heightened interest in the identification of cellular and molecular mechanisms responsible for the lack of an anabolic response of aged muscle to hypertrophic stimuli. The use of satellite cells to treat loss of skeletal muscle mass is considered a promising therapeutic strategy given their stem cell characteristics and essential role in post- natal muscle growth and regeneration. The results of our studies have prompted us to perform mechanistic analyses of both of the well-known function of satellite cells; fusion to myofibers to provide additional nuclei for hypertrophic growth, to other functional consequences of satellite cell expansion that occurs in response to various exogenous stimuli such as exercise, as the increase in satellite cell abundance in response to mechanical overload far exceeds myonuclear accretion associated with increased myofiber size. We reported satellite cells were necessary for optimal long-term hypertrophic growth of skeletal muscle by regulating the extracellular matrix. Activated satellite cells repressed fibroblast collagen production via extracellular vesicle (EV) delivery of miR-206, revealing a previously unrecognized function of satellite cells, in addition to providing a mechanism through which satellite cells communicate with other cells within muscle. We now have in vivo single-cell (sc)RNA-seq evidence from Pax7-tdT reporter mice of a satellite cell intercellular communication network in which satellite cells communicate with FAPs/fibrogenic cells and endothelial cells during hypertrophic growth. Aim 1 will test the hypothesis that aging negatively impacts this communication network inhibiting proper remodeling of the extracellular matrix thereby inhibiting hypertrophy. We have also developed a novel mouse model that allows us to simultaneously deplete satellite cells and label resident myonuclei, Aim 2 will use this model and single-myonuclear (smn)RNA-seq to characterize age-dependent changes in the resident myonuclear transcriptome and identify mechanisms that enable short term hypertrophy in the absence of satellite cells in adult mice, which is lost in old age. Aim 3 will use an additional newly developed reporter mouse that enables specific and stable labeling of satellite cell nuclei to determine how aging alters the satellite cell-derived myonuclear transcriptome in response to a hypertrophic stimulus. We hypothesize that in aged muscle, satellite cell-derived myonuclei have altered transcriptional output that does not promote a hypertrophic response, and that impaired fusion of satellite cells or defective satellite cells may negatively impact resident myonuclear transcriptional activity contributing to impaired growth. Defining fusion-dependent and -independent roles of satell...