PROJECT SUMMARY Understanding the molecular wiring that controls the myogenic process in adult muscle repair and regeneration will have significant impact on human health issues ranging from muscular dystrophies to disease- and aging-related loss of regenerative capacity. Work from our laboratory in the last grant cycle has uncovered novel signaling mechanisms in skeletal myogenesis. Of direct relevance to the current proposal is our most recent discovery that knockout of ARHGEF3 (a RhoGEF) enhances muscle regeneration in young adult and aged mice, while also protecting muscle quality during aging. Our findings reveal autophagy as the link between ARHGEF3 activation of RhoA/ROCK, and muscle regeneration and maintenance. This is the first demonstration of a role of an endogenous RhoGEF in muscle regeneration/maintenance. We propose studies in the next grant cycle to investigate the mechanisms of ARHGEF3 regulation in its newly discovered function, which will be important for the ultimate exploration of therapeutic potential of this new class of regulators. With a combination of innovative approaches in biochemistry, cell biology, mouse genetics and muscle biology, and utilizing both in vitro and in vivo experimental systems, we aim to investigate regulation of ARHGEF3 by PI(4,5)P2 and phosphorylation, as well as crosstalk of those regulatory pathways, in muscle regeneration, autophagy, and age-dependent loss of regenerative capacity and muscle quality. We will also establish a muscle- specific CRISPR/Cas9-dependent gene editing mouse model and use it to definitively probe the muscle- autonomous nature of ARHGEF3 function. Our expertise in biochemical characterization of signal transduction mechanisms as well as in muscle biology, our strong preliminary data, and unique animal models put us in an ideal position to pursue the proposed studies. Information gained will likely lead to novel mechanistic insights into the regulation of autophagy and muscle regeneration, and shed light on new therapeutic targets for muscle repair and maintenance of muscle strength in healthy aging.