SUMMARY The rotator cuff is composed of 4 muscles that stabilize the shoulder and controls upper arm range of motion. Tears in the tendons of these muscles, known as rotator cuff tears (RCTs), are among the most common debilitating shoulder injuries. While RCTs can be surgically repaired, the rate of retear is significant and has been correlated to high levels of rotator cuff muscle fatty infiltration, fibrosis, and muscle atrophy. A heterogeneous population of muscle resident non-myogenic mesenchymal cells, known as fibro-adipogenic progenitors (FAPs), have been implicated in many skeletal muscle pathologies in which intramuscular fat and fibrosis are abundant. We and others have recently demonstrated that PDGFRa-expressing FAPs expand during a 2-week period following massive RCTs in mice and contribute to the accumulation of intramuscular fat and fibrosis that accompanies rotator cuff muscle atrophy by 8-weeks post injury (wpi). While these findings are important, virtually nothing is known regarding the molecular signals associated with the promotion of FAP differentiation into fibrogenic and adipogenic fates, nor the factors that may be secreted by FAPs to drive rotator cuff muscle atrophy following RCT. Utilizing a murine model of massive RCTs, genetic lineage tracing/reporter analysis, as well as sophisticated bulk RNA-sequencing assays from isolated FAPs and other gene expression studies in muscle fibers, we identified several important molecular regulators of adipogenesis, fibrosis, and muscle atrophy that are likely key factors in promoting the RCT pathology. Validation of signaling and functionality of these molecules/pathways were demonstrated in our mouse model of RCTs (in vivo). In this application we will utilize conditional genetic approaches to delete the relevant genes specifically in FAPs and muscle fibers following RCTs, while also performing genetic reporter analysis, histology, IHC/IF, bulk-RNA sequencing, and sophisticated single cell RNA-sequencing assays, to determine whether cell-type specific signaling activation is necessary to promote the RCT pathology following massive RCTs in mice and determine whether the presence or absence of these molecular regulators affects FAP heterogeneity of rotator cuff muscles following RCTs. Further, we will perform proof-of-concept studies demonstrating the utility of prophylactic and therapeutic pharmacologic approaches to the inhibition of this pathway following RCTs. This work will provide vital information to our understanding of the cellular and molecular roles for FAPs in RCT induced fatty infiltration, fibrosis, and the muscle atrophy associated with massive RCTs, while also testing clinically relevant therapies for the treatment of the RCT pathology.