Abstract The rotator cuff muscles allow for movement of the arm and shoulder and are particularly susceptible to tear with age. Age-related muscle degeneration is alarming as an ever-increasing aging US population burdens our health care system. While surgical intervention strategies for rotator cuff tears exist, in many instances the muscle fails to regenerate after surgery, leading to a significant and variable rate of retear for 10-50% of patients, depending on the study. Risk factors for retear include age, gender, and pre-operative tear size; however, the interdependency of each risk factor is unknown. Moreover, at the cellular and molecular level, myofibers, satellite cells, stromal cells, and immune cells dynamically regulate the regeneration processes. While its known cell populations shift occurs with age, gender, and rotator cuff tear, no study has evaluated the dynamics of cell pathology across patient populations. In this proposal, the Hicks and Gupta labs will test two hypothesis that 1) the cellular composition of chronic human RCTs inhibits SC regeneration through inflammatory factors and/or fibrosis, and 2) chronic human muscle injury leads to exhaustion and senescence of the SC pool, thereby leading to accelerated degeneration. In Aim 1, we will use high-dimensional imaging mass cytometry to quantify immune-stromal- muscle heterogeneity as well as phenotypic markers of senescence and activation. Imaging mass cytometry can achieve multiplex staining upwards of 40 antibodies on a single tissue, enabling significant refinement of complex cellular phenotypes and histopathological classification of clinical tissue samples. We expect to find will unique molecular signatures clustered by age, gender, and Goutallier grade, thus providing predictive measures of muscle regeneration following rotator cuff tear. In Aim 2, we will test the functional status of SCs and stromal cells in vitro using three main assays, senescence: via live cell imaging, trans differentiation: via fibrotic and adipogenic stimulating factors, and stromal cell regulation of muscle regeneration: via co- culturing stromal populations with satellite cells. Results from these assays will guide the use of anti-senescent or anti-fibrotic drugs in vitro. This study serves as a basis for understanding the intrinsic and extrinsic defects in skeletal muscle repair and will shed light on both predicting outcomes of rotator cuff tear surgery and identifying new treatments that could improve muscle regeneration for aging and muscle tears.