PROJECT SUMMARY Mineralocorticoid receptor (MR) antagonists (MRAs) have long been considered as anti-fibrotic drugs for treatment of heart failure. Although MRAs also have therapeutic benefits on skeletal muscle pathology in mouse models of Duchenne muscular dystrophy (DMD), their effects on skeletal muscle fibroblasts have never been defined. Fibroblasts produce extracellular matrix proteins that constitute fibrosis that replaces muscle tissue that leads to loss of ambulation, respiratory insufficiency and cardiomyopathy in DMD. Both MRAs and genetic deletion of MR in myofibers lead to reduced fibrosis in dystrophic models. In contrast, deletion of MR from myeloid cells increases fibrosis in dystrophic muscles. These data support the overall hypothesis that MR signaling has complex cross-talk between cell types in the injured muscle microenvironment that ultimately regulates fibrosis. The endogenous MR agonist aldosterone increases proliferation and migration of cardiac and renal fibroblasts, which is prevented by MRAs. Recent data support that fibroblasts differ between organs, preventing extrapolating conclusions from one tissue to another. In addition to its role in disease, fibrosis is essential for homeostasis of organ structure and wound healing. MRAs reduce the kinetics of acute injury repair in normal muscle, supporting a normal role for MR signaling in skeletal muscle wound healing. In this application, we will identify fibroblast regulation through non-fibroblast MR signaling and directly through fibroblast MR signaling in muscle wound healing and muscular dystrophy. We will compare molecular signatures, proliferation, migration, and activity between fibroblasts isolated from dystrophic mice treated with MRAs or untreated. We will compare these mechanisms in more fibrotic dystrophic diaphragm with less fibrotic limb muscles. To define mechanisms of MR signaling in essential fibrosis during wound healing versus chronic fibrosis during disease, we will compare fibroblasts isolated from mice with myofiber and myeloid MR knockouts in dystrophic muscles and in acutely injured wild-type muscles. Single-cell RNA-sequencing will be used to identify MRA effects on mononuclear cell populations in acute injury versus dystrophy. We will perform co-culture and conditioned media experiments with MR knockout and control myeloid cells or myotubes with fibroblasts to identify MR-regulated cross talk between these cell types. Direct MR signaling in skeletal muscle fibroblasts, different signaling in resolving fibrosis in wound healing versus chronic fibrosis in dystrophy, between dystrophic muscles with different fibrosis levels, or between young and old muscles have never been studied. Fibroblasts isolated from young and old mdx diaphragms and limb muscles and acutely-injured wild- type muscles will be treated with increasing dosages of aldosterone with or without an MRA and gene expression, proliferation, migration and activity will be compared. A fib...