Distinct Cellular Communication from Aldosterone-treated Myotubes to Fibroblasts between Wild-type and Dystrophic Skeletal Muscle

Chronic fibrosis is a hallmark of the fatal striated muscle disease, Duchenne muscular dystrophy (DMD), caused by the absence of functional dystrophin. In dystrophic muscles, aldosterone, an endogenous mineralocorticoid that activates mineralocorticoid receptor (MR), is locally produced by infiltrated myeloid inflammatory cells. Aldosterone secretion into the muscle microenvironment leads to nuclear translocation of MR and signaling in surrounding cell types, including expression of profibrotic genes. Although aldosterone is present transiently in normal muscle wound healing, its chronic presence in a dystrophic microenvironment may contribute to chronic fibrosis. MR antagonists (MRA) have a long history of safety and efficacy in treating heart failure. We have repeatedly demonstrated MRA efficacy for both early cardiomyopathy and skeletal muscle pathogenesis in DMD mouse models. However, dystrophic mice with cell-specific MR ablation in myofibers or myeloid cells developed to dissect mechanisms of MR antagonism, demonstrated contrasting fibrotic consequences, further indicating MR-regulated cell communication. We hypothesized that MR-regulated cellular crosstalk from myofibers to fibroblasts contributes to chronic fibrosis in a dystrophic skeletal muscle microenvironment and to wound healing in normal muscles. A conditioned media (CM) experiment using primary myogenic progenitor cells and fibroblasts isolated from neonatal C57 (wild-type) and mdx (DMD model) muscles, followed by RNA sequencing and functional assays, was performed. Differential expression (DE) analyses suggested CM from aldosterone-treated C57 myotubes caused DE (padj<0.1;1.5-fold) in C57 fibroblasts compared to vehicle-treated C57. The low DE in aldosterone-treated mdx compared to vehicle-treated mdx suggests that the presence of local aldosterone signaling in early stages of disease development may prime fibroblasts for activation. We also found an altered transcriptional profile in mdx myotubes and CM-treated fibroblasts compared to C57, intrinsic to dystrophin deficiency. Transcript analyses following Kallisto suggested the dystrophin isoforms Dp427 and Dp71 to be prevalent in fibroblasts. Gene-set enrichment analyses on DE genes from CM-treated C57 fibroblasts suggested processes related to proliferation and migration to be influenced (|NES|>1.5) by aldosterone treatment in myotubes. Increased proliferation was validated in CM-treated C57 fibroblasts by an Edu proliferation assay (p<0.05). In conclusion, the distinct transcriptional profiles observed in myotubes and CM-treated fibroblasts suggest the differential impact of MR signaling in a healthy vs dystrophic microenvironment. These findings support the presence of distinct cellular communication regulating wound healing versus fibrosis in dystrophic muscles. Funded by NIH R01 AR072574, NIH R01 NS124681 and OSU Presidential Postdoctoral Scholars Program This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.