Direct aldosterone stimulation of skeletal muscle fibroblasts changes gene expression and differentially affects fibroblast functions from normal and diseased muscles.

INTRODUCTION

Fibroblasts are critical for stabilizing skeletal muscle and facilitating wound healing after injury but become overactivated and lead to fibrotic replacement of muscle tissue in chronic degenerative diseases such as Duchenne muscular dystrophy (DMD). We have previously shown that the mineralocorticoid receptor (MR) is present in skeletal muscle and MR antagonist drugs reduce fibrosis and chronic inflammation in dystrophic mouse models. Indirect MR signaling from other cell types in the muscle microenvironment affects fibroblast gene expression and function. However, the direct effects of MR activation of skeletal muscle fibroblasts are unknown.

METHODS

To determine whether direct stimulation with the endogenous MR agonist aldosterone changes gene expression in skeletal muscle fibroblasts, we performed RNA sequencing comparing fibroblasts isolated from neonatal wild-type skeletal muscles treated with aldosterone or vehicle. To further investigate the effects of aldosterone treatment of fibroblasts in skeletal muscle health and disease, we then performed in vitro proliferation and migration assays on fibroblasts isolated from neonatal and adult wild-type and dystrophic muscles.

RESULTS

Treatment with aldosterone leads to differential expression of 492 genes in fibroblasts isolated from neonatal wild-type mouse muscles. Protein levels of differentially expressed genes Fkbp5, p57 and c-Fos were also increased by direct aldosterone stimulation of fibroblasts from both wild-type and dystrophic muscles. Surprisingly, cultured fibroblasts from both neonatal wild-type and dystrophic muscles retain a higher proliferation rate compared to adult muscle fibroblasts. Direct aldosterone treatment represses proliferation and slows scratch-wound closure kinetics only in fibroblasts isolated from adult dystrophic skeletal muscle.

DISCUSSION: This study shows that aldosterone treatment of skeletal muscle fibroblasts alters gene expression. However, fibroblasts from adult dystrophic muscle appear most sensitive to gene expression changes after short-term aldosterone treatment. These data suggest that MR signaling in the skeletal muscle microenvironment may differentially affect fibroblasts in wound healing and in chronic fibrotic diseases such as muscular dystrophies.