Effect of SWELL1 (LRRC8a) gene deletion on gene expression in differentiated myotubes

Maintenance of skeletal muscle is beneficial in obesity and Type 2 diabetes. Mechanical stimulation can regulate skeletal muscle differentiation, growth and metabolism, however the molecular mechanosensor remains unknown. Here, we show that SWELL1 (LRRC8a) functionally encodes a swell-activated anion channel that regulates PI3K-AKT, ERK1/2, mTOR signaling, muscle differentiation, myoblast fusion, cellular oxygen consumption, and glycolysis in skeletal muscle cells. SWELL1 over-expression in SWELL1 KO myotubes boosts PI3K-AKT-mTOR signaling to supra-normal levels and fully rescues myotube formation. Skeletal muscle targeted SWELL1 KO mice have smaller myofibers, generate less force ex vivo, and exhibit reduced exercise endurance, associated with increased adiposity under basal conditions, and glucose intolerance and insulin resistance when raised on a high-fat diet, compared to WT mice. These results reveal that the SWELL1-LRRC8 complex regulates insulin-PI3K-AKT-mTOR signaling in skeletal muscle to influence skeletal muscle differentiation in vitro and skeletal myofiber size, muscle function, adiposity and systemic metabolism in vivo. Overall design: We used RNA-Sequencing to quantify the changes in global mRNA expression in wild-type as compared to SWELL1 (LRRC8a) knock out differentiated myotubes. We generated isogenic wild-type (WT) and SWELL1 (LRRC8a) KO myoblast cell lines from mouse C2C12 myoblast cell. CRISPR/Cas9 was used to knockout the LRRC8a gene in the parental C2C12 cell line. These cell lines were grown in culture and RNA was extracted for sequencing under basal culture conditions. RNA was sequenced on an Illumina Hi-Seq at the University of Iowa Core Facility.