A Novel in vitro Model of Muscle Atrophy

Abstract

PURPOSE: Skeletal muscle disuse is associated with decreases in muscle size and function resulting from decreases in protein synthesis and increases in protein degradation. Current in vitro models do not exhibit gene expression consistent with disuse in vivo, possibly because the atrophic phenotype is elicited through potent cytokine or glucocorticoid treatment. A cell culture model using methods more physiologically analogous to disuse, such as the modulation of growth factor availability, may provide a better tool to study disuse atrophy. The purpose of this study was to measure cell size and levels of protein following a novel means to induce atrophy using growth-factor stimulation and subsequent withdrawal in C C myotubes. METHODS: C2 C12 myoblasts were incubated in Dulbeccoâ™s modified Eagleâ™s medium (DMEM) supplemented with 10% Fetal Bovine Serum, a growth factor rich medium (GM). At ~80% confluency, cells were transferred to DMEM supplemented with 2% horse serum, a relatively growth factor deficient differentiation medium (DM). Following 4 days in DM, fused myotubes were re-introduced into GM. After 48 hours, the GM was replaced again with DM. Immunocytochemistry, fluorescence microscopy, and a Metavue software package were used to visualize and quantify myotube area 48 hours post GM treatment and again following 48 hours of DM treatment. At these time points cells were also harvested for total cell lysate. Western Blotting was used to determine protein levels of myosin heavy chain (MHC). RESULTS: Growth factor stimulated C2 C12 myotubes had a mean area of 16,750 μM2, which represented a 34% greater area than control cells (11,025 μM2). Furthermore, subsequent growth factor withdrawal for 48 hours resulted in a decrease in myotube area to a mean area of 8295 μM2, which represented a decrease in area of 50% from growth factor stimulated myotubes. Western blotting results were inconclusive. CONCLUSION: These data indicate that growth factor stimulation and removal produce a hypertrophy and atrophy response respectively in C2 C12 myotubes. Future planned experiments to explore gene expression during this stimulus will help determine the extent to which this model may mimic disuse. Funded by the NASA Space Physiology Research Grant (NASA) from the American College of Sports Medicine Foundation.