Acute Knockdown of MG29 Alters Skeletal Muscle Cells Differentiation and Leads to Cellular Atrophy

Abstract

Sarcopenia is defined as the progressive loss of skeletal muscle mass and development of force/power that occurs during aging. It is estimated that more than 30% of humans over 60 years of age suffer from sarcopenia and age‐related loss of muscle function that contributes to more than half of all chronic conditions in people over 50 and accounts for 1.5% of total U.S. healthcare expenditures (>$40 billion). Mitsugumin 29 (MG29), is a muscle‐specific protein belonging to the synaptophysin and MAL and related proteins for vesicle trafficking and membrane link (MARVEL) families of proteins which are involved in cholesterol binding and formation of oligomers of these proteins. MG29 is essential for the proper formation of the transverse‐tubule (TT) system, maintenance of lipid content of the sarcolemmal membrane, and efficient signaling between RyR1 and the store‐operated Ca2+ entry (SOCE) machinery. MG29 null (mg29−/−) mouse muscle displays abnormalities in the membrane ultrastructure of the triad junction where the TT are swollen and the sarcoplasmic reticulum (SR) networks show a vacuolated and fragmented structure. Clear phenotypic changes are seen in mg29−/− skeletal muscle at young ages (3–6 months) that resemble those seen in aged wildtype muscle. Our previous work clearly demonstrated that SOCE is compromised in an MG29‐associated manner in aged skeletal muscle and that reduced SOCE associates with a drastic decrease in MG29 content (~50%), altered intracellular Ca2+ homeostasis and decreased specific contractile force. But the function of MG29 on muscle differentiation remains unclear. In this study, we knocked down MG29 expression in mouse primary skeletal muscle cells by using MG29 siRNA. Our data shows that 48h after MG29 siRNA (10nM) transfection, the expression of MG29 decreased by ~ 80%, compared to negative control siRNA. After MG29 siRNA treatment, at day 3 of differentiation, thinner, longer myotubes were observed compared to negative controls (diameter of myotubes: negative control = 24.9 ± 2.08μm; MG29 siRNA = 19.1 ± 0.65μm, p < 0.05; Length of myotubes: negative control = 424.9 ± 50.2μm; MG29 siRNA = 580.4 ± 39.9 μm, p < 0.05). Surprisingly, fusion index (FI) was significantly increased compared to control (FI values: negative control= 53.2% ± 6.8; MG29 siRNA = 70.3% ± 7.9, p < 0.05), in agreement with increased mRNA expression of myogenenic markers MyoG and MyoD detected by real time PCR. These results suggest that the acute knock down of MG29 leads in just three days to complex myogenic changes characterized by increased myogenic differentiation but with abnormally thinner and longer muscle cells. These studies further indicate specific roles of MG29 in maintaining normal muscle mass and function, and its importance for cellular homeostasis during aging.Support or Funding InformationThis work was supported by NIH‐National Institutes of Aging R01AG056504 (NW, MB), and the George W. and Hazel M. Jay and Evanston Research Endowments (MB).