Effects of neutral sphingomyelinase activation associated factor (Nsmaf) gene knock‐down on myogenesis in murine C2C12 myoblast

Neutral sphingomyelinase activation associated factor (Nsmaf) modulate ceramide production by activating neutral sphingomyelinase enzymes. Nsmaf also regulates tumor necrosis factor-α (TNF-α)-induced signaling pathway. Since both ceramide metabolisms and TNF-α signaling are key events during inflammation, Nsmaf might play a nodal role in inflammation-induced events, such as muscle wasting. In the present study, we examined whether Nsmaf gene knock-down effects on myogenesis in murine C2C12 myoblasts. C2C12 myoblasts were used in this study. In the growth phase, cells were maintained in DMEM and 10% FBS (growth medium: GM). For cell differentiation, the culture medium was switched to DMEM containing 1% HS (differentiation medium: DM). Small interfering RNA (siRNA) was used to induce Nsmaf gene Knock-down (NsmafKD, n = 6). siRNA with a scrambled sequence was used as negative controls (Scr, n = 6). Gene knockdown was performed both in myoblasts and myotubes. After two-day siRNA treatment, Western blotting was performed to examine protein expression levels. Laser scanning microscopy was used to investigate cell morphology. Even in the GM condition, differentiation markers, such as myogenin and myosin heavy chain (slow) were upregulated in the NsmafKD cells. In transfected myotubes, myotube diameter of the NsmafKD was about 50% smaller than that of the Scr. Both in myoblasts and myotubes, contents of a cell fusion mediator, myomixer, in the NsmafKD was significantly lower than that in the Scr. These data indicate that Nsmaf is a novel regulator of myogenesis.