Abstract: The study was aimed to provide a theoretical basis for investigating the effect of sphingomyelin on development of skeletal muscle of UCP1 knock-in (UCP1-KI) pigs, through detection the function of sphingomyelin in myogenic differentiation of C2C12 mouse satellite cells. ELISA method was used to measure the content of total sphingomyelin in the back muscle and serum from both wild type and UCP1-KI pigs. CCK8 was used to detect the effects of different concentrations of sphingomyelin (0, 5, 20, 50 and 100 μg/mL) on the proliferation of C2C12 cells. The successful differentiation was established and confirmed by morphological observation, the immunofluorescence of Myogenin and the mRNA levels of the myogenic differentiation markers, including Myf5, MyoD, Myogenin and MRF4. Then, the C2C12 mouse satellite cells were treated with different concentrations of sphingomyelin during myogenic differentiation. Cells were harvested after 6 days of differentiation and the optimal sphingomyelin concentration was determined based on the formation of myotubes, and immunofluorescence staining signal of Myogenin and the upregulation of differentiation markers (Myf5, MyoD, Myogenin and MRF4). Furthermore, the gene expression levels of cyclins (CyclinD1, CyclinE, CDK2 and CDK4) in C2C12 cells at 2, 4 and 6 d of myogenic differentiation were detected by Real-time quantitative PCR, and cell viability was detected by CCK8 at 2 d of myogenic differentiation. The results showed that the content of total sphingomyelin in the back muscle of UCP1-KI pigs was significantly increased (P<0.05) compared with those from wild-type pigs after cold stimulation. No significant difference in serum sphingomyelin content (P>0.05) was observed. Sphingomyelin had no effect on the proliferation of C2C12 cells before myogenic differentiation (P>0.05). After 6 days of myogenic differentiation, myotubes was formed obviously and the mRNA levels of myogenic differentiation markers (Myf5, MyoD, Myogenin and MRF4) were significantly up-regulated (P<0.01). Compared with control group without sphingomyelin, the Myogenin positive cells and myotube fusion index were significantly increased in the 20 μg/mL sphingomyelin group (P<0.05). Furthermore, the expression levels of Myogenin and MRF4 genes were significantly increased (P<0.05). With 20 μg/mL sphingomyelin, the expression levels of CyclinD1 and CDK4 in the treatment group were significantly higher than those from control group at 2 d of differentiation (P<0.05), and the cell viability of the treatment group was significantly higher than that of the control group (P<0.05). The mRNA expression levels of CyclinD1, CyclinE, CDK2 and CDK4 in the treatment group were significantly lower than those of control group at 6 d of differentiation (P<0.05). In this study, the results revealed that the addition of 20 μg/mL sphingomyelin could improve the cell viability at the early stage of myogenic differentiation and promote the myogenic differentiation efficiency of C2C12 cells, providing some references for the effect of sphingomyelin on muscle formation.

Key words: sphingomyelin; C2C12; myogenic differentiation