Regulatory Effects And Influence On Differentiation Of CaMKⅡ On MEF2C During C2C12 Cells Myogenic

The essence of muscle cell differentiation is skeletal muscle satellite cells (cells, SCS) exiting the cell cycle and fusing to form multinucleated myotubes. Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a key role in myoblast differentiation, and the activity and transcriptional activity of its downstream target proteins can be regulated through the changes of its phosphate level. Myocyte enhancer factor 2C (MEF2C), found in skeletal muscle, with muscle specific binding activity of DNA, cells is a transcriptional regulator that combines with promoter regions and modulates the expression of muscle cell differentiation-related genes. In a previous study, we found that CaMKII exerts a regulatory effect on MEF2C during myogenic differentiation, but the underlying mechanism remains unclear. Here, the effects of CaMKII on MEF2C and myogenic differentiation of C2C12 cells into myotubes were explored. At the same time, the expression of MEF2C muscle related downstream genes of myogenin (myogenin, MyoG) and muscle creatine kinase (muscle creatine kinase, MCK) were detected, so that to provide experimental basis for the understanding of the mechanism of skeletal muscle development.In this study, Ionophore A23187 as the active agent of CaMKⅡ and Kn62 as specific inhibitor of CaMKII were used for C2C12 cells treatment. During the induced differentiation of C2C12 cells into myoblasts,0.4μM A23187 and 5μM Kn62 were respectively used to treat the cells. At Day 7 of induced differentiation, the myoblasts formed amounts of myotubes, and muscle myosin (Fast muscle myosin, FMM) assessed by the immunostaining was expressed. Compared with the control group, the amounts of myotubes generated by induced differentiation was increased after C2C12 cells were treated with Ionophore A23187. Besides, after Kn62 treatment, myotube formation was inhibited and their number decreased. C2C12 cells were successfully induced the formation of myotubes, indicating that the induced differentiation of myotubes was not affected.Next we explore the role of CaMKII on regulating MEF2C. C2C12 cells were treated as described above, and the total protein content of CaMKII and MEF2C were unchanged after A23187 treatment (P> 0.05). However, CaMKII and MEF2C phosphorylation levels were significantly increased compared with the control group. The phosphorylation level of CaMKII and MEF2C significantly increased 1.43 times (1.4312±0.094, P<0.01; Control:1.0335±0.022, P>0.05) and 1.27 times (1.2671±0.066, P<0.05; Control:1.004±0.077, P>0.05) respectively. MEF2C initiated the transcription of downstream muscle specific genes, including MyoG and MCK, which were also detected. The transcription levels of MyoG was significantly enhanced 2.64 times (2.6397±0.118, P<0.05; Control:1.0126+0.070) and MCK enhanced 1.43 times (1.4307±0.043, P<0.05; Control:1.010±0.063), which compared with the control group.In presence of the CaMKII specific inhibitor Kn62, phosphorylation levels of CaMKⅡ and MEF2C were significantly decreased 0.55times (0.5500±0.067, P<0.01; Control:1.0093±0.055, P>0.05) and 0.58times (0.5809 ±0.087, P<0.01; Control: 1.0011±0.040, P>0.05) compared with the control group. However CaMKⅡ and MEF2C protein contents in cells were also unchanged. CaMKII affected the expression level of MyoG and MCK by regulating the transcriptional activity of MEF2C, and the results were consistent with the Western blot analysis. The transcription levels of MyoG and MCK were respectively significantly reduced 0.47 times (0.4661±0.068, P<0.01; Control:1.0126±0.070, P>0.05) and 0.53times (0.5337±0.054, P<0.01; Control:1.0100±0.064, P>0.05). By co-immunoprecipitation, kinase assays and Western blot, ATP was found to activate the kinase activity of CaMKII in vitro, further phosphorylating MEF2C.In conclusion, during the myogenic differentiation of C2C12 cells, CaMKII directly positively regulates the activity and transcriptional activity of MEF2C, further regulats the expression of C2C12 cell myogenic differentiation-related genes, and promots myoblast differentiation. Those results will be benefit to understand the activation mechanism and physiological function of CaMKII on MEF2C, and has important significance for treatment of muscle hypertrophy related diseases.