The Role Of ARF6 On Differentiation And Fusion Of Skeletal Myoblasts

Skeletal muscle cells,also known as muscle fibers,are formed by the differentiation and fusion of embryonic mesoderm mononuclear muscle precursor cells.Skeletal muscle repair and regeneration is a process in which satellite cells proliferate and differentiate into myoblasts,which fuse to form new muscle fibers or fuse with damaged muscles fibers.The key step of these processes is the myoblasts fusion.Induction of mammalian myoblasts fusion in vitro and animal muscle injure models are common methods for studying myoblasts fusion.This study explored the mechanism of myoblasts fusion provide more theoretical basis for improving the efficiency of myoblasts fusion.Our previous study found that PLD1 was recruited to the cell membrane and affected the secondary fusion during myoblasts fusion,while its mechanism is still not very clear.Studies have shown that ARF6 is one of the upstream regulatory factors of PLD1,and the expression of ARF6-GTP increased in the early stages of myoblasts fusion,while the mechanism of it is not clear and the role of ARF6 activation PLD1 needs to be explored during myoblasts fusion.Therefore,the role of ARF6 and ARF6 activation PLD1 will be explored to study the mechanism of myoblasts fusion.Objective: 1.The specific role of ARF 6 in the fusion of skeletal myoblasts.2.The role of ARF6 activation PLD1 in the fusion of skeletal myoblasts.Methods: 1.The expression of ARF6 mRNA was detected by RT-PCR(means of half-quantitative PCR and real-time fluorescence quantitative PCR)during the repair of skeletal muscle injury and myoblasts fusion.2.The fusion process and fusion index of ARF6-Q67L-HA(keep activation)/ WT-HA(wild type)overexpressed L6 of and normal L6 was observed separately,and immunofluorescence and Western blot were applied to detected the expression of related functional genes.3.The fusion process and fusion index of ARF6-Q67L-HA(keep activation)/ ARF6-N48I-HA(unable to interact with PLD1)overexpressed C2C12 of and normal C2C12 was observed separately,and immunofluorescence and Western blot were applied to detected the expression of related functional genes.Results: 1.The results of RT-PCR showed that the expression level of ARF6 increased significantly during the repair of skeletal muscle injury,while there was no significant change during myoblasts fusion.2.The fusion index of nascent and mature myotubes in the early stage and mature myotubes in the late stage significantly increased of ARF6-Q67L-HA overexpressed rat myoblasts L6,and there was also a significant increase in the proportion of nuclei distributed within the myotubes.And western blot showed the advanced expression time and increased expression level of myogenin and myosin.However,there was no significant change in the fusion index after overexpression of ARF6-WT-HA in L6,and the expression time of myogenin and myosin was advanced at d1-2.3.There was a significant increase in the fusion index of nascent and mature myotubes in the early stage and mature myotubes in the late stage of ARF6-Q67L-HA overexpressed mouse myoblasts C2C12,and there was also a significant increase in the proportion of nuclei distributed within the myotubes.However,the fusion index of mature myotubes in the early and late stage significantly decreased of ARF6-N48I-HA overexpressed C2C12.Conclusions: 1.ARF6 participates in the repair of skeletal muscle injury.2.The overexpression of ARF6-GTP promotes the formation of nascent and maturemyotubes in the early stage and mature myotubes in the late stage of myoblasts fusion.3.The formation of mature myotubes is inhibited during myoblasts fusion if the PLD1 isn't activated by ARF6.