Myostatin promotes proliferation of bovine muscle satellite cells through activating TRPC4/Ca²⁺/calcineurin/NFATc3 pathway

Myostatin (MSTN) is principally expressed in skeletal muscle and negatively regulates muscle growth and development. MSTN mutation can induce muscle overgrowth in cattle by activating cell proliferation, presenting a "double-muscle" phenotype. However, the specific regulatory mechanism is still unclear. Here, we found that Ca²⁺ content in muscle tissue and muscle satellite cells of MSTN mutated (MSTN^–/–) cattle were significantly increased compared to wild-type (WT). Furthermore, transcriptome analysis of muscle satellite cells revealed that TRPC4 was significantly increased in MSTN^–/– cattle. And the expression of TRPC4 in muscle tissue of MSTN^–/– cattle was detected by RT-qPCR and Western blot, which was significantly higher than that of WT. These results suggested that MSTN mutation promoted muscle satellite cells proliferation through activation of TRPC4 channel. To further verify, ML204, a specific inhibitor of TRPC4, was used to treat MSTN^–/– muscle satellite cells. We found that cell proliferation was inhibited, calcineurin expression was downregulated, and the entry of NFATc3 into nuclei was reduced, which was similar to WT group. Thus, MSTN mutation leads to the activation of TRPC4 channel, which increases intracellular Ca²⁺ content, further activates calcineurin/NFATc3 pathway, and ultimately promotes the proliferation of muscle satellite cells.