Myostatin Mutation Regulates Bovine Muscle Satellite Cells Of Myogenic Differentiation Via PIGF1R-NM ⅡA Pathway

High meat yield is the main selection direction of cattle genetic breeding,which depends on the growth and development of skeletal muscle.Bovine muscle satellite cells(BMSCs)affect the development of skeletal muscle through differentiation and fusion of muscle tubes.The growth and development of skeletal muscle is regulated by a complex network of molecular mechanisms,in which Myostatin(MSTN)is a negative regulator of muscle development,and the gene mutation will produce a "double-muscle" phenotype.Our laboratory used CRISPR/Cas9 gene editing technology to breed "double muscle" Luxi yellow cattle with MSTN mutation,greatly increasing meat production.In this study,BMSCs of MSTN edited cattle and control cattle were used as experimental materials to explore the effects of MSTN mutation on differentiation of BMSCs at the cellular and molecular levels.The main results are as follows:1.MSTN gene mutation promotes BMSCs differentiation.BMSCs were differentiated and cultured,and the fusion rate and length of muscle tubes in MSTN gene edited cattle were significantly improved compared with control cattle.The enrichment analysis of MSTN gene edited bovine up-regulated genes in RNA-seq data showed that GO function was enriched to cell differentiation,migration and other biological functions.According to the results of RNA-seq,the migration ability of BMSCs during differentiation was detected.The results showed that the mutation of MSTN promoted the migration ability of BMSCs during differentiation.2.MSTN mutation promotes migration and differentiation of BMSCs through IGF1 R.Firstly,the up-regulated differential genes in RNA-seq were enriched,and IGF1 R was significantly up-regulated.The levels of IGF1 R protein and mRNA during BMSCs differentiation were detected,and MSTN edited cattle were higher than control cattle.Inhibition of IGF1 R attenuated migration and differentiation of MSTN edited bovine BMSCs.To further explore the mechanism of MSTN and IGF1 R.Western blot analysis showed that MSTN mutation reduced the expression of downstream transcription factor Smad2+3 during BMSCs differentiation.ChIP-qPCR confirmed that MSTN mutation down-regulates Smad2+3 enrichment in IGF1 R promoter.Activation of Smad2+3 inhibited the expression of IGF1 R mRNA and protein during MSTN edited bovine BMSCs differentiation,and also reduced the content of pIGF1 R.IGF1R is activated to pIGF1 R by phosphorylation,which further regulates downstream proteins.Western blot analysis showed that the expression level of pIGF1 R was higher than that of control cattle during the differentiation of MSTN edited bovine BMSCs,which was determined by IGF1 R.3.MSTN mutation promotes the migration and differentiation of BMSCs through pIGF1R-MYH9.The downstream protein of pIGF1 R was screened by Co-IP protein profile,and the NM ⅡA heavy chain(MYH9)was significantly up-regulated,and the protein regulated cell migration.Western blot detected the expression level of MYH9 during BMSCs differentiation and found no difference between MSTN edited cattle and control cattle.The Co-IP experiment showed that MSTN mutation promoted pIGF1R-MYH9 binding during differentiation.pIGF1 R regulates cell growth by affecting downstream proteins through phosphorylation.Detection of MYH9 phosphorylation during BMSC differentiation showed that MSTN mutation promoted tyrosine phosphorylation of MYH9 by pIGF1 R during differentiation,but did not affect seronine phosphorylation.4.MSTN mutation promotes the binding of MYH9 to NMRLC during BMSCs differentiation through pIGF1 R.Phosphorylation of MYH9 affects binding to light chains(NMRLC).Co-IP experiment demonstrated that MSTN mutation promoted the binding of MYH9 to NMRLC during the differentiation of BMSCs.At the same time,the binding ability of pIGF1 R with NMRLC was weak.Inhibition of pIGF1 R decreased tyrosine phosphorylation of MYH9 in MSTN edited cattle,and downregulated MYH9 binding to NMRLC,thereby inhibiting the migration and differentiation of BMSCs.5.MSTN mutation promotes the migration and differentiation of BMSCs through pIGF1R-NM ⅡA.MYH9 can be combined with NMRLC to form Non myosin ⅡA(NM ⅡA).The expression level of NM ⅡA during BMSCs differentiation was detected.The results showed that MSTN gene edited cattle were significantly higher than control cattle.Inhibition of NM ⅡA can reduce migration and differentiation of MSTN edited bovine BMSCs.The regulation of NM ⅡA during BMSCs differentiation by MSTN mutation was further investigated.It was found that inhibition of NM ⅡA did not affect the content of pIGF1 R,while inhibition of pIGF1 R down-regulated the expression of NM ⅡA.The results indicated that MSTN mutation promoted the migration and differentiation of BMSCs by affecting the content of NM ⅡA through pIGF1 R.In conclusion,MSTN gene mutation can increase the expression of IGF1 R by down-regulating Smad2+3,while up-regulating the content of pIGF1 R can increase the tyrosine phosphorylation of MYH9,promote the binding of MYH9 to NMRLC,thus increasing the formation of NM ⅡA,and up-regulating the migration and differentiation of BMSCs.It provides a theoretical basis for increasing beef production.