Molecular Mechanism Of Myotilin,a Differentially Expressed Protein In Chicken Muscle At Different Stages During Embryonic Development,Regulating Skeletal Muscle Growth And Development

With the large-scale development of poultry industry,meat production performance,one of the important indicators of economic value,is closely related to the growth and development of skeletal muscle,which is a complex and regulated process involving the proliferation,migration,adhesion of myoblasts and the fusion of skeletal muscle tubes.Proteomics is the technical application of the identification and quantification of the total protein content of a cell,tissue or organism.It complements omics techniques such as genomics and transcriptome to elucidate the proteins of an organism,as well as to understand the structure and function of specific proteins,and to define the overall protein content of a cell’s localization,interaction,post-translational modification and transformation at a given time.However,many vital life processes are controlled not only by the relative abundance of proteins but,more importantly,by the purposefully spatially distributed and reversible post-translational modifications.Phosphorylation is the most common post-translational modification of proteins.In recent years,with the development of proteomics technology,the analysis of proteomics and phosphorylated proteomics based on mass spectrometry has revealed the biochemical basis of important physiological processes of muscle proteins,and protein phosphorylation has been extensively studied in various muscle samples.In order to explore the molecular mechanism of chicken embryonic leg muscle development from proteome and phosphoproteome and explore the differentially expressed proteins,phosphorylated peptides,proteins and metabolic pathways of differentially expressed proteins at different stages of chicken embryonic leg muscle development,we we selected chicken embryonic leg muscle at E7,E12,E18 and D3 stages,then screened important differentially expressed proteins(DEPs)and phosphorylated peptides at different stages of chicken embryonic leg muscle development by using label-free techniques.Differential phosphorylated peptides and related differential signaling pathways were screened for common proteins by combined analysis and significantly enriched.According to the proteome and phosphoproteome results,we then verified the molecular mechanism of the important differential protein Myotilin regulating the proliferation and differentiation of chicken skeletal muscle myoblasts at the cellular level,and further explored the signaling pathway and potential molecular mechanism of Myotilin in myoblasts by RNA-seq technology.The main conclusions are as follows:(1)Label-free sequencing results showed that a large number of proteins were expressed during embryonic leg muscle development and were involved in the proliferation and differentiation of chicken myoblasts,the fusion of muscle tubes and the formation of muscle fibers.(2)KEGG analysis showed that the abundance of differently-expressed proteins enriched in adhesion patch signaling pathway,glycolysis/gluconeogenesis pathway,arginine and proline metabolic pathway was higher,indicating that these signaling pathways play an important role in the growth and development of skeletal muscle.ACTG1,PKM,ENO3,MYLPF,ACTC1,ACTA1,GOT1 and PGAM1 and so on regulated skeletal muscle growth and development through mediating these differential enrichment signaling pathways.(3)Sequencing results of phosphorylated proteins showed that the expression levels of most of the proteins corresponding to the differential phosphorylated modification sites were mainly in the above regulation during development and were highly phosphorylated during the period of proliferation,differentiation and fusion of chicken myoblasts.(4)Myofibrogenic proteins such as MYLPF,PKM,CDKN1 B,TNNI2 and Myl2 were up-regulated in E12 vs E17,and interacted with myocardial contraction and FAK pathways,suggesting that these myofibrogenic proteins mediate phosphorylation of related signaling pathways to regulate myoblast proliferation,differentiation and myogenesis.(5)The combined analysis of proteomics and phosphorylated proteomics sequencing revealed that FAK pathway is an important signal pathway that regulates leg muscle development.(6)Myot is specifically expressed in chicken muscle tissue and the expression level of Myot is significantly up-regulated during embryonic leg muscle development.Interference and overexpression of Myot in chicken myoblasts significantly affected the proliferation and differentiation of chicken myoblasts.Myot positively regulated the proliferation and differentiation of chicken myoblasts.(7)The deletion of Myot expression results in the down-regulation of m RNA expression levels of MEF2 C,Mb,Tnnt3,CSRP3,Myo G,CAV3,MYL1,MYL2,MYOM2 and other genes.Rna-seq results indicated that DEGs was significantly enriched in myocardial cell contraction pathway,calcium signaling pathway,Apelin signaling pathway and MAPK signaling pathway.The expression of Myot can affect the myogenesis through a variety of molecular pathways.(8)Myot mediates the proliferation and differentiation of chicken myoblasts through the FAK-MAPK signaling pathway,affects the fusion of myotubes and myogenesis.In conclusion,this study explored the differences of protein expression and phosphorylation sites in embryonic chicken leg muscle at different stages of development from the proteome and phosphoproteome perspectives.At the same time,the potential molecular mechanism and function of Myotilin,a key regulatory protein affecting chicken embryonic leg muscle development,as well as the regulated signaling pathway were further discussed,laying a theoretical foundation for a systematic and comprehensive exposition of the molecular mechanism of chicken skeletal muscle development.