Potential Regulatory Mechanisms Underlying Distinct Fate Commitment Of Myogenic And Adipogenic Cells In Skeletal Muscle

Mechanism controlling myo-adipogenic balance in skeletal muscle is of great significance for human skeletal muscle dysfunction and myopathies as well as livestock meat quality.Four experiments were conducted in this study to investigate the molecular mechanisms that underlying distinct fate commitment of myogenic and adipogenic cells in pig skeletal muscle.(1)In Exp.1,two cell subpopulations with particular potency of adipogenic or myogenic differentiation were isolated from neonatal porcine longissimus dorsi using the preplate method and flow cytometry analysis based on cell surface marker profile were conducted to identify their mesenchymal origin.(2)In Exp.2,RNA-seq were conducted to detect the distinct gene expression profile between myogenic and adipogenic cells.A total of 448 differentially expressed genes(DEGs)(FDR<0.05 and |log2 FC|>1)between two distinct cells were identified via RNA-seq,including 358 up-regulated and 90 down-regulated genes in myogenic cells compared with adipogenic cells.The results of functional annotation and enrichment showed that 42 DEGs were implicated in cell differentiation,among them PDGFRa,ITGA3,ITGB6,MLCK and MLC acted as hubs between environment information processing and cellular process,indicating that the interaction of the two categories exerts an important role in distinct fate commitment of myogenic and adipogenic cells.Particularly,we are first to show that up-regulation of intracellular Ca2+-MLCK and Rho-DMPK,and subsequently elevated MLC,may contribute to the distinct commitment of myogenic and adipogenic lineages via mediating cytoskeleton dynamics.(3)In Exp.3,we profiled the porcine skeletal-muscle derived myogenic and adipogenic cells DNA methylome.Comparative methylome analysis identified 11,361 differentially methylated regions(Differentially methylated regions,DMRs)(FDR<0.05,δ>0.03),which were associated with 5,333 genes,among which including lineage specific genes related to myogenic and adipogenic.Beside,153 genes were identified to be differentially methylated and expressed.Enrichment analisis indicated that tyrosine kinase receptor(FGFR2,FGFR4,MET and PDGFRa)and ECM-receptor(ITGA9)as hubs connecting intercellular and intracellular information,which plays important role in the commitment of MSCs into specific lineages.This result indicated that DNA methylation regaluates the expression of tyrosine kinase receptors and ECM-receptor,and thus resulting distinct fate commitment of myogenic and adipogenic cells.(4)In Exp.4,we studied the effects of(Transcription factors,TFs)TFs expression levels and the DNA methylation of their binding sites on distinct fate commitment of myogenic and adipogenic cells in skeletal muscle.Myogenic-specific TFs including MYODI,MYOG,MEF2C,SIX1 and SIX2 were highly expressed in myogenic cells;Adipogenic-specific TFs including C/EBPa,PPARγ,ZNF423 and EBF2 were highly expressed in adipogenic cells.Besides,results of analysis the TFBS of DMRs indicated that the DNA methylation status of(Transcription factors binding sites,TFBSs)were different between myogenic and adipogenic cells.Additionally,The DMR existing in tyrosine kinase receptor(PDGFRa,MET and FGFR2)and cell membrane calcium channel(ATP2B3)includs the binding site of myogenic specific TFs(MYOD1 and MYOG)and adipogenic specific TFs(KLF5 and PPARG),and these TFs might mediate the distinct fate commitment of myogenic and adipogenic cells in skeletal muscle.In conclusion,nyogenic and adipogenic cells were isolated from pig skeletal muscle by preplate method,indicating the distinct gene expression profiling between myogenic and adipogenic cells.Additionally,TFs expression level accompanied the DNA methylation status of their binding sites influences the gene expression of myogenic and adipogenic cells,thus mediate the distinct fate commitment of myogenic and adipogenic cells,and finally influencs the myo-adipogenic balance in the development of pig skeletal muscle.