The Differentiation And Regulation Mechanism Of Skeletal Muscle Satellite Cells At Different Ages

Skeletal muscle is the most important motor organ of organisms as a kind of striated muscle.The skeletal muscle of livestock and poultry can not only be used as an important motor organ,but also be one of the important sources of human intake of high-quality protein.Therefore,the research on skeletal muscle development of livestock and poultry is particularly important.Satellite cells are a kind of undifferentiated monocytes in skeletal muscle which located between the myofibrillary basal membrane and the muscle cell membrane,and closely adhered to the surface of muscle fiber.Satellite cells participate in the development of the muscle fiber,and they are the nuclear source of of the muscle fibers.In generally,satellite cells gradually enter a quiescence state after birth.Satellite cells are activated and asymmetrically divided when damage occure on muscle fiber.Some daughter cells enter the cell cycle,and then undergo proliferation and myogenic differentiation to participate in the regeneration of muscle fibers.Another part of the daughter cells quit cell cycle and enter a quiescence state to renewal themselves and maintain tthe pool of satellite cells.Therefore,satellite cells have the property of stem cells as a kind of myogenic progenitor cells.In this research,C57/BL6 mice were used as models to explore the differentiation and regulation mechanism of satellite cells during skeletal muscle development at both cellular and tissue levels.And the development rule of pig skeletal muscle satellite cells was preliminarily studied.The results were state as follows:(1)The number of PAX7~+cells in skeletal muscle decreased gradually after birth.The gastrocnemius muscle of mice at Day 1,Day 8,Week 2,Week 4,Week 6,Week 8,Week 10,Week 12,Week 24,and Week 52 postnatal was obtained to perform immunofluorescence detection.The immunofluorescence results indicated that PAX7~+cells accounted for 19.7%on Day 1,and this value markedly decreased during development,accounting for less than 0.5%after Week 10 in gastrocnemius muscle tissues.(2)The dynamic changes of the expression of MYF5,MYOD,and myogenin in satellite cell after birth.The immunofluorescence results indicated that the number of MYF5~+cells decreased slightly from Day 1 to Week 8 postnatal which maintained at a high level,but decreased significantly after Week 10.The number of MYOD~+cells remained at a low level at different ages after birth.The myogenin+cells significantly decreased from Day1 to Week 2 after birth,but significantly increased at Week 4 and Week 6,followed by significant downregulation.(3)The differentiation ability of satellite cells decreased with age.Satellite cells were isolated from hind-limb muscle of mice at six different ages(Week 2,Week 4,Week6,Week 8,Week 10,and Week 12).Immunofluorescence and Q-PCR were performed after cells were induced differentiation for 24 h and 48 h.The results showed that the expression of Mck and myosin,marker gene of myogenic,showed a downward trend with age.(4)Identification of differentially expressed genes in satellite cells at different period.Satellite cells were isolated from hind-limb muscle of mice at six different ages(Week 2,Week 4,Week 6,Week 8,Week 10,and Week 12),and the transcriptome profiles were detected using RNA-seq.A total of 2907 DEGs were identified.Furthermore,WGCNA analysis revealed that the 2907 DEGs were enriched in six main expression modules,of which 1739 DEGs were enriched to the red module.And the DEGs in the red module were mainly involved in skeletal muscle development processes(P<0.01).Pathways analysis indicated that the DEGs included in the red module were enriched in 12 signaling pathways(P<0.01),including cell adhesion,hypertrophic cardiomyopathy,Wnt,and MAPK.Combined with Q-PCR validation analysis and co-expression network analysis,we screened Tgf?2,Wnt9a,and Fgfr4 as key genes responsible for the development of satellite cells.(5)TGF?2,TGF?3,and WNT9a could effectively inhibite the differentiation of satellite cells.At the cell level,the expression of Tgf?2,Tgf?3,Wnt9a,Akt2,and Mknk2were significantly up-regulated during the differentiation of satellite cells.The inhibition of the expression level of TGF?2,TGF?3,and WNT9a protein using si-RNA or Pirfenidone could significantly promote the formation of myotubes and the expression of MyHC2d and Mck.At the individual level,the inhibition of the expression of TGF?2protein using Pirfenidone could effectively promote the activation and the differentiation of satellite cells which could accelerate the fusion of satellite cells to myotube and the regeneration of skeletal muscle.(6)FGFR4 could effectively promote the differentiation of satellite cells.The expression of Fgfr4 gene was significantly up-regulated in the differentiation stage compared with the proliferation stage of satellite cells.Furthermore,the down-regulation of the expression level of FGFR4 protein could significantly inhibit the formation of myotubes as well as the expression of MyHC2d and Mck.(7)TGF?2,WNT9a,and FGFR4 could regulate the skeletal muscle development by adjusting the activation status of satellite cells.The RNAi technology was used to inhibit the expression level of TGF?2,WNT9a,and FGFR4 protein in satellite cell;Pirfenidone was used to inhibit the expression level of TGF?2 protein both in satellite cells and tissue.Western blotting results showed that TGF?2,TGF?3,and WNT9a could promote the expression level of PAX7 protein and inhibit the expression level of MYOD protein.FGFR4 could inhibit the expression level of PAX7 protein and promote the expression level of MYOD protein.(8)There were regulation interaction between TGF?2,WNT9a,and FGFR4 protein.Western blotting results showed that TGF?2 could inhibite the expression level of FGFR4,WNT9a,AKT2,and MKNK2 protein;FGFR4 could inhibite the expression level of TGF?2 and WNT9a protein and promote the expression level of AKT2 and MKNK2protein;WNT9a could promote the expression level of TGF?2 and FGFR4 protein,but had no significant effect on AKT2 and MKNK2 protein levels.(9)Preliminary study on pig skeletal muscle satellite cells.The dorsal longest muscle of large white pig at Day 35 embryo,Day 55 embryo,Day 1,Month 1,Month 2,Month 3,Month 6,and Month 12 postnatal was obtained to perform immunofluorescence detection.The results showed that the number of MYF5~+cells gradually decreased with the development of skeletal muscle.The number of MYOD~+cells were highest at Day 55 of the the embryo stage,but was lower at other periods.The number of myogenin~+cells gradually decreased from the embryo stage to the postnatal stage.And then the isolated satellite cells were induced differentiation.The expression of MYOD and myogenin protein was up-regulated at the initiation of cell differentiation,followed by significant down-regulation.