Effect Of GPX8 On Myogenic Differentiation Of Porcine Skeletal Muscle Satellite Cells

Skeletal muscle is the largest tissue and organ in the body,accounting for 40-60% of the body composition,and is the main organ of energy metabolism in the body.Pig is one of the most important livestock.Pork is the main source of protein in human diet.The growth and development of skeletal muscle is closely related to meat quality and meat yield,and the growth and development process of skeletal muscle is also affected by many factors.The development process of skeletal muscle mainly includes the proliferation,migration,differentiation and fusion of muscle cells to form muscle tubes,and then mature to form muscle fibers.Different muscle fiber types have different mitochondrial quantity and metabolic type,which can be transformed into each other under different nutritional and environmental conditions,resulting in changes in meat color,p H value,tenderness and nutritional value.Therefore,muscle fiber type is the key factor affecting meat quality.Glutathione peroxidase 8(GPX8)is a member of the family of Glutathione peroxidase.The existing researches mainly focus on cancer.The study of pig GWAS found that the variation of GPX8 gene was significantly correlated with economic traits such as meat yield,so this study speculated that it might be a regulatory factor related to muscle development.In this study,the role of GPX8 in myoblastic differentiation of porcine skeletal muscle satellite cells was explored through interference and overexpression.The binding proteins were screened by mass spectrometry to explore the downstream mechanism.The main results are as follows:1.GPX8 is highly expressed in porcine skeletal muscle,and the expression of GPX8 is decreased in both proliferative and differentiated skeletal muscle satellite cellsAnalysis of RNA-seq data published online showed that GPX8 expression was related to growth rate,myogenic differentiation and muscle fiber type of pigs.RT-q PCR analysis showed that GPX8 was highly expressed in porcine skeletal muscle,and gradually decreased in porcine skeletal muscle satellite cell proliferation stage,and gradually decreased with myoblast differentiation.2.GPX8 promotes the proliferation of pig skeletal muscle satellite cellsCompared with the control group,transfection of GPX8 si-RNA significantly inhibited the number of S-phase cells of porcine skeletal muscle satellite cells,decreased the proportion of Ed U positive cells,inhibited the m RNA and protein expression levels of Cyclin E and Cyclin D genes related to positive regulation of proliferation,and significantly increased the expression level of p21 gene related to negative regulation of proliferation.Overexpression of GPX8 was reversed.3.GPX8 inhibits the differentiation of pig skeletal muscle satellite cellsInterference with GPX8 significantly increased the number of My HC positive cells,increased the differentiation index and m RNA and protein expression levels of differentiation-related marker genes My HC,My OD and My OG.The overexpression results were reversed verified.4.GPX8 promotes the formation of muscle tubes in slow muscles and inhibits the formation of muscle tubes in fast musclesThe expression of GPX8 in slow muscle SOL was higher than that in fast muscle LD,and the formation of Fast-My HC muscle tubes increased and Slow-My HC muscle tubes decreased after interference with GPX8.In addition,after the interference of GPX8,the number of mitochondria decreased,morphosis occurred,ATP level decreased,mitochondrial copy number decreased,and mitochondrial related proteins ATP5 A,MTCO1,UQCRC2,SDHB,NDUFB8 decreased.Overexpression of GPX8 gives the opposite result.5.Inhibition of GPX8 on myoblastic differentiation of porcine skeletal muscle satellite cells depends on TMD domain activityBy constructing GPX8 TMD domain mutant vector,the conserved amino acid Cys28 in TMD domain was mutated to Ala and Cys79 to Ser.It was found that My HC positive cells increased and differentiation index increased after mutation.m RNA and protein expressions of differentiation-related marker genes My HC,My OD and My OG were increased,indicating that the inhibition effect of GPX8 on myoblastic differentiation of porcine skeletal muscle satellite cells was restored after TMD domain mutation.Mass spectrometry preliminarily identified GPX8 interacting proteins mainly enriched in metabolism,motor neuron and other muscular-related signaling pathways,and preliminarily screened the possible interacting proteins such as LRPPRC,NAA15 and EIF3 M.In conclusion,this study revealed the regulatory role of GPX8 in myoblast differentiation of pig skeletal muscle satellite cells at the cellular level,preliminarily identified the regulatory function of GPX8 in myoblast differentiation of pig skeletal muscle satellite cells,and provided a new target for genetic improvement of meat quality.