Studies On The Relationship Between Intracellular Calcium Ion Homeostasis And The Myogenic Or Adipogenic Differentiation Of Precursors In Skeletal Muscle

The balance of myo-adipogenic differentiation during skeletal muscle development is of great significance for livestock meat quality.Intracellular Ca2+ concentration is strictly regulated during cell differentiation.Three experiments were conducted in this study to investigate the interrelations between the intracellular Ca2+ homeostasis and cell differentiation.In Exp.1,myogenic and adipogenic cells isolated from longissimus dorsi of neonatal large white pigs were identified for their potency of adipogenic or myogenic differentiation using qRT-PCR method and inducing of differentiation in vitro.proteomics TMT technique was used to detect differentially expressed proteins between myogenic and adipogenic cells.The results showed that myogenic and adipogenic cells have their particular potency of adipogenic or myogenic differentiation,respectively.A total of 433 proteins(339 proteins upregulated and 94 ones downregulated in myogenic cells)among 6199 quantifiable proteins identified by Proteomics analysis were differentially expressed(P<0.05&(FC<0.83 or FC>1.20)).Differentially expressed proteins analysed by bioinformatics showed that myogenic and adipogenic cells have a distinct metabolism profile for three main nutrients.Molecular function analysis of proteins indicated that differentially expressed proteins most enriched in some cellular functions,such as cell development,cell migration,hormone regulation,ion homeostasis,immunity,and gene expression.Moreover,six signaling pathway were significantly enriched by differentially expressed proteins,including PPAR signaling pathway,lysosome,protein digestion and absorption,mineral absorption,phosphatidylinositol signaling system,and pathogenic Escherichia coli infection.In Exp.2,neonatal large white and Laiwu pigs were used to reveal the difference of regulatory mechanism involved in muscle development and intramuscular fat deposition between lean and obese pigs.Myogenic and adipogenic cells were isolated from the two species of pig and identified for their potency of adipogenic or myogenic differentiation.Total protein shamples extracted from the two kind of myogenic cells was used for Proteomics analysis.There are 181 differentially expressed proteins(P<0.05&(FC<0.83 or FC>1.20)),including 47 proteins upregulated and 131 ones downregulated in myogenic cells from Laiwu pigs.GO enrichment analysis of differentially expressed proteins showed that 13 biological processes(BP)were related to sugar metabolism,14 BP related to immune regulation,12 BP related to the regulation of DNA replication and mRNA expression,11 BP related to lipid metabolism,and 10 BP involved in cell apoptosis.The BP which maybe responsible for potency of cell differentiation mainly falled into three categories,namely ion homeostasis,cell proliferation and differentiation,and muscle development.The result of KEGG analysis showed that five signaling pathways were significantly enriched by differentially expressed proteins,including Glycerolipid metabolism,Lysosome,GABAergic synapse,Phosphatidylinositol signaling system,and Valine,leucine and isoleucine degradation.In Exp.3,the interrelations between the intracellular Ca2+ homeostasis regulated by Ryrl and cell myogenic differentiation were studied comprehensively.CRISPR-Cas9 gene-editing approach was conducted to build Ryrl-knockout cell line and its function domain(responsible for Ca2+ transportation)knockout cell line for the revelation of the regulatory effect of intracellular Ca2+ homeostasis regulated by Ryrl during cell myogenic differentiation of C2C12.Endoplasmic reticulum stress models were established by the treatment of Dantrolene(Ryrl inhibitor)and Thapsigargin(SERCA inhibitor)during the period of proliferation or differentiation of C2C12.The mRNA expressions of myogenic transcription factors and channel proteins related to Ca2+ homeostasis in endoplasmic reticulum stress models were detected.The abundance of proteins related to endoplasmic reticulum stress,MAPK signal pathways,andapoptosis was analysed by Western Blot.Results show that Ca2+ signaling pathway mediated by Ryr1 plays an important role in regulating muscle development.The apoptosis triggered by Ryr1-knockout not entirely resulted from endoplasmic reticulum stress caused by the disruption of intracellular Ca2+homeostasis.In conclusion,cell fate commitment and the strength of differentiation potential were regulated by many differentially expressed proteins which mainly involved in gene expression,hormone regulation,ion homeostasis,cell migration,and cell proliferation and differentiation,especially the regulation of intracellular Ca2+ concentration.This study provides extensive research ideas to explore the muscle development regularities and molecular mechanism of intramuscular fat deposition...