Tet2-mediated Calcium Signaling Pathway Regulates Muscle Growth And Development

Meat production is an important trait in livestock production to assess the capacity of livestock.The growth and development of animal muscle plays a crucial role in meat production.Muscle stem cell(Mu SC)plays a crucial role in muscle development and regeneration as myogenic progenitor cells.Skeletal muscle growth and development is a sophisticated process involving endogenous regulatory factors,epigenetic and various signaling pathway factors,of which DNA methylation is the most common and prominent epigenetic regulation.TET2 is a pivotal enzyme in the DNA demethylation process and its main function is to perform the demethylation of methylated DNA molecules.Current research on DNA methylation and muscle development has focused on the association between DNA methylation-related enzymes such as DNMT and muscle development,but has generally overlooked the impact of the DNA demethylation enzyme TET on skeletal muscle growth and development.Although TET2 is essential for myogenesis,the mechanisms of TET2-regulated muscle formation,particularly its effects on muscle stem cells,remain unclear.In our research,we validated the impact of TET2 deficiency on cellular properties in porcine muscle stem cells.We selected the TET2 knockout(TET2-KO)mouse model to investigate the function of TET2 in muscle development and regeneration.We compare body weight,muscle tissue and muscle stem cell traits in TET2-KO and WT mice.We also exhibit the differences between the two types of mice during muscle regeneration.To address these phenotypic differences,we further analysed the transcriptome and methylation data of TET2-KO and WT Mu SCs and identified a number of key regulators.We found that Slc8a3,one of these key regulators,could restore the muscle stem cell and muscle development defects caused by TET2 deficiency.The results are as follows:1.TET2 knockdown inhibits proliferation and differentiation of porcine muscle stem cellsOur analysis of transcriptomic data from proliferating and differentiating muscle stem cells revealed that the TET2 gene is upregulated during myoblast differentiation.According to the changes in the expression of this gene in different states of muscle stem cells,we designed si RNA sequences to interfere with TET2 expression in porcine muscle stem cells.Furthermore,we compared the proliferation and differentiation characteristics of TET2 knockdown porcine muscle stem cells(si TET2)and control cells(Control).The result showed that TET2 knockdown had a remarkable negative effect on the proliferation and differentiation of porcine muscle stem cells.2.Absence of TET2 affects body weight and muscle tissue in miceIn terms of morphological changes,TET2-KO mice were significantly smaller than WT mice.Regarding body weight changes,the body weight of TET2-KO mice at 2 and 8 weeks of age was considerably lower than that of WT mice.In terms of muscle tissue changes,we found that the size of the tibialis anterior(TA)tissue from TET2-KO mice was smaller than that of the WT mice,and that this deficiency was reflected specifically in the cross-sectional area of the muscle tissue as well as in the number of muscle fibers.We further discovered that both neonatal and adult TET2-KO mice had fewer muscle stem cells than WT mice at the same developmental stage,and the neonatal TET2-KO mice displayed diminished muscle stem cell proliferation capacity compared to WT mice.3.TET2 deletion impairs Mu SC propertiesWe isolated fresh muscle stem cells from TET2-KO and WT mice for detection.In terms of proliferation characteristics,the proportion of proliferating cells in TET2-KO Mu SC was markedly decreased compared to WT cells and proliferation-related transcription factors such as MCM2 and CDKN1 B were also affected.In terms of cell differentiation characteristics,TET2-KO Mu SCs were significantly impaired in their ability of differentiating into multinucleated myotubes compared to WT cells,and the expression levels of myogenic differentiation-related regulatory factors(MYOG,MYH1)were decreased in TET2-KO Mu SCs.4.TET2 deficiency affects muscle regeneration in miceRegarding muscle regeneration,TET2-KO mice exhibited deficiencies and delays in muscle regeneration and repair compared to WT mice.In addition,our research compared the activation of muscle stem cells in vivo at different post-injury time points and found that the number of activated muscle stem cells in TET2-KO mice was remarkably less than the corresponding WT mice at both 3 and 20 days after injury.5.Transcriptome analysis for TET2-KO Mu SCWe performed RNA-seq sequencing for TET2-KO and WT Mu SCs.We identified 987 differentially expressed genes(DEGs),including 431 up-regulated DEGs and 556 downregulated DEGs.GO analysis of DEGs revealed that up-regulated DEGs were mainly enriched in biological functions such as negative regulation of tissue morphogenesis and cell proliferation,while down-regulated DEGs were mainly enriched in the regulation of muscle tissue development and calcium transport.Given that calcium signaling pathwayrelated genes displayed distinct repression in TET2-KO Mu SCs,we detected intracellular calcium concentrations in TET2-KO and WT cells and found that the calcium levels were significantly decreased in TET2-KO Mu SCs compared to WT Mu SCs.the expression levels of calcium signaling pathway-related regulatory factors were remarkably reduced in TET2-KO Mu SCs.Similarly,we detected a remarkable decrease in calcium ion concentration after TET2 knockdown and a corresponding downregulation of calcium signaling pathway-related regulatory factors in porcine muscle stem cells and control cells.6.Methylation profiling of TET2-KO Mu SCWe performed WGBS-seq for TET2-KO and WT Mu SCs and identified 11,502 differentially methylated regions(DMRs),64.4% of which were hypermethylated.The promoter region and gene body region of the TET2-KO group possessed stronger DNA methylation levels than the corresponding regions of the WT group.Moreover,most of the hypermethylated DMRs were located in introns,distal intergenic regions and promoter regions.In addition,hypermethylated genes were primarily enriched in biological progress regarding the regulation of ion transport,cell adhesion and skeletal muscle tissue development.We identified 175 candidate genes from overlap analysis of hypermethylated and down-regulated genes,and these candidates were predominantly enriched in the calcium signaling pathway.We further analysed methylation levels in the ±2KB region around the transcription start site(TSS)of calcium pathway genes and found that methylation levels in the promoter and gene body regions were dramatically increased in the TET2-KO group compared to the WT group.Subsequently,correlation analysis of m RNA expression levels and methylation levels of calcium pathway genes revealed that Prkcb,Slc8a3,Avpr1 a and Ednra were more pronounced in the hypermethylated and downregulated genes.7.Exogenous Slc8a3 restores the function of TET2-KO Mu SCsWe first demonstrated that Slc8a3 can affect the proliferation capacity of Mu SCs by maintaining cellular calcium homeostasis through treatment of WT Mu SCs with the Slc8a3 inhibitor KB-R7943.We used a constructed exogenous Slc8a3 expression vector to enable Slc8a3-induced expression in TET2-KO Mu SCs and compared phenotypic differences in TET2-KO Mu SCs and Control Mu SCs and TET2-KO Mu SCs overexpressing Slc8a3.In terms of calcium regulation,we found that intracellular Ca2+ concentration was clearly increased in TET2-KO-Slc8a3 Mu SCs compared to TET2-KO Mu SCs.And the expression of calcium signaling-related genes was increased significantly.In terms of cell proliferation,the proliferation rate of TET2-KO-Slc8a3 group was dramatically increased than that of TET2-KO group.Similarly,the expression levels of proliferative and myogenic-related transcription factors were elevated in TET2-KO-Slc8a3 Mu SC expression.In terms of differentiation capacity,overexpression of Slc8a3 restored the ability of TET2-KO Mu SCs to differentiate into multinucleated myotube.The expression levels of typical myogenic differentiation-related genes were also distinctly increased in TET2-KO Mu SCs overexpressing Slc8a3.In this study,we investigated the mechanism of TET2 gene and muscle stem cell development to reveal the essential regulatory mechanism of TET2 in muscle development and its crucial effects on porcine muscle stem cells,as well as its potential role on meatproducing traits in livestock animals.