| Chinese cattle generally have the advantages of strong stress resistance,rough feeding resistance and tender meat quality,but there are defects such as slow growth rate,underdeveloped hindquarters and low meat yield.Myostatin(MSTN)is a negative regulator of muscle growth,and its deletion mutation causes animals to produce a "double muscle" phenotype of increased muscle mass.Lactylation is a newly discovered modification on histone(Histone lysine lactylation,Kla)that promotes gene transcription and affects cellular metabolism.Our research group used CRISPR/Cas9 editing technology to breed "double muscle" Luxi cattle with increased meat yield of MSTN knockout.We previously tested the lactate(LA)content in the muscle tissue of double muscle Luxi cattle,and the results showed that the LA content in muscle increased significantly after MSTN knockout,but the regulatory relationship between increased LA content and improved meat production performance is unknown.Based on the above research background,this study selected MSTN edited(MT)and nonedited(WT)Luxi cattles as research objects to explore the effect and mechanism of MSTN knockout on Kla modification during the differentiation and fusion of muscle satellite cells(MuSCs).The results are as follows:1.MSTN edited promotes the production of LA in glycolytic pathways and improves the differentiation and fusion ability of MuSCs.The induced differentiation of MuSCs in the MT group and the WT group showed that the key time node for the significant difference in the differentiation and fusion of MuSCs in the MT group and the WT group was 72 h after induced differentiation.Muscle histology and molecular testing found that after MSTN edited,glycolyzed muscle fibers increased.The results of glycolytic products and enzyme activities of MT-MuSCs and WT-MuSCs at this node showed that pyruvate dehydrogenase(PK)activity in MT-MuSCs was upregulated,thereby significantly increasing pyruvate(PA)content,and upregulating lactate dehydrogenase(LDH)activity catalyzed the production of a large amount of LA by PA.After inhibiting the glycolytic pathway of MT-MuSCs,the content of PA and LA decreased significantly,and the length and fusion rate of the myotube also decreased.Activating another pentose phosphate pathway for WT-MuSCs to produce PA and LA can increase the content of PA,but does not increase the content of LA and the differentiation and fusion ability of MuSCs.The above results have shown that LA produced by glycolytic pathway is a key product affecting the differentiation and fusion of MuSCs.2.MSTN edited increases the degree of Kla and improves the differentiation and fusion ability of MuSCs.The results of western blot and product content detection showed that Kla had the same trend as LA and LDH during MuSCs differentiation,and the increase of LA content was also accompanied by the increase of Kla degree in skeletal muscle tissues of MT group.Increasing the LA content in MuSCs simultaneously increased the degree of Kla and increased the length and fusion rate of the myotube.Inhibition of LDH production of LA simultaneously reduced the degree of Kla,the length of the myotube and the rate of fusion.Therefore,it was determined that MSTN edited led to an increase in LA content during myogenic differentiation and fusion,thereby increasing the degree of Kla and promoting the differentiation and fusion of MuSCs.3.MSTN edited enhances the degree of Kla in the Mymk promoter region and improves the differentiation and fusion ability of MuSCs.The Kla degree of MuSCs with 72 h differentiation of the MT group and WT component was analyzed using CUT&Tag,and the results showed that the lactylation signals of the two groups were mainly enriched near TSS,and the peak enhancement of the MT group compared to the WT group was mainly in the promoter region of the myoblast fusion gene.RNAseq was performed on MuSCs with 72 h differentiation between MT group and WT group,and the results showed that 1764 genes with differential expression upregulation were screened at this node,which were related to myoblast fusion,cell differentiation and glucose metabolism pathways.The results of Ch IP-q PCR confirmed that the degree of Kla in the promoter region of the MT component myocyte fusion gene was enhanced,and the Kla of the membrane fusion gene Mymk was found to be significantly enhanced.MYMK showed significant differences in the protein expression and m RNA expression at 72 h of MuSCs differentiation between the two groups.Overexpression of Mymk in the WT group MuSCs increases myotube length and fusion rate,while interference with Mymk decreases MT bovine myotube length and fusion rate.In summary,MSTN deletion led to an increase in the degree of glycolysis of MuSCs,an increase in Kla modification on the membrane fusion gene Mymk,enhanced the differentiation and fusion ability of MuSCs,and ultimately promoted bovine muscle growth.This result clarifies the role of Kla modification in the differentiation and fusion of MSTN edited cattle MuSCs,and provides a new idea and direction for improving Chinese cattle through epigenetic modification. |
