Effects Of CSRP3 On Myofiber Type Differentiation Of Mouse And Its Mechanism

Pork is the most in demand commodity on the meat consumer market in China,and it is an indispensable food in the daily lives of residents.With the upgrading and transformation of the consumer market,the goal of animal husbandry practitioners has gradually shifted from meat production to meat quality,and the type of muscle fiber has an important impact on meat tenderness,moisture,fat content and other indicators.The molecular mechanism that regulates the types of muscle fibers is very complex and involves the participation of many signals.A large number of studies have shown that CSRP3 has a positive regulatory effect on muscle differentiation,but it’s role in myofiber type conversion is not clear.In this study,transcriptome sequencing was performed on the soleus and longissimus dorsi muscles of 3 large white pigs.The CSRP3 gene was screened from the differentially expressed genes,and its conservation between pigs and mice was analyzed;The function gain(pc DNA3.1-CSRP3)and slience(Si-CSRP3)tests of CSRP3 were carried out on C2C12 cells.And the effects of CSRP3 on the differentiation of skeletal muscle fiber types and mitochondrial function were explored.The target protein of CSRP3 was verified by immunoprecipitation technology,and the regulatory effect of CSRP3 on the target protein was verified at the same time;An overexpression vector of the target protein was constructed to clarify its effect on mitochondrial function and its interaction with CSRP3.The following main results were obtained:1.CSRP3 was highly expressed in the slow muscle of pig and mouse,and was highly expressed in the late during differentiationTranscriptome sequencing and RT-q PCR verification of longissimus dorsi and soleus muscle of 180-day-old large white pigs showed that the expression of CSRP3 in soleus muscle was significantly higher than that in longissimus dorsi muscle of pigs;By comparing the CDS sequence and amino acid sequence of CSRP3 in pig and mouse,it was found that CSRP3 was conserved in pig and mouse;CSRP3 was highly expressed in the slow muscle of mouse,but hardly expressed in the fast muscle of mice;CSRP3 was highly expressed after differentiation and overlapped with the myotube.2.CSRP3 promotes the differentiation of oxidative muscle fibers of C2C12 myoblastOverexpression of CSRP3 can markedly promote the expression of oxidative muscle fibers(p < 0.05),and inhibit the expression of glycolytic fibers(p < 0.05).The opposite result was obtained after interference with CSRP3.3.CSRP3 promotes mitochondrial function of C2C12 myoblastOverexpression of CSRP3 can significantly increase the mitochondrial copy number(p< 0.05),and up-regulate the expression of PGC1a(p < 0.05);Overexpression CSRP3 promotes mitochondrial biogenesis and membrane potential(p < 0.05);Overexpression of CSRP3 can significantly increase mitochondrial complex-related genes ATPase6(p < 0.01),Mt-nd1(p < 0.05),Ndufa1(p < 0.01)and Uqcrfs1(p < 0.01)m RNA levels;Overexpression of CSRP3 can significantly increase the production of reactive oxygen species(p < 0.05);After overexpress CSRP3,the expression of CV-ATP5A(p < 0.05)and CI-NDUFB8(p <0.05)were significantly upregulated.Conversely,interference with CSRP3 resulted in the opposite of overexpression.4.CSRP3 regulates mitochondrial function by interacting with D lactate dehydrogenaseThe immunoprecipitation test found that CSRP3 can target D lactate dehydrogenase.Overexpression of CSRP3 can enhance the activity of D lactate dehydrogenase and reduce the concentration of D lactate in the cell,but the opposite result was obtained after interference.Overexpression of D lactate dehydrogenase can significantly increase the expression of PGC1a(p < 0.05);Overexpression of DLDH significantly up-regulates the mitochondrial OXPHOS-related gene ATPase6(p < 0.01),Mt-nd1(p < 0.01),Ndufa1(p <0.05)and Uqcrfs1(p < 0.05).The protein results showed that overexpression of DLDH significantly up-regulated CV-ATP5A(p < 0.01)and CI-NDUFB8(p < 0.01).The above results indicate that CSRP3 promotes mitochondrial function by promoting the oxidation of D lactic acid.In conclusion,this study found that,at the cellular level,CSRP3 can promote mitochondrial function by targeting D lactate dehydrogenase,and further promote the differentiation of slow muscle fibers through the enhancement of oxidative metabolism.This study not only clarified the effect of CSRP3 on muscle fiber type conversion and mitochondrial function,but also provided a theoretical basis for improving pork quality and treating mitochondrial dysfunction diseases.