| Skeletal muscle is an important part of the body,which is the main animal products.Skeletal muscle is the largest metabolic and endocrine organ of the body,which is crucial to the metabolic regulation of the body.Meanwhile,skeletal muscle is composed of different muscle fiber types,it’s composition ratio and distribution determines the meat quality.The ratio is regulated by a complex regulatory molecular network,long non-coding RNA(LncRNA)plays an important role in regulation.H19 is highly expressed in muscle tissue and plays an important role in muscle growth and development.Furthermore,single cell sequencing results showed that the expression of H19 in slow muscle fibers was significantly higher than that in fast muscle fibers.In this study,the regulatory effect of H19 on muscle fiber type composition was explored both in vivo and vitro of mice and pig satellite cells(PSCs).The inhibition and overexpression of H19 in C2C12 cell were firstly investigated to determine the effect of H19 on muscle fiber type composition.Subsequently,an adenoassociated virus vector inhibition with H19 was constructed and injected into the tibialis anterior of 2-month-old mice to detect its effects on body condition,exercise ability,muscle fiber type,mitochondrial number and morphology,metabolism-related enzyme activities,and antioxidant capacity of mice.Finally,the regulatory effect of H19 on pig muscle fiber type composition was explored.The main results are as follows:1.H19 promotes the generation of slow muscle fibers in C2C12 cells,inhibits the generation of fast muscle fibers,and affects the cell metabolism.The expression level of H19 in soleus(slow muscle)was significantly higher than that in extensor digitorum longus(fast muscle)(P<0.05).The expression of H19 was inhibited by si-RNA in C2C12 cell lines,and the protein expression levels of MyHCI and MyHCIIA were significantly decreased compared to the control(P<0.05);The expression levels of COX5 A,COX2,UQCRFS1,FABP3,CD36 were significantly lower than those in the control(P<0.05);In addition,H19 overexpression was constructed using pcDNA3.1 plasmid vector,and H19 was overexpressed in C2C12 cell.Compared with the control,the protein expression level of slow muscle related gene MyHCI was significantly increased(P<0.05),and the protein expression level of fast muscle related gene MyHCIIb was significantly down-regulated(P<0.05);Only CD36 expression level of metabolism-related genes was significantly higher than control(P<0.05).2.Muscular knockdown of H19 did not affect the normal physiological condition of mice,but changed the muscle fiber type of tibial anterior in mice.The muscle-specific adenoassociated virus vector sh H19 was constructed and intramuscular injected into 2-month-old mice.here were no significant differences in body weight and the index of muscle,fat,liver and heart to body weight between the sh H19 treated group and the control(P>0.05);The glucose tolerance test(GTT)showed no significant difference between the sh H19 treated group and the control(P>0.05);There was no significant difference in serum routine indexes among different groups(P>0.05);The endurance exercise duration of mice in the sh H19 treated group was significantly lower than that in the control(P<0.05),but it’s not muscular atrophy;The expression levels of genes and proteins associated with different muscle fiber types in the tibial anterior of mice were detected,and the results showed that the m RNA and protein expression levels of genes associated with slow muscle in the sh H19 treated group were significantly decreased(P<0.05),m RNA and protein expression levels of fast muscle related genes were significantly increased(P<0.05);HE staining showed that the area of single muscle fiber area in the control was significantly smaller than that in the sh H19 treated group(P<0.05).3.H19 knockdown affected the number and morphology of mitochondria in the tibialis anterior.The expression of genes related to mitochondria and oxidative metabolism in the tibial anterior of 3-month-old mice in the sh H19 treated group was significantly lower than that in the control(P<0.05),but it is no difference of 4-month-old mice;Transmission electron microscopy(TEM)showed that the number of mitochondria decreased,mitochondrial vacuoles appeared,and the structure of mitochondrial intima cristae changed in the sh H19 treated group.The results of enzyme activity test showed that there was no significant difference in malate dehydrogenase between the sh H19 treated group and the control(P>0.05),the activities of lactate dehydrogenase,succinate dehydrogenase,superoxide dismutase in the sh H19 treated group were significantly lower than those in the control(P<0.05);The contents of lactic acid,triglyceride and malondialdehyde in the sh H19 treated group were significantly higher than those in the control(P<0.05).4.si-H19 inhibited the generation of slow muscle fibers in PSCs.The results of RT-PCR showed that the expression level of H19 in slow muscle was significantly higher than that in other tissues and organs(P<0.05);In the process of myoblast differentiation of PSCs,the expression of H19 increased gradually and remained at a high level.The results of the nucleus and cytoplasm RNA isolation and FISH showed that H19 was mainly present in the cytoplasm of pig skeletal muscle cells,and only a small amount was present in the nucleus.si-RNA was used to inhibit the expression of pig H19 in PSCs.The results showed that the composition of muscle fiber types in PSCs was significantly changed,and the expression level of slow muscle associated protein was significantly decreased in the si-H19 treated group(P<0.05).In conclusion,H19 can regulate the composition of muscle fiber types in vivo and vitro of mice and affects mitochondrial number and metabolic process.Meanwhile,H19 was highly expressed in slow muscle,mainly in the cytoplasm of pig skeletal muscle cells and involved in the regulation of muscle fiber type composition.These results provide a research basis for the exploration of LncRNA that related to the regulation of muscle fiber type composition,and also give theoretical support for the improvement of pork quality. |
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