A Study On Muscle Characteristics In Newborn Heterozygous Myostatin-knockout Pigs

Myostatin(MSTN)is a highly conserved negative regulator of skeletal muscle growth development.Naturally-occurring mutations in the MSTN gene have been reported to cause increased muscling in cattle,sheep,dogs.It is found that there was a trend for Follistatin gene expression to be higher when MSTN gene mutation,potentially change fiber-type distribution and the expression level of myosin heavy chain in muscles.At present,there are relatively few studies on heterozygous MSTN-mutant animals.Pig is similar to humans in terms of anatomy and physiology,it able to be animal model for exploring potential human muscle function.In this study,we used the established MSTN+/-pig cell lines to produce MSTN+/-pigs by somatic cell nuclear transplantation and embryo transfer,exploring the impact for muscle weight,fiber cross-sectional area and numbers,fiber-type distribution and expression levels of MHC isoforms of semitendinosus(ST)and semimembranosus(SM)in heterozygous MSTN-knockout pigs.Main contents and results of this study are as follows:1.Generation of heterozygous myostatin-knockout pigs.We used established MSTN+/-pig cell lines that have a 5 bp deletion in one MSTN allele to proceed with somatic cell nuclear transfer.The rates of cleavage,blastocyst formation and the mean numbers of cells were similar in embryos reconstructed with MSTN+/-and WT donor cells.Cloned embryos were transferred to surrogates in estrus,MSTN+/-piglets were obtained.The mRNA levels of MSTN and Follistatin were significantly lower and higher,respectively,in ST and SM from MSTN+/-pigs than WT pigs(P<0.05).2.Detection of histomorphological characteristics of ST and SM of newborn MSTN+/-pigs.The whole cross-sectional area(CSAs),absolute muscle masses and the relative weights of two muscles were larger in MSTN+/-pigs than in WT pigs.We analyzed muscle fibers by HE staining.The muscle fiber CSA was also significantly larger in MSTN+/-pigs than in WT pigs for two muscles(P<0.05)?But the fiber numbers were similar in newborn MSTN+/-and WT pigs for two muscles.Our observations demonstrate that the MSTN+/-mutation primarily leads to fiber hypertrophy in both ST and SM muscles of pigs.3.Observe the muscle fibers distribution.We labeled MHCI protein to distinguish between slow-and fast-type fibers by immunofluorescence staining.The proportion of slow-type fibers was lower in newborn MSTN+/-pigs than in WT pigs for both ST and SM muscles(P<0.05).SDH activity was lower in the ST and SM muscles of MSTN+/-pigs than in those of WT pigs,indicating that more fibers were fast glycolytic.The results show that there was a change in the fiber-type distribution to more fast.glycolytic fibers.4.Compare the level of myosin heavy chain isoforms.We examined the expression level of MHCI,MHCIIA and MHCIIB in MSTN+/-pigs and WT pigs for ST and SM muscles by quantitative RT-PCR.In both ST and SM muscles,the MHCIIB mRNA level was significantly higher in MSTN+/-pigs than in WT pigs(P<0.05),while the MHCIIA mRNA level did not differ.The MHCI mRNA level in the ST muscle was higher in MSTN+/-pigs than in WT pigs.This finding confirmed that fibers tend to be fast glycolytic in MSTN+/-pigs.The above results show that,the competency of reconstructed embryos cultured in vitro were similar in embryos recon-structed with MSTN+/-and WT donor cells.The mRNA levels of MSTN and Follistatin were significantly lower and higher of MSTN+/-pigs than in those of WT pigs.Body and muscle weight were markedly larger in MSTN+/-pigs than in WT pigs,primarily due to fiber hypertrophy in both ST and SM muscles of pigs.In addition,MSTN+/-mutation change the fiber-type distribution in two muscles of newborn pigs,that increase the proportion of fast-type fibers and the expression level of MHCIIB,also decrease the SDH activity.Change the type of muscle fiber into fast glycolytic fibers.