Functional Of FHL3 Gene In The Regulation Muscle Growth And Fiber Type Conversion

Four and a half LIM domain protein 3(FHL3),belong to the LIM protein and are mainly expressed in skeletal muscle.FHL3 can regulate myoblast differentiation,cytoskeletal structure,muscle cell formation,and downstream gene expression through the interaction of LIM domains with various proteins such as MyoD,actin,and cell cycle regulators.In this study,the FHL3 gene transgenic mouse model established in the laboratory was used to study the effect of overexpression of FHL3 gene on muscle fiber type conversion and muscle growth.The results were as follows:1.The F1 transgenic mice obtained previously in the laboratory were identified,and the construction of three FHL3-transgenic mice was completed and expanded to the fifth generation.The sex ratio of the offspring of the transgenic mice was balanced,and the positive rate gradually increased with the passage,reaching 90% of the 5th generation,and the foreign genes could be stably inherited.At the same time,the litter size of the transgenic mice was not significantly different from that of the wild type mice.Real-time PCR and Western Blot techniques were used to analyze the expression of FHL3 gene in heart,liver,kidney,muscle and testis of 2-month-old transgenic mice.The results showed that FHL3 gene was specifically and highly expressed in the muscle tissue of transgenic mice.2.The body weight gain of the transgenic mice was examined.The results showed that there was no significant difference between the primary weight of the transgenic mice and the wild type mice,and the growth rate and muscle ratio were significantly higher in the 1-8 weeks after birth than in the wild mice.HE staining and immunofluorescence results showed that the number of muscle fibers in the quadriceps,tibialis anterior,and gastrocnemius muscles increased,and the cross-sectional area increased and the density decreased.The results of ATPase staining and immunohistochemistry showed that type II fast muscle fibers increase in the gastrocnemius muscle and type I slow muscle fibers decrease.Mechanical tests on transgenic mice showed that their muscle tension increased and their fatigue resistance decreased.The heart size and weight were the same as those of wild mice.HE staining showed that the transgenic mice had normal myocardium development and there was no significant difference in the size of the myocardium from wild mice.At the same time,it was found that the mRNA level of FHL3 gene in the hearts of transgenic mice was significantly up-regulated,but there was no significant change in protein levels.3.The transcriptional and protein levels of FHL3 and muscle fiber-related genes in gastrocnemius muscle of transgenic mice were analyzed.The results showed that expression of FHL3 and type II myofiber-related genes MyHC IIa,IIb,and IIx were significantly up-regulated,and the type I muscle fiber-related genes MyHC I/slow was significantly down-regulated in the gastrocnemius muscle at the mRNA level in 1,2 and 3 months old mice.At the protein level,FHL3,MyHC,MyHC IIa and IIb proteins were significantly up-regulated in the gastrocnemius muscle of 2-month-old transgenic mice,and MyHC I/slow and Myoglobin were significantly down-regulated.Mitochondrial function-related genes PGC1-?,COX2,COX4,AMPK,and CS,glycolytic-related gene PDK4,and ubiquitin-ligases Atrogin-1,Murf1,and Musa1 that mediate protein degradation and the expression changes of muscle growth and development related genes Myf5,Myf6,MSTN,and MEF2 c were detected in the gastrocnemius muscle of 2-month-old transgenic mice.The expression of PGC1-?,COX4,PDK4,Myf5,Myf6,MEF2 c was significantly up-regulated,and MSTN was significantly down-regulated.These results demonstrate that transgenic mice have a significant effect on muscle fiber type conversion,mitochondrial function enhancement,and energy conversion and utilization.4.Muscle satellite cells were isolated and identified from FHL3 transgenic mice.The proliferation of muscle satellite cells was analyzed by real-time quantitative PCR,Western Blot,flow cytometry and RTCAxCELLigence system.The results showed that overexpression of FHL3 gene promoted the proliferation of muscle satellite cells and promoted the increase of muscle fiber number and muscle mass in transgenic mice.In summary,this study demonstrated that the FHL3 gene has the function of promoting muscle fiber type conversion and improving muscle growth and weight at the living level,and provides a new idea for further studying the mechanism of FHL3 gene regulation of muscle fiber type transformation.