The Molecular Mechanism Of Filamin C Regulation Of Skeletal Muscle Development Through Wnt/β-catenin Signaling Pathway And Balance Autophagy In Chicken

Chicken is an important source of protein in human diet.How to further improve meat production performance has always been a hot spot in poultry genetics and breeding research.The largest edible part of meat animals is muscle,which can be divided into skeletal muscle,smooth muscle and cardiac muscle according to the morphology and structure of muscle cells.Skeletal muscle accounts for about 50% of the carcass weight of animals,and the quantity and quality of skeletal muscle directly determine the economic value of meat animals.The basic constituent unit of muscle is muscle fiber,whose number is basically fixed in embryo stage,and muscle growth after birth mainly comes from muscle fiber hyperplasia and hypertrophy.Embryogenesis is a complex biological process,which is regulated by muscle specific genes,transcription factors and signaling pathways.Therefore,the discovery of important candidate genes for the regulation of skeletal muscle growth and development is the key to the rapid improvement of animal meat production performance by molecular means.Therefore,the discovery of important candidate genes for the regulation of skeletal muscle growth and development is the key to the rapid improvement of animal meat production performance by molecular methods.Filament C(Flnc),a member of the actin crosslinking protein family,which is specifically expressed in skeletal muscle and myocardium,mainly located near the z-line of sarcomeres,and plays an important role in the process of myofiber and repair after injury.Previous studies have shown that deletion or mutation of Flnc gene in mice leads to different forms of myogenic diseases such as delayed myasthenia,muscular atrophy and muscular dysplasia,proving that Flnc is an important candidate gene for regulating skeletal muscle growth and development.To explore whether Flnc can regulate the growth and development of chicken skeletal muscle and its molecular regulation mechanism,this study detected the effects of Flnc on the development of chicken breast muscle and myoblast and its molecular signal regulation pathway by RNA-seq,q RT-PCR,western-blot,flow cytometry,immunofluorescence,immunoprecipitation and other molecular biological methods.The main results are as follows:1.RNA-seq and q RT-PCR showed that Flnc was significantly higher in breast muscle of fast-growing chicken(Ros-308 broiler)than that of slowly-growing chicken(White Leghorn layer),and specifically expressed in skeletal muscle and myocardium of chicken.The expression level of Flnc in myotubes was significantly higher than that in myoblasts.In addition,We use myoblasts to silenced and overexpression Flnc found that Flnc could promote myoblast differentiation into myotubes.2.Flnc could inhibited the expression of atrophy related genes atrogin-1 and Mu RF1 by culturing myoblasts in vitro.Flnc can alleviate myotubes atrophy by treating with dexamethasone to structure atrophy model.By injecting lentivirus-packed Flnc silenced and overexpression vector into chicken breast muscle in vitro,it was found that the absence of Flnc resulted in the decrease of skeletal muscle mass and muscle fiber cross-sectional area,whereas the overexpression of Flnc promoted the development of skeletal muscle and the increase of muscle fiber cross-sectional area.3.RNA-seq and cell assay showed that interference with Flnc expression in chicken primary cells could inhibit wnt/β-catenin signaling pathway.Azakenpaullone(1-AKP),a activator of wnt signaling pathway,we found Flnc requires the mediating of Dishevelled-2(Dvl2)to regulate wnt/β-catenin signaling pathway after treating with 1-AKP,thus affecting the control of myogenesis.4.The number of autophagosomes and mitochondria increased significantly in Flnc-silenced cells,which activated autophagy.Our further study found that Flnc could directly bind Dvl2 protein and protect Dvl2 protein from being ubiquitinated,thus reducing the combination of ubiquitinated Dvl2 with LC3 to form autophagosomes,and ultimately inhibiting the autophagy-lysosome system to reduce skeletal muscle loss.In addition,the production of reactive oxygen species and ATP in Flnc-silenced cells increased significantly,while dexamethasone induced myotube atrophy increased mitochondrial autophagy activity,suggesting that muscle atrophy induced by Flnc silenced may be mediated by mitochondrial autophagy.In conclusion,this study found that Flnc promotes the growth and development of chicken skeletal muscle,and its molecular regulation pathway is mediated by Dvl2 antagonistic autophagy and mitochondrial autophagy system,thus activating wnt/β-catenin signaling pathway to affect the growth and development of chicken myoblasts.This study provided an important theoretical basis for revealing the molecular mechanism of Flnc regulating the growth and development of chicken skeletal muscle,providing support for molecular assisted breeding to improve chicken yield,and possibly providing a reference for the prevention and treatment of human metabolic and neuromuscular diseases.