The Mechanism And Application Of Aryl Hydrocarbon Receptor Translocator(ARNT) To Promote Skeletal Muscle Regeneration

Background and Objective:To maintain muscle volume and strength,which declines with aging,requires muscle regeneration.Hypoxia signaling,required for robust muscle regeneration,is known to similarly decrease with aging.However,the mechanism of reduced muscle regeneration in hypoxic pathway is still not fully understood.Aryl hydrocarbon receptor translocator(ARNT),a key component of the hypoxic pathway,may be the key to regulate skeletal muscle regeneration.The purpose of this study is to provide theories for improving the quality of life in aging population by exploring mechanism of muscle regeneration.Methods:1.Analyze the effect of aging on skeletal muscle regeneration.Muscle regeneration was determined by HE staining between young and old mice using cross-sectional area of regenerating fibers on the 5th and 10th day following cryoinjury.Whole muscle was obtained and analyzed with ELISA,Western,qRT-PCR,PCR array analysis to evaluate the expressions of hypoxia,Notch pathway and skeletal muscle regeneration related protein/genes.Immunofluorescence and Doppler ultrasound were utilized to visualize the distribution of blood vessels and blood flow in the lower extremities of mice.Skeletal muscle precursors(SMPs)from different aged mice were cultured and underwent differentiation test to their differentiation capacity.2.Cells and mice with deletion of ARNT were utilized to explore the role of ARNT in the process of skeletal muscle regenerationSiRNA was utilized to knock down ARNT gene in C2C12 cells to determine the role of ARNT gene in cell differentiation.Transgenic mice,whose ARNT expressions in skeletal muscle were knocked out,were used for animal study.HE,Western,qRT-PCR,Immunofluorescence and Doppler ultrasound were utilized to evaluate muscle regenerative potential and signaling between transgenic and wild-type mice.SMPS were extracted for culture and differentiation in vitro3.Find ways to improve skeletal muscle regenerationHypoxia pathway activator(ML228)was added in the aging mice,young mice,transgenic and wild-type mice to simulate the actions of ARNT.Then the changes of skeletal muscle regeneration ability and related pathways between groups were analyzed.Results:1.The regeneration ability of skeletal muscle decreased in aging mice.The expressions of HIF-1?,intracellular ARNT content,NICD,Myogenin,Myf5,lower extremity blood flow and SMPs quantity all decreased as well.No differences could be found in the vascular density and SMPs differentiation between young and old mice.2.After deletion of ARNT gene,the differentiation ability of C2C12 cells and expressions of related genes decreased.ARNT knockout in young mice hindered skeletal muscle regeneration,as well as the expressions of HIF-1?,intracellular ARNT content,NICD and related genes(MyoD,Myogenin,Myf5)without limiting vasculogenesis or SMPs.3.Old and transgenic mice treated with ML228 exhibited increased muscle regeneration ability,as well as higher levels of nuclear HIF-1? and whole muscle NICD without vasculogenesis changes.No differences in skeletal muscle regeneration were observed following ML228 supplementation in young and wild-type mice.Conclusion:Loss of ARNT and hypoxia signaling in skeletal muscle limits muscle regeneration in aging.The restoration of ARNT may improve myogenesis,possibly through restoring the whole skeletal muscle levels of NICD.Therefore,the pharmacological activator of hypoxia signal to improve the regeneration of skeletal muscle in the elderly is promising.