Bmal1 Plays An Important Role In Mouse Muscle Regeneration

Background:Bmall is an important circadian gene that is highly expressed in both muscle and brain.Studies have found that the circadian clock gene plays a key role in maintaining the maturation of skeletal muscle.The deletion of Bmall gene changes a variety of physiological functions,but its specific role in skeletal muscle remains unclear.Objective:This study aims to investigate the role of Bmall in muscle regeneration.Methods:Mice with systemic knockout of Bmall gene were used to investigate the role of Bmall in muscle maintenance and regeneration.We measured body weight of Bmal1-/-mice and Bmall+/-mice to see if the deletion of Bmall has an effect on the weight of mice.To further investigate whether Bmall has an effect on the myogenic capacity,we used immunofluorescence staining on frozen sections of gastrocnemius muscle from Bmal1-/-mice and Wild type mice to determine whether loss of Bmal1 has an impact on the proportions and CSA of muscle fibers.We isolated satellite cells from the tibialis anterior muscle of Bmal1-/-mice and Bmal1+/-mice,and compared their ability to differentiate into myotubes in medium containing 2%horse serum.In order to identify genes that are regulated by Bmall,we performed microarray analysis using satellite cells from Bmal1-/-mice and Wild Type mice,and differentially expressed genes that are related to myogenesis were further validated by real time PCR.We used a muscle injury model by injecting BaCl2 into the TA muscles of Bmal1-/-and Wild Type mice in order to evaluate the role of Bmall in skeletal muscle regeneration.,Immunofluorescence staining of eMyHC was used to identify newly formed fibers after injury.Results:(1)Both the Bmal1-/-and Bmal1+/-mice continued to grow,but the body weight of the Bmal1-/-mice were consistently lower than that of the Bmal1+/-mice.(2)Immunofluorescence analysis of gastrocnemius muscle frozen sections revealed that the mean cross-sectional area(CSA)of type 2a,2b and 2x myofibers are reduced in Bmal1-/-mice compared to age and sex matched Wild Type mice,but no difference was found in type 1 fiber.In addition,the proportion of type 2a and 2x fibers was increased in Bmal1-/-mice compared to Wild Type mice.(3)The number of myotubes formed by satellite cells from Bmal1-/-and Bmal1+/-mice were similar,but the myotube length and diameter were significantly lower in Bmal1-/-compared to Bmal1+/-mice,suggesting incomplete myogenic differentiation when Bmal1 is totally absent.(4)In order to assess the role of Smal1 in skeletal muscle regeneration,we injected BaCl2 into the tibial anterior(TA)muscle.Five days after injection,newly formed centronucleated fibers(CNF)occupied the regenerating TA muscle of Wild Type mice.By contrast,TA muscle of corresponding Bmal1-/-mice showed poor signs of regeneration,evident by the reduction in myofiber CSA,number of CNF,and number of myofibers containing more than 1 central nuclei per field.Immunohistochemical analysis revealed that the CSA of eMyHC+ fibers in regenerating muscle from Bmal1-/-mice was smaller compared with corresponding Wild Type at day 5 after injury.(5)The microarray data revealed increased expression of myostatin(Mstn)but decreased expression of Smad3 and Pax7 in Bmal1-/-mice.These findings were confirmed by real-time PCR.The increased expression of Mstn in Bmal1-/-satellite cells is consistent with our findings that Bmall deficiency led to impaired myogenesis.Conclusion:Bmal1 plays an important role in muscle maintenance and regeneration.Loss of Bmal1 led to delayed satellite cell differentiation and impaired muscle regeneration.In summary,we for the first time demonstrated a potential link between Bmall and myostatin.Our findings shed new light into the role of TGF-?signaling pathway in myogenesis.