Per1/2 Plays Important Roles In Mouse Muscle Maintenance And Regeneration

Background:Skeletal muscle is one of the peripheral organs regulated by the circadian clock.Maintaining normal circadian rhythm is essential for the growth,maintenance and repair of skeletal muscle.As a core component of the circadian clock,Per 1/2 is involved in various physiological functions,but its specific roles in skeletal muscle remains largely unknown.Objective:This study aimed to investigate the role of Per 1/2 in muscle maintenance and regeneration.Methods:(1)Using the systemic Perl and Per2 double knock-out mice(Per1-/-Per2-/-)and wild-type(WT)mice(C57BL/6),we assessed the impact of deletion of Perl and Per2 on body and muscle weight in age and sex-matched mice.(2)We performed immunofluorescence staining on frozen sections from gastrocnemius muscles of Per1-/-Per2-/-and WT mice to determine whether loss of Perl/2 could affect on the proportion and cross-sectional area of muscle fibers.(3)Using BaC12 induced muscle injury model,we investigated the effect of Perl/2 deletion on muscle regeneration after injury.(4)Using satellite cells isolated from the tibialis anterior muscle of Per1-/-Per2-/-mice and WT mice,we explored the effect of the deletion Perl/2 gene on the proliferation and differentiation of muscle satellite cells.(5)Based on our RNA-seq and Q-PCR results that the expression of Mybph was significantly reduced in skeletal muscles of Per1-/-Per2-/-compared to WT mice,we generated Mybph overexpression and CRISPR/Cas9 knockout C2C12 cell lines to investigate the effect of Mybph modulation on the proliferation and differentiation of C2C12 cells in order to identify the potential correlation between Per1/2 and Mybph.Results:The body weight and the wet weight of skeletal muscles of Per1-/-Per2-/-mice are consistently lower compared to that of the age and sex-matched wild-type mice,indicating that loss of Per 1/2 led to impairment of the growth of the mice and maintenance of skeletal muscle.Immunofluorescence staining of the gastrocnemius muscle showed that the proportion of type ?(slow-twitch)muscle fiber of Per1-/-Per2-/-mice was higher,and the cross-sectional area of type ? b(fast-twitch)muscle fiber was smaller than that of age and sex-matched wild-type mice.The increase of slow-twitch muscle fiber and the decline of fast-twitch muscle fiber are important characteristics of muscles undergoing development toward aging.The muscle injury model showed that loss of Per 1/2 resulted in impairment of muscle regeneration,accompanied by significantly reduced expression of myogenic markers.Loss of Per1/2 led to the inhibition of proliferation,differentiation and myogenic potential of muscle satellite cells.The RNA-seq results revealed that the expression of Mybph was down-regulated in gastrocnemius muscle of Per1-/-Per2-/-mice compared to that of the WT mice and the data was verified by Q-PCR.The proliferation and differentiation of C2C12 cell were enhanced by Mybph overexpression and were inhibited by Mybph deletion.Conclusions:Per1/2 plays significant roles in muscle maintenance and regeneration.Loss of Per1/2 resulted in inhibition of the myogenic potential of satellite cells,leading to impaired muscle regeneration.The expression of Mybph gene was reduced in gastrocnemius muscles with Per1/2 deletion.Overexpression of Mybph promotes C2C12 cell proliferation and differentiation.This study for the first time identified a potential link between Per1/2 and Mybph in the process of myogenesis.