Molecular Mechanism Of Retinoic Acid And Its Gamma Receptor Regulating Quiescence Maintenance Of Muscle Stem Cells

In animal husbandry,the development and growth of skeletal muscle is a major factor in determining meat production.Skeletal muscle stem cells(Mu SCs)play an important role in the growth of embryonic and postnatal skeletal muscle,and their cellular status is closely related to the rate of nuclei accretion in skeletal muscle fibers.In muscle tissue,quiescent Mu SCs sustainably supply myofibers with nuclei through self-renewal to promote muscle development and rapid growth.The capacity of Mu SCs to maintain quiescent state is important for the embryonic and postnatal myonuclei accretion in muscle fibers,and for promoting the meat production of animals in livestock husbandary.Although many studies have revealed the molecular mechanisms underlying the proliferation and differentiation of Mu SCs,the precise mechanism of how Mu SCs maintain a quiescent state remains unclear.Vitamin A and its metabolites can regulate the quiescence,activation and proliferation of many types of cells,and the addition of vitamin A has a significant effect on the growth traits of skeletal muscle in livestock and poultry,and animals show a decrease in meat production when vitamin A is deficient.Retinoic acid is an oxidative metabolite of vitamin A that binds genomic DNA in conjunction with retinoic acid receptors to regulate the transcription of target genes.However,the mechanisms by which vitamin A and retinoic acid affect muscle cell status and muscle growth and development need to be further investigated.Based on the regulatory effect of vitamin A on skeletal muscle growth in pigs,this study used quiescent and activated Mu SCs transcriptomic data in model animals validated the key role of vitamin A signaling pathways in the maintenance of the quiescent state.In addition,this study investigated the molecular mechanisms by which candidate genes regulate the maintenance of the quiescent state of Mu SCs in pigs and mice through molecular biology and cell biology approaches,with the aim of providing a theoretical basis for genetic breeding and nutritional regulation of skeletal muscle growth traits in pigs.The main results of this study were obtained as follows:(1)Retinoic acid inhibited the proliferation and differentiation of porcine Mu SCs,and suppressed the expression level of Myo D protein,a marker gene related to Mu SC activation and early differentiation,presumably retinoic acid may promote the quiescent state of porcine Mu SCs.(2)A comparative analysis of in vivo transcriptome data from quiescent and different activated Mu SCs revealed that the retinol metabolic pathway was significantly enriched,suggesting that retinoic acid signaling is important for quiescent state maintenance in Mu SCs.(3)The inhibitory effects of overexpressing RARβ as well as RARγ on cell proliferation as well as differentiation were further enhanced than retinoic acid treatment,suggesting that retinoic acid may promote Mu SC quiescence through RARs binding to the genome and regulation of downstream target gene transcription.(4)Treatment of Mu SCs with retinoic acid and its receptor RARγ had no significant effect on Pax7 and Myo D gene transcript levels,while Myo D protein synthesis was significantly inhibited,suggesting that retinoic acid and its receptor RARγ inhibit Myo D protein synthesis through a transcription-independent pathway.(5)Combining RNA-seq and CUT&Tag data of RARγ overexpression in Mu SCs and experimental validation revealed that retinoic acid and its receptor RARγ inhibited e IF4EBP1 phosphorylation by suppressing Akt activity,while the hypophosphorylated e IF4EBP1 prevented Myo D protein synthesis,further leading to a quiescent state with reduced proliferation and differentiation capacity of Mu SCs.(6)Injection of retinoic acid receptor inhibitor BMS493 into young mice induced Mu SCs to exit the quiescent state,while it was almost unaffected in senescent mice.The presence of Ed U-positive nuclei in the center of tibialis anterior muscle fibers on day 14 after BMS493 injection in young mice indicated that the Mu SCs exited the quiescent state and entered a proliferative state and fused with muscle fibers after BMS493 injection..(7)Similar to the mouse model,retinoic acid and its receptor RARγ inhibited Myo D protein levels,Akt activity,e IF4EBP1 phosphorylation,cell proliferation and differentiation in porcine Mu SCs,indicating that retinoic acid and its receptor RARγcould inhibit Myo D expression in porcine Mu SCs through the Akt/e IF4EBP1 signaling cascade and induce them to enter a quiescent state.In summary,this study considered the correlation between vitamin A and muscle growth traits in animal husbandary and hypothesized that vitamin A and its derivative RA have an important role in the quiescent state of Mu SCs.We found that RA and RARγ contribute to the maintenance of the quiescnet state of Mu SCs through the Akt/e IF4EBP1 signaling cascade in porcine and mouse.This study provided new clues to elucidate the molecular regulatory mechanism of quiescence maintenance in Mu SCs,and new targets for genetic breeding to enhance meat production in livestock husbandry.