| Skeletal muscle is one of the most important tissues of human body,which attaches to bone by tendon.Skeletal muscle accounts for about 40%weight of adult weight,and produces contraction force for human body,as well as being the tissue with abundantly metabolic activity.Thus,how to maintain and improve the function of skeletal muscle is crucial for human health.Muscle satellite cells(SCs)are a population of muscle stem cells originating from embryonic mesoderm.The maintenance of SCs is the basis of muscle growth and regeneration.Notably,the exhaustion of SCs is correlated with several muscle diseases,including sarcopenia and duchenne muscular dystrophy(DMD).However,maintaining the homeostasis and function of SCs still lacks effective interventions.Exercise(resistance training and cardio)triggers a remodeling program in skeletal muscles that progressively improves muscle mass and strength,and enhances performance in both,young and elderly individuals.Physical exercise is the most effective intervention for sarcopenia in elderly individuals.Voluntary exercise has also been shown to improve muscle function in DMD mice.Due to the rare number of SCs,most researches focus on muscle cells,rather than SCs.Thus,exploring how exercise regulate SCs and the underlying mechanisms will benefit the understanding of homeostasis of SCs,and do help to screen exercise analogues to improve muscle function.In this study,we investigated the effect of 4-week treadmill training on skeletal muscle and SCs in C57BL/6 mice,and we found that exercise negatively regulated Akt-mTOR signaling to preserve SCs homeostasis.Moreover,we demonstrated that inhibition of Akt-mTOR activity(utilizing NVP-BEZ235 and MK-2206)showed protective effect on the SC pool and regenerative potential of SCs.The detailed results are listed as follows:(1)Exercise promoted muscle hypertrophy and regenerationTo explore the precise effect of exercise on skeletal muscle,C57BL/6 mice underwent 4 weeks of training on a treadmill.The tibialis anterior(TA)was observed to be substantially larger in exercise than control mice.The average myofiber cross-sectional area(CSA)of the TA was significantly larger in exercise than control mice,and histological analysis revealed larger regenerated myofibers in exercise than control mice at 14 and 30 days post-injury(DPI).Together,these data suggested that exercise induced muscle hypertrophy in mice and promoted muscle regeneration.(2)Exercise promoted SCs to enter into cell-cycle and maintains number of SCsThe Pax7 staining and flow cytometry results indicated that exercise promoted SCs expansion,and the Ki67+DAPI staining revealed that exercise induced quiescent SCs enter into cell-cycle.Mice that were subjected to long-term training or muscle injuries still have more SCs than control mice,suggesting that exercise promoted SC activation and preserved the expanded SC pool without exhaustion.(3)Exercise inhibited Akt-mTOR signaling to protect proliferative SCs against exhaustionThrough RNA-Sequence,we found that the gene sets encoding products correlated with Akt-mTOR signaling were enriched.Using q PCR and flow cytometry,we found that exercise inhibited transcription of Akt and decreased phosphorylation of Akt.Mice being trained were subjected to synchronized injection of a mTOR activator,MHY1485,and showed increased numbers of SCs at 1 week but strikingly decreased numbers of SCs at 4 weeks,indicating that exercise inhibited Akt-mTOR signaling to protect proliferative SCs against exhaustion.(4)Exercise upregulted expression of Igfbp7 to block phosphorylation of AktUsing RNA sequencing and flow cytometry,we found that the expression of Igfbp7 was considerably elevated in SCs induced by exercise.The western blot results showed that Igfbp7 protein evidently decreased the phosphorylation of Akt.Knockdown of Igfbp7 in SCs partially increased mTOR activity but negatively affected SC maintenance in exercise mice,indicating that exercise inhibited Akt-mTOR signaling partially by upregulating Igfbp7,which blocked the phosphorylation of Akt.(5)Exercise inhibited mitochondrial metabolism to repress acetylation of H3K27,resulting in reduced transcription of AktThe intensity of H3K27me3(triple methylated H3K27),H3K27ac(acetylated H3K27)and H3K56ac(acetylated H3K56)in SCs isolated from exercise mice showed marked differences compared with control and injured mice.Through MNase analysis,we found that chromosomes in SCs from exercise mice had lower accessibility than those from control mice.Next,we conducted a ChIP-PCR assay and found that SCs from exercise mice showed significantly decreased occupancy of H3K27ac at both promoter regions of Akt1 and Akt2 compared with their counterparts.Histone acetylation relies on the substrate,nuclear acetyl-CoA,which is converted by ATP citrate lyase(Acly)utilizing mitochondrial citrate.SCs from exercise mice had the lower content of acetyl-CoA compared with control mice.We supplied acetyl-CoA in the culture medium and found that the acetyl-CoA treatment dramatically rescued the intensity of H3K27ac and transcription of Akt in SCs of exercise mice,indicating that exercise inhibited mitochondrial metabolism to repress acetylation of H3K27,resulting in reduced transcription of Akt.(6)NVP-BEZ235 and MK-2206 protected SCs against exhaustionIn the absence of inhibitor treatment,repeatedly injured mice showed a considerably decreased number of Pax7~+SCs compared with uninjured mice.Mice exposed to triple injuries showed a preserved SC pool with NVP-BEZ235 or MK-2206treatment.Moreover,they also showed improved muscle regeneration upon treatment with NVP-BEZ235 or MK-2206,suggesting that NVP-BEZ235 and MK-2206 were effective exercise analogues.The findings of our study show that exercise protects proliferative SCs against exhaustion via the Igfbp7-Akt-mTOR axis.These findings establish a link between mechanical signaling,mitochondrial metabolism,epigenetic modification,and stem cell fate decisions;thus,present potential therapeutic targets for muscle diseases correlated with SCs exhaustion. |
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