GSE211885
Skeletal muscle fiber-derived small extracellular vesicle miR-690 inhibits satellite cell differentiation and muscle atrophy during aging
Background: Sarcopenia is a common and progressive skeletal muscle disorder characterized by atrophic muscle fibers and contractile dysfunction. Accumulating evidence shows that the number and function of satellite cells (SCs) decline and become impaired during aging, which may contribute to impaired regenerative capacity. A series of myokines/ small extracellular vesicles (sEVs) released from muscle fibers regulate metabolism in muscle and extramuscular tissues in an autocrine/paracrine/endocrine manner during muscle atrophy. It is still unclear whether myokines/sEVs derived from muscle fibers can affect satellite cell function during aging.                                                                                                               Methods: Aged mice were used to investigate changes in the myogenic capacity of SCs during aging-induced muscle atrophy. The effects of atrophic myotube-derived sEVs on satellite cell differentiation were investigated by biochemical methods and immunofluorescence staining. Small RNA sequencing was performed to identify differentially expressed sEV microRNAs (miRNAs) between the control myotubes and atrophic myotubes. The target genes of the miRNA were predicted by bioinformatics analysis and verified by luciferase activity assays. The effects of identified miRNA on the myogenic capacity of SCs in vivo were investigated by intramuscular injection of adeno-associated virus (AAV) to overexpress or silence miRNA in skeletal muscle.   Results: Our study showed that the myogenic capacity of SCs was significantly decreased (50%, n = 6, P < 0.001) in the tibialis anterior muscle of aged mice. We showed that atrophic myotube-derived sEVs inhibited satellite cell differentiation in vitro (n = 3, P < 0.001) and in vivo (35%, n = 6, P < 0.05). We also found that miR-690 was the most highly enriched miRNA among all the screened sEV miRNAs in atrophic myotubes [Log2 (Fold Change) = 7, P<0.001], which was verified in the atrophic muscle of aged mice (3-fold, n = 6, P < 0.001) and aged humans (2.8-fold, n = 10, P < 0.001). miR-690 can inhibit myogenic capacity of SCs by targeting myocyte enhancer factor 2, including Mef2a, Mef2c, and Mef2d, in vitro (n=3, P < 0.05) and in vivo (n=6, P < 0.05). Specific silencing of miR-690 in the muscle can promote satellite cell differentiation (n = 6, P < 0.001) and alleviate muscle atrophy in aged mice (n = 6, P < 0.001).   Conclusions: Our study demonstrated that atrophic muscle fiber-derived sEV miR-690 may inhibit satellite cell differentiation by targeting myocyte enhancer factor 2 during aging. Overall design: For the myogenic differentiation experiment, when confluence was reached to 80–90%, mouse C2C12 myoblasts  were induced with differentiation medium-DMEM containing 2% horse serum (Hyclone). Then we constructed a starvation-induced myotube atrophy model by starvating induction in Earle's balanced salt solution (EBSS) for 7 h. We isolated the small extracellular vesicle (sEVs) from control myotubes and atrophic myotubes.  MiRNA components in either control myotubes-EVs or atrophic myotubes-EVs were profiled by single-end sequencing (1x50bp) on an Illumina platform (NEB, USA) at Novogene (Beijing, China).