Identification of protein markers for skeletal muscle‐derived extracellular vesicles (SkM‐EVs) by quantitative proteomics reveals how SkM‐EVs function in vivo

Abstract

BackgroundSkeletal muscle serves as a secretory organ; it releases various soluble factors called myokine. They act as mediators for cell‐cell communications in autocrine, paracrine, and endocrine fashions. Extracellular vesicles (EVs) have been proposed to be a partial paracrine and/or endocrine effectors by facilitating the exchange of biological cargoes among cells and tissue. Some in vitro study showed that skeletal muscle released EVs and skeletal muscle‐derived EVs (SkM‐EVs) could induce biological effects in recipient cells. However, whether the skeletal muscle actively releases EVs in vivo, how much proportion of plasma EVs are derived from this tissue, and where SkM‐EVs are delivered and exert their roles remain largely unknown. Here, we aimed to identify SkM‐EV marker proteins by quantitative proteomics on human and mouse myocyte‐derived EVs to evaluate SkM‐EVs abundance in vivo. In addition, we explored whether SkM‐EVs isolated from mice could exert biological functions.MethodsProteomic analysis of EVs released from mouse C2C12 myotubes and human iPS‐derived myocytes was performed to identify potential SkM‐EVs markers. The levels of SkM‐EVs in plasma and muscle interstitium were evaluated by the abundance of identified SkM‐EVs protein markers. Based on previous methods, we developed a modified protocol to isolated SkM‐EVs from muscle interstitium. We confirmed that muscle interstitium‐EVs were incorporated into C2C12 myoblast and investigated the expression levels of myogenesis‐related genes.Results& DiscussionWe first determined proteomic profiles of EVs released from C2C12 myoblasts, C2C12 myotubes, and hiPSC‐derived myocytes and identified several proteins that serve as potential markers for SkM‐EVs. Among them, we identified ATP2A1, β‐enolase, and desmin as reliable SkM‐EV marker proteins. Using these protein markers, we confirmed that SkM‐EVs were present in muscle interstitium but little in the blood, which were supported by our electron microscopy. Unexpectedly, the abundance of SkM‐EVs present in plasma and muscle interstitium was not changed in response to acute exercise. Furthermore, we showed that SkM‐EVs accumulated in muscle interstitium space were incorporated into C2C12 myoblast and promoted muscle differentiation by regulating gene expression of Pax7 and myosin heavy chain at least partially through myomiRs (miRs‐1, ‐206, ‐431, and ‐486). We expect that molecular characterization and in vivo distribution of SkM‐EVs could provide a conceptually new basis how skeletal muscle EVs exert their functions.