Skeletal muscle releases extracellular vesicles with distinct protein and miRNA signatures that accumulate and function within the muscle microenvironment

Extracellular vesicles (EVs) contain various regulatory molecules and mediate intercellular communications. Although EVs are secreted from various cell types, including skeletal muscle cells, and present in the blood, their identity is poorly characterized in vivo, limiting the identification of their origin in the blood. Since the skeletal muscle is the largest organ in the body, it could substantially contribute to circulating EVs as their source. However, due to the lack of defined markers that distinguish SkM-EVs from others, whether the skeletal muscle releases EVs in vivo and how much the skeletal muscle-derived EVs (SkM-EVs) account for plasma EVs remain poorly understood. In this work, we perform quantitative proteomic analyses on EVs released from C2C12 cells and human iPS cell-derived myocytes and identify potential marker proteins that mark SkM-EVs. These markers we identified apply to in vivo tracking of SkM-EVs. The results show that skeletal muscle makes only a subtle contribution to plasma EVs as their source in both control and exercise conditions in mice. On the other hand, we demonstrate that SkM-EVs are concentrated in the skeletal muscle interstitium. Furthermore, we show that interstitium EVs are highly enriched with the muscle-specific miRNAs and repress the expression of the paired box transcription factor Pax7, a master regulator for myogenesis. Taken together, our findings reveal that the skeletal muscle releases exosome-like small EVs with distinct protein and miRNA profiles in vivo and that SkM-EVs mainly play a role within the muscle microenvironment where they accumulate.