Identification of novel myokines and putative protein targets that mediate functional adaptations in response to chronic contractile activity induced skeletal muscle-extracellular vesicle treatment

Abstract We have previously shown that skeletal muscle-derived extracellular vesicles (EVs) released post-chronic contractile activity (CCA) increased mitochondrial biogenesis in murine myoblasts, and decreased cell viability and induced apoptosis and senescence in non-small cell lung cancer cells. While the underlying mechanisms are unknown, the effects perpetuated were dependent on membrane-bound proteins. Here, we performed an extensive LC-MS/MS proteomic analysis on EVs from control and CCA myotubes. A total of 2900 proteins were identified in CON-EVs and CCA-EVs, including EV-associated proteins such as TSG101, tetraspanins (CD9, CD81, and CD63), flotillin-1, and annexins. Of these, 856 proteins are novel and not listed in EV databases (ExoCarta and Vesiclepedia), indicating that myotube-EVs harbor proteins not yet identified in EVs of different origin. Additionally, we identified 2062 unique proteins that have not yet been previously reported in myotube-EVs to date. Remarkably, of the 2900 total proteins identified, we observed 46 upregulated, and 25 downregulated differentially expressed proteins (DEPs) in CCA-EVs vs . control-EVs. Most of upregulated DEPs include EV-associated proteins. Comparing the 71 DEPs with proteins expressed in skeletal muscle indicated 61 of these as potential myokines. We identified actin cytoskeleton signaling, integrin signaling and muscle contraction as the most enriched pathways among the DEPs using different databases/software including FunRich, KEGG, STRING and Ingenuity Pathway Analysis. Using a relevance score that prioritized membrane-bound proteins with known function in mitochondrial biogenesis and inhibition of cancer growth, we identified top-scoring highly enriched DEPs of interest: IGF1R, ATP7A, PFN1, GJA1, PRKCA and ITGA6. We confirmed upregulation of these targets in EVs using immunoblotting. Among these top-scoring DEPs, PFN1, and ITGA6 are associated with EVs, with expression upregulated following acute exercise. In summary, we report the first comprehensive analysis of skeletal muscle-EV proteome following CCA, with identification of putative protein targets and signaling pathways that may execute the pro-metabolic and anti-tumorigenic effects of CCA-EVs.