## 4 Discussion
To follow up this finding, we investigated the effects of MuSC-EVs on mitochondrial function. Our data demonstrate MuSC-EVs reverse mitochondrial dysfunction caused by oxidative stress. Mitochondria are critical in cellular energy production as well as the regulation of overall cellular viability [69]. Mitochondrial dysfunction plays a central role in numerous muscle disorders and pathologies, including Duchenne Muscular Dystrophy (DMD) [30; 70]. For example, work by [PERSON] et al. (2019) demonstrated that mitochondrial dysfunction, attributed to impaired oxidative phosphorylation and elevated oxidant production, contributes to early stage pathology in the D2.mdx mouse model [21]. Additionally, in the mdx mouse mode mitochondrial dysfunction is one of the earliest cellular consequences of DMD and is associated with the impaired ability of dystrophic muscle cells to respond to sarcolemmal damage [70]. Adverse alterations in mitochondrial morphology and function are also known to play a central role in the pathology of muscle dysfunction and wasting in murine models of cancer cachexia, attributed to decreased adenosine triphosphate production and uncoupling of oxidative phosphorylation [71]. The contribution of mitochondrial dysfunction to disuse atrophy is well documented [35]. For example, work by [PERSON] et al. (2011) demonstrated a mitochondrial-targeted antioxidant was able to ameliorate ventilation-induced diaphragm weakness [37]. Given that mitochondrial dysfunction is associated with or contributes to an array of muscle pathologies, MuSC-EVs may alleviate a significant disease burden and provide therapeutic relief to a large number of patients. MuSC-EVs may have distinct advantages over cell transplantation including, most notably, the abundance of EVs released and the efficiency with which they deliver molecular cargo. While it is clear that MuSC-EVs attenuate mitochondrial dysfunction caused by acute oxidative stress, the underlying mechanism is decidedly less apparent. Speculatively, these EVs may deliver some factor (RNA and/or protein) that serves to reverse H\({}_{2}\)O\({}_{2}\)-mediated mitochondrial damage and restore mitochondrial function. However, the identity of this factor or factors remains unknown. Given the 24 h period of MuSC-EV exposure to myotubes there is ostensibly sufficient duration to allow translation of delivered RNA or incorporation of delivered protein into the appropriate cellular compartments, including the mitochondria themselves. To provide context to our data demonstrating the ability of MuSC-EVs to efficiently deliver fluorescently-labeled protein cargo and reverse mitochondrial dysfunction in myotubes in vitro, we analyzed a sample of MuSC-EVs via UPLC MS/MS. As previously mentioned, the identified list of proteins was analyzed via enrichment analysis using the database DAVID for significant enrichment of GO terms falling under biological processes, molecular function and cell component, as well as KEGG pathways. The top GO terms (lowest FDR value) under biological processes represent processes involving the regulation and organization of structural proteins, such as intermediate filament organization, extracellular structure organization, and cytoskeleton organization. These findings are particularly interesting in light of previous work by [PERSON] et al. (2017) [PERSON] et al. (2017) [PERSON] et al. (2020) [PERSON] et al. (2020) demonstrating miRNAs, such as miR-206, contained within MuSC-EVs regulate extracellular matrix (ECM) remodeling. The top molecular function GO terms also reflect proteins involved in the regulation and organization of the cytoskeleton and ECM, with structural molecule activity, structural constituent of the cytoskeleton and ECM, and structural constituent as the top results. Given our data demonstrating the involvement of MuSC-EVs in regulating metabolic processes in myotubes, two other interesting molecular function GO terms that were significantly enriched are carbohydrate derivative binding and oxidoreductase activity.