## 4 Discussion
In accordance with the functions associated with identified proteins, the top cell component result was the GO term extracellular region part. The cell component results also correctly identified the proteins as being associated with EVs. The top KEGG pathways were ECM-receptor-interaction and focal adhesion further suggesting a role of the MuSC-EV proteins in ECM regulation and organization. Other interesting pathways that appear in the results are the phosphoinositol-3-kinase/Akt (PI3K/Akt) and phagosome pathways. These may provide interesting mechanistic insights, given that these pathways regulate critical anabolic (PI3K/Akt) [PERSON] et al. (2017) and catabolic (phagosome) [PERSON] et al. (2020) processes in myotubes. In addition to the enrichment analysis data, which analyzes the identified proteins more broadly based on function and localization, we identified a particularly interesting protein in the dataset in relation to our discoveries regarding MuSC-EVs and mitochondrial function. We identified peroxisome proliferatorator-activated receptor gamma coactivator 1-beta (PGC1-beta), which activates the transcriptional activity of transcription factors, such as estrogen-related receptor and nuclear respiratory factor, promoting mitochondrial biogenesis [PERSON] et al. (2020). Further, PGC1-beta has been shown to coordinate with peroxisome proliferatorator-activated receptor gamma coactivator 1-alpha (PGC1-alpha) in the promotion of mitochondrial biogenesis [PERSON] et al. (2020). This is a promising association as PGC1-alpha gene transfer has consistently attenuated disease severity and promoted a type I, oxidative phenotype in the skeletal muscle of mdx mice [PERSON] et al. (2020). Based on the available data, we cannot be certain that this protein cargo indicates a definitive mechanism. However, these data provide important insights into the molecular cargo contained within MuSC-EVs when interpreted alongside the therapeutic effects of MuSC-EVs on mitochondrial function. Overall, these proteomics findings should be interpreted as a descriptive identification of potential targets for further, more definitive mechanistic investigations of the contents and therapeutic potential of MuSC-EVs. Other groups have also shed light on potential mechanisms by which MuSCs and related stem cell types confer therapeutic effects beyond the well-documented fusion-mediated role of these cells. [PERSON] et al. (2020) showed MuSC-derived IGF-2 induces an anti-inflammatory phenotype in macrophages, potentially due to modulation of oxidative phosphorylation [26]. This is a particularly interesting finding in line with our data demonstrating metabolic reprogramming in cultured myotubes. [PERSON] et al. (2019) demonstrated that exosomes from wild type and dystrophin-deficient induced pluripotent stem cell-derived cardiomyocytes exert cardioprotective effects on dystrophic cardiomyocytes [77]. The authors attributed these findings to a decrease in reactive oxygen species and delay in the formation of the mitochondrial transition pore following oxidative injury with H\({}_{2}\)O\({}_{2}\), mediated by ERKI/2 and p38 MAPK signaling. Alternatively, MuSC-EVs may deliver intact mitochondria to recipient cells. This mechanism is attractive as EV-mediated delivery of mitochondria to damaged neurons has been demonstrated previously [78]. Our data indicate that MuSC-EVs delivered to undamaged myotubes did not alter mitochondrial respiration. If mitochondria were being delivered, we would expect to see increased respiration in this group. Given that there was only an increase in mitochondrial respiration in the group that was damaged first, it appears that the MuSC-EVs are delivering cargo that helps restore mitochondrial function but does not expand the overall mitochondrial capacity of otherwise healthy myotubes.