Extracellular vesicles from skeletal muscle cells: mediators of physical adaptation in chronic low frequency exercise and new players for efficient CoQ10 delivery

Skeletal muscle (SkM) has emerged as an important secretory organ releasing myokines and extracellular vesicles (EVs), which contribute to exercise-induced muscle adaptations. Currently, there is a substantial interest in how exercise can promote EV release and its role in mediating the systemic effects of SkM activity. Nevertheless, the difficulty to isolate SkM EVs from serum and plasma led to set up system to reproduce the exercise in vitro, such as electrical pulse stimulation (EPS), to recover EVs directly from cultured contracting SkM cells. We found that a chronic low frequency session of EPS in C2C12 myotubes influences the secretory activity of contracting SkM cells and the molecular cargo of contracting SkM EVs. In detail, two different populations of EVs released from contracting myotubes, 10k-EVs and 100k-EVs were isolated with differential ultracentrifugation, quantified with nanoparticle tracking analysis and characterized for their protein and DNA cargo. A consistent change of protein phenotype occurs only in 10k-EVs. ADAM10 and Calnexin resulted to be more expressed in chronic EPS, and this change may be related to Ca2+ transients produced in myotubes during contraction. Interestingly, two subpopulations were characterized in EPS-derived 10K-EVs: a CD81+/α7-INT+ subpopulation shared in common with no EPS negative control 10k-EVs (EPS-Comm) and a CD81-/α7-INT+ subpopulation secreted only during chronic EPS (EPS-Spec). Moreover, qPCR analysis in EV mitochondrial DNA content presents a significant loading of Cox1 and ND1 in EPS-derived 10k-EVs, which could drive the upregulation of pro-inflammatory cytokines observed in murine macrophages treated with EPS-derived 10k-EVs. Recently, EVs are also proposed as a new drug delivery system for their ability to transport biomolecules to recipient cells. In particular, bio-inspired exosome-like nanovesicles could be a promising tool to enhance CoQ10 bioavailability in muscle cells, which is essential for the treatment of metabolic disorders related to CoQ10 secondary deficiency, including sarcopenia and strenuous exercise. Hence, a second aim is to evaluate the ability of engineered nanovesicles (eNVs) to transport CoQ10 to recipient muscle cells. For the studies of CoQ10 bioavailability, HeLa cells, myocytes C2C12, and cardiomyocytes H9C2 and AC16 were treated with CoQ10-loaded eNVs decorated with membrane proteins from C2C12 myotubes to determine their uptake levels compared to liposomes. FACS analysis in C2C12 cells have unveiled a more efficient internalization of eNVs compared to liposomes after 24-hour incubation, whereas H9C2 cells were able to take up a huge amount of eNVs or liposomes than the other cell types. The CoQ10 levels measured in C2C12 and H9C2 cells after treatment with Q10-eNVs are higher compared to Q10-lipo treatment and, moreover, C2C12 and H9C2 can uptake more CoQ10 than HeLa cells from eNVs. Microscopy imaging in wounded C2C12 or H9C2 cells showed an almost complete migration in 18 hours after peroxide treatment, demonstrating that Q10-eNVs are able to protect skeletal and cardiac muscle cells against oxidative stress. In conclusion, the contraction activity elicited by chronic sessions of EPS can stimulate the secretion of a specific CD81-/α7-INT+ EV subpopulation from C2C12 myotubes. Of note, SkM-derived EVs may have a role in regulating physical adaptations to chronic low frequency exercise through the EV loading of mitochondrial DNA (mtDNA). Furthermore, the studies of CoQ10 delivery in muscle cells suggest that eNVs decorated with membrane proteins from C2C12 myotubes can serve as novel drug transporters and they are able to preserve CoQ10 bioactivity in skeletal and cardiac muscle cells.