Satellite cell-derived extracellular vesicles reverse peroxide-induced mitochondrial dysfunction in myotubes

ABSTRACT Satellite cells (SCs) are muscle-specific stem cells that have a central role in muscle remodeling. Despite their therapeutic potential, SC-based therapies have been met with numerous logistical challenges, limiting their ability to effectively treat systemic muscle diseases, such as Duchenne muscular dystrophy (DMD). Delivery of SC-derived extracellular vesicles (SC-EVs) may unlock the potential offered by SCs and overcome their numerous limitations. Purpose The purpose of this investigation was to determine the extent to which SC-EVs could restore mitochondrial function in cultured myotubes following oxidative injury. Methods SC-EVs were isolated from cultured SCs from C57 mice and quantified using nanoparticle tracking analysis (NTA). C2C12 myotubes were cultured and divided into four treatment groups: untreated control, treated for 24 h with SC-EV, 24 h exposure to 50 μM H 2 O 2 followed by a 24 h recovery period with no treatment, or 24 h exposure to 50 μM H 2 O 2 followed by a 24 h treatment with SC-EV. Inter-group differences in mitochondrial function were assessed via one-way ANOVA with Tukey post hoc analysis (p<0.05). Results Given the seeding density used, we calculated that each SC releases approximately 2.35 × 10 5 ± 3.10 × 10 4 EVs per 24 h. Further, using fluorescent microscopy, we verified SC-EVs deliver cargo into myotubes, some of which was localized to the mitochondria. H 2 O 2 exposure resulted in a 42% decline in peak mitochondrial respiration (p=0.0243) as well as a 46% reduction in spare respiratory capacity (p=0.0185) relative to the untreated control group. Subsequent treatment with SC-EVs (3.12×10 8 SC-EV; 24 h) following H 2 O 2 exposure restored 76% of peak mitochondrial respiration (p=0.0187) and 84% of spare respiratory capacity in the damaged myotubes (p=0.0198). SC-EVs did not affect mitochondrial function in the undamaged myotubes. Conclusion Collectively, these data demonstrate SC-EVs may represent a novel therapeutic approach for treatment of myopathies associated with mitochondrial dysfunction.