Muscle Stem Cell-Derived Extracellular Vesicles Reverse Hydrogen Peroxide-Induced Mitochondrial Dysfunction in Mouse Myotubes

Abstract

Muscle stem cells (MuSCs) hold great potential as a regenerative therapeutic but have met numerous challenges in treating systemic muscle diseases. Muscle stem cell-derived extracellular vesicles (MuSC-EVs) may overcome these limitations. We assessed the number and size distribution of extracellular vesicles (EVs) released by MuSCs ex vivo, determined the extent to which MuSC-EVs deliver molecular cargo to myotubes in vitro, and quantified MuSC-EV-mediated restoration of mitochondrial function following oxidative injury. MuSCs released an abundance of EVs in culture. MuSC-EVs delivered protein cargo into myotubes within 2 h of incubation. Fluorescent labeling of intracellular mitochondria showed co-localization of delivered protein and mitochondria. Oxidatively injured myotubes demonstrated a significant decline in maximal oxygen consumption rate and spare respiratory capacity relative to untreated myotubes. Remarkably, subsequent treatment with MuSC-EVs significantly improved maximal oxygen consumption rate and spare respiratory capacity relative to the myotubes that were damaged but received no subsequent treatment. Surprisingly, MuSC-EVs did not affect mitochondrial function in undamaged myotubes, suggesting the cargo delivered is able to repair but does not expand the existing mitochondrial network. These data demonstrate that MuSC-EVs rapidly deliver proteins into myotubes, a portion of which co-localizes with mitochondria, and reverses mitochondria dysfunction in oxidatively-damaged myotubes.

A high-throughput methodology for the efficient isolation of highly pure extracellular vesicles from skeletal muscle myoblasts

“…The ability to identify additional SM-EV markers and map their biological functions is confounded by the fact that no optimal method has been developed for the isolation of SM-EVs. To date, studies have applied multiple isolation methods that often lack speci city, such as dUC (14,15,41,42,(16)(17)(18)(19)(20)(38)(39)(40), PEG or commercial isolation kits (21,39,43,44). While neutral on a functional level, any EV isolation protocol can affect sample quality, introducing impurities or a variety of co-isolated particles (lipoproteins and protein/RNA complexes) (77)(78)(79).…”

“…The content and function of heterogenous SM-EV fractions are also dependent on the local physiological environment, with SM-EVs derived from myotubes under an induced in ammatory environment found to inhibit myogenesis and induce atrophy in C2C12 cells (19). While, SM-EVs recovered from both myoblasts and myotubes had effects on mitochondria and energy metabolism (20,21). Finally, murine hindlimb SM bre derived EVs contained high levels of some miRNAs, such as miR-133a, which could have downstream effects on myogenic and osteogenic activities through the targeting of Smarcd1 and Runx2, respectively (22).…”

“…mass spectrometry). When focusing on the skeletal muscle eld, the majority of in vitro studies have applied dUC (14,15,41,42,(16)(17)(18)(19)(20)(38)(39)(40) or commercial isolation kits (21,39,43,44) for this purpose. In vivo, the majority of studies have applied dUC (26,45-48), polyethylene glycol (PEG) (49) or commercial isolation kits (27,29,50) with blood plasma as the main reported source of SM-EVs.…”

Extracellular Vesicle-Mediated Mitochondrial Reprogramming in Cancer

“…Furthermore, this mitochondria-mediated therapeutic action is not limited to apoptosis. In the context of cancer cachexia, muscle stem cell derived-EVs can reverse mitochondrial injury through their protein cargo by improving maximal oxygen consumption rate and spare respiratory capacity [222,223], which is a parameter indicative of mitochondrial adaptability to stress conditions [224].…”

Fusion and beyond: Satellite cell contributions to loading‐induced skeletal muscle adaptation

“…Maintenance of the myonuclear domain and restoration of muscle fiber membrane integrity via satellite cell fusion are likely important aspects of satellite cell participation in exercise adaptation, but there is recent growing appreciation for the non‐fusion roles satellite cells can play in adult muscle 47,48 . Indeed, the ability to specifically deplete satellite cells in an inducible fashion in adult skeletal muscle of mice confirmed their indelible role in myonuclear addition to adult muscle fibers via direct fusion, 21,32 but also revealed effects on ambulatory coordination as well as the behavior of non‐satellite cells throughout muscle which has a marked effect on muscle fiber phenotype 23,32,49–53 ; this includes secretory communication to muscle fibers, fibrogenic cells, and endothelial cells during adaptation.…”