Related Experiment Video
Updated: Oct 26, 2026

Enzymatic Isolation of Skeletal Muscle Interstitial Extracellular Vesicles
Published on: February 7, 2025
Contracting Skeletal Muscle Releases Antioxidant-Enriched Extracellular Vesicles and Non-vesicular Extracellular
Abstract:
While the benefits of exercise are well-documented, the underlying molecular mechanisms remain to be fully elucidated. Previous research in our lab demonstrated that electrical pulse stimulation (EPS) of cultured C2C12 myotubes activates the Nrf2/antioxidant pathway. In the study presented in this manuscript, we hypothesized that these upregulated antioxidant enzymes are secreted into the conditioned medium (CdM) via extracellular vesicles (EVs) and non-vesicular extracellular nanoparticles (nvEPs). We also hypothesized that these antioxidant-enriched particles protect H9c2 cardiomyocytes from H 2 O 2 -induced damage. EVs/nvEPs were isolated from the CdM of EPS-treated C2C12 cells and compared to those from non-stimulated (NS) cells using differential ultracentrifugation. Transmission electron microscopy (TEM) revealed that the isolated particles displayed a typical morphology, with EVs showing a cup-shaped structure and lipid bilayer membrane, and nvEPs exhibiting a pill-like and amembranous structure with high electron density. Western blot analysis demonstrated that EVs/nvEPs from EPS-C2C12 myotubes contained higher levels of antioxidant proteins (NQO1 and GSTA2) than those isolated from non-stimulated myotubes. Using a CCK-8 viability assay, we found that H9c2 cells pretreated with C2C12-derived nvEPs showed varying degrees of survival following H 2 O 2 challenge. Our findings demonstrate that the EVs/nvEPs released from EPS-C2C12 myotubes contain enriched antioxidant enzymes, which protect cardiomyocytes from oxidative stress.
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