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

Figures

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  Figure 1. Characterization of MuSC-EVs. (A) Nanoparticle tracking analysis of extracellular vesicles (EVs) released during the first 24 h of SC culture, demonstrating the concentration and size profile of SC-EVs released in vitro. (B) Table of MuSC-EV characterization data. (C) Image of MuSC-EVs released during the first 24 h of cell culture taken via TEM. (D) Image of pooled MuSC-EVs released during days 2–6 of cell culture taken via TEM. N = 3 (biological replicates).

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  Figure 2. Fluorescence Microscopy of MuSC-EV Protein Uptake in Myotubes. (A) 40× negative control (No MuSC-EV treatment) of C2C12 myotubes with DAPI stain (blue). (B) Representative 63× image of carboxyfluorescein succinimidyl ester (CFSE)-labeled SC EV protein (green) delivery to C2C12 myotubes following 24 h incubation period in vitro. (C) Representative 40× image of CFSE-labeled MuSC-EV protein (green) delivery into C2C12 myotubes with mitochondrial stain (red) suggesting interaction of MuSC-EV protein with portions of the mitochondrial network. (D–J) 20× images of C2C12 myotubes incubated with 1 × 109 CFSE-labeled SC-EVs/well in a 96-well plate for varying time periods. (D) Negative control (E) 5 min (F) 30 min (G) 1 h (H) 2 h (I) 6 h (K) 24 h (J) 48 h of incubation in vitro. N = 3 (technical replicates).

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  Figure 3. Fluorescence Intensity of CFSE-Labeled MuSC-EV Protein Delivered to C2C12 Myotubes In Vitro Following Varying Incubation Periods. * p < 0.05 vs. control. N = 3 (technical replicates).

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  Figure 4. Mitochondrial Stress Test in C2C12 Myotubes. Myotubes were divided into one of four groups: untreated control, treatment with 3.12 × 108 SC-EV for 24 h, treatment with H2O2 for 24 h, or treatment with H2O2 for 24 h followed by treatment with SC-EV for 24 h. (A) Oxygen consumption rate over the duration of the mitochondrial stress assay. O = oligomycin, F = 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP), R = Rotenone and Antimycin A. (B) Energy map depicting reliance on aerobic vs. glycolytic energy systems and energetic state of each treatment group. (C) Basal respiration of myotubes in each treatment group. (D) Maximal respiration of myotubes in each treatment group. (E) Spare respiratory capacity of myotubes in each treatment group (calculated as difference between maximal respiration and basal respiration). * p < 0.05. N = 3 (technical replicates).

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  Figure 5. Proteomic Analysis of MuSC-EVs. Significantly enriched (p < 0.05) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms identified using DAVID enrichment analysis following UPLC MS/MS of MuSC-EVs. (A) Biological processes (B) Molecular function (C) Cellular component (D) KEGG Pathway results.