Exercise is associated with various health benefits, including the prevention and management of obesity and cardiometabolic risk factors. However, a strong heterogeneity in the adaptive response to exercise training exists. The objective of this study was to evaluate if changes in extracellular vesicles (EVs) after acute aerobic exercise (AE) were associated with the responder phenotype following 6-weeks of resistance exercise training. This is a secondary analysis of plasma samples from the EXIT trial (clinical trial #02204670). Eleven sedentary youth with obesity (15.7±0.5 years, BMI ≥ 95th percentile) underwent an acute bout of AE (60% heart rate reserve, 45 min). Blood was collected before exercise [at time (AT) 0 min], during [AT15, 30, 45 min], and 75 min after exercise [AT120]. Afterward, youth participated in 6-week resistance training program, and were categorized into responders (RE) or non-responders (NRE) based on changes in insulin sensitivity as measured by the Matsuda Index. EVs were isolated using size exclusion chromatography (Izon®). The primary outcome variable was EV biophysical profile, which includes size, zeta potential, protein yield and expression of markers associated with EV subtypes. The variables were analyzed in a single-blind fashion. Overall, there was a general increase in EV production in both groups. Average EV size was larger in RE (~147 nm) vs. NRE (~124 nm; p<0.05). Average EV size at AT0 was associated with absolute change in Matsuda index following 6-weeks of resistance training (r=0.44, p=0.08). EV size distribution revealed RE preferentially expressed EVs between 150 – 250 nm in size, whereas NRE expressed EVs between 50 – 100 nm (p<0.05). At baseline, RE-EVs contained ~25% lower Tsg101 protein, ~85% higher MMP2 content, while CD63 levels remained unchanged between the groups. Total protein yield in RE-EVs was higher than NRE at AT15 (p<0.05). Our data suggest that youth with obesity that respond to exercise training produce larger EVs, with lower exosome- and higher microvesicle-specific protein expression. RE-EVs also had higher EV protein yield during AE. The relationship between larger EV subtypes and/or cargo, and the individual response to exercise has yet to be fully elucidated.

Extracellular vesicles (EVs), released from all cells, are essential to cellular communication and contain biomolecular cargo that can affect recipient cell function. Studies on the effects of contractile activity (exercise) on EVs usually rely on plasma/serum-based assessments, which contain EVs from many different cells. To specifically characterize skeletal muscle–derived vesicles and the effect of acute contractile activity, we used an in vitro model where C2C12 mouse myoblasts were differentiated to form myotubes. EVs were isolated from conditioned media from muscle cells at pre-differentiation (myoblasts) and post-differentiation (myotubes) and also from acutely stimulated myotubes (1 h @ 14 V, C-Pace EM, IonOptix, Westwood, MA, USA) using total exosome isolation reagent (TEI, ThermoFisher (Waltham, MA, USA), referred to as extracellular particles [EPs]) and differential ultracentrifugation (dUC; EVs). Myotube-EPs (~98 nm) were 41% smaller than myoblast-EPs (~167 nm, p < 0.001, n = 8–10). Two-way ANOVA showed a significant main effect for the size distribution of myotube vs. myoblast-EPs (p < 0.01, n = 10–13). In comparison, myoblast-EPs displayed a bimodal size distribution profile with peaks at <200 nm and 400–600, whereas myotube-Eps were largely 50–300 nm in size. Total protein yield from myotube-EPs was nearly 15-fold higher than from the myoblast-EPs, (p < 0.001 n = 6–9). Similar biophysical characteristics were observed when EVs were isolated using dUC: myotube-EVs (~195 nm) remained 41% smaller in average size than myoblast-EVs (~330 nm, p = 0.07, n = 4–6) and had comparable size distribution profiles to EPs isolated via TEI. Myotube-EVs also had 4.7-fold higher protein yield vs. myoblast EVs (p < 0.05, n = 4–6). Myotube-EPs exhibited significantly decreased expression of exosomal marker proteins TSG101, CD63, ALIX and CD81 compared with myoblast-EPs (p < 0.05, n = 7–12). Conversely, microvesicle marker ARF6 and lipoprotein marker APO-A1 were only found in the myotube-EPs (p < 0.05, n = 4–12). There was no effect of acute stimulation on myotube-EP biophysical characteristics (n = 7) or on the expression of TSG101, ARF6 or CD81 (n = 5–6). Myoblasts treated with control or acute stimulation–derived EPs (13 µg/well) for 48 h and 72 h showed no changes in mitochondrial mass (MitoTracker Red, ThermoFisher, Waltham, MA, USA), cell viability or cell count (n = 3–4). Myoblasts treated with EP-depleted media (72 h) exhibited ~90% lower cell counts (p < 0.01, n = 3). Our data show that EVs differed in size, distribution, protein yield and expression of subtype markers pre vs. post skeletal muscle–differentiation into myotubes. There was no effect of acute stimulation on biophysical profile or protein markers in EPs. Acute stimulation–derived EPs did not alter mitochondrial mass or cell count/viability. Further investigation into the effects of chronic contractile activity on the biophysical characteristics and cargo of skeletal muscle–specific EVs are warranted.

Extracellular vesicles (EVs) are small lipid membrane-bound structures that are secreted by all cells, and play a central role in cellular communication. EVs are released from skeletal muscle during exercise, but the effects of contractile activity on skeletal muscle-derived EVs (Skm-EVs) are poorly understood due to the challenges in distinguishing Skm-EVs derived from exercising muscle in vivo. To specifically characterize Skm-EVs, C2C12 myoblasts were differentiated into myotubes, and electrically paced (3h/day x 4days @14V, C-PACE EM, IonOptix) to mimic chronic exercise in vitro. EVs were isolated from conditioned media from control and stimulated myotubes using differential ultracentrifugation. Isolated EVs were characterized biophysically (size, zeta potential, yield, protein markers and by transmission electron microscopy; TEM). Chronic stimulation increased markers of mitochondrial biogenesis such as MitoTracker Red staining, cytochrome c oxidase activity and expression of cytochrome c (p<0.05, N=7-8) in stimulated vs. non-stimulated myotubes. The average size of EVs from chronically stimulated myotubes (CS-EVs, 132 nm) was 26% smaller than control (CON-EVs, 178 nm) (p<0.05, N=8). Size distribution analysis revealed that CS-EVs were enriched with 100-150 nm sized small EVs, while CON-EVs were largely composed of 200-250 nm sized vesicles (p<0.05, main interaction effect, N=8). TEM confirmed the presence of round-shaped vesicles of about 30-100 nm with an intact lipid bilayer in CON-EVs and CS-EVs. Zeta potential was 27% lower in CS-EVs vs. CON-EVs (p<0.05, N=8), and total EV protein yield remained unchanged between groups. Protein-based EV characterization showed that both CON-EVs/CS-EVs were CD81+ but CD63-, and expressed hallmark cytosolic proteins recovered in EVs: Tsg101, Flotillin-1, HSP70, and Alix. CD81 and HSP70 expression increased in CS-EVs vs. CON-EVs (p<0.05, N=9). We evaluated if chronic stimulation affected whole cell expression of transmembrane and cytosolic proteins used in EV origin/purity analysis. CD63 and ApoA1 were reduced with chronic stimulation in myotube lysates (p<0.05, N=7), whereas Tsg101, CD81, Flotillin-1 and HSP70 levels remained constant. Taken together, our study revealed that chronic stimulation triggers the release of more stable, smaller sized EVs, enriched with transmembrane and cytosolic protein markers of small EVs, and are CD81+/CD63- indicating the origin of these EVs might be ectosomal rather than endosomal. The upstream signaling cascades that regulate biogenesis of EVs with chronic stimulation, and whether skeletal muscle-EVs are released through endosomal or ectosomal pathways remains to be elucidated. Our findings support the role of chronic contractile activity in the modulation of EV biophysical characteristics. Whether this affects biological cargo recruitment into CS-EVs, and their subsequent biological activity remains to be established.