Skeletal muscle-derived extracellular vesicles are altered with chronic contractile activity

Abstract

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.

Defining the influence of size‐exclusion chromatography fraction window and ultrafiltration column choice on extracellular vesicle recovery in a skeletal muscle model

“…Since this initial study, EV‐enriched fractions have been isolated from immortalised C2C12 cell lines at different stages of the myogenic differentiation process, as well as from primary myoblasts, myotubes and murine SM fibres (Forterre et al., 2014; Romancino et al., 2013). However, due to the lack of availability of bespoke protocols for the isolation of EVs from SM, the majority of publications to date have studied EV‐enriched fractions that also contain additional co‐isolated particles such as lipoproteins and protein aggregates (Obi et al., 2022; Rome, 2022; Rome et al., 2019). Consequently, while outcomes from previous studies have provided highly interesting observations concerning the potential functions of EVs in process such as myogenic regulation and energy metabolism (Kim et al., 2018; Shuler et al., 2020; Sork et al., 2018; Takafuji et al., 2020), they have frequently not accounted for the presence and contribution of lipoproteins and RNA binding proteins (e.g., AGO2) commonly recovered in EV preparations, which due to their overlapping diameters (low density lipoproteins: 20–200 nm) and densities (high density lipoproteins: 1.06–1.21 g/mL), can lead to inaccuracies if not depleted in the isolation protocol (Yuana et al., 2014).…”