Characterizing Extracellular Vesicles and Particles Derived from Skeletal Muscle Myoblasts and Myotubes and the Effect of Acute Contractile Activity

Abstract

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.

Pulsed Electrical Stimulation Affects Osteoblast Adhesion and Calcium Ion Signaling

“…The purpose of this study is threefold: (i) characterizing the electric fields; (ii) the influence of ES (AC) on liquid characteristics (DMEM); and (iii) influence of ES (AC) on the initial cell adhesion and calcium ion (Ca 2+ ) signaling of MG-63s. ES generated by IonOptix is described in studies with, e.g., myoblasts [ 46 ], or osteoblasts [ 12 , 14 ]. Ercan et al [ 12 ], showed a positive effect of IonOptix AC stimulation on osteoblast (hFOB 1.19) proliferation, but they technically modified the system with potentiometers to achieve a physiological current.…”

“…The electric field is generated between two parallel graphite plates (i.e., a plate capacitor) 1.1 cm apart and in direct contact with the cells. We utilized a commercially available device from IonOptix (voltage controlled), which has been described earlier [ 12 , 14 , 46 ]. The IonOptix stimulator generates AC electric fields with constant biphasic rectangular pulses at a frequency of 7.9 or 20 Hz and voltage of 1 and 5 V, respectively.…”

Exploiting the therapeutic potential of contracting skeletal muscle-released extracellular vesicles in cancer: Current insights and future directions

“…It is important to acknowledge that determining the proportion of SkM-derived EVs to total EVs in circulation poses several challenges. First, the presence of markers specific to the SkM origin, such as β- or α-sarcoglycans, integral components of the dystrophin-glycoprotein complex, cannot be guaranteed in all SkM-derived EVs [ 17 ], making it difficult to definitively confirm EVs originating from SkM contractile activity. Second, α-sarcoglycan has been detected not only in SkM but also in cardiac muscle [ 58 ].…”

“…Second, α-sarcoglycan has been detected not only in SkM but also in cardiac muscle [ 58 ]. Third, intramuscular injections of fluorescently labeled EVs or genetic manipulation, which are excellent options in rodent models, are impractical in human studies [ 17 ]. Despite the difficulties in studying SkM-derived EVs in vivo, several in vitro studies have clearly demonstrated that both myoblasts and myotubes are capable of releasing EVs whose biological role in recipient cells is unclear [ 59 , 60 ].…”

“…However, in the late 1990s, it was discovered that EVs can transfer proteins, RNA, and even organelles from one cell to another, making them mediators of cell–cell communication [ 14 – 16 ]. EVs are secreted by all types of cells and are present in biological fluids such as blood, cerebrospinal fluid, urine, and saliva [ 17 ]. Recent studies have shown that exercise can stimulate the release of EVs from the SkM into the bloodstream, thereby delivering biomolecules to recipient cells [ 9 , 10 ].…”