Skeletal muscle cell growth alters the lipid composition of extracellular vesicles

We sought to characterize the lipid profile of skeletal muscle cell-derived Extracellular Vesicles (EVs) to determine if a hypertrophic stimulus would affect the lipid composition of C2C12 myotube-derived EVs. Analyses included C2C12 murine myoblasts differentiated into myotubes and treated with Insulin-Like Growth Factor 1 (IGF-1) for 24 h to induce hypertrophic growth. EVs were isolated from cell culture media, quantified using Nanoparticle Tracking Analysis (NTA) and analyzed using Transmission Electron Microscopy (TEM). EVs were homogenized and lipids extracted for quantification by Mass Spectrometry followed by downstream lipid class enrichment and lipid chain analysis. IGF-1 treatment elicited an increase in CD63 and CD81 levels (39% and 21%) compared to the controls (16%), respectively. Analysis revealed that skeletal muscle-derived EVs are enriched in bioactive lipids that are likely selectively incorporated into EVs during hyper-trophic growth. IGF-1 treatment of myotubes had a significant impact on the levels of diacylglycerol (DG) and ceramide (Cer) in secreted EVs. Specifically, the proportion of unsaturated DG was two-to three-fold higher in EVs derived from IGF-treated cells, as compared to those from control cells. The levels of saturated DG were unaffected. Selective increases were similarly seen in C16-and C24-Cer but not in other species. Levels of free sphingoid bases tended to decrease, while those of sphin-gosine-1-phosphate was unaffected. Our results suggest that the lipid composition and biogenesis of skeletal muscle-derived EVs, are specific and highly selective during hypertrophic growth.