Amplitude dependence in extracellular vesicle release from cultured myotubes under vibration stimulation

Skeletal muscle-derived extracellular vesicles (SkM-EVs) are promising mediators of intercellular communication, yet practical strategies to safely and efficiently enhance their release remain limited. Vibration is a non-invasive, parameter-controlled mechanical stimulus that is already widely used in musculoskeletal rehabilitation, making it an attractive candidate for enhancing SkM-EV release. However, it remains unclear whether vibration increases EV release and which vibration parameter (amplitude, frequency, or acceleration) regulates this effect. Accordingly, we investigated the parameter dependence of this process and the corresponding effective intensity.

Differentiated C2C12 myotubes were exposed to sinusoidal vibration using a custom-built device. Two parameter-decoupled series were designed: an acceleration-variation series and an amplitude-variation series. EVs were isolated by ultracentrifugation and analyzed by western blotting and tunable resistive pulse sensing (TRPS). RNA sequencing with gene set enrichment analysis (GSEA) was used to identify pathways associated with vibration-induced EV secretion.

Vibration did not reduce cell viability. SkM-EV concentration remained unchanged across acceleration conditions but significantly increased under medium-amplitude (MAm) stimulation. Transcriptomic analysis showed that MAm downregulated Wnt signaling and upregulated the NOD-like receptor pathway, with specific upregulation of Rab27a and Nlrp4e.

Vibration promotes SkM-EV secretion in an amplitude-dependent manner, with the medium-amplitude (MAm) condition being the most effective. This effect appears to be mediated by modulation of the Wnt-Rab27a axis and NLR-Nlrp4e signaling.