Biomechanical strain alters the microRNA cargo of primary myogenic cell-derived extracellular vesicles associated with a differentiation phenotype.

Extracellular vesicles (EVs) are membrane-bound vesicles that regulate intercellular signaling by transporting cellular cargo including RNAs, proteins, and lipids. In recent years, EVs have emerged as promising biologic therapeutics for musculoskeletal repair, recapitulating many of the benefits of mesenchymal stromal cells. However, strategies to enhance the therapeutic potential of EVs remains limited. Here, we investigated how cyclic mechanical strain influences the microRNA (miRNA) cargo and function of EVs produced by primary C57BL/6 murine myogenic cells. Specifically, we aimed to determine how biomechanical strain regimens alter myogenic EV cargo and regulate the transcriptome of recipient myoblasts. We identified miR-222 as a miRNA that was significantly and selectively enriched in low-strain long-duration (LSLD) mechanically strained EVs compared to Static EVs and to high-strain short-duration (HSSD) EVs. Recipient primary myoblasts treated with LSLD EVs displayed distinct transcriptomic changes, characterized by a statistically significant overrepresentation of downregulated predicted target genes associated with miR-222-5p and miR-222-3p at 24 h and 72 h after LSLD EV treatment, respectively. This transcriptomic shift correlated with an increase in Myosin Heavy Chain (MyHC) expression in recipient myoblasts. Together, these findings demonstrate that biomechanical strain regulates the packaging of miRNAs within myogenic EVs, and that LSLD EV delivery is associated with a differentiation-related phenotype in recipient myoblasts. This work provides a foundation for future studies utilizing biomechanical cues to tune EV cargo for potential therapeutic applications in muscle repair and regeneration.