C2C12‐Derived ApoVs Promote Skeletal Muscle Development and Ameliorate Age‐Related Muscle Loss Through Igf1r/PI3K/AKT/mTOR Pathway

BACKGROUND: Apoptosis coincides with the differentiation of skeletal myoblasts, and numerous studies have shown that the apoptotic activity is required for myogenic differentiation. Although the role of apoptosis in skeletal muscle differentiation has been well documented, its mechanism is largely unknown. METHODS: Apoptotic extracellular vesicles (apoVs) were extracted from differentiated C2C12 cells or STS-treated undifferentiated C2C12 myoblasts. C2C12 myoblasts, 8-week-old male mice or 15-month-old male mice were used as in vitro and in vivo models, respectively. These models were treated with C2C12-derived apoVs to explore the biological function and mechanism of apoVs in myogenic differentiation, skeletal muscle development and aging. RESULTS: Proteomic analysis revealed that inhibition of apoptotic activity by Z-VAD-FMK (ZVAD) affected extracellular components. Using immunofluorescence staining, western blotting and transmission electron microscopy analysis, our results demonstrated the generation of apoVs during myogenic differentiation. C2C12-derived apoVs exhibited a typical double-membrane spherical structure, phosphatidylserine exposure and were highly positive for the general apoV markers cleaved caspase 3 (CASP3), Alix and TSG101. Inhibition of apoptotic activity significantly reduced (p = 0.0029) the protein level of myosin heavy chain (1.05 in the Con group vs. 0.38 in the ZVAD group) accompanied by a marked decrease (p < 0.0001) in apoV production (5.91e+10 ± 8.93e+09 in the Con group vs. 1.77e+10 ± 1.36e+09 in the ZVAD group). Proteomic analysis of apoVs suggested that C2C12-derived apoVs contain multiple pro-differentiation proteins, including insulin-like growth factor 1 receptor (Igf1r). ApoVs could be taken up by recipient cells and subsequently rescued the impaired C2C12 differentiation induced by ZVAD (p < 0.0001) and promoted the normal myogenic differentiation process (p = 0.0081) by carrying Igf1r and promoting PI3K/AKT/mTOR activation. Knockdown of Igf1r or inhibition of PI3K activation diminished the positive role of apoVs. In addition, apoV treatment promoted skeletal muscle development in 8-week-old male mice (n = 6, Cohen's d = 1.993, power = 0.874) and relieved age-related muscle loss (n = 6, Cohen's d = 3.97, power = 0.999). CONCLUSIONS: In summary, this study demonstrates the generation of apoVs during myogenic differentiation. The apoVs derived from skeletal muscle cells promote skeletal muscle cell differentiation and delay age-related muscle loss. These results provide a theoretical basis for elucidating the mechanism of skeletal muscle development and treating skeletal muscle-related diseases.