Release of micro-vesicles during C2C12 myogenic differentiation process

Skeletal muscle is highly plastic tissue able to adapt to different stress, in part due to its remarkable regenerative capacity. Many growth factors have been implicated in the regulation of myoblast proliferation and differentiation to promote muscle regeneration and repair. Tissue specific micro-vesicles (MVs) are exciting candidates for novel signalling factors, in fact, MVs are able to carry proteins, mRNAs and miRNAs contributing to modifying the microenvironment. The C2C12 cell line was used as a model to investigate a potential role of MV secretion in the myogenic differentiation process. Western blotting analysis, using antibodies against well-defined MV markers, showed that Tsg101, Hsp60 were more abundant in T0 MV extracts than T2; while an opposite trend was found for LAMP-1 and RAB5. Histone 2B appeared only at T1 and T2 demonstrating the presence of apoptotic bodies. Furthermore, TEM analysis of MV size distribution showed that myoblasts released MVs of about 42±8 nm in diameter, while differentiating cells released significant larger MVs. These data clearly demonstrate that during differentiation myoblasts release a complex mixture of MVs. Moreover, it was investigated whether MVs contained the myogenic microRNA miR-1, miR-133a, miR-133b and miR-206. This analysis showed different microRNA ratios in MVs respect to cell body. These results represent an important step forward the understanding of muscle regeneration process shedding light on an underestimated aspect as MV cell-to-cell communication.