[Autophagy induced by cyclic mechanical stretch inhibited caspase-dependent apoptosis of myoblasts].

PURPOSE: The aim of this study was to investigate the molecular mechanism of autophagy and apoptosis induced by cyclic mechanical stretch and the potential role of autophagy in stretch-induced apoptosis of myoblasts. METHODS: Loading model of L6 myoblasts was established in vitro. The cells were then subjected to cyclic mechanical stretch involving 3 s of 15% stretch alternating with 3 s of relaxation. The cells were collected after mechanical stretch for 6 h, 12 h and 24 h, respectively. Control cells were cultured on the same plates without mechanical strain. Apoptosis of myoblasts was assessed by Hoechst 33258 staining and Annexin V binding and propidium iodide staining. Autophagy was determined by MDC staining and transmission electron microscopy(TEM). The level of proteins associated with apoptosis and autophagy was detected by Western blot. The data were analyzed with SPSS 17.0 software package. RESULTS: The results of Hoechst 33258 staining and Annexin V binding and propidium iodide staining indicated that mechanical stretch notably induced apoptosis of myoblasts. Caspase inhibitor z-VAD-fmk effectively abrogated apoptosis of myoblasts, indicating mechanical stretch induced caspase-dependent apoptosis. In addition, the results of TEM, MDC staining and Western blot proved that mechanical stretch elicited autophagy of myoblasts. Inhibition of autophagy using 3-MA enhanced caspase-dependent apoptosis induced by mechanical stretch. CONCLUSIONS: Cyclic mechanical stretch induced apoptosis and autophagy of myoblasts time-dependently. Protective autophagy, acting as the compensatory mechanism, inhibited caspase-dependent apoptosis induced by mechanical stretch.

[Role of calcineurin-nuclear factor of activated T cells signaling pathway in myoblast apoptosis induced by cyclic tensile strain].

OBJECTIVE: This study investigated the role and mechanism of calcineurin (CaN)-nuclear factor of activated T cells (NFAT) pathway in the myoblast apoptosis induced by cyclic tensile strain. METHODS: Myoblasts were cultured using an in vitro-mechanical stimulation model and imposed with tension for different hours with a multi-channel cell stress loading system. Cyclosporine (CsA) was used as CaN inhibitor to clarify the role of CaN in the apoptosis induced by cyclic stress. Hochest 33258 staining and flow cytometry detection were performed to detect the apoptotic cells. Real-time polymerase chain reaction was conducted to detect the mRNA expression of CaN and NFAT. Protein levels of NFAT3 were evaluated by Western blot. RESULTS: The apoptosis rate increased with the extension of loading time. The mRNA expression of the CaN subunits, CnA and CnB, and the protein levels of NFAT3 also increased. When the myoblasts were incubated with CsA, the apoptosis rate decreased, the mRNA expression of CnA and NFAT3 significantly decreased, and the NFAT3 protein expression levels became significantly lower than those of the groups without CsA. CONCLUSION: Continuous cyclic tensile stress can induce myoblast apoptosis. The CaN-NFAT signaling pathway may be involved in the cyclic stretch-induced apoptosis of myoblasts.