Hsp70 On Hydrogen Peroxide - Induced C - ₂ C ₁₂ Smac Muscle Cells Release From Mitochondria And Apoptosis

Reactive oxygen species (ROS) are important mediators which contribute to acute myocardial injury. ROS can result in not only cadiomyocyte necrosis, but also cadiomyocyte apoptosis by damaging the chromatin and DNA. Smac/DIABLO is a novel pro-apoptotic protein which is normally located in mitochondria and released into cytosol when cells undergo apoptosis. Smac/DIABLO in cytosol can neutralize inhibitors of apoptosis proteins (lAPs) and play important roles in the mitochondria-initiated caspase activation. The expression of Smac/DIABLO is very high in the myocardium. However, whether ROS can induce the release of Smac/DIABLO from mitochondria and promote apoptosis in cardiomyocyte is still unclear. In addition, there are growing evidences showing that heat shock proteins (HSPs) can protect cells against injury and several HSPs can exert their anti-apoptotic functions downstream or upstream of caspase-3 like proteases. But it is still obscure whether HSPs protect against apoptosis through interfering with Smac/DIABLO.In order to elucidate these problems mentioned above, C2C12 myogenic cells were exposed to H2O2 (0.5mmol/L). Apoptotic morphological changes and percentage of apoptotic nuclei were assayed with Hoechst 33258 staining. DNA ladder was observed by agarose gel electrophoresis. The release of Smac/DIABLO from mitochondria to cytoplasm was analysed by Western blotting and indirectimmunofluorescence. Activities of caspase-9, 3 were assayed with Caspase Colorimetric Assay Kit and Western blotting. Smac and HSP70 genes were overexpressed by the transfection of their full length cDNA plasmids so as to further analyze the effects of HSP70 on the release of Smac from mitochondria, activation of caspase-9 and caspase-3, and accurance of apoptosis in C2C12 myogenic cells.The results showed that: (1)H2O2 (0.5mmol/L) resulted in a marked release of Smac/DIABLO from mitochondria to cytoplasm 1hr after treatment, activation of caspase-9, caspase-3 4hr after treatment and specific morphological changes of apoptosis 24hr after treatment; (2) Heat shock pretreatment (42C, 1h) could increase the expression of HSP90, HSP70, and aB-crystallin and could inhibit H2O2-induced release of Smac/DIABLO from mitochondria to cytoplasm, activation of caspase-9, 3 and apoptosis in C2C12 myogenic cells; (3) Smac/DIABLO overexpression significantly promoted H2O2-induced apoptosis in C2C12 myogenic cells as showed by specific DNA ladder pattern, increase of percentage of apoptotic nuclei, and by activities of caspase-9, caspase-3; (4) HSP70 overexpression could inhibit H2O2-induced and Smac/DIABLO-promoted activation of caspase-9, 3 and apoptosis in C2C12 cells; (B)HSP70 overexpression could inhibit the release of Smac/DIABLO from mitochondria induced by H2O2.It was concluded that: (1)Mitochondria! signal transduction pathway was involved apoptosis induced by H2O2 in C2C12 myogenic cells; (2) The release of Smac/DIABLO frommitochondria to cytoplasm and the subsequent activation of caspase-9, 3 played important roles in H2O2-induced apoptosis in C2C12 myogenic cells; (1) HSP70 inhibited Smac/DIABLO release from mitochondria, activations of caspase-9, 3 and protected C2C12 myogenic cells against H2O2-induced apoptosis.