Objective: To investigate the effects of Sirtuin 3 on cytochrome C (CytC) release and caspase-3 activation in myocardiac H9c2 cells with calcium overload.
Methods: H9c2 cells were randomly divided into four groups: control group, Sirtuin 3 group, ionomycin group, ionomycin & Sirtuin 3 group. Myocyte calcium overload models were established by ionomycin revulsion. The levels of CytC and caspase-3 in mitochondria and cytoplasm were examined by Western blotting. Intracellular caspase-3 activity was examined by spectrophotometry. H9c2 cell apoptosis rate in each group was detected by annexin V-FITC/PI double staining combined with flow cytometry.
Results: Calcium overload models of H9c2 cells were successfully established. Western blotting showed that CytC level in mitochondria of the ionomycin group was significantly lower than that in the control group, and that it was significantly higher in the ionomycin & Sirtuin 3 group than that in the ionomycin group. Changes of CytC level in cytoplasm was just opposite to that in mitochondria. Caspase-3 level in the ionomycin group was significantly higher than that in the control group, and it was significantly lower in the ionomycin & Sirtuin 3 group than that in the ionomycin group. Spectrophotometry showed that caspase-3 activity in the ionomycin group was significantly higher than that in the control group, and that it was obviously lower in the ionomycin & Sirtuin 3 group than that in the ionomycin group. Flow cytometry combined with double staining revealed that the apoptosis rate in the ionomycin group was significantly higher than that in the control group, and that it was significantly lower in the ionomycin & Sirtuin 3 group than that in the ionomycin group.
Conclusion: Sirtuin 3 could inhibit cell apoptosis by reducing CytC release and inhibiting caspase-3 activation in cardiomyocyte models with calcium overload.