Effects And Molecular Mechanisms Of Sirtuin 3 On Apoptosis Of Calcium Overload H9C2 Cardiomyocytes

Objective To explore the effects and molecular mechenisms of exogenous Sirtuin 3 on apoptosis of calcium overload H9c2 cardiomyocytes.Methods H9c2 cardiomyocytes were randomly divided into four groups including group 1 as control group, group 2 as Sirtuin 3 group, group 3 as ionomycin group and group 4 as ionomycin & Sirtuin 3 group. Calcium overload H9c2 cardiomyocyte models in group 3 and group 4 were established by ionomycin revulsion.The intracellular Ca2+concentration of cardiomyocytes([Ca2+]i) in each group were examined by Spectrophotometry. Cyt C expressions in mitochondria and cytoplasm of cardiomyocytes in each group were detected by Western blot. Caspase-3 expressions of cardiomyocytes in cytoplasm of each group were examined by Western blot. The intracellular caspase-3 activities of cardiomyocytes in each group were examined by Spectrophotometry. The cardiomyocyte apoptosis ratios in each group were inspected by flow cytometry combined with Annexin V-FITC/PI double staining.Results [Ca2+]i of cardiomyocytes in each group (nmol/L)were (112.31±20.12), (110.28±19.83), (374.82±26.29), and (186.45±23.14). [Ca2+]i in Sirtuin 3 group had no significant increase compared with that in control group(P>0.05), [Ca2+]i in ionomycin group was significantly increased compared with that in control group(P<0.05). And [Ca2+]i in ionomycin & Sirtuin 3 had significantly decreased compared with that in ionomycin group(P<0.05). Calcium overload models of H9c2 cardiomyocyte were successfully established by ionomycin revulsion and the cardiomyocytes calcium overload had significantly decreased after treated by exogenous Sirtuin 3.Cyt C expressions of cardiomyocytes in each group detected by Western blot were as follows (1) intramitochondria Cyt C expression in Sirtuin 3 group was no difference compared with that in control group(P>0.05), Cyt C in the ionomycin group was significantly lower than that in control group(P<0.05) and Cyt C in the ionomycin & Sirtuin 3 group was significantly higher than that in ionomycin group(P<0.05). (2) Cyt C expression in cytoplasm of Sirtuin 3 group was no difference compared with that in control group(P>0.05), Cyt C in the ionomycin group was significantly higher than that in control group(P<0.05) and Cyt C in the ionomycin & Sirtuin 3 group was significantly lower than that in ionomycin group(P<0.05).Caspase-3 expressions of cardiomyocytes were detected by Western blot showed that there was no difference between Sirtuin 3 and control group(P>0.05), caspase-3 in ionomycin group was much higher than that in control group(P<0.05), caspase-3 in the ionomycin & Sirtuin 3 group was much lower than that in ionomycin group(P<0.05). Caspase-3 activities (nmol/h) detected by Spectrophotometry were (1.231±0.13), (1.128±0.19), (4.825±0.21) and (2.253±0.23). There was no difference of caspase-3 activity between Sirtuin 3 and control group(P>0.05). Caspase-3 activity in ionomycin group was much higher than that in control group(P<0.05) and caspase-3 activity in ionomycin & Sirtuin 3 group was significantly lower than that in ionomycin group(P<0.05).The apoptosis ratios of cardiomyocytes in each group detected by Flow cytometry combined double staining were (29.22±0.3)%, (28.30±0.2)%, (67.24±0.5)% and (38.78±0.7)%. There was no difference of apoptosis ratio between Sirtuin 3 and control group(P>0.05). The apoptosis ratio in ionomycin group was much higher than that in control group(P<0.05) and the apoptosis ratio in the ionomycin & Sirtuin 3 group was significantly lower than that in ionomycin group(P<0.05).Conclusions Exogenous Sirtuin 3 could alleviated apoptosis of calcium overload cardiomyocyte. It was suggested that the possible mechanisms might includ reducing the intramitochondria Cyt C release to cytoplasm, furtherly inhibiting the caspase-3 activation and restraining the initiation of mitochondria apoptosis pathway.