Mitochondrial Pathway Involved In H9c2 Cardiomyocytes Apoptosis Induced By N, N-dimethylformamide

Objective To investigate whether mitochondrial pathway was involved in H9c2 cardiomyocytes apoptosis induced by dimethylformamide(DMF), and whether the change of intracellular redox level was the trigger of mitochondrial pathway. Methods We observed the morphological changes of H9c2 cardiomyocytes and nucleus after DMF exposure. Western Blotting was used to detect the the levels of Procaspase-3, Cleaved caspase-3, Procaspase-9, Bax, Bcl-2 and Cytochrome C(Cyt-c) in cytoplasm after different doses of DMF(0 m M, 50 m M, 100 m M, 200 m M) exposure for 24 h, the levels of Cleaved caspase-3, Bcl-2, Cyt-c in cytoplasm after 200 m M DMF exposure for different time intervals(0h, 2h, 4h, 8h, 12 h, 24h) and 4 m M NAC intervention for 1h. Immunofluorescence(IFC) was used to observe the location of Cyt-c after 200 m M DMF exposure for different time intervals and 4 m M NAC intervention for 1h. Results Most of the cells became shrunken, some even became rounded and suspended in the culture medium. Some nucleus showed different degrees of chromatin condensation after DMF exposure. After different doses of DMF(0 m M, 50 m M, 100 m M, 200 m M) exposure for 24 h, the levels of Procaspase-3 and Bax increased. The level of Procaspase-9 and Bax decreased, while Cleaved caspase-3 and Cyt-c increased. The differences between groups were statistically different(Procaspase-3: F=16.11, P=0.0009; Bax : F=60.31, P < 0.0001; Procaspase-9: F=16.66, P=0.0008; Bcl-2: F=577.8, P<0.0001; Cleaved caspase-3: F=23.33, P=0.0003; Cyt-c: F=104.0, P<0.0001). The levels of Procaspase-3 and Bax increased after 50 m M and 100 m M DMF exposure compared with control group(P<0.05), while the difference between 200 m M DMF exposure and control group were not statistically significant. The level of Procaspse-9 decreased in 200 m M exposure group compared with control group, the difference was statistically significant(P<0.05). The level of Bcl-2 exposed under 50 m M, 100 m M and 200 m M DMF decreased compared with the control group, the differences were statistically significant(P < 0.05). Cleaved caspase-3 and Cyt-c increased after 100 m M and 200 m M exposure compared with control group, the differences were significant(P<0.05). The ratio of Cleaved caspase-3/Total caspase-3 and Bax/Bcl-2 in 100 m M and 200 m M exposure group increased compared with control group, the differences were significant(P<0.05). After 200 m M DMF exposure for different time intervals(0h, 2h, 4h, 8h, 12 h, 24h), the levels of Cleaved caspase-3 and Cyt-c increased, Bcl-2 decreased, the differences were statistically significant(Cleaved caspase-3: F=503.7, P<0.0001; Cyt-c: F=51.42, P<0.0001; Bcl-2: F=73.37, P<0.0001). The levels of Cleaved caspase-3 increased when exposed for different time intervals(2h, 4h, 8h, 12 h, 24h) compared with the control group and the differences were statistically significant(P<0.05). The level of Cyt-c increased after 12 h and 24 h exposure compared with control group(P<0.05). Bcl-2 level decreased when exposed for different time intervals(2h, 4h, 8h, 12 h, 24h) compared with the control group, the differences were statistically significant(P < 0.05). The differences of Cleaved caspase-3, Cyt-c, Bcl-2 levels between 4 m M N-Acetyl-L-cysteine(NAC) intervention for 1h followed by 200 m M DMF exposure group, only 200 m M DMF exposure group, only 4 m M NAC intervention for 1h group and the control group were statistically significant(Cleaved caspase-3: F=28.98, P<0.0001; Cyt-c: F=26.14, P=0.0002; Bcl-2: F=18.52, P=0.0006). The levels of Cleaved caspase-3 and Cyt-c after NAC intervention decreased compared with 200 m M DMF exposure group(P < 0.05). Bcl-2 level increased compared with 200 m M DMF exposure group, while the difference was not statistically significant. Immunofluorescence(IFC) showed a punctate distribution of Cyt-c in the cytoplasm of normal cells, with the extension of exposure time intervals Cyt-c dispersed into the cytoplasm, the outline of the nucleus were not as clear as control group. For 4 m M NAC intervention for 1h followed by 200 m M DMF exposure group, the dispersion of Cyt-c was improved compared with DMF exposure group, and some cells showed clearer outline of the nucleus. Conclusion DMF exposure up-regulated the levels of Cleaved caspase-3 and Cyt-c, down-regulated the level of Bcl-2. Eventually, the ratio of Cleaved caspase-3/Total caspase-3 and Bax/Bcl-2 increased. Mitochondrial pathway was involved in H9c2 cardiomyocytes apoptosis induced by DMF; moreover, the change of intracellular redox level was the trigger of mitochondrial pathway apoptosis...