Study On The Apoptosis In H9c2 Cardiomyocytes Induced By Cadmium Chloride And Its Mechanisms

Cadmium, one of nonessential elements, is a very important environment pollutant. It is showed in recent researches that myocardiocytes are so sensitive to cadmium of which lower dosage could produce toxicity, while it may be harmless to other organs. The generation of some heart diseases, such as ischemia/reperfusion injury, myocardial infarction, ventricular wall reconstruction and cardiac failure, is related to cell apoptosis, hence, cell apoptosis and necrosis may be the major mechanism of cardiac toxicity causing by cadmium. This study is designed to establish cell apoptotic model from rat myocardiocytes cell line H9c2 induced by cadmium chloride to discuss related mechanisms and finally provide theoretical basis for research on cardiovascular toxic effect caused by cadmium.1.Toxicity of CdCl2 on H9c2 cellsMTT assay was performed to detect the toxicity after 6, 12 and 24 h exposure to CdCl2 (0, 5, 10, 30, 50 and 80μmol/L). It was showed the survival rate decreased with the increase of dosage and duration of exposure to CdCl2. Six h after exposure to 10μmol/L CdCl2,the A value decreased to 0.41 and survival rate to 87.4%, there was significant difference in survival rate between CdCl2 group and control group (P < 0.05). However, within a certain dosage and duration of exposure to CdCl2 (6 h and 12 h after exposure to 10~ 30μmol/L CdCl2), the change of survival rate was not obvious; with 80μmol/L CdCl2 or prolonged time exposure to it, half of the cells died. The results suggest that cardiomyocytes are sensitive to cadmium and a certain tolerance in certain dosage.2.DNA damage of H9c2 cellsSingle cell gel electrophoresis (SCGE) assay was used to examine DNA damage of H9c2 cells 6, 12 and 24 h after exposure to CdCl2 (0, 5, 10, 30, 50 and 80μmol/L). When exposed to 5μmol/L CdCl2 for 6 h, there was no damage in H9c2 cells and the comet tail was not observed. However, the comet tail more prolonged and DNA damage more aggravated with the increase of CdCl2 12 h after exposure to 30μmol/L CdCl2, and there was significant difference between CdCl2 group and control group (P﹤0.05), suggesting CdCl2 could cause DNA damage of myocardiocytes with a dose-effect manner. The mechanisms of above changes could be the direct or indirect interaction between Cd and DNA. Cd could influence DNA phosphoric acid or basic groups to induce DNA damage, or injure DNA repair system or inhibit activities of DNA repairase.3.Apoptosis detected by AO/EB double fluorescent dye methodThere were distinctive changes on cell apoptosis observed under fluorescence microscope 6, 12 and 24 h after exposure to CdCl2 (0, 5, 10, 30, 50 and 80μmol/L). The nucleus was in the center with flavovirens fluorescence in the normal cells; while it was concentrated into crescent-shape in the side of cells dyed with flavovirens fluorescence within apoptotic cells. When cell necrosis happened, myocardiocytes have already or near disintegrated with enlarged body, blurred boundary and nucleus dyed with asymmetrical almon pink fluorescence.4.Apoptosis detected by FCM with AnnexinⅤ/PI double fluorescent staining Cell apoptosis was observed by FCM 6, 12 and 24 h after exposure to CdCl2 (0, 5, 10, 30, 50 and 80μmol/L). When exposed to 5μmol/L CdCl2 for 6 h, cell apoptotic rate increased to 47.0%, and there was significant difference between CdCl2 group and control group (P﹤0.001). The apoptotic rate only increased slightly 24 h and there was not significant difference between group and control group. It was indicated that CdCl2 could cause H9c2 apoptosis within a certain dosage and time of exposure while cell itself has tolerance against CdCl2. This changes of cell apoptotic rate after exposure to CdCl2 could be explained by the detoxicant effect of metallothionein (MT). However, when chelate ability of MT reaches maximum, it could not antagonize the toxicity of cadmium, and myocardiocytes would change from apoptosis to necrosis because of the loss of protection.In this study, two methods were applied to detect cell apoptosis and the cell apoptosis rates detected with both were same. AO/EB double fluorescent dye method could distinguish early and late cell apoptosis from cell necrosis according to morphological changes of cell nucleus while early cell apoptosis mainly detected with flow cytometer (FCM).5.PARP clearage of H9c2 cellsWestern blot was applied to detect PARP activity 24 h after exposure. The characteristic 89 kD fragment of PARP was observed in 30 ~ 80μmol/L CdCl2 groups, with the increase of dosage, more fragments appeared and PARP almost completely clearage at 80μmol/L group.6.Changes of expression of Bcl-2, Bax, Cyt c, caspase– 9 and AIF Immunocytochemical method was used in this experiment. With the increase of CdCl2, the expressions of both Bcl-2 and Bax appeared a ascend tendency and increased to the highest at 10μmol/L group, then Bcl-2 expression decreased while Bax expression maintained at the previous level resulted in the value of Bcl-2/Bax increased at first then decreased from 10μmol/L group leading to the occurrence of apoptosis. The expression of Cyt c enhanced with the adding of CdCl2 while weakened at 10μmol/L group, this probably was related to Bcl-2 inhibiting Bax expression while promoting the expression of Cyt c. Expression of caspase -9 also increased with the increase of CdCl2, and apoptosis probably was initiated by the Cyt c - caspase- 9 complex.The expression of AIF increased with the adding of CdCl2 and the expression could be observed in nucleus at 5μmol/L groups while at 80μmol/L group, positive AIF expression in nucleus could appear in almost all cells. AIF could trigger non-caspases dependent apoptosis. When mitochondrial membrane permeability increased, AIF was released to cytoplasm from mitochondria then translocated to nucleus resulting in abnormal chromatin clumping and DNA gragmentation finally lead to the occurrence of apoptosis.7.Changes of mRNA expression of Bcl-2RT-PCR was performed in this experiment. Total RNA of myocardiocytes 24 h after exposure to CdCl2 was extracted to do RT-PCR. In 10μmol/L CdCl2 group, mRNA expression of Bcl-2 enhanced and then weakened; in both 30 and 80μmol/L CdCl2 groups, the expression decreased; in 50μmol/L CdCl2 group, the expression increased as compared with that in control group. It was indicated that Cd could induce Bcl-2 expression alteration to influence cell apoptosis.8.Molecular mechanisms of cell apoptosis of rats myocardiocytes H9c2 induced by cadmium chlorideDuring the early exposure to CdCl2, the stress reaction of myocardiocytes was triggered and PARP activated then bind to DNA for reparation; anti-apoptotic factor Bcl-2 increased to resist this external irritant; meanwhile, MT was activated to inhibit toxic effect of Cd2+ by chelating it and forming Cd2MT. Therefore, within a certain dosage and duration of exposure to CdCl2, the cell damage and apoptosis do not change, which indicate that the self defense mechanism of myocardiocytes has be triggered by apoptotic signals. With the increase of dosage and time of exposure to CdCl2, the cells suffered from heavier and heavier damages which could no longer repair DNA. During the process, PARP changed its role from maintaining the stability of cells to inducing apoptosis and removing damage cells. During apoptosis, PARP was deactivated to avoid unnecessary DAN repair in dead cells and save energy for completing a series of biochemistrical cascade reaction to ensure the successful apoptosis process and avoiding formulation of mutant cells, eliminating potential threat of tumor. At the same time, the expression of Bcl-2 in myocardiocytes weakened, in order mitochondrial membrane permeability enhances, and AIF releases from mitochondrial to cytoplasm then gets into nucleus and causes abnormal chromatin clumping and DNA gragmentation, finally leading to the occurrence of apoptosis.During cell apoptosis, all kinds of factors interact together. Cell apoptosis induced by Cd might depend on nuclear transfer of AIF. AIF could trigger non-caspases dependent apoptosis, while the activation of PARP had a close relationship with caspase-3. The inhibition of PARP expression could prevent the nuclear transfer of AIF, and bcl-2 was closely related to cytochrome (Cyt c) in mitochondrial pathway.In this study, it was proved that Cd could induce cell apoptosis, nuclear transfer of AIF, cleavage of PARP and changes in Bcl-2 expression in rat H9c2 myocardiocytes. The results suggest that there are several relating and interacting pathways in myocardial cell apoptosis, which provide theoretical basis for researches on mechanisms of Cd toxicity.