Aldehyde Dehydrogenase-2 Inhibition Of Oxidative Stress Caused By Cardiac Myocyte Apoptosis And Its Molecular Mechanism

Multiple risk factors are involved in the cardiac ischemia reperfusion injury, and increasing evidences have suggested that a burst of reactive oxygen species (ROS) in reperfusion is a major contributor in triggering myocardial injury. Approaches to reduce excess ROS are important to attenuate myocardial injury and keep cardiac function. Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme and belongs to one of the members of the aldehyde dehydrogenase gene family. The best known role for ALDH2 was to catalyze the oxidation of acetaldehyde to acetic acid in ethanol metabolism. Recently it has been proved that ALDH2 protects the heart from ischemia and reperfusion injury. Our experiment showed that inhibition of activation of ALDH2 enhanced the cardiomyocytes apoptosis induced by antimycin A. Although multiple mechanisms are involved in the myocyte apoptosis induced by ischemia and hypoxia, ALDH2 prevented cardiomyocytes from apoptosis induced by ischemia and hypoxia through detoxification of 4-hydroxy-nonenal (4-HNE) and suppression of p53.Aims:The role of aldehyde dehydrogenase 2 (ALDH2) in antimycin A induced cardiomyocytes apoptosis was investigated by suppressing ALDH2 activity with a specific ALDH2 inhibitor daidzin.Methods:Cultured cardiomyocytes of neonatal rats were used. Antimycin A was imposed to the cardiomyocytes with or without daidzin pretreatment beyond the optional daidzin concentration. ALDH2 activity was measured by the method of acetaldehyde metabolism, ROS was measured by C400. Apoptosis was measured by Fluorescence Activated Cell Sorting system (FACS) and Hoechest 33324 staining methods. Western blot was used to detected MAPK signaling pathway.Results:Daidzin (60μM) effectively inhibited ALDH2 activity by 50% without own effect on cell apoptosis and significantly enhanced antimycin A-induced cardiomyocytes apoptosis from 33.5±4.4% to 56.5±6.4%(Hochest method, p<0.05), and from 57.9±1.9% to 74.0±11.9%(FACS, p<0.05). Phosphorylation of activated MAPK signaling pathway, including extracellular signal-regulated kinase (ERK1/2), c-Jun NH2-terminal kinase (JNK) and p38 was also increased in antimycin A and daidzin treated cardiomyocytes compared to the cells treated with antimycin A alone.Conclusion:Suppression of mitochondrial ALDH2 activity by daidzin could aggravate antimycin A-induced cardiomyocytes apoptosis through activation of MAPK signaling pathway. These findings indicated that modifying mitochondrial ALDH2 activity/expression might be a potential therapeutic option on reducing oxidative insults induced cardiomyocytes apoptosis.Aim:To explore the molecular mechanism involved in the protection of ALDH2 from myocytes apoptosis induced by ischemia and hypoxiaMethods:Wild type and ALDH2 gene knockout mice were subjected to myocardial infarction. Immunohistochemistry staining was used to examine the expression of 4-hydroxy-nonenal (4-HNE) and p53 in the infarcted area. Using a Langendorff apparatus, hearts of mice were perfused with 4-HNE or subjected to a period of 30min ischemia followed by a perfusion period of 60 min. Cross-sectional slices derived from the hearts were stained by Nagar-Olsen and TUNEL assay to measure the infracted area and apoptosis cells. Expression of p53 in the slice was stained by immunostaining. The cultured neonatal rat cardiomyocytes transfected with ALDH2 gene or empty adenovirus vectors were exposed to hypoxia, the expression and activity of p53 were examined by western blot. The cultured cardiomyocytes were treated with 4-HNE with or without pifithrin-a, the inhibitor of p53, apoptosis cells were measured by TUNEL staining. Western blot were used to detect the expression of p-ERK and p-JNK in neonatal rat cardiomyocytes treated with 4-HNE. The expression of p53 was monitored when the 4-HNE treated cardiomyocytes were pretreated with the inhibitor of ERK or JNK.Results:The expression of 4-HNE and p53 in the infarcted heart were obviously increased in ALDH2 knockout mice than those in the wild type mice's infarcted heart, while the wild type mice transfected with ALDH2 gene had a less expression of 4-HNE and p53. The infarcted area and apoptosis cells in the hearts perfused with 4-HNE were increased significantly, which were similar to the cardiac ischemia reperfusion injury.4-HNE increased the expression of p53 ex vitro. Overexpression of ALDH2 gene in cardiomyocytes in vitro reduced the expression of p53 induced by hypoxia. Our experiment showed that inhibition of p53 attenuated cardiomyocyte injury induced by 4-HNE. Western blot revealed activation of ERK, JNK in 4-HNE treated myocytes, the inhibitor of JNK attenuated the expression of p53 induced by 4-HNE.Conclusion:The expression of 4-HNE in the cardiomyocytes is induced by ischemia and hypoxia. The enhanced 4-HNE is harmful to myocardium and induces cardiomyocyte apoptosis. The myocyte apoptosis induced by 4-HNE is through the activation of JNK and p53. The molecular mechanisms by which ALDH2 protect the heart from ischemia and hypoxia injury are mediated by detoxification of 4-HNE and inhibition of p53.