ALDH2 Prevents Hypoxia Myocardial Injury In Rat

Cardiac mitochondria plays a key role in the ischemic pathogenesis of heart failure. The apoptotic regulatory proteins mediated by injuried mitochondria have a multifactorial and multistage pathogenesis. Despite extensive studies, the fundamental mechanisms responsible for the development and progression of heart failure have not yet been fully elucidated. Recent technological advances in proteomics allow us for the first time to examine the expression profiles at protein level on a genome-wide scale. In our study, we used 2-DE and LC- MS to identify the proteins with different expressing level in rat infarction hearts. We got a group of significant differential proteins including ALDH2 protein, which may be related to the pathogenesis of heart failure. We also drew a positive conclusion through further biological analysis and validation with the specific inhibitor and transgene, and performed a series experiments related to cell apoptosis and signal route with cellular and molecular techniques.PART ONE Aldehyde dehydrogenase 2 (ALDH2) proteomic analysis of ischemic myocardialmitochondrial protein expression in ratsAbstract Aim To determine the metabolic changes in rat ischemic heart, we applied the method of proteomics to discover the possible protein variation in rat myocardial mitochondria. Methods Myocardial infarction model was established by left anterior descending coronary artery ligation. 4 weeks after operation, we used LVEF 46.4±10.9% as myocardial infarction model. The rats were killed and their left ventricules were cut off. Those mitochondrial morphologies were observed by electron microscope, and the mitochondrial proteins were extracted. Weight-age matched rats acted as control. The mitochondrial protein expression in two groups were analyzed by two-dimensional gel electrophoresis and LC-mass spectrometry, and were identified using database comparision. After found the significant changes, RT-PCR and western blot methods would be used to prove this expression difference at the time of 3d 7d 14d and 28d's myocardial infarction. On the other hand, we made the hypoxia model from myocytes with enzymatic digestion and their mitochondrial protein activity from mycytes was measured at different hypoxia time. Results The mitochondria showed significant pathological changes in rat ischemicheart tissue under the transmission electron microscope, including shape swelling, matrix thinning and cristea breakdown. And 3 protein spots altered significantly were found. Aldehyde dehydrogenase 2 (ALDH2) was identified with database comparision. Expression of ALDH2 decreased remarkably in ischemic heart tissue compared with control. Furthermore, we detected the ALDH2 mRNA and its protein expression of rats at 3d, 7d, 14d, 28d after LAD ligation, and the results showed it decreased gradually after operation. The decrease of ALHD2 enzyme activity also showed hypoxia-time dependant in vitro by the method of acetaldehyde metabolism (the purity of cardiac mitochondria was more than 90% under the electron microscope) . Conclusion The expression of mitochondrial ALDH2 in rat ischemic myocytes decreased and also its enzyme activity showed hypoxia-time dependant in vitro.PART TWO The mechanism study of Daidzin, inhibitor of Aldehyde dehydrogenase 2, ininjuring the rat cardiomyocytes induced by hypoxiaAbstract Aim To observe and verificate the effect of inhibited ALDH2 on cardiac myocyte induced by hypoxia, including apoptosis, MAPK signal route, and the role of ALDH2. Methods Cultured cardiomyocytes of neonatal rats were used. Hypoxia 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 by Hoechest 33324, Fluorescence Activated Cell Sorting (FACS) and The DeadEnd^TM Fluorometric TUNEL System, and MAPK signal pathway (ERK1/2 JNK P38) by western blotting. Results ALHD2 enzyme activity of myocytes could be inhibited by daidzin (24h, 20-100 μ M) without induction of apoptosis, the most effective but no apoptotic concentrantion was 60 μ M. When exposed to hypoxia, however, the ROS and apoptisis was significantly increased in the cells pretreated with daidzin compared to those without the pretreatment (P<0.05), also an increased MAPK phospholyrated activation, including ERK1/2, JNK and P38 (P<0.05). Conclusion Myocytes showed decreased tolerance to hypoxia after ALDH2 specifically inhibited by daidzin, The reduction of ALDH2 activity might increase the susceptivity of myocytes to apoptosisfollowing hypoxia and the increase of ROS may related to MAPK activation, suggesting a protective role of ALDH2 in hypoxia-induced myocardial injury. PART THREE The mechanism study of Aldehyde dehydrogenase 2 transgene in preventing ratcardiomyocytes from apoptosis induced by hypoxiaAbstract Aim To observe the effect of wild and mutant type of ALDH2 gene on cardiomyocyte induced by hypoxia, including apoptosis and MAPK cell signal route. Methods Cultured cardiomyocytes of neonatal rats were used. Hypoxia was imposed to the cardiomyocytes with transgene ALDH2 (wild type-ALDH2*1 and mutant type-ALDH2*2) to observe the change of ROS, 4-HNE, apoptosis, MAPK(ERK1/2 and JNK), bcl2 and P53. ALDH2 activity was measured by the method of acetaldehyde metabolism, ROS was measured by C400, 4-HNE by immonohistology, apoptosis by Hoechest 33324, Fluorescence Activated Cell Sorting (FACS) and The DeadEnd^TMColorimetric TUNEL System, and MAPK signal pathway by western blotting, bcl2 and P53 by RT-PCR and western blot. Results myocytes had a different tolerance to hypoxia after transgene of ALDH2 wild and mutant type. The wild and mutant ALHD2 transgene (ALDH2*1/ALDH2*2) had no effect on ROS and MAPK phosphoryrated activation including ERK1/2 and JNK (P>0. 05) . But in transgenic mutant type-ALDH2*2 myocytes induced by hypoxia (compared with its inhibitor daidzin), besides the apoptotic cells, including Hoechest 33324 staining (karyopyknosis and karyorrhexis), FACS and TUNEL, 4-HNE increased significantly (P<0.05). Furthermore the transgenic wild type-ALDH2*1' induced by hypoxia, their expression of higher bcl2 and lower P53 were quite different from mutant type-ALDH2*2's(P<0.05). Conclusion 4-HNE might be an important factor in myocytes oxidative injury. ALDH2 Transgene had no obvious effect on ROS and MAPK cell signal route. And the anti-apoptotic function from ALDH2*1 might be related to bcl2-P53 without ROS-MAPK dependant route. Myocytes showed decreased tolerance to hypoxia after transgene of ALDH2*2 and might increase the susceptivity of myocytes to apoptosis, suggesting a protective role of ALDH2 in hypoxia-induced myocardial injury.Conclusion1. The expression of ALDH2 in rat ischemic hearts decreased and also its enzyme activity showed hypoxia-time dependant in vitro.2. Myocytes showed decreased tolerance to hypoxia after ALDH2 specifically inhibited by daidzin, The reduction of ALDH2 activity might increase the susceptivity of myocytes to apoptosis following hypoxia and the increase of ROS may related to MAPK activation.3. The transgenic ALDH2*1/ALDH2*2 myocytes induced by hypoxia had no obvious effect on ROS and MAPK(ERK1/2 and JNK) cell signal route. ALDH2*1 could degrade 4-HNE and its anti-apoptotic function might be related to bcl2- P53 dependant route without ROS-MAPK's.4. Under the lower ALDH2 enzyme activity circumstances, myocytes showed decreased tolerance to hypoxia and might increase the susceptivity of myocytes to apoptosis, suggesting a protective role of ALDH2*1 in hypoxia-induced myocardial injury.The potential application and novelty of this project1. Recent technological advances in proteomics allow us for the first time to find the decreased ALDH2 expression profiles in ischemic and hypoxia rat myocytes. And our study confirmed the ALDH2 protective function in heart.2. Those with myocardial infarction and hypertension increase the susceptivity to heart failure due to the reduction of ALDH2 activity and also have a worse prognosis. We put forward that ALDH2*1 can prevents myocytes from hypoxia injury, which might discover new possible therapeutic targets in clinic.