| Bachground:Diabetes mellitus is a common metabolic disorder that can affect patient survival and quality of life because of acute and chronic complications. Cardiovascular complications, including diabetic cardiomyopathy, are the major causes of morbidity and mortality in diabetic patients. This common and serious comorbidity has an asymptomatic onset and is characterized by impaired contractility and relaxation of the left ventricle independent of coronary artery disease or hypertension. However, the molecular mechanisms underlying diabetic cardiomyopathy remain incompletely understood.Hyperglycemia induces the production of reactive oxygen species (ROS), including superoxide anion (O2-), hydroxyl radicals (·OH) and hydrogen peroxide (H2O2). These free radicals play a crucial role in the pathogenesis of diabetic complications. Mitochondrial aldehyde dehydrogenase 2 (ALDH2), the main enzyme responsible for acetaldehyde oxidation in ethanol metabolism, is considered responsible for oxidation and detoxification of aromatic and aliphatic aldehydes such as 4-hydroxy-2-nonenal (4-HNE).4-HNE is a highly cytotoxic aldehyde that is generated from free radicals as a result of lipid peroxidation. ALDH2 has recently been considered a key cardioprotective enzyme during ischemia-reperfusion injury, probably because of its detoxification of reactive aldehydes. Nonetheless, previous studies also indicated that ALDH2 had redox-sensitive thiol group in the active site of the enzyme and thus was susceptible to oxidative inactivation. Objectives:We aimed to investigate the effect of chronic hyperglycemia-induced oxidative stress on ALDH2 activity in diabetic rats; and investigate the association between ALDH2 activity and left ventricular dysfunction of the diabetic heart and possible underlying mechanisms.Methods:Male Wistar rats weighing 200 to 250 g were used in this study. Diabetes was induced by intraperitoneal (i.p.) injection of 60 mg/kg streptozotocin (STZ). Rats were divided randomly into four groups (n=8):control, untreated diabetic, diabetic treated with N-acetylcysteine (NAC) and diabetic treated with a-lipoic acid (a-LA). One week after diabetes induction, NAC and a-LA were administered to the diabetic treated groups by oral gavage for 8 weeks.Rat left ventricular end-diastolic internal diameter (LVEDD), interventricular septal end-diastolic thinkness (IVSD), left ventricular posterior wall end-diastolic thinkness (LVPWD), left ventricular end-systolic internal diameter (LVESD), interventricular septal end-systolic thinkness (IVSS) and left ventricular posterior wall end-systolic thinkness (LVPWS) were measured by echocardiography. Left ventricular ejection fraction (LVEF) and left ventricular short axis fraction shortening (LVFS) were calculated using these measurements. Cardiac structure was detected by H&E staining.Plasma glucose concentration was measured by use of the Glucose analyzer and plasma HbA1c was determined by high performance liquid chromatography (HPLC). Total plasma antioxidant concentration, content of malondialdehyde (MDA) (a reliable index of ROS-induced lipid peroxidation), glutathione (GSH) content and Mn-superoxide dismutase (Mn-SOD) activity in rat heart homogenates were measured by commercially available kits. Intracellular ROS levels were monitored by flow cytometry by use of a peroxide-sensitive fluorescent probe,2’,7’-dichlorofluorescin diacetate (DCFH-DA). The activity of ALDH2 in isolated cardiac mitochondria was determined by measuring the conversion of acetaldehyde to acetic acid and/or the conversion of propionaldehyde to propionic acid. To determine whether ALDH2 inactivation was resulted from thiol-group oxidation, the heart mitochondrial fractions from diabetic and control rats were sonicated and incubated with the selective thiol-reducing agent dithiothreitol (DTT) at room temperature. ALDH2 expression was determined by western blot and 4-HNE content was determined by immunohistochemistry.The role of ALDH2 activity in change in hyperglycemia-induced mitochondrial membrane potential (△ψm) was tested in cultured neonatal cardiomyocytes. Briefly, Cardiomyocytes were treated for 48h, with a normal concentration of glucose (5.5 mmol/L); a high concentration of glucose (30 mmol/L); low glucose combined with a selective ALDH2 activity inhibitor, daidzin; or high glucose combined with daidzin. After 48-h treatment, mitochondrial△ψm was detected by a unique fluorescent cationic dye JC-1(5,5,6,6-tetrachloro-1,1,3,3-tetraethyl- benzamidazolocarbocyanin iodide).Results:STZ-administered rats showed characteristic symptoms of diabetes, including polydipsia, polyuria and increased food intake, along with reduced body weight gain as compared with controls. At the end of the experiment, plasma glucose and HbA1c levels were markedly higher in diabetic than control rats (P＜0.05). Supplementation with NAC orα-LA for 8 weeks significantly ameliorated these changes (P<0.05). Body weight was significantly lower in diabetic than control rats (P<0.05). NAC treatment increased the body weight of rats as compared with untreated diabetic group, but the differences were not statistically significant (P>0.05). No significant effect ofα-LA on body weight of diabetic rats. Myocardial MDA content and ROS were significantly higher in diabetic rats than in controls (P＜0.05), whereas GSH content and Mn-SOD activity were decreased in diabetic rats (P<0.05). NAC orα-LA treatment attenuated these changes (P＜0.05).H&E staining showed that myocardial cells of diabetic rats were larger than controls, NAC orα-LA treatment attenuated these changes. Compared with controls, diabetic rats exhibited significant reduction in left ventricular EF and FS (P<0.05). Both NAC andα-LA treatment attenuated these changes (P＜0.05). ALDH2 activity in diabetic rat heart mitochondria was markedly decreased (P<0.05), by approximately 40%, as compared with the control group. NAC orα-LA treatment significantly improved mitochondrial ALDH2 activity (P<0.05), although it remained lower in treatment groups than in the control group (P＜0.05). The ALDH2 activity was positively correlated with function of hearts, as indicated by EF and FS (r2=0.680, P＜0.01, and r2=0.733, P＜0.01, respectively).The heart mitochondrial fractions from diabetic and control rats were incubated with DTT in vitro. ALDH2 activity in diabetic and control groups was not significant increased by 0.5 mmol/L DTT (P>0.05), but ALDH2 activity was markedly enhanced in the presence of 1 or 2 mmol/L DTT in both groups (P<0.05). Western blot analysis revealed a decrease in ALDH2 expression in diabetic rat hearts as compared with control hearts (P<0.05), accompanied by an increase in the formation of HNE-protein adducts as shown by immunohistochemical staining results (P<0.05). NAC or a-LA treatment ameliorated these changes in diabetic treatment groups (.P<0.05).Mitochondrial membrance potential (△ψm) was decreased greatly with high glucose treatment as compared with low glucose (P＜0.05). Daidzin supplementation in low glucose medium did not have a significant effect on△ψ, but daidzin combined with high glucose treatment produced a further decrease in△ψas compared with high glucose treatment alone (P＜0.05).Conclusions:Hyperglycemia-induced oxidative stress could reduce the activity and expression of ALDH2 in the STZ-induced diabetic rat heart, and antioxidants ameliorated these changes. ALDH2 inhibition was associated with reduced left ventricular contractility, and mitochondrial impairment aggravated by ALDH2 inhibition might reflect an underlying mechanism which causes cardiac dysfunction in diabetic rats. |
PDF Full Text Request