Inhibition of Aldehyde Dehydrogenase 2 by Oxidative Stress Is Associated with Cardiac Dysfunction in Diabetic Rats Jiali Wang,1,2* Haigang Wang,3* Panpan Hao,1,2 Li Xue,1,2 Shujian Wei,1,2 Yun Zhang,2,4 and Yuguo Chen1,2 1Department of Emergency, Qilu Hospital, Shandong University, Jinan, China; 2Key Laboratory of Cardiovascular Remodeling and Function Research affiliated to Ministry of Education of the China and Ministry of Health of the China, Shandong University, Jinan, China; 3Department of Pharmacy, Qilu Hospital, Shandong University, Jinan, China; and4Department of Cardiology, Qilu Hospital, Shandong University, Jinan, China Left ventricular (LV) dysfunction is a common comorbidity in diabetic patients, although the molecular mechanisms underlying this cardiomyopathic feature are not completely understood. Aldehyde dehydrogenase 2 (ALDH2) has been considered a key cardioprotective enzyme susceptible to oxidative inactivation. We hypothesized that hyperglycemia-induced oxidative stress would influence ALDH2 activity, and ALDH2 inhibition would lead to cardiac functional alterations in diabetic rats. Diabetes was in- duced by intraperitoneal (i.p.) injection of 60 mg/kg streptozotocin. Rats were divided randomly into four groups: control, untreated diabetic, diabetic treated with N-acetylcysteine (NAC) and diabetic treated with a-lipoic acid (α-LA). Cardiac contractile func- tion, oxidative stress markers and reactive oxygen species (ROS) levels were assessed. ALDH2 activity and expression also were de- termined. The role of ALDH2 activity in change in hyperglycemia-induced mitochondrial membrane potential (Δψ) was tested in cultured neonatal cardiomyocytes. Myocardial MDA content and ROS were significantly higher in diabetic rats than in controls, whereas GSH content and Mn-SOD activity were decreased in diabetic rats. Compared with controls, diabetic rats exhibited sig- nificant reduction in LV ejection fraction and fractional shortening, accompanied by decreases in ALDH2 activity and expression. NAC and α-LA attenuated these changes. Mitochondrial Δψ was decreased greatly with hyperglycemia treatment, and high glu- cose combined with ALDH2 inhibition with daidzin further decreased Δψ. The ALDH2 activity can be regulated by oxidative stress in the diabetic rat heart. ALDH2 inhibition may be associated with LV reduced contractility, and mitochondrial impairment aggra- vated by ALDH2 inhibition might reflect an underlying mechanism which causes cardiac dysfunction in diabetic rats. © 2011 The Feinstein Institute for Medical Research, www.feinsteininstitute.org Online address: http://www.molmed.org doi: 10.2119/molmed.2010.00114 INTRODUCTION 30% of patients with type 1 diabetes documented to play a crucial role in the Diabetes mellitus is a common meta- have this specific cardiomyopathy (6). pathogenesis of diabetic complications bolic disorder that can affect patient sur- However, the molecular mechanisms un- (7,8). vival and quality of life because of acute derlying diabetic cardiomyopathy re- Mitochondrial aldehyde dehydroge- and chronic complications (1–3). Cardio- main incompletely understood. nase 2 (ALDH2), the main enzyme re- vascular complications, including dia- Shortly after the onset of hyperglyce- sponsible for acetaldehyde oxidation in betic cardiomyopathy, are the major mia, the production of reactive oxygen ethanol metabolism, is considered re- causes of morbidity and mortality in dia- species (ROS)—including superoxide sponsible for oxidation and detoxifica- betic patients. This common and serious anion, hydroxyl radicals and hydrogen tion of aromatic and aliphatic aldehydes comorbidity has an asymptomatic onset peroxide (H2O2)—increases by glucose such as 4-hydroxy-2-nonenal (4-HNE) and is characterized by impaired contrac- autoxidation, the electron transport chain (9–11). 4-HNE is a highly cytotoxic alde- tility and relaxation of the left ventricle in mitochondria and membrane-bound hyde generated during oxidative stress independent of coronary artery disease NADPH oxidase (7). Oxidative stress as a result of lipid peroxidation (12,13). or hypertension (4,5). Approximately caused by these free radicals has been ALDH2 recently has been considered a key cardioprotective enzyme, probably because of its detoxification of reactive *JW and HW contributed equally to this work. aldehydes. Transgenic overexpression of Address correspondence and reprint requests to Yuguo Chen or Yun Zhang, Qilu Hospital, ALDH2 was found to attenuate ethanol Shandong University, No. 44 Wenhuaxi Road, Jinan 250012, China. Phone: +86 531 exposure-induced myocardial dysfunc- 82169307; Fax: +86 531 86927544; E-mails: [email protected] or [email protected]. tion (14,15). Enhanced ALDH2 activity Submitted July 15, 2010; Accepted for publication October 14, 2010; Epub by ethanol preconditioning or the direct (www.molmed.org) ahead of print October 15, 2010. effect of the ALDH2 activator-1 led to 172 | WANG ET AL. | MOL MED 17(3-4)172-179, MARCH-APRIL 2011 RESEARCH ARTICLE cardioprotection against ischemia- duction, NAC and α-LA were adminis- tion), glutathione (GSH) content and Mn- reperfusion injury (16–18). Nonetheless, tered to the diabetic treated groups by superoxide dismutase (Mn-SOD) activity previous studies also indicated that oral gavage for eight wks. The concentra- were measured by commercially avail- ALDH2 had redox-sensitive thiol group tions of NAC and α-LA were adjusted for able kits according to the manufacturer’s in the active site of the enzyme and thus a daily intake of 1.4 g/kg and 60 mg/kg, instructions (Jiancheng Co., Nanjing, was prone to an oxidative-based inacti- respectively, according to previous stud- China). vation (19–21). More specifically, long- ies (22,23). term nitroglycerin treatment caused Measurement of Intracellular ROS ALDH2 inactivation because of overpro- Measurement of Cardiac Function by Production in Isolated Rat Hearts duction of mitochondrial ROS (19). Echocardiography Intracellular ROS levels were moni- From this knowledge, we hypothe- Transthoracic echocardiography was tored by flow cytometry by use of a per- sized that chronic hyperglycemia-in- performed noninvasively with a Vevo 770 oxide-sensitive fluorescent probe, 2’,7’- duced oxidative stress would have an ef- high-resolution imaging system equipped dichlorofluorescin diacetate (DCFH-DA) fect on ALDH2 activity, and that ALDH2 with a 30-MHz transducer (RMV-707B; (Sigma) (25). Heart tissue was dissected inhibition would involve left ventricular VisualSonics, Toronto, Canada). Rats and immediately frozen in liquid nitro- (LV) dysfunction of the diabetic heart. were lightly anesthetized (0.3 mL of a gen. After rapid thawing, tissue was Accordingly, we evaluated cardiac con- cocktail containing 100 mg/mL ketamine homogenized in 1 mL ROS buffer tractile function, oxidative stress levels and 10 mg/mL acepromazine given i.p.) (150 mmol/L KCl, 20 mmol/L Tris, and ALDH2 activity and expression in for the duration of the recordings. The 0.5 mmol/L EDTA, 1 mmol/L MgCl2, the diabetic rat heart in this study. heart rate was monitored simultaneously 5 mmol/L glucose and 0.5 mmol/L by electrocardiography (ECG). Left ven- octanoic acid, pH 7.4). The homogenate MATERIALS AND METHODS tricular (LV) end diastolic diameter was exposed to 10 μmol/L DCFH-DA (LVEDD) and end systolic diameter and incubated at 37°C for 30 min in the Animals and Induction of Diabetes (LVESD) were used to calculate fractional dark. After being washed twice with Male Wistar rats weighing 200 to 250 g shortening (FS) by the following formula: PBS, the homogenate was made into a were purchased from the Department of FS (%) = [(LVEDD – LVESD)/LVEDD] single-cell suspension through a screen Experimental Animals of Shandong Uni- ×100%. LV end diastolic volume (LVEDV) filter, and intracellular ROS levels were versity (Jinan, China). The rats were fed and end systolic volume (LVESV) were measured by use of FACS (Becton Dick- normal chow and had free access to calculated as described previously (24). inson, San Jose, CA, USA) and CellQuest water. Housing was at a constant tem- Ejection fraction (EF) was calculated by software (Becton Dickinson). perature of 21°C ± 1°C with a fixed 12-h the following formula: EF (%) = light/dark cycle. All animal procedures [(LVEDV – LVESV)/LVEDV] ×100%. All Assessment of Mitochondria were in accordance with NIH Guide and echocardiographically derived measures Rat mitochondria were prepared from were approved by the Animal Use and were obtained by averaging the readings freshly excised hearts by differential cen- Care Committee of Shandong University. of three consecutive beats. trifugation as described previously (26). Diabetes was induced in overnight- Briefly, hearts were cut into small pieces fasted rats by administering a single in- Biochemical Measurements in Plasma and homogenized in HEPES buffer. The traperitoneal (i.p.) injection of 60 mg/kg and Heart Tissue resulting homogenate was centrifuged at streptozotocin (STZ) (Sigma-Aldrich, St. Plasma glucose concentration was 1,500g for 10 min and 2,000g for 5 min at Louis, MO, USA) freshly dissolved in measured by use of the Glucose Ana- 4°C. The pellet at the bottom of the cen- 0.1 mol/L sodium citrate buffer (pH 4.5). lyzer (Yellow Spring Instrument, Yellow trifuge tube was discarded and the su- The control group was injected with a Springs, OH, USA), and plasma Hb A1c pernatant was then centrifuged at similar volume of sodium citrate buffer was determined by HPLC (Roche Diag- 12,000g for 15