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Inhibition of 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 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 oxidation in betic cardiomyopathy, are the major mia, the production of reactive oxygen ethanol , is considered re- causes of morbidity and mortality in dia- species (ROS)—including 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 (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

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cardioprotection against - 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 (Mn-SOD) activity previous studies also indicated that oral gavage for eight wks. The concentra- were measured by commercially avail- ALDH2 had -sensitive thiol group tions of NAC and α-LA were adjusted for able kits according to the manufacturer’s in the 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 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 min. The resulting pellet alone. We considered STZ-treated rats nostics, Indianapolis, IN, USA). At the was washed twice by resuspension in with blood glucose levels > 15.0 mmol/L end of the experimental period, rats were 1 mL HEPES buffer and centrifuged after 72 h of injection as diabetic. killed, hearts were excised and heart tis- again at 12,000g for 15 min, followed by sues were weighed (wet weight) and ho- further purification by discontinuous Experimental Protocol mogenized in ice-cold PBS. The ho- gradient centrifugation using 30% Animals were randomly divided into mogenates were centrifuged at 3,000g for (wt/vol.) Percoll. Mitochondrial purity four groups (n = 8): control, untreated dia- 15 min at 4°C to obtain the supernatant. was assessed by Western blot analysis betic, diabetic treated with N-acetylcysteine Total antioxidant concentration, content with the mitochondrial marker pro- (NAC) and diabetic treated with α-lipoic of malondialdehyde (MDA) (a reliable hibitin, the plasma membrane marker acid (α-LA). One wk after diabetes in- index of ROS-induced lipid peroxida- Na+K+ATPase and the cytosolic marker

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Table 1. General characteristics of experimental groupsa glyceraldehyde 3-phosphate dehydroge- nase (GAPDH). Protein concentration Control Diabetic Diabetic + NAC Diabetic + α-LA was determined by the bicinchoninic Parameter (n = 8) (n = 8) treatment (n = 6) treatment (n = 6) acid protein assay. The mitochondrial Plasma glucose (mmol/L) 3.7 ± 0.2 21.7 ± 1.5b 16.1 ± 3.1b,c 18.2 ± 1.3b,c samples were frozen at –80°C until use. b c c Hb A1c (% of Hb) 5.7 ± 0.3 8.8 ± 0.6 7.2 ± 0.5 6.8 ± 0.5 Body weight (g) 448 ± 25 270 ± 21b 316 ± 15b 273 ± 17b ALDH2 Enzymatic Activity Heart weight (g) 1.17 ± 0.08 0.86 ± 0.05b 0.90 ± 0.05b 0.81 ± 0.04b The activity of ALDH2 in isolated mi- Heart weight/body weight 2.62 ± 0.10 3.25 ± 0.15b 2.86 ± 0.08 3.02 ± 0.16 tochondria was determined by measur- (mg/g) ing the conversion of acetaldehyde to aData are presented as mean ± SEM. and/or the conversion of pro- bP < 0.05 versus control group. pionaldehyde to propionic acid. The mi- cP < 0.05 versus diabetic group. tochondria were sonicated, centrifuged at 12,000g for 10 min at 4°C, and the su- pernatant was used to detect ALDH2 ac- stripped and reblotted with antibody to After 48-h treatment, mitochondrial tivity at room temperature by monitor- β-actin (1:1,000; Santa Cruz Biotechnol- membrane potential (Δψ) was detected ing NADH formation from NAD+ at ogy) as an internal control. Membranes (28). Briefly, cardiomyocytes were sus- 340 nm in a spectrophotometer (Beckman were washed 3x and then incubated pended in HEPES buffer and incubated Coulter, Chaska, MN, USA). The assay with 1:10,000 dilution of horseradish with 5 μmol/L JC-1 (Invitrogen, Carls- mixture (0.2 mL) contained 100 mmol/L peroxidase–conjugated secondary bad, CA, USA), a dual-emission mito- Tris-HCl (pH 8.5), 1 mmol/L NAD+, antibody for 1 h. Protein bands were chondrial potentiometric dye, for 30 min 1 mmol/L 4-methylpyrazole and 50 μg identified by a standard enhanced at 37°C. Then cells were washed 3x with protein. The reaction was started by the chemiluminescence method. HEPES buffer. The fluorescence intensity addition of 1 mmol/L acetaldehyde or of each sample was analyzed (excitation propionaldehyde to the cuvette. Enzyme- Immunohistochemistry 490 nm, emission 530 and 590 nm) using specific activity was expressed as nmol We performed immunohistochemistry a spectrofluorimeter (Spectra Max Gem- NADH min–1 mg–1 protein. for 4-HNE, a marker of lipid peroxida- ini; Molecular Devices, Sunnyvale, CA, The mitochondria from control and dia- tion and substrate of ALDH2 enzyme. USA). betic hearts were sonicated and incubated Formalin-fixed myocardial sections were with dithiothreitol (DTT) for 30 min at deparaffinized and rehydrated as de- Statistical Analysis room temperature. Then, 1 mmol/L ac- scribed previously in detail (27). Primary Data are presented as mean ± SEM. Sta- etaldehyde or propionaldehyde, together antibody against 4-HNE (1:50; Santa tistical significance was determined with with 1 mmol/L NAD+ and 1 mmol/L Cruz Biotechnology) and biotinylated one-way analysis of variance (ANOVA) 4-methylpyrazole were added, and ab- secondary antibody (1:200; Beyotime, followed by Student– Newman–Keuls post sorbance changes were recorded. Nanjing, China) were used. Images were hoc analysis. Correlation analysis was per- captured using a microscope (Eclipse formed using the Pearson correlation. P < Western Blot Analysis E800; Nikon, Tokyo, Japan) and digital 0.05 was considered statistically significant. Ventricular tissues were homogenized sight camera (Nikon). Data were analyzed with use of GraphPad and sonicated in a lysis buffer contain- Prism 5 (GraphPad Software Inc., San ing 20 mmol/L Tris (pH 7.4), 150 Studies of Neonatal Cardiomyocytes Diego, CA, USA). mmol/L NaCl, 1 mmol/L EDTA, 1 in Primary Culture mmol/L EGTA, 1% Triton, 0.1% SDS and Neonatal rat cardiomyocytes were iso- RESULTS 1% protease inhibitor cocktail. Equal lated from hearts of 1–3-day-old Wistar amounts (20 μg) of proteins were sepa- rat pups and cultured in medium contain- General Observations rated by SDS-PAGE on a 12% gel and ing 5.5 mmol/L glucose. Cardiomyocytes STZ-administered rats showed charac- transferred to PVDF membrane. The were treated for 48 h, with a normal con- teristic symptoms of diabetes, including membranes were blocked for 2 h in a so- centration of glucose (5.5 mmol/L, LG); a polydipsia, polyuria and increased food lution of 5% (wt/vol) milk and then in- high concentration of glucose (30 mmol/L, intake, along with reduced body weight cubated overnight at 4°C with anti- HG); LG combined with a selective gain as compared with controls (data not ALDH2 (1:1,000; Santa Cruz ALDH2 activity inhibitor, daidzin shown). At the end of the experiment, μ Biotechnology, Santa Cruz, CA, USA) (Sigma), at 20 mol/L (LG + daidzin); or plasma glucose and Hb A1c levels were and anti-SOD2 (1:300; Santa Cruz HG combined with 20 μmol/L daidzin markedly higher in diabetic than control Biotechnology). The same blot was (HG + daidzin). rats (Table 1). Supplementation with NAC

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Table 2. Plasma and tissue markers of oxidative stress in experimental groupsa treated diabetic rats, the differences were Control Diabetic Diabetic + NAC Diabetic + α-LA not statistically significant. Heart rate Parameter (n = 8) (n = 8) treatment (n = 6) treatment (n = 6) was significantly lower in untreated dia- Total antioxidant 1.40 ± 0.20 0.73 ± 0.09b 1.18 ± 0.09c 1.16 ± 0.08c betic than control rats (288.2 ± 18.3 ver- concentration (mmol/L) sus 399.4 ± 24.8 beats/min), and antioxi- Mn-SOD (U/mg prot) 64.53 ± 1.82 55.81 ± 2.71b 64.31 ± 2.70c 61.58 ± 2.41c dant treatment enhanced the heart rate: GSH (mg/g prot) 5.08 ± 0.28 3.40 ± 0.15b 4.40 ± 0.26c 4.12 ± 0.22b,c 353.6 ± 28.9 beats/min for NAC-treated MDA (nmol/mg prot) 2.10 ± 0.21 2.96 ± 0.27b 2.20 ± 0.10c 2.31 ± 0.17c diabetic rats; 342.2 ± 19.8 beats/min for α-LA–treated diabetic rats (P < 0.05). a Data are presented as mean ± SEM. Compared with control, diabetic rats bP < 0.05 versus control group. showed a significant reduction in EF and cP < 0.05 versus diabetic group. FS (P < 0.05) (Figure 1). Both NAC and α-LA treatment restored FS (Figure 1C). or α-LA for 8 wks significantly amelio- rated these changes. Body weight was sig- nificantly lower in diabetic than control rats (P < 0.05). NAC treatment increased the body weight of rats as compared with the untreated diabetic group, but the dif- ferences were not statistically significant. The body weight was significantly lower in NAC and α-LA treatment groups than in the control group (Table 1).

Plasma and Heart Tissue Markers of Oxidative Stress Table 2 shows the results of oxidative stress markers in experimental groups. Plasma total antioxidant concentration was significantly lower in diabetic rats than in control rats (P < 0.05), in parallel with a significant reduction in the tissue activity of Mn-SOD. Both NAC and α-LA treatment restored total antioxidant concentration and Mn-SOD activity. The myocardial MDA content was signifi- cantly higher in diabetic than control rats, whereas the GSH content in diabetic rats was decreased. NAC and α-LA treat- ment attenuated these changes.

Myocardial Parameters and Systolic Function As shown in Table 1, heart weight was significantly lower in diabetic than con- trol rats (P < 0.05), whereas heart weight Figure 1. Left ventricular functional analysis with echocardiography. (A) Representative M-mode echocardiograms from control (Con), untreated diabetic (Dia), diabetic did not differ between NAC- and treated with NAC (NAC) and diabetic treated with α-LA (α-LA) rats. LVESD, left ventricular α-LA–treated rats and untreated diabetic end systolic diameter; LVEDD, left ventricular end diastolic diameter. (B) The mean left rats. The ratio of heart-to-body weight ventricular ejection fraction (EF) from Con, Dia, NAC and α-LA rats. *P < 0.05 versus con- was higher in diabetic than control rats. trol group; **P < 0.05 versus diabetic group. Data are presented as mean ± SEM (n = Although the ratio of heart-to-body 6–8). (C) The mean left ventricular fractional shortening (FS) from Con, Dia, NAC and α-LA weight decreased in NAC- or α- rats. *P < 0.05 versus control group; **P < 0.05 versus diabetic group. Data are presented LA–treated rats as compared with un- as mean ± SEM (n = 6–8).

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Figure 2. Reactive oxygen species (ROS) production in isolated rat hearts from con- trol (Con), untreated diabetic (Dia), dia- betic treated with NAC (NAC) and dia- betic treated with α-LA (α-LA) rats. Intracellular ROS levels were determined by measuring the fluorescence of DCF with FACS (excitation 488 nm, emission 530 nm). *P < 0.05 versus control group; **P < 0.05 versus diabetic group. Data are presented as mean ± SEM of the geomet- ric mean fluorescence (n = 6–8).

Only NAC treatment improved EF as compared with untreated diabetic rats (Figure 1B). The mean LVEDD and Figure 4. Correlation between myocardial LVESD were significantly higher in dia- ALDH2 activity and EF (A) and FS (B). betic than control rats (LVEDD, 6.04 ± Data were analyzed by linear regression. 0.24 versus 5.45 ± 0.34; LVESD, 3.20 ± *P < 0.05. 0.31 versus 2.59 ± 0.31). NAC and α-LA treatment ameliorated these changes. control group (Figure 3A). Importantly, the decrease in ALDH2 dehydrogenase Intracellular ROS Production activity was correlated positively with To determine ROS levels, DCFH-DA decreased function of hearts, as indicated was used because it permeates into cells Figure 3. ALDH2 dehydrogenase activity by the reduction in EF and FS (r2 = 0.680, analysis. (A) The effect of NAC and α-LA and is oxidized to the fluorescent de- P < 0.01, and r2 = 0.733, P < 0.01, respec- treatment on mitochondrial aldehyde de- rivate DCF by ROS. Intracellular ROS tively; Figure 4). hydrogenase 2 (ALDH2) activity in isolated was higher in diabetic than control hearts rat hearts from control (Con), untreated To determine whether ALDH2 inactiva- (Figure 2). The diabetes-associated in- diabetic (Dia), diabetic treated with NAC tion resulted from thiol-group oxidation, crease in ROS was ameliorated markedly (NAC) and diabetic treated with α-LA the heart mitochondrial fractions from by NAC or α-LA treatment (Figure 2). (α-LA) rats. ALDH2 activity was determined diabetic and control rats were coincu- by measuring the conversion of propi- bated with the selective thiol-reducing ALDH2 Enzymatic Activity Analysis onaldehyde to its propionic acid product. agent DTT. ALDH2 dehydrogenase activ- ALDH2 activity in diabetic rat heart *P < 0.05 versus control group; **P < 0.05 ity in diabetic and control groups was not mitochondria was decreased markedly, versus diabetic group. Data are mean ± significantly increased by 0.5 mmol/L by approximately 40% and 30%, as com- SEM (n=6–8). (B) The effect of dithiothreitol DTT, but the activity was markedly en- (DTT) on ALDH2 activity in isolated heart pared with the control group, as mea- hanced in the presence of 1 or 2 mmol/L mitochondria from control or diabetic rats. sured by the conversion of propionalde- DTT in both groups (Figure 3B). In the *P < 0.05 versus control group; **P < 0.05 hyde (Figure 3A) and acetaldehyde, versus diabetic group. Data are mean ± diabetic group, the addition of 2 mmol/L α respectively. NAC and -LA treatment SEM (n=4). (C) Representative Western blot DTT could not completely restore significantly improved mitochondrial showing various cellular markers in total ALDH2 activity to the level of the con- ALDH2 activity, although it remained homogenate (Homo) and isolated mito- trol group, which suggests that other lower in treatment groups than in the chondria (Mito) prepared from rat hearts. mechanisms account for enzymatic inac-

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Figure 6. The change in Δψ was quantified from normal glucose (LG), normal glu- cose+daidzin (LG+daidzin), high glucose (HG) and high glucose+daidzin (HG+daidzin) group. *P < 0.05 versus LG group; #P < 0.05 versus HG group. Data are presented as mean ± SEM (n = 4).

nificant effect on Δψ, but daidzin com- bined with HG treatment produced a further decrease in Δψ as compared with HG treatment alone (Figure 6).

DISCUSSION Figure 5. (A) Western blots of ALDH2 expression from control (Con), untreated diabetic The aim of this study was to examine (Dia), diabetic treated with NAC (NAC) and diabetic treated with α-LA (α-LA) rats. β-actin whether chronic hyperglycemia-induced was an internal control. *P < 0.05 versus control group; **P < 0.05 versus diabetic group. Results are presented as mean ± SEM (n=6-8). (B) Representative immunohistochemistrical oxidative stress would have an effect on staining for 4-hydroxy-2-nonenal (HNE)-protein adducts from Con, Dia, NAC and α-LA rats. ALDH2 activity and expression in dia- Scale bar, 50 μm. (C) Western blots of Mn-SOD protein expression from Con, Dia, NAC and betic rat hearts, and whether ALDH2 in- α-LA rats. β-actin was an internal control. *P < 0.05 versus control group; **P < 0.05 versus hibition would involve impaired LV diabetic group. Results are presented as mean ± SEM (n = 6–8). function, using a STZ-induced diabetic rat model. Diabetic rat hearts showed a tivation. Western blot analysis of mito- groups. Western blots of Mn-SOD ex- significant increase in myocardial tissue chondrial preparations from rats with pression did not differ between diabetic content of MDA, as well as intracellular different cellular markers revealed a high and control rats (Figure 5C). NAC or ROS production. Enhanced MDA and degree of mitochondrial enrichment, as α-LA treatment did not influence the ROS were accompanied by compromised indicated by the mitochondrial marker production of Mn-SOD. Mn-SOD activity, GSH content and total prohibitin, and the absence of contami- antioxidant concentration. These findings nation with cellular membrane or Study of Cultured Neonatal are similar to those reported in the previ- as shown by Na+K+ATPase and GAPDH Cardiomyocytes ous literature (22,30), indicating in- results (Figure 3C). Given that mitochondrial function is creased levels of oxidative stress in dia- essential to cardiac function (28,29), we betic rats. ALDH2 Expression investigated cardiomyocyte mitochondr- We used two antioxidants, NAC and Western blot analysis revealed a de- ial function using a lipophilic and α-LA, to investigate the effect of oxida- crease in ALDH2 expression in diabetic cationic dye JC-1. The result was ex- tive stress in vivo in rats. Interestingly, rat hearts as compared with control pressed as the ratio between red (aggre- these two antioxidants reversed ALDH2 hearts (Figure 5A), accompanied by an gated JC-1) and green (monometric form protein expression in treated diabetic an- increase in the formation of HNE-protein of JC-1) fluorescence. Quantitative analy- imals but had no effect on Mn-SOD ex- adducts as shown by immunohisto- sis showed cardiomyocyte Δψ greatly de- pression. In fact, we found Mn-SOD ex- chemistrical staining results (Figure 5B). creased during treatment with HG as pression did not change with either NAC or α-LA treatment ameliorated compared with LG. Daidzin supplemen- treatment. Results in the literature these changes in diabetic treatment tation in LG medium did not have a sig- showed a varied effect of hyperglycemia

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on Mn-SOD expression (31–33), and it is changes in response to antioxidants. and function have been reported to be difficult to explain this discrepancy cur- Song et al. showed that incubation with controlled by the Δψ across the inner rently. From our findings, we speculate reducing agents such as DTT restored the membrane (44,45). The decrease in Δψ that the changes in Mn-SOD activity in suppressed ALDH2 activity in alcohol- will lead to changes in mitochondrial experimental groups we found are not fed rats (37). However, in other reports, Ca2+ and ATP content, with deleterious due to the alteration of its expression. ALDH2 activity was restored only par- effects. These data might reflect an un- However, a posttranslational modifica- tially by DTT (38,39). A possible explana- derlying mechanism by which hypergly- tion might explain at least in part the ob- tion for incomplete normalization of cemia leads to cardiac dysfunction. How- served changes in Mn-SOD activity. In ALDH2 activity in this study could be ever, given the nature of the associated addition, supplementation with NAC that oxidants cause irreversible modifica- rather than causal relationship of our re- and α-LA moderately reduced plasma tion of the thiol group by prolonged ex- sults, further study is warranted to better glucose. Both antioxidants previously posure to hyperglycemia. In addition, elucidate the role of ALDH2 in mitochon- were found to prevent hyperglycemia-in- modification of ALDH2 other than oxi- drial and cardiac function. duced insulin resistance or improve in- dation may contribute to enzymatic inac- In summary, we have shown that hy- sulin sensitivity in diabetic rats (34,35). tivation; one example is phosphorylation perglycemia-induced oxidative stress The partial lowering of glucose levels by (39), which needs further investigation. could reduce the activity and expression these two antioxidants might be attribut- The present study suggests that re- of ALDH2 in the STZ-induced diabetic able to such effects. duced ALDH2 activity is associated with rat heart, and antioxidants ameliorated A key finding of the current investiga- impaired cardiac contractile function in these changes. We also suggest that tion is the significant inhibition of the diabetic rats, consistent with previous ob- ALDH2 inhibition aggravates mitochon- myocardial ALDH2 activity in the dia- servations (16,17). Chen et al. found that drial impairment in response to hyper- betic heart. Of note, propionaldehyde ALDH2 activity was correlated inversely glycemia, which might represent a mech- and acetaldehyde oxidation was mark- with cardiac infarct size in rat hearts sub- anism underlying LV contractile edly lower, by about 40% and 30%, re- jected to ischemia and reperfusion ex vivo dysfunction in diabetic rats. These find- spectively, in diabetic than control rat (16). Increasing ALDH2 activity by etha- ings might provide new knowledge hearts. Because ALDH2 functions as an nol or other treatments was accompanied about diabetic cardiomyopathy. antioxidant enzyme, this protein may be by a reduction in infarct size, suggesting easily inactivated by free radicals. Re- the involvement of ALDH2 in cardiopro- ACKNOWLEDGMENTS cently, ALDH2 was identified as a target tection. Because of the protective role of This study was supported by two for oxidative modification during glyc- ALDH2 in the metabolism of toxic alde- grants from the Department of Science eryl trinitrate tolerance (19,21) and he- hydes, inactivation of ALDH2 likely and Technology of Shandong Province patic ischemia-reperfusion (36). There- causes accelerated accumulation of alde- (Y2007C075 and 2008RKB060), the Shan- fore, oxidative modification of the thiol hydes, and thus leads to increased sus- dong Provincial Outstanding Medical group may be responsible for ALDH2 in- ceptibility to myocardial damage. 4-HNE, Academic Professional Program, and the activation under hyperglycemic condi- for example, which was significantly in- 1020 Program (Excellent Medical Subject tions. We further hypothesized that inhi- creased in the diabetic rat hearts in our Leaders) and a key grant (2009HD011) bition of ALDH2 activity might be due to study, reacts with , hidtidine and from the Health Department of Shan- an alteration in its expression. Indeed, residues and forms protein dong Province. we found the ALDH2 protein level sig- adducts, thus resulting in inhibition of nificantly downregulated in the diabetic proteins such as the GAPDH (40), DISCLOSURE heart as compared with the control Na+K+ATPase (41) and 20S proteosome The authors declare that they have no group, and this might explain the ob- (42). At high concentrations, 4-HNE also competing interests as defined by Molecu- served decrease in ALDH2 activity in re- directly inhibits ALDH2 activity (43), lar Medicine, or other interests that might sponse to hyperglycemia. thus contributing to a vicious cycle. To be perceived to influence the results and In this study, we observed that ALDH2 further investigate the role of ALDH2 ac- discussion reported in this paper. activity was improved significantly, but tivity in the development of myocardial only partially restored by NAC or α-LA. injury, we examined the decrease in REFERENCES In vitro, DTT dose-dependently im- ALDH2 activity by use of the selective in- 1. Adeghate E, Schattner P, Dunn E. (2006) An up- proved ALDH2 activity in isolated mito- hibitor daidzin on mitochondrial function date on the etiology and epidemiology of dia- betes mellitus. Ann. N. Y. Acad. Sci. 1084:1–29. chondria from diabetic and control rats in cultured neonatal cardiomyocytes. Re- 2. Gremizzi C, Vergani A, Paloschi V, Secchi A. but also moderately rescued ALDH2 ac- duced ALDH2 activity contributed to the (2010) Impact of pancreas transplantation on tivity in diabetic rats. 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