Mitochondrial Aldehyde Dehydrogenase (ALDH2) Protects

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Zhang et al. BMC Medicine 2012, 10:40 http://www.biomedcentral.com/1741-7015/10/40

RESEARCHARTICLE Open Access Mitochondrial aldehyde dehydrogenase (ALDH2) protects against streptozotocin-induced diabetic cardiomyopathy: role of GSK3b and mitochondrial function Yingmei Zhang1,2, Sara A Babcock2, Nan Hu2, Jacalyn R Maris2, Haichang Wang1 and Jun Ren1,2*

Abstract Background: Mitochondrial aldehyde dehydrogenase (ALDH2) displays some promise in the protection against cardiovascular diseases although its role in diabetes has not been elucidated. Methods: This study was designed to evaluate the impact of ALDH2 on streptozotocin-induced diabetic cardiomyopathy. Friendly virus B(FVB) and ALDH2 transgenic mice were treated with streptozotocin (intraperitoneal injection of 200 mg/kg) to induce diabetes. Results: Echocardiographic evaluation revealed reduced fractional shortening, increased end-systolic and -diastolic diameter, and decreased wall thickness in streptozotocin-treated FVB mice. Streptozotocin led to a reduced respiratory exchange ratio; myocardial apoptosis and mitochondrial damage; cardiomyocyte contractile and intracellular Ca2+ defects, including depressed peak shortening and maximal velocity of shortening and relengthening; prolonged duration of shortening and relengthening; and dampened intracellular Ca2+ rise and clearance. Western blot analysis revealed disrupted phosphorylation of Akt, glycogen synthase kinase-3b and Foxo3a (but not mammalian target of rapamycin), elevated PTEN phosphorylation and downregulated expression of mitochondrial proteins, peroxisome proliferator-activated receptor g coactivator 1a and UCP-2. Intriguingly, ALDH2 attenuated or ablated streptozotocin- induced echocardiographic, mitochondrial, apoptotic and myocardial contractile and intracellular Ca2+ anomalies as well as changes in the phosphorylation of Akt, glycogen synthase kinase-3b, Foxo3a and phosphatase and tensin homologue on chromosome ten, despite persistent hyperglycemia and a low respiratory exchange ratio. In vitro data revealed that the ALDH2 activator Alda-1 and glycogen synthase kinase-3b inhibition protected against high glucose- induced mitochondrial and mechanical anomalies, the effect of which was cancelled by mitochondrial uncoupling. Conclusions: In summary, our data revealed that ALDH2 acted against diabetes-induced cardiac contractile and intracellular Ca2+ dysregulation, possibly through regulation of apoptosis, glycogen synthase kinase-3b activation and mitochondrial function independent of the global metabolic profile. Keywords: ALDH2, cardiac contraction, diabetes, GSK3b?β?, mitochondrial function

Background others has revealed a rather singular role for ALDH2 in The mitochondrial isoform of aldehyde dehydrogenase cardioprotection against ischemic injury, arrhythmias (ALDH2) has been shown to play a pivotal role in the and alcoholism [2,3,5-7]. However, the role of ALDH2 metabolism of acetaldehyde and other toxic aldehydes in myopathic anomalies associated with metabolic disor- [1-4]. Ample evidence from our laboratory as well as ders, including diabetes mellitus, has not been elucidated. The prevalence of diabetes and associated heart * Correspondence: [email protected] diseases has been steadily increasing, particularly in 1Department of Cardiology, Xijing Hospital, Fourth Military Medical Asian countries, with approximately 50% of populations University, Xi’an, 710032, China carrying one copy of the mutant ALDH2 gene [4,8-11]. Full list of author information is available at the end of the article

© 2012 Zhang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Zhang et al. BMC Medicine 2012, 10:40 Page 2 of 17 http://www.biomedcentral.com/1741-7015/10/40

A plethora of studies have depicted significant contribu- hour dark circadian cycle with access to water and food tion from genetic variants, such as peroxisome prolifera- ad libitum. Five-month-old male FVB (used as wild-type) tor-activated receptors (PPARs), in the predisposition of and ALDH2 transgenic mice received intraperitoneal diabetes [12]; however, very few have examined the role injections of streptozotocin (STZ, 200 mg/kg). All mice of ALDH2 in the onset and progression of diabetes and were maintained for four weeks with free access to stan- its complications. Recent evidence revealed that ALDH2 dard laboratory chow and tap water before their blood polymorphism is closely associated with an increased glucose levels were monitored. Mice with fasting blood risk of diabetes [11] while experimental findings showed glucose levels > 13 mM were deemed diabetic [17]. reduced ALDH2 expression and activity associated with ALDH2 activity was measured in 33 mM sodium pyro- oxidative stress and cardiac dysfunction in diabetes [13]. phosphate containing 0.8 mM NAD+,15μMpropionalde- These observations are somewhat consistent with the hyde and 0.1 mL protein extract. Propionaldehyde, the notion that inactive ALDH2 promotes hyperglycemia substrate of ALDH2, was oxidized in propionic acid, while [9], while genotypes of ALDH2 can modify diabetes risk NAD+ was reduced to NADH to estimate ALDH2 activity. irrespective of alcohol intake [14]. To this end, this NADH was determined by spectrophotometric absorbance study was designed using a unique murine model to at 340 nm. ALDH2 activity was expressed as nanomoles examine the impact of ALDH2 overexpression in the NADH per minute per milligram of protein [18]. pathogenesis of diabetic cardiomyopathy and the under- lying cellular mechanism(s) involved. Recent evidence Serum free fatty acid, plasma insulin and blood glucose from our group has revealed a pivotal role for the essen- levels tial survival factor Akt and its downstream signaling Plasma free fatty acids were measured three hours after molecules, including glycogen synthase kinase-3b mice were denied access to food using a Free Fatty Acid (GSK3b), PPAR (mTOR) and the forkhead transcrip- Assay Kit (Cayman Chemical, MI, USA). In brief, mouse tional factor in ALDH2, in cardioprotection against blood samples were centrifuged at 2000 × g for 15 minutes alcoholism and ischemia-reperfusion [3,7,15]. To better at 4°C. An excitation wavelength of 530 nm and an emis- elucidate the interplay between these signaling cascades sion wavelength of 590 nm were used to detect the quan- and mitochondrial function in diabetes and/or ALDH2- tity of free fatty acids. Plasma insulin levels were measured induced cardiac responses, we scrutinized apoptosis and using a mouse insulin ELISA kit from Diagnostic System mitochondrial integrity, including mitochondrial mem- Laboratory (Webster, TX, USA). Fasting (overnight) and brane potential, as well as cell signaling of Akt, GSK3b, postprandial (two hours after re-feeding following the mTOR and Foxo3a in wild-type FVB and ALDH2 trans- overnight fasting) blood glucose levels were determined genic mice with or without the induction of experimen- using a glucometer (Accu-ChekII, model 792, Boehringer tal diabetes. Given that Akt signaling is under the Mannheim Diagnostics, Indianapolis, IN, USA) [17]. negative control of phosphatase and tensin homologue on chromosome ten (PTEN) in a wide variety of disease Metabolic measurement conditions, including myocardial hypertrophy, heart fail- Indirect calorimetry and total physical activity were ure and preconditioning [16], we monitored pan protein measured in light (10 a.m.) and dark (10 p.m.) phases expression and phosphorylation of PTEN. To evaluate if using the Comprehensive Laboratory Animal Monitor- ALDH2 affects myocardial morphometric and functional ing System (Oxymax/CLAMS; Columbus Instruments, anomalies in diabetes through any potential effect sec- Columbus,OH,USA).Volumeofoxygenintake(VO2), ondary to global metabolic alterations, we scrutinized volume of Carbon Dioxide exhaled (VCO2), the RER whole body metabolism, including the respiratory (VCO2/VO2) and physical activity were measured. All exchange ratio (RER), and total physical activity as well the parameters were measured every 10 minutes for six as plasma levels of free fatty acid, insulin and glucose hours during daytime and six hours during nighttime. (fasting and postprandial) in control and diabetic mice. Result recorded in the first and last half hour was not be used. For simplicity, only the RER is presented with- Methods out displaying original data from VO2 and VCO2 [19]. Experimental animals, experimental diabetes and ALDH2 activity Echocardiographic assessment All animal procedures were approved by our Institutional Cardiac geometry and function were evaluated in Animal Care and Use Committee at the University of anesthetized mice using a two-dimensional guided M- Wyoming (Laramie, WY, USA). Production of ALDH2 mode echocardiography (Sonos 5500; Phillips Medical transgenic mice using the chicken b-actin promoter was System, Andover, MA, USA) equipped with a 15-6 MHz as described previously [5]. All mice were housed in a linear transducer. Left ventricular (LV) wall thickness temperature-controlled room under a 12 hour light-12 and diastolic and systolic dimensions were recorded Zhang et al. BMC Medicine 2012, 10:40 Page 3 of 17 http://www.biomedcentral.com/1741-7015/10/40

from the M-mode images. Fractional shortening was cal- velocities of shortening (+dL/dt) and relengthening (-dL/ culated from end-diastolic diameter (EDD) and end-sys- dt), the maximal velocities of ventricular pressure rise and tolic diameter (ESD) using the equation: fall. − × (EDD ESD)/EDD 0.01 Intracellular Ca2+ transient measurement Myocytes were loaded with fura-2-acetoxymethyl ester Estimated echocardiographic LV mass was calculated (0.5 μM) for 10 minutes and fluorescence measurements as: were recorded with a dual-excitation fluorescence photo-

[(LVEDD + septal wall thickness + posterior wall thickness)3 − LVEDD3] × 1.055 multiplier system (IonOptix). Cardiomyocytes were placed on an Olympus IX-70 inverted microscope and 3 where 1.055 (in mg/mm ) represents the density of imaged through a Fluor × 40 oil objective. Cells were the myocardium. Heart rate was calculated from 20 con- exposedtolightemittedbya75Wlampandpassed secutive cardiac cycles [20]. through either a 360 or a 380 nm filter, while being stimulated to contract at 0.5 Hz. Fluorescence emissions Isolation of murine cardiomyocytes were detected between 480 and 520 nm by a photomulti- After intraperitoneal administration of a sedative (keta- plier tube after first illuminating the cells at 360 nm for mine 80 mg/kg and xylazine 12 mg/kg), the hearts were 0.5 seconds then at 380 nm for the duration of the removed and digested for 20 minutes with Liberase recording protocol (333 Hz sampling rate). The 360 nm Blendzyme 4 (Hoffmann-La Roche Inc., Indianapolis, excitation scan was repeated at the end of the protocol IN, USA). Cardiomyocyte yield was approximately 75% and qualitative changes in intracellular Ca2+ concentra- and was not affected by STZ treatment or ALDH2 over- tion were inferred from the ratio of fura-2 fluorescence expression. Only rod-shaped myocytes with clear edges intensity (FFI) at the two wavelengths (360 and 380 nm). were selected for the mechanical study [21]. Fluorescence decay time was measured as an indication For the in vitro study, cardiomyocytes from control of the intracellular Ca2+ clearing rate. Both single- and FVB mice were exposed to high extracellular glucose bi-exponential curve fit programs were applied to calcu- (25.5 mM) [22] in the absence or presence of the ALDH2 late the intracellular Ca2+ decay constant [5]. activator Alda-1 (20 μM), the mitochondrial uncoupler carbonyl cyanide 4-trifluoromethoxyphenylhydrazone Histological examination (FCCP, 1 μM) or the GSK3b inhibitor SB216763 (10 μM) After anesthesia, hearts were excised and immediately [23,24] for 12 hours before an assessment of their placed in 10% neutral-buffered formalin at room tem- mechanical and biochemical properties. perature for 24 hours after a brief rinse with PBS. The specimens were embedded in paraffin, cut into 5 μm Cell shortening and relengthening sections and stained with H & E as well as fluorescein The mechanical properties of cardiomyocytes were isothiocyanate (FITC)-conjugated wheat germ agglutinin. assessed using a SoftEdge MyoCam system (IonOptix Cor- Heart sections were stained with H & E for gross mor- poration, Milton, MA, USA) [5]. In brief, cells were placed phology analysis. Thereafter, the slides were washed in a Warner chamber mounted on the stage of an inverted three times with PBS, mounted with aqueous mounting microscope (Olympus, IX-70, Olympus Corporation, media and cover-slipped. Cardiomyocyte cross-sectional Tokyo, Japan) and superfused (approximately 1 mL/min at areas were calculated on a digital microscope (× 400) 25°C) with a buffer containing 131 mM NaCl, 4 mM KCl, using the Image J (version1.34S) software [5,20]. 1 mM CaCl2,1mMMgCl2, 10 mM glucose and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Caspase-3 assay (HEPES), at pH 7.4. The cells were field-stimulated with a Tissue homogenates were centrifuged (10,000 g at 4°C, 10 supra-threshold voltage at a frequency of 0.5 Hz for 3 ms minutes) and pellets were lysed in an ice-cold cell lysis using a pair of platinum wires placed on opposite sides of buffer. The assay was carried out in a 96-well plate with the chamber connected to a FHC stimulator (Brunswick, each well containing 30 μL cell lysate, 70 μL of assay buf- NE, USA). The studied myocyte was displayed on the fer (50 mM HEPES, 0.1% 3-([3-cholamidopropyl]- computer monitor using an IonOptix MyoCam camera. dimethyllammonio)-1-propanesulfonate (CHAPS), 100 IonOptix SoftEdge software was used to capture changes mM NaCl, 10 mM dithiothreitol and 1 mM ethylenedia- in cell length. Cell shortening and relengthening were minetetraacetic acid) and 20 μL of caspase-3 colorimetric assessed using the following indices: peak shortening (PS), substrate Ac-DEVD-pNA. The 96-well plate was incu- the peak ventricular contractility; time-to-PS (TPS; con- bated at 37°C for one hour, during which time the caspase traction duration) and time-to-90% relengthening (TR90), in the sample was allowed to cleave the chromophore the cardiomyocyte relaxation duration; and maximal pNA from the substrate molecule. Caspase-3 activity was Zhang et al. BMC Medicine 2012, 10:40 Page 4 of 17 http://www.biomedcentral.com/1741-7015/10/40

expressed as picomoles of pNA released per microgram of was detected as previously described [27]. Briefly, after protein per minute [5]. incubation with JC-1 (5 μM) for 10 minutes at 37°C, cells were rinsed twice by sedimentation using the HEPES sal- Aconitase activity ine buffer free of JC-1 before being examined under a Mitochondrial aconitase, an iron-sulfur enzyme located confocal laser scanning microscope (Leica TCS SP2, in the citric acid cycle, is readily damaged by oxidative Leica Microsystems Inc. Buffalo Grove, IL, USA) at an stress via removal of an iron from the [4Fe-4S] cluster. excitation wavelength of 490 nm. The emission of fluor- Mitochondrial fractions prepared from whole heart escence was recorded at 530 nm (monomer form of JC-1, homogenate were resuspended in 0.2 mM sodium citrate. green) and at 590 nm (aggregate form of JC-1, red). An aconitase activity assay (Aconitase Activity Assay Kit, Results in fluorescence intensity were expressed as the Aconitase-340 Assay; OxisResearch, Portland, OR, USA) 590 nm to 530 nm emission ratio. The mitochondrial was performed according to the manufacturer instruc- uncoupler carbonyl cyanide m-chlorophenylhydrazone tions with minor modifications. Briefly, the mitochon- (10 μM) was used as a positive control for the mitochondrial sample (50 μL) was mixed in a 96-well plate with 50 drial membrane potential measurement. μL trisodium citrate (substrate) in Tris-HCl pH 7.4, 50 μL isocitrate dehydrogenase (enzyme) in Tris-HCl, and Western blot analysis 50 μLNADPinTris-HCl.Afterincubatingfor15min- The myocardial protein was prepared as previously utes at 37°C, the absorbance was dynamically recorded at described [27]. The antibodies used for western blotting 340 nm every minute for five minutes with a spectro- included anti-PGC-1a, anti-UCP-2 (EMD Millipore Bill- photometer. During the assay, citrate is isomerized by erica, MA, USA), anti-ALDH2 (gift from Dr. Henry aconitase into isocitrate and eventually a-ketoglutarate. Weiner, Purdue University, West Lafayette, IN, USA), The Aconitase-340 Assay measures NADPH formation, a anti-GSK3b, anti-phosphorylated(p)-GSK3b (Ser9), anti- product of the oxidation of isocitrate to a-ketoglutarate. Akt, anti-pAkt (Thr308), anti-Foxo3a, anti-pFoxo3a Tris-HCl buffer (pH 7.4) was served as the blank [25]. (Thr32), anti-mTOR, anti-pmTOR (Ser2448), anti-PTEN, anti-pPTEN (Ser380), anti-sarcoendoplasmic reticulum Terminal deoxynucleotidyl transferase mediated dUTP Ca2+-ATPase (SERCA2a; Affinity Bioreagents Inc., nick end labeling assay Golden,CO,USA),anti-Na+-Ca2+ exchanger (1:1000; Terminal deoxynucleotidyl transferase mediated dUTP Sigma, St. Louis, MO, USA), anti-phospholamban nick end labeling (TUNEL) staining of myonuclei positive (1:500; Abcam, Cambridge, MA, USA) and anti-glyceral- for DNA strand breaks was determined using a fluores- dehyde 3-phosphate dehydrogenase (GAPDH; loading cence detection kit (Roche, Indianapolis, IN, USA) and control). Antibodies for GSK3b,pGSK3b,Akt,pAkt, fluorescence microscopy. Paraffin-embedded sections (5 Foxo3a, pFoxo3a, mTOR, pmTOR, PTEN and pPTEN μm) were incubated with Proteinase K solution for 30 were purchased from Cell Signaling Technology (Bev- minutes. TUNEL reaction mixture containing terminal erly, MA, USA) while antibody for PGC-1a was deoxynucleotidyl transferase and fluorescein-dUTP was obtained from Santa Cruz Biotechnology (Santa Cruz, added to the sections in 50 μL drops and incubated for CA, USA) unless otherwise indicated. The membranes 60 minutes at 37°C in a humidified chamber in the dark. were incubated with horseradish peroxidase-coupled The sections were rinsed three times in PBS for five min- secondary antibodies. After immunoblotting, films were utes each. Following embedding, sections were visualized scanned and detected with a Bio-Rad Calibrated Densit- with an Olympus BX-51 microscope equipped with an ometer Hercules, CA, USA) Olympus MaguaFire SP digital camera. DNase I and label solution were used as positive and negative controls. To Data analysis determine the percentage of apoptotic cells, micrographs Data are presented as the mean ± standard error of the of TUNEL-positive and 4’-6-dia!midino-2-phenylindole- mean (SEM). Statistical comparison was performed by a stained nuclei were captured using an Olympus fluores- one-way analysis of variance (ANOVA), with a two-way cence microscope and counted using the ImageJ software ANOVA for RER and physical activity studies, followed (ImageJ version 1.43r; National Institutes of Health) fol- by Tukey’s post hoc test. Significance was set as P < lowed by manual exclusion of the false-positive staining 0.05. from 10 random fields at 400 × magnification. At least 100 cells were counted in each field [26]. Results General features, mitochondrial and metabolic properties Measurement of mitochondrial membrane potential of normal and diabetic mice Murine cardiomyocytes were suspended in HEPES saline STZ treatment significantly reduced body but not organ buffer and the mitochondrial membrane potential (ΔΨm) weights in FVB and ALDH2 mice. Although STZ Zhang et al. BMC Medicine 2012, 10:40 Page 5 of 17 http://www.biomedcentral.com/1741-7015/10/40

treatment did not affect the size of the kidney (organ-to- Echocardiographic properties of normal and diabetic body weight ratio), it overtly increased heart and liver mice sizes in FVB but not ALDH2 mice. ALDH2 transgene Heart rate and absolute LV mass (calculated using echo- did not affect the body and organ weights or organ cardiography) were comparable among all mouse sizes. As expected, blood glucose (fasting and postpran- groups, regardless of diabetic or ALDH2 state. However, dial) and free fatty acid levels were significantly elevated diabetes increased the normalized LV mass value in the whereas plasma insulin levels were severely decreased in FVB but not ALDH2 group, consistent with its effect on STZ-challenged mice compared with the non-diabetic gross heart weights. Experimental diabetes significantly FVB mice. ALDH2 did not affect the levels of fasting or increased EDD and ESD and decreased wall thickness, postprandial blood glucose, serum free fatty acids or the effect of which was mitigated by ALDH2. Interest- plasma insulin in either normal or diabetic groups ingly, STZ treatment significantly depressed fractional (Table 1). Furthermore, experimental diabetes decreased shortening in FVB mice. ALDH2 mitigated the diabetes- both protein level and enzymatic activity of ALDH2, the induced decrease in fractional shortening without elicit- effect of which was masked by ALDH2 overexpression. ing any effect itself (Figure 3). STZ elicited overt apoptosis (as evidenced by caspase-3 activity) and mitochondrial damage (reduced aconitase Cardiomyocyte contractile and intracellular Ca2+ activity). Although ALDH2 enzymatic overexpression properties itself had little effect on apoptosis and mitochondrial Neither experimental diabetes nor ALDH2 overexpression function, it significantly attenuated or nullified diabetes- affected resting cell length, as depicted in Figure 4. Experi- induced apoptosis and mitochondrial damage (Figure 1). mental diabetes significantly reduced PS and maximal To evaluate the impact of ALDH2 overexpression on velocity of shortening and relengthening (± dL/dt) as well metabolic indices, calorimetric parameters were obtained as prolonged TPS and TR90 in FVB cardiomyocytes, remi- in control and diabetic FVB and ALDH2 mice using the niscent of our earlier findings [28,29]. Importantly, six-chamber Oxymax system (Columbus Instruments, ALDH2 abolished diabetes-induced mechanical abnormal- Columbus, OH, USA) as previously described [19]. Dia- ities without eliciting any notable effect by itself. To betic FVB and ALDH2 mice had a significantly lower explore the potential mechanism of action involved in the RER (VCO2/VO2), especially during the dark cycle, com- ALDH2-elicited beneficial effects against experimental dia- pared with control FVB and ALDH2 mice. This diabetes- betes, fura-2 fluorescence microscopy was employed to associated change in RER was not due to altered physical monitor the intracellular Ca2+ homeostasis. Data pre- activity. There was little difference in physical activity sented in Figure 5 reveal a significantly depressed intracel- among the four mouse groups tested (Figure 2). These lular Ca2+ rise in response to electrical stimulus (ΔFFI) results indicate that STZ-induced diabetic mice oxidized and reduced intracellular Ca2+ decay rate (single or bi- a lower proportion of carbohydrate compared with con- exponential curve fit) along with unchanged baseline intra- trol mice, reflecting a higher fractional reliance on lipid cellular Ca2+ in cardiomyocytes from STZ-treated mouse rather than glucose as the main energy source. ALDH2 hearts. ALDH2 overexpression negated STZ-induced pro- overexpression did not alter the energy source globally in longationinintracellularCa2+ decay and depression in control or diabetic mice. ΔFFI with little effect on baseline FFI. Last but not least,

Table 1 Biometric parameters of control or diabetic FVB and ALDH2 mice Parameter FVB FVB-STZ ALDH2 ALDH2-STZ Body weight (g) 30.3 ± 1.3 24.2 ± 0.7a 29.5 ± 1.1 26.1 ± 0.8a Heart weight (mg) 165 ± 9 171 ± 8 163 ± 6 159 ± 5 Heart/body weight ratio (mg/g) 5.45 ± 0.19 7.23 ± 0.43a 5.66 ± 0.26 6.16 ± 0.18b Liver weight (g) 1.54 ± 0.07 1.44 ± 0.07 1.52 ± 0.05 1.46 ± 0.06 Liver/body weight (mg/g) 51.2 ± 1.4 59.4 ± 2.3a 51.9 ± 1.9 54.9 ± 1.9 Kidney weight (g) 0.45 ± 0.03 0.39 ± 0.02 0.42 ± 0.02 0.40 ± 0.03 Kidney/body weight (mg/g) 14.8 ± 0.4 16.1 ± 0.8 14.4 ± 0.4 15.4 ± 0.8 Plasma insulin (ng/mL) 0.27 ± 0.03 0.05 ± 0.01a 0.28 ± 0.03 0.06 ± 0.02a Fasting blood glucose (mM) 5.50 ± 0.26 19.7 ± 1.5a 5.43 ± 0.18 18.5 ± 1.7a Postprandial blood glucose (mM) 9.94 ± 0.67 25.3 ± 1.5a 10.57 ± 0.54 26.5 ± 1.8a Plasma free fatty acids (mM) 1.00 ± 0.11 1.30 ± 0.04a 1.00 ± 0.15 1.25 ± 0.08a Mean ± SEM, n = 21 to 22 mice per group. aP < 0.05 versus FVB group; bP < 0.05 versus FVB-STZ group. Zhang et al. BMC Medicine 2012, 10:40 Page 6 of 17 http://www.biomedcentral.com/1741-7015/10/40

Figure 1 Influence of streptozotocin and ALDH2 on expression and activity of ALDH2, apoptosis and mitochondrial function.(A) ALDH2 expression; (B) ALDH2 enzymatic activity measured using spectrophotometry; (C) caspase-3 activity; (D) mitochondrial aconitase activity. Inset: Representative gel blots of ALDH2 and GAPDH (loading control) using specific antibodies. Mean ± SEM, n = 5 to 7 per group. *P < 0.05 versus FVB group; #P <0.05 versus FVB-STZ group.

Figure 2 Oxygen consumption and total activity during day and night time in FVB and ALDH2 transgenic mice treated with or without streptozotocin. (A) Respiratory exchange ratio (RER) during day time; (B) RER at night; (C) total activity during day time; (D) total activity at night. Mean ± SEM, n = 4 to 5 mice per group. *P < 0.05 versus FVB group for both STZ groups. Zhang et al. BMC Medicine 2012, 10:40 Page 7 of 17 http://www.biomedcentral.com/1741-7015/10/40

Figure 3 Echocardiographic properties in FVB and ALDH2 transgenic mice treated with or without streptozotocin. (A) Representative echocardiographic images; (B) heart rate; (C) wall thickness; (D) left ventricular (LV) end-diastolic diameter; (E) LV end-systolic diameter; (F) LV mass; (G) LV mass normalized to body weight; (H) fractional shortening. Mean ± SEM, n = 12 to 13 mice per group. *P <0.05 versus FVB group; #P <0.05 versus FVB-STZ group. the ALDH2 transgene itself did not affect the intracellular examined. Low magnification transverse heart sections Ca2+ indices tested. indicated reduced LV wall thickness and enlarged chamber size in mice with experimental diabetes, with lesser Effect of ALDH2 on diabetes-induced change in alteration in ALDH2 transgenic mice. Findings from myocardial histology FITC-conjugated wheat germ staining sections revealed To assess the impact of ALDH2 overexpression on myo- increased cardiomyocyte area after the induction of cardial histology after STZ treatment, heart gross mor- experimental diabetes, consistent with increased normal- phology and the cardiomyocyte cross-sectional area were ized LV mass, EDD and ESD in FVB-STZ mice. The Zhang et al. BMC Medicine 2012, 10:40 Page 8 of 17 http://www.biomedcentral.com/1741-7015/10/40

Figure 4 Cardiomyocyte contractile properties in FVB and ALDH2 transgenic mice treated with or without streptozotocin. (A) Resting cell length; (B) peak shortening (PS), normalized to cell length; (C) maximal velocity of shortening (+ dL/dt); (D) maximal velocity of

relengthening (- dL/dt); (E) time-to PS (TPS); (F) time-to-90% relengthening (TR90). Mean ± SEM, n = 101 to 102 cells from four mice per group. *P <0.05 versus FVB group; #P <0.05 versus FVB-STZ group.

experimental diabetes-induced change in cardiomyocyte TUNEL staining, and mitochondrial integrity, using JC-1 size was effectively ablated by ALDH2 overexpression fluorescence microscopy. Results shown in Figure 7 while the ALDH2 transgene itself did not affect cardio- (panels A-I) indicate that the TUNEL-positive cells were myocyte size (Figure 6). more abundant in STZ-treated FVB mice, the effect of which was significantly attenuated by ALDH2 overex- Effects of ALDH2 on diabetes-induced apoptosis and pression. ALDH2 itself did not affect myocardial apop- mitochondrial damage tosis. Our fluorescence data displayed in Figure 7 To further examine the mechanism(s) of action behind (panels J and K) revealed loss of mitochondrial mem- ALDH2-elicited protection against STZ-induced cardiac brane potential in cardiomyocytes from STZ-treated mechanical dysfunction, the myocardium and cardio- FVB mice, the effect of which was reconciled by ALDH2 myocytes from normal or diabetic FVB and ALDH2 overexpression. ALDH2 itself did not affect the mito- mice were examined for myocardial apoptosis, using chondrial membrane potential. These findings indicate a Zhang et al. BMC Medicine 2012, 10:40 Page 9 of 17 http://www.biomedcentral.com/1741-7015/10/40

Figure 5 Cardiomyocyte intracellular Ca2+ handling properties in FVB and ALDH2 transgenic mice treated with or without streptozotocin. (A) Resting fura-2 fluorescence intensity (FFI); (B) electrically-stimulated rise in FFI (ΔFFI); (C) intracellular Ca2+ decay rate (single exponential); (D) intracellular Ca2+ decay rate (bi-exponential). Mean ± SEM, n = 76 to 77 cells from four mice per group. *P <0.05 versus FVB group; #P<0.05 versus FVB-STZ group.

corroborative role of apoptosis and mitochondrial func- Western blot analysis for Akt, GSK3b, Foxo3a, mTOR and tion in ALDH2-offered cardioprotection against experi- PTEN signaling mental diabetes. To examine possible signaling mechanism(s) involved in the ALDH2-offered protection against diabetes-induced Expression of UCP-2, PGC1a, SERCA2a, Na+-Ca2+ myocardial anomalies, the expression and phosphoryla- exchanger and phospholamban tion of post-insulin receptor signaling, including Akt To explore the possible mechanism behind ALDH2 and and the Akt downstream signaling molecules GSK3b, diabetes-induced responses on cardiac function, particu- Foxo3a and mTOR, were evaluated. Western blot find- larly on mitochondrial function and intracellular Ca2+ ings revealed that diabetes overtly dampened phosphory- homeostasis, western blot analysis was performed to lation of Akt, GSK3b and Foxo3a without affecting that assess the levels of the key mitochondrial proteins UCP-2 of mTOR, the effect of which was mitigated by ALDH2 and PGC1a as well as the essential intracellular Ca2+ reg- overexpression. Neither diabetes nor ALDH2 affected ulatory proteins SERCA2a, Na+-Ca2+ exchanger and the pan protein expression of Akt, Foxo3a or mTOR phospholamban. Our data shown in Figure 8 depict that (Figure 9). To explore the possible mechanisms respon- diabetes significantly downregulated the expression of sible for ALDH2- and experimental diabetes-elicited UCP-2, PGC1a,SERCA2aandNa+-Ca2+ exchanger changes in Akt phosphorylation, levels of pan and while upregulating the level of phospholamban in FVB pPTEN, a negative regulator of Akt signaling, were mice. Although the ALDH2 transgene itself did not alter examined in control and diabetic FVB and ALDH2 the expression of UCP-2, PGC1a,SERCA2a,Na+-Ca2+ mice. Our data shown in Figure 10 revealed that STZ exchanger or phospholamban, it nullified STZ-induced treatment significantly increased phosphorylation of changes in all five. PTEN (both absolute and normalized value) without Zhang et al. BMC Medicine 2012, 10:40 Page 10 of 17 http://www.biomedcentral.com/1741-7015/10/40

Figure 6 Histological analyses in hearts from FVB and ALDH2 transgenic mice treated with or without streptozotocin. (A-D) Representative photomicrographs from gross morphological view of transverse myocardial sections (scale bar = 1 mm). (E-H) Representative fluorescein isothiocyanate-conjugated wheat germ agglutinin staining depicting cardiomyocyte size (× 200; scale bar = 100 μm). (I) Quantitative cardiomyocyte cross-sectional (transverse) area from 60 cells from three mice per group. Mean ± SEM, *P < 0.05 versus FVB; #P < 0.05 versus FVB-STZ group. affecting pan protein expression of PTEN, the effect of in diabetes, cardiomyocytes from control FVB mice were which was mitigated by ALDH2 overexpression. The exposed to high glucose (25.5 mM) in the absence or pre- ALDH2 transgene itself did not affect pan or phos- sence of the ALDH2 activator Alda-1 (20 μM), the mito- phorylated levels of PTEN. chondrial uncoupler FCCP (1 μM) or the GSK3b inhibitor SB216763 (10 μM) for 12 hours [6,9,30] prior to an assess- Influence of ALDH2 activation, mitochondrial uncoupling ment of mechanical and biochemical properties. Figure 11 and GSK3b inhibition on high glucose-induced depicts that high glucose significantly dampened mito- cardiomyocyte mitochondrial and contractile responses chondrial function (shown as loss of aconitase activity) To further examine the causal relationships between and cardiomyocyte contractile function (shown as reduced ALDH2-induced mechanical and mitochondrial responses PS, ± dL/dt and prolonged TR90), the effect of which was Zhang et al. BMC Medicine 2012, 10:40 Page 11 of 17 http://www.biomedcentral.com/1741-7015/10/40

Figure 7 Effect of streptozotocin and ALDH2 on myocardial apoptosis and mitochondrial membrane potential using TUNEL staining and JC-1 fluorescence. TUNEL-positive nuclei were visualized with fluorescein (green) in panels (A) FVB, (C) FVB-STZ, (E) ALDH2 and (G) ALDH2-STZ. All nuclei were stained with 4’-6-diamidino-2-phenylindole (blue) in panels (B) FVB, (D) FVB-STZ, (F) ALDH2 and (H) ALDH2-STZ. Original magnification × 400. (I) Quantified data. (J) Quantitative analysis of cardiomyocyte mitochondrial membrane potential using JC-1 ratio in FVB and ALDH2 mice treated with or without STZ (10 μM carbonyl cyanide m-chlorophenylhydrazone was used as a positive control). (K) Representative JC-1 fluorochrome images depicting mitochondrial membrane potential in cardiomyocytes. Mean ± SEM, n = 12 and 7 fields from three mice per group for panel I and J, respectively. *P < 0.05 versus FVB group; #P < 0.05 versus FVB-STZ group. abolished by Alda-1 and SB216763, without any additive including phosphorylation of Akt, GSK3b and Foxo3a effect between the two. Interestingly, FCCP abolished transcriptional factor. These data favor a likely role of the Alda-1-induced beneficial mitochondrial and mechanical activation of Akt and GSK3b as well as inactivation effects. There was little effect on mitochondrial integrity (phosphorylation) of Foxo3a in ALDH2-elicited preserva- and mechanical function by the pharmacological inhibitors tion of mitochondrial and mechanical function in dia- themselves (data not shown for FCCP). betes. Our data further reveal that ALDH2 may preserve Akt activation in diabetes through ablation of diabetes- Discussion induced mitochondrial injury and/or increasing the phos- Earlier findings from our group indicated that ALDH2 phorylation of PTEN, a negative regulator of Akt [16]. An may rescue against ischemic and alcoholic injuries to the analysis of global metabolism indicated that ALDH2 heart [3,7,15]. Data from this study provides, for the first failed to alter diabetes-induced changes in plasma levels time, compelling evidence that ALDH2 protects against of glucose (fasting and postprandial), insulin and serum diabetes-induced myocardial remodeling and contractile free fatty acids, the RER or total physical activity, exclud- defect through lessened apoptosis, preserved mitochon- ing the possibility of a potential cardiac protective effect drial function and post-insulin receptor signaling, secondary to any ALDH2-elicited global metabolic Zhang et al. BMC Medicine 2012, 10:40 Page 12 of 17 http://www.biomedcentral.com/1741-7015/10/40

Figure 8 Western blot analysis of the mitochondrial proteins UCP-2 and PGC1a as well as the Ca2+ regulatory proteins SERCA2a, Na +-Ca2+ exchanger and phospholamban in myocardium from FVB and ALDH2 mice treated with or without streptozotocin. (A) Representative gel blots of UCP-2, PGC1a, SERCA2a, Na+-Ca2+ exchanger, phospholamban and GAPDH (loading control) using specific antibodies; (B) UCP-2; (C) PGC1a; (D) SERCA2a; (E) Na+-Ca2+ exchanger; (F) phospholamban. All proteins were normalized to the loading control GAPDH. Mean ± SEM, n = 5 to 6 mice per group. *P < 0.05 versus FVB group; #P < 0.05 versus FVB-STZ group. benefits. Taken together, these findings should lead to a stress [28,29]. In our study, the impaired intracellular better understanding of the role of ALDH2 in myocardial Ca2+ handling (reduced intracellular Ca2+ clearance and anomalies in diabetes. intracellular Ca2+ rise (ΔFFI)) may likely underscore the Reduced contractility and prolonged duration of systole prolonged duration of contraction and relaxation and the as well as diastole are hallmarks of diabetic cardiomyopa- reduced PS, maximal velocity of shortening and relength- thy [17,28,29]. Findings from our present study revealed ening and fractional shortening observed in STZ-induced reduced fractional shortening; enlarged EDD and ESD; diabetic mouse hearts. The fact that the ALDH2 transgene decreased wall thickness, PS and ± dL/dt; and prolonged reconciled STZ-induced intracellular Ca2+ mishandling 2+ TPS and TR90 in whole hearts and isolated cardiomyo- favors a possible role of intracellular Ca homeostasis in cytes in diabetic mice. These findings are similar to our diabetes-induced myocardial dysfunction and ALDH2- previous findings [17,28,29]. Several mechanisms may be offered protection, somewhat reminiscent of the beneficial postulated for diabetes-related abnormalities such as role of mitochondrial protection against diabetes or obe- impaired intracellular Ca2+ homeostasis and oxidative sity-induced myocardial dysfunction [31,32]. Our findings Zhang et al. BMC Medicine 2012, 10:40 Page 13 of 17 http://www.biomedcentral.com/1741-7015/10/40

Figure 9 Phosphorylation of Akt, GSK3b, mTOR and Foxo3a in myocardium from FVB and ALDH2 mice treated with or without streptozotocin. (A) pAkt-to-Akt ratio; (B) pGSK3b-to-GSK3b ratio; (C) pmTOR-to-mTOR ratio; (D) pFoxo3a-to-Foxo3a ratio. Insets: representative gel blots of pan and phosphorylated Akt, GSK3b, mTOR and Foxo3a (GAPDH as loading control) using specific antibodies. Mean ± SEM, n = 6 to 7 mice per group. *P < 0.05 versus FVB group; #P < 0.05 versus FVB-STZ group. revealed a loss of mitochondrial membrane potential and preserved myocyte survival and mitochondrial integrity. overt apoptosis (demonstrated by caspase-3 and TUNEL) These beneficial effects of ALDH2 in cardiac geometry along with downregulated levels of PGC1a and UCP-2 in and function, cell survival and mitochondrial integrity STZ-induced diabetic hearts, suggesting a corroborative were seen despite the persistent hyperglycemic and role of mitochondrial dysfunction and apoptosis in dia- hyperlipidemic environments in STZ-induced experi- betic cardiomyopathy, as reported previously [31]. mental diabetes, thus excluding a possible secondary In addition, our observations that the ALDH2 trans- effect for ALDH2-induced protection against diabetic gene restored downregulated expression of SERCA2a and cardiomyopathy. This is further supported by the fact Na+-Ca2+ exchanger as well as upregulated phospholam- that ALDH2 failed to alter global metabolism (RER and ban in diabetes also support a role of intracellular Ca2+ physical activity) in diabetes. In our study, STZ failed to homeostasis in diabetes-induced cardiac contractile dys- elicit any hypertrophic effect as evidenced by absolute function and ALDH2-offered protection. heart weight and LV mass, although it enhanced cardio- Perhaps our most significant finding is that ALDH2 myocyte size and heart-to-body weight ratio, and nor- overexpression reconciled diabetes-induced cardiac malized LV mass. These effects were likely due to an remodeling (represented by cardiomyocyte cross-sec- STZ-induced loss in body weight. Interestingly, ALDH2 tional area, changes in LV wall thickness, ESD and overexpression attenuated diabetes-induced changes in EDD) and contractile dysfunction in association with cardiomyocyte, heart and LV sizes, possibly due to the Zhang et al. BMC Medicine 2012, 10:40 Page 14 of 17 http://www.biomedcentral.com/1741-7015/10/40

Figure 10 Total and phosphorylated PTEN in myocardium from FVB and ALDH2 mice treated with or without streptozotocin. (A) Representative gel blots of pan and phosphorylated PTEN and GAPDH (used as loading control) using specific antibodies; (B) pan PTEN expression; (C) pPTEN levels; (D) pPTEN-to-PTEN ratio. Mean ± SEM, n = 6 mice per group. *P < 0.05 versus FVB group; #P < 0.05 versus FVB-STZ group. antagonism of ALDH2 against diabetes-induced cardiac accumulate and interact with functional signaling systems apoptosis and mitochondrial damage. Both apoptosis to impose oxidative damage and associated gene altera- and mitochondrial damage are known to regulate car- tions in response to the stress challenge [34]. The notion diac remodeling in diabetes and obesity [31,33]. Our that ALDH2 protects against diabetic cardiomyopathy observation of preserved levels of the mitochondrial pro- through preservation of mitochondrial integrity was teins PGC1a and UCP-2 as well as of aconitase activity further substantiated by our in vitro findings. Our results and mitochondrial membrane potential in ALDH2 mice revealed that the ALDH2 activator Alda-1 effectively res- after STZ treatment strongly supported a role of mito- cued against high glucose-induced mitochondrial and chondrial function in ALDH2-offered cardioprotection. mechanical dysfunctions, and this effect was nullified by The therapeutic role of the mitochondrial protein the mitochondrial uncoupling compound FCCP. These ALDH2 in diabetes is consistent with the fact that the data convincingly support the permissive role of mito- protein level and enzymatic activity of ALDH2 are both chondria in ALDH2-offered cardioprotection against reduced in experimental diabetes [13] (also seen in our hyperglycemia-induced anomalies. study) while inactive ALDH2 promotes hyperglycemia Data from our study showed dampened phosphoryla- and enhances the risk of diabetes [14]. tion of the post-insulin receptor signaling Akt, GSK3b It is noteworthy that the reduction in the ALDH2 and Foxo3a in STZ-treated diabetic hearts, in line with expression and activity is relatively minor although such mitochondrial injury in diabetes and observations from subtle loss of ALDH2 may be sufficient to trigger overt our earlier studies [28,35]. These signaling molecules changes in mitochondrial integrity and cell survival. play an essential role in the maintenance of cardiac survi- Although it is beyond the scope of our current study, the val, structure and function. Akt, GSK3b and mTOR are main substrate for ALDH2 detoxification, aldehydes, serve essential post-insulin receptor signaling molecules, which as the main source for oxidative stress and pathological may be compromised after mitochondrial injury and con- changes in disease condition. Even with a moderately tribute to apoptosis and cardiac dysfunction in pathologi- reduced ALDH2 level, sublethal levels of aldehydes may cal conditions [10,14,36]. Our data revealed that diabetes Zhang et al. BMC Medicine 2012, 10:40 Page 15 of 17 http://www.biomedcentral.com/1741-7015/10/40

Figure 11 Influence of the ALDH2 agonist Alda-1 on high glucose-induced responses of aconitase activity and cardiomyocyte contractile properties. Cardiomyocytes from control FVB mice were exposed to high glucose (HG; 30 mM) in the absence or presence of Alda- 1 (20 μM), the mitochondrial uncoupler FCCP (1 μM) or the GSK3b inhibitor SB216763 (10 μM) for 12 hours prior to assessment of mitochondrial and mechanical properties. (A) Aconitase activity; (B) peak shortening (PS; normalized to cell length); (C) maximal velocity of shortening (+ dL/

dt); (D) maximal velocity of relengthening (- dL/dt); (E) time-to PS (TPS); (F) time-to-90% relengthening (TR90). Mean ± SEM, n = 5 isolations (panel A) or 72 to 73 cells per group (Panel B-F). *P < 0.05 versus control group; #P < 0.05 versus HG group. dampened phosphorylation of Akt and its downstream staining) after STZ treatment, as reported previously by signaling molecules Foxo3a and GSK3b (although not our group using the same diabetic model [35]. GSK3b,a mTOR), the effect of which was nullified by ALDH2 serine/threonine kinase downstream of Akt that is inacti- transgene. The decrease in the phosphorylation of vated by oxidative stress through the phosphorylation of Foxo3a and GSK3b is expected to result from dampened Ser9, serves as a negative regulator of cardiac hypertro- Akt phosphorylation. The reduced phosphorylation of phy and mitochondrial function through mitochondrial Foxo3a appears to coincide with overt mitochondrial permeation pore opening [24,25,37]. Data from our study injury (as evidenced by mitochondrial membrane poten- revealed that ALDH2 abrogated the diabetes-induced tial and levels of PGC1a, UCP-2 and aconitase) and decrease in GSK3b phosphorylation, aconitase activity apoptosis (demonstrated by caspase-3 and TUNEL and levels of PGC1a and UCP-2, favoring a possible role Zhang et al. BMC Medicine 2012, 10:40 Page 16 of 17 http://www.biomedcentral.com/1741-7015/10/40

of GSK3b signaling and mitochondrial protection in 90% relengthening; TUNEL: terminal deoxynucleotidyl transferase mediated ALDH2-offered cardioprotection. This is supported by dUTP nick end labeling assay. the finding that GSK3b inhibition using SB216763 and Acknowledgements mitochondrial uncoupling using FCCP respectively This work was supported by NIAAA 1R01 AA013412 and NCRR ablated high glucose and Alda-1-induced mitochondrial 5P20RR016474 to JR. and mechanical changes. Author details Mitochondrial injury is known to compromise insulin 1Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an, 710032, China. 2Center for Cardiovascular Research and signaling at both insulin receptor and post-receptor Alternative Medicine, University of Wyoming College of Health Sciences, levels [30]. A recent report from our group revealed Laramie, WY 82071, USA. that protection of mitochondrial integrity using cardiac- Authors’ contributions specific overexpression of insulin-like growth factor 1 YZ, SAB, NH and JRM participated in data collection; YZ, HW, JR designed effectively alleviates high fat diet intake-induced loss of the study, secured the research funding and wrote the manuscript. All insulin sensitivity, oxidative stress and contractile dys- authors have read and approved the final manuscript. function in the heart [38], supporting the pivotal role of Competing interests mitochondria in the maintenance of cardiac insulin sig- The authors declare that they have no competing interests. naling. Nonetheless, our data also depicted elevated Received: 18 January 2012 Accepted: 23 April 2012 phosphorylation of the Akt negative regulator PTEN in Published: 23 April 2012 experimental diabetes, the effect of which was mitigated by ALDH2. 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