Aldehyde dehydrogenase 2 ameliorates doxorubicin-induced myocardial dysfunction through detoxification of 4-HNE and suppression of autophagy

Journal of Molecular and Cellular Cardiology 71 (2014) 92–104

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Original article

Aldehyde dehydrogenase 2 ameliorates doxorubicin-induced myocardial dysfunction through detoxification of 4-HNE and suppression of autophagy Aijun Sun a,b,⁎,1, Yong Cheng a,d,1, Yingmei Zhang c,1, Qian Zhang a,e, Shijun Wang a,b, Shan Tian f, Yunzeng Zou a,b, Kai Hu a, Jun Ren a,c, Junbo Ge a,b a

Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai 200032, China Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China c Center for Cardiovascular Research and Alternative Medicine, University of Wyoming College of Health Sciences, Laramie, WY 82071, USA d Heart Centre of Zhengzhou Ninth People's Hospital, Zhengzhou, Henan 450000, China e Department of Cardiology, Branch of Shanghai First People's Hospital, Shanghai 200050, China f Department of Rehabilitation, Huashan Hospital, Fudan University, Shanghai 200040, China b

a r t i c l e

i n f o

Article history: Received 14 October 2013 Received in revised form 6 December 2013 Accepted 3 January 2014 Available online 13 January 2014 Keywords: ALDH2 Dilated cardiomyopathy Doxorubicin 4-HNE Autophagy

a b s t r a c t Mitochondrial aldehyde dehydrogenase (ALDH2) protects against cardiac injury via reducing production of 4hydroxynonenal (4-HNE) and ROS. This study was designed to examine the impact of ALDH2 on doxorubicin (DOX)-induced cardiomyopathy and mechanisms involved with a focus on autophagy. 4-HNE and autophagic markers were detected by Western blotting in ventricular tissues from normal donors and patients with idiopathic dilated cardiomyopathy. Cardiac function, 4-HNE and levels of autophagic markers were detected in WT, ALDH2 knockout or ALDH2 transfected mice treated with or without DOX. Autophagy regulatory signaling including PI-3K, AMPK and Akt was examined in DOX-treated cardiomyocytes incubated with or without ALDH2 activator Alda-1. DOX-induced myocardial dysfunction, upregulation of 4-HNE and autophagic proteins were further aggravated in ALDH2 knockout mice while they were ameliorated in ALDH2 transfected mice. DOX downregulated Class I and upregulated Class III PI3-kinase, the effect of which was augmented by ALDH2 deletion. Accumulation of 4-HNE and autophagic protein markers in DOX-induced cardiomyocytes was significantly reduced by Alda-1. DOX depressed phosphorylated Akt but not AMPK, the effect was augmented by ALDH2 knockout. The autophagy inhibitor 3-MA attenuated, whereas autophagy inducer rapamycin mimicked DOX-induced cardiomyocyte contractile defects. In addition, rapamycin effectively mitigated Alda-1-offered protective action against DOX-induced cardiomyocyte dysfunction. Our data further revealed downregulated ALDH2 and upregulated autophagy levels in the hearts from patients with dilated cardiomyopathy. Taken together, our findings suggest that inhibition of 4-HNE and autophagy may be a plausible mechanism underscoring ALDH2-offered protection against DOX-induced cardiac defect. This article is part of a Special Issue entitled “Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy”. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Mitochondrial aldehyde dehydrogenase (ALDH2) is an allosteric tetrameric enzyme responsible for mitochondrial oxidative ATP generation [1,2]. Recent evidence has depicted a pivotal role for ALDH2 in the regulation of cardiac homeostasis under both physiological and pathological conditions such as ischemia/reperfusion injury, alcoholic cardiomyopathy and diabetes mellitus, possibly via alleviated accumulation of reactive

⁎ Corresponding authors at: Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai 200032, China. Tel.: + 86 2164041990 2745; fax: +86 2164223006. E-mail address: [email protected] (A. Sun). 1 These authors contributed equally to this work. 0022-2828/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.yjmcc.2014.01.002

oxygen species (ROS) in particular 4-hydroxynonenal (4-HNE) [3–7]. Along the same line, ALDH2 gene variation has been associated with an increased susceptibility to stroke and alcohol-associated dilated cardiomyopathy (DCM) [2,4,8,9]. Doxorubicin (DOX), on the other hand, may trigger a unique form of dilated cardiomyopathy through ROS accumulation [10] and oxidant-mediated DNA damage [11,12]. Nonetheless, it remains elusive if ALDH2 or ALDH2 gene mutation affects the pathogenesis and manifestation of dilated cardiomyopathy. Autophagy, a cellular process for degrading and recycling macromolecules, organelles and nutrients, is known to play an important role in the regulation of myocardial structure and function [13–15]. Dysregulated autophagy has been demonstrated in various forms of cardiac diseases including ischemia/reperfusion injury, cardiac hypertrophy, cardiac aging, alcoholic cardiomyopathy and heart failure [4,16–20]. In addition,

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autophagy may contribute to the maintenance of genomic integrity in the face of metabolic stress, drug treatment or radiation damage [17,21,22]. Recent experimental and clinical evidence has depicted a pivotal role of autophagy in failing myocardium [5,16,23–25]. In particular, autophagy has been shown to serve as a target for treatment of heart failure. It was demonstrated that left ventricular assist device (LVAD) improves cardiac function in association with retarded mRNA and protein levels of autophagy in patients with dilated cardiomyopathy [26]. Findings from our group showed that ALDH2 rescued against myocardial ischemia/reperfusion injury through either promoting autophagy during ischemia or inhibiting autophagy during reperfusion, suggesting a unique role for autophagy in the beneficial response of ALDH2 in cardiac pathology [27]. Recent evidence revealed that ALDH2 polymorphism is closely associated with an increased risk of heart disease and heart failure [28–30]. These observations are consistent with the notion that inactive ALDH2 gene dampens myocardial function under pathological conditions with overt cardiac stress [6,27]. To this end, this study was designed to examine the impact of ALDH2 knockout and viral overexpression on the pathogenesis of dilated cardiomyopathy and the underlying mechanism(s) involved. Given that recent evidence from our group revealed a pivotal role of autophagy regulation in ALDH2-offered cardioprotection against ER stress, alcoholism and ischemia–reperfusion [27,31,32], special attention was given to autophagy in dilated cardiomyopathy and ALDH2 genetic manipulation. 2. Methods 2.1. ALDH2 knockout and viral overexpression Sixty adult male C57BL/6 mice (6–8 weeks of age, weighing 20–22 g) were obtained from the Shanghai Animal Administration Center (Shanghai, China) or the University of Wyoming College of Health Science Animal Facility (Laramie, USA). ALDH2 knockout (KO) mice were generated as described previously [6]. Mice were randomly assigned to 8 groups including wild-type (WT), ALDH2 knockout, WT mice transfecting vehicle vector, and ALDH2 viral transfection mice treated with or without DOX (15 mg/kg, i.p. for 6 days) or equal volume saline (i.p.) for 6 days [33]. All animal study protocols were performed in accordance with the guidelines of the Animal Care and Use Committees at the Fudan University (Shanghai, China) and University of Wyoming, and were in compliance with the Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, revised 1996). 2.2. Adenoviral transfection of ALDH2 in vivo Adenoviral vectors encoding ALDH2 or empty vector (5 × 109 PFU in 50 μl medium) were injected into left ventricular cavity through apex of the heart under temporary clamping of ascending aorta and pulmonary artery for 30 s 2 days prior to intraperitoneal injection of DOX [34]. Ratio of adenoviral vector transfection into myocardium evaluated by eGFP vector was N 50%. 2.3. Echocardiography Echocardiography was performed using an animal specific instrument (Vevo707B, Visual Sonics Inc., Toronto, Canada) at pre-operation and 4 weeks post DOX or saline treatments. Animals were anesthetized by 1.5% isoflurane and M-mode images were recorded when the heart rate (HR) of the mice was maintained at 450–500 bpm. Left ventricular ejection fraction (LVEF), left ventricular end-diastolic dimension (LVEDD) and left ventricular fractional shortening (LVFS) were measured as previously described [35]. All measurements were averaged from five consecutive cardiac cycles and were carried out by three experienced technicians who were unaware of the identities of animal groups.

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2.4. Histological examination Following anesthesia, hearts were excised and immediately placed in 10% neutral-buffered formalin at room temperature for 24 h after a brief rinse with PBS. The specimen were embedded in paraffin, cut in 5 μm sections and stained with hematoxylin and eosin (H&E). Five to 8 random fields (× 400) were selected from each slide and imaged with a color digital camera [36]. 2.5. Human tissue acquisition Tissue samples from failing human hearts were obtained from endstage (NYHA III–IV) DCM patients (2 males and 1 female, average age: 46 years) underwent heart transplantation in Zhongshan Hospital (Shanghai, China). Left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were 72.3 ± 26.5 and 59.0 ± 24.8 mm, respectively, with an ejection fraction (EF) of 26.3 ± 7.5% prior to heart transplantation (Table 1). Three healthy adult donor hearts (2 males, 1 female, average age: 43 years) from accidental death where hearts could not be transplanted due to technical reason were used as control. Immediately after explanation, left ventricular free wall was stored at − 70 °C for protein analysis. The study protocol was in compliance with the principles outlined in the Declaration of Helsinki and was reviewed and approved by the local Ethical Committee. 2.6. Western blot analysis Total protein extracted from heart tissues, cardiomyocytes or cardiac mitochondria were fractionated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore). The membranes were immunoblotted with primary anti-ALDH2, anti-Beclin-1, anti-Atg5, antiLC3, anti-4-HNE (OXIS International, Inc., Beverly Hills, CA), anti-PI3K Class III, anti-PI3K Class I (p110α), anti-LKB1, anti-AMPK, antiphosphorylated AMPK (pAMPK, Ser9), anti-Akt, anti-phosphorylated Akt (pAkt, Ser473) and anti-GAPDH (loading control) antibodies. All antibodies were obtained from Cell Signaling (Danvers, MA) or Santa Cruz (Santa Cruz, CA) biotechnology companies unless otherwise stated. Quantification was performed using a Bio-Rad equipped with Image software Basic Quantity one. 2.7. Isolation of murine cardiomyocytes After ketamine/xylazine (ketamine 80 mg/kg and xylazine 12 mg/kg i.p.) sedation, hearts were removed and digested for 20 min with Liberase Blendzyme 4 (Roche Diagnostics Inc. Indianapolis, IN). Cardiomyocyte yield was ~75%. Only rod-shaped cardiomyocytes with clear edges were selected for mechanical study. For in vitro study, cardiomyocytes from WT mice were exposed to DOX (1 μM) [37,38]

Table 1 Anthropometric data for patients with idiopathic dilated cardiomyopathy and healthy donors.

Gender Ethnicity Mean age (year-old) Cause of death Mean LVEDD (mm) Mean LVESD (mm) Mean LAD (mm) Mean LVEF (%)

Healthy donors

Dilated cardiomyopathy

2 male, 1 female Chinese origin, Han ethnic 43 ± 2 (range 38–50) Drowning, blood loss N/A N/A N/A N/A

2 male, 1 female Chinese origin, Han ethnic 46 ± 2 (range 44–48) Terminal stage heart failure 72.3 ± 26.5 59.0 ± 24.8 41.3 ± 5.0 26.3 ± 7.5

N/A: not available; LVEDD: left ventricular end-diastolic diameter; LVESD: left ventricular end-systolic diameter; LAD: left atrial dimension; LVEF, left ventricular ejection fraction; Mean ± SEM.

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in the absence or presence of the ALDH2 activator Alda-1 (20 μM, EMD Millipore Corporation, Billerica, MA) [31], the autophagy inducer rapamycin (5 μM) [31] or the autophagy inhibitor 3-methyladenine (3-MA, 10 mM) [31] for 4 h prior to assessment of mechanical properties.

2.8. Cell shortening/relengthening Mechanical properties of cardiomyocytes were assessed using a SoftEdgeMyoCam® system (IonOptix Incorporation, Milton, MA) [4]. In brief, cells were placed in a Warner chamber mounted on the stage of an inverted microscope (Olympus, IX-70, Tokyo, Japan) and superfused (~ 1 ml/min at 25 °C) with a buffer containing (in mM): 131 NaCl, 4 KCl, 1 CaCl2 , 1 MgCl2 , 10 glucose, 10 HEPES, at pH 7.4. The cells were field stimulated with a supra-threshold voltage at a frequency of 0.5 Hz, 3 ms duration, using a pair of platinum wires placed on opposite sides of the chamber connected to a FHC stimulator (Brunswick, NE). The cardiomyocyte being studied was displayed on the computer monitor using an IonOptixMyoCam camera. IonOptixSoftEdge software was used to capture changes in cell length. Cell shortening and relengthening were assessed using the following indices peak shortening (PS), time-to-PS (TPS), time-to90% relengthening (TR90), maximal velocities of shortening (+ dL/ dt) and relengthening (− dL/dt).

3. Results 3.1. Echocardiographic properties of mice with ALDH2 knockout and ALDH2 transfection Echocardiographic examination showed that heart rate and LV mass were unaffected by DOX and were similar among WT, ALDH2 knockout and ALDH2 overexpression groups (data not shown). However, DOX significantly decreased LV ejection fraction and fractional shortening while it overtly enlarged LVEDD, the effect of which was further accentuated by ALDH2 knockout (Figs. 1A–D) or partially reversed by ALDH2 viral transfection (Figs. 1E–H). 3.2. Effect of ALDH2 knockout or overexpression on DOX-induced myocardial histological changes Western blot analysis shown in Fig. 2A validated murine models of ALDH2 knockout and overexpression. Effective ALDH2 viral transfection was further verified by fluorescence detection using the GFP-ALDH2 (Fig. 2B). In H&E stained myocardial samples, disarray of myofilament arrangement and focal tissue lysis became visible following DOX treatment. DOX-induced myocardial histological changes were significantly attenuated and accentuated, by ALDH2 overexpression and knockout, respectively. Neither ALDH2 viral transfection nor knockout produced any notable effect on myocardial histology (Figs. 2C–D).

2.9. Neonatal cell culture

3.3. Effects of ALDH2 on DOX-induced 4-HNE accumulation and autophagy

Neonatal cardiomyocytes were prepared from ventricles of 1–2 day-old rats and were cultured in 100-mm dishes at a density of 1 × 105 cells/cm2 in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 0.1% penicillin and streptomycin at 37 °C in a 5% CO2 humidified atmosphere [35]. Neonatal cardiomyocytes were exposed to DOX (1 μM) for 18 h [39,40] at 37 °C in a 5% CO2 humidified atmosphere. A cohort of cardiomyocytes was cultured at 37 °C in a 5% CO2 humidified atmosphere in DMEM for 6 h in the absence or presence of 4-HNE (50 μM dissolved in ethanol with a final ethanol concentration b0.1%) [27,41] supplemented with 10% inactivated FBS. 4-HNE-free cells incubated in the presence or absence of 0.1% ethanol served as untreated and control cells. The GFP-labeled-LC3 vector (GFP-LC3, 3 × 109 PFU) was transfected into cardiomyocytes for 18 h. Translocation of GFP-LC3 was examined in cardiomyocytes using a fluorescence microscope (Leica, Wetzlar, Germany).

To explore the potential mechanism(s) behind DOX- and ALDH2elicited cardiac contractile responses, the lipid peroxidation end product 4-HNE, a key ALDH2 substrate, and autophagy protein markers (Beclin-1, Atg5 and LC3-II) were evaluated in myocardium from ALDH2 knockout and overexpression mice. Our results depicted that DOX promoted 4-HNE accumulation, the effect of which was significantly augmented and attenuated, respectively, by ALDH2 knockout and overexpression (Figs. 3A–D). DOX treatment significantly enhanced myocardial autophagy (except Beclin1 in WT mice), the effects of which were accentuated and mitigated (or significantly attenuated), by ALDH2 knockout and overexpression, respectively (Figs. 3A, B, E–J).

2.10. Cell viability assay Cells were seeded into 96-well fat-bottomed plates at 5 × 103 cells per well and were incubated with Alda-1 (20 μM) [31] for 0.5 h prior to treatment with DOX (1 μM) [37,38] for 18 h in DMEM supplemented 10% FBS, at 37 °C in a 5% CO2 humidified atmosphere. Cell viability was assessed using a Cell Count Kit-8 (CCK-8, Beyotime Institute of Biotechnology, Haimen, China). After 2 h of incubation with 10% CCK8, the solutions from each sample were aspirated and the absorbance was measured at 450 nm.

3.4. Effect of ALDH2 activator Alda-1 on DOX-induced 4-HNE build-up and autophagy To further elucidate ALDH2-offered protective effects against DOXinduced cardiac dysfunction, accumulation of 4-HNE and autophagy markers were assessed following DOX challenge (1 μM for 18 h) [37,38] in the absence or presence of the ALDH2 activator Alda-1 (20 μM) [31]. DOX significantly enhanced 4-HNE accumulation and upregulated the autophagy protein markers including Beclin-1, Atg5 and LC3-II in neonatal cardiomyocytes, the effects were obliterated by Alda-1 (Figs. 4A–E). Cell viability assay revealed that DOX overtly dampened cell survival in neonatal cardiomyocytes, the effect of which was rescued by co-treatment of Alda-1 (Fig. 4F). In line with these findings, green fluorescent protein (GFP) examination further depicted that DOX treatment significantly promoted autophagy as evidenced by increased translocation of GFP-labeled-LC3-II in neonatal cardiomyocytes, the effect of which was negated by Alda-1 (Fig. 4G).

2.11. Statistical analysis All data are presented as Mean ± SEM. Multiple group comparison was made using repeated-measures analysis of variance (ANOVA) followed by a Tukey's post hoc analysis or the two-tailed student's t-test (wherever appropriate). A p value less than 0.05 was considered statistically significant.

3.5. Effect of the ALDH2 activator Alda-1 on 4-HNE-induced autophagy changes Expression of autophagy protein markers was examined following a 6-hr incubation of 4-HNE (50 μM) in neonatal cardiomyocytes in the presence or absence of Alda-1 (20 μM) [31]. Our result indicated that

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Fig. 1. Effect of doxorubicin (DOX) on cardiac geometry and function in WT, ALDH2 KO or ALDH2 overexpression mice. A–D: Echocardiographic properties of WT and ALDH2 KO mice challenged with or without DOX (15 mg/kg, i.p., for 6 days); E–H: Echocardiographic properties of adult mice transfected with vehicle or ALDH2 virus prior to DOX (15 mg/kg, i.p., for 6 days) challenge; A and E: Representative M-mode images; B and F: LV ejection fraction; C and G: LV fractional shortening; D and H: LV end diastolic diameter (LVEDD). Mean ± SEM, n = 4 mice per group, * p b 0.05 vs. WT or vehicle group; # p b 0.05 vs. respective DOX-treated group.

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Fig. 2. Myocardial ALDH2 levels and morphology in ALDH2 KO or overexpression mice. A: ALDH2 protein expression in mouse hearts from ALDH2 KO or viral transfection mice; B: Validation of adenovirus-eGFP-ALDH2 (5 × 109 PFU) fluorescence in mice transfected with negative control or ALDH2 virus; C: H&E staining in WT and ALDH2 KO mice changed with or without doxorubicin (DOX, 15 mg/kg, i.p., for 6 days); and D: H&E staining in vehicle and ALDH2 virus transfected mice changed with or without DOX (15 mg/kg, i.p., for 6 days). Original magnification = 200×.

4-HNE significantly upregulated expression of Beclin-1, Atg5 and LC3-II. Consistent with its effects on DOX-induced change in autophagy, Alda-1 ablated or significantly attenuated 4-HNE-induced upregulation of autophagy markers (Fig. 5).

responses on Class I and III PI3K as well as Akt phosphorylation (Fig. 6). These data suggested a role of Class I and Class III PI3K as well as Akt activation but unlikely the LKB1-AMPK cascade of ALDH2- and DOX-induced autophagy changes.

3.6. Effect of ALDH2 knockout on DOX-induced autophagy regulatory signaling proteins

3.7. Role of autophagy and Alda-1 on DOX-induced cardiomyocyte contractile dysfunction

To examine the impact of ALDH2 knockout on DOX-induced changes of autophagy regulatory proteins, myocardial expression of the autophagic regulatory signaling molecules including Class III PI3 kinase, AMPK (pan and phosphorylated) and its upstream activator LKB1, as well as Akt (pan and phosphorylated) and its upstream activator Class I PI3K were evaluated in WT and ALDH2 knockout mice treated with or without DOX (15 mg/kg, i.p., for 6 days). Our data revealed that DOX significantly up- and downregulated the expression of Class III and I PI3K, respectively. DOX challenge also dampened Akt phosphorylation without affecting LKB1, Akt, AMPK and AMPK phosphorylation. ALDH2 knockout itself did not elicit any notable effect on these autophagy signaling molecules although it overtly augmented DOX-induced

To further examine the role of autophagy on DOX-induced myocardial mechanical dysfunction, isolated cardiomyocytes from adult WT mice were exposed to DOX (1 μM) in the absence or presence of the autophagy inhibitor 3-MA (10 mM) [31] or autophagy inducer rapamycin (5 μM) [31] along with the ALDH2 activator, Alda-1 (20 μM) [31] for 4 h prior to assessment of mechanical properties. Our results showed that DOX significantly dampened cardiomyocyte contractile function (shown as reduced PS, ±dL/dt and prolonged TR90) in a manner reminiscent of the in vivo echocardiographic results. Although 3-MA and Alda-1 did not have any significant effect on cardiomyocyte contractile function themselves, they effectively abolished DOX-induced cardiomyocyte contractile anomalies. Autophagy induction with rapamycin

Fig. 3. Doxorubicin-induced expression of cardiac 4-HNE and autophagic proteins in vivo. A and B: Representative gel bands of 4-HNE, Beclin-1, Atg5, LC3-II and GAPDH (as loading control) using specific antibodies in ALDH2 KO and transfection models; C and D: 4-HNE expression in ALDH2 KO and transfection models; E and F: Beclin-1 expression in ALDH2 KO and transfection models; G and H: Atg5 expression in ALDH2 KO and transfection models; I and J: LC3-II expression in ALDH2 KO and transfection models; Mean ± SEM, n = 4, *p b 0.05 vs. WT or vehicle group, #p b 0.05 vs. respective DOX-treated group.

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Fig. 4. Effect of doxorubicin (DOX) on levels of 4-HNE and autophagic protein markers in the presence or absence of the ALDH2 activator Alda-1 in vitro. Neonatal cardiomyocytes were incubated with Alda-1 (20 μM) for 0.5 h prior to treatment with DOX (1 μM) for 18 h in DMEM supplemented 10% FBS, at 37 °C in a 5% CO2 humidified atmosphere. A: Representative gel blots depicting expression of 4-HNE, Beclin-1, Atg5, LC3-II and GAPDH (used as loading control) using specific antibodies; B: 4-HNE; C: Beclin-1; D: Atg5; E: LC3-II; F: Cell viability; and G: Detection of autophagy using GFP-fusion protein (green color denoted by red arrows) in cardiomyocytes treated with DOX in the absence or presence of Alda-1. Mean ± SEM, n = 4 independent isolations per group, *p b 0.05 vs. Control group, # p b 0.05 vs. DOX group.

mimicked DOX-induced cardiomyocyte contractile defects without producing any additive or synergistic effects with DOX. Interestingly, rapamycin abolished Alda-1-induced beneficial mechanical effects against DOX (Fig. 7).

3.8. Levels of ALDH2 and autophagy in patients with dilated cardiomyopathy Anthropometric data of healthy individuals and patients with dilated cardiomyopathy were displayed in (Table 1). The mean age, gender

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Fig. 5. Impact of 4-HNE on autophagic proteins expression in cardiomyocytes treated with or without Alda-1. Neonatal cardiomyocytes were incubated with 4-HNE (50 μM) for 6 h in the presence or absence of Alda-1 (20 μM) in DMEM supplemented 10% FBS, at 37 °C in a 5% CO2 humidified atmosphere. A: Representative gel blots depicting levels of Beclin-1, Atg5, LC3-II and GAPDH (used as loading control) using specific antibodies; B: Beclin-1; C: Atg5; and D: LC3-II; Mean ± SEM, n = 4 independent isolations per group, *p b 0.05 vs. Control group, #p b 0.05 vs. 4-HNE group.

and body weight were comparable for both groups. Myocardial ALDH2 expression was significantly lower in heart failure patients with endstage dilated cardiomyopathy compared to that of normal individuals. Furthermore, expressions of autophagy proteins including Beclin-1, Atg5 and LC3-II were significantly upregulated in hearts from DCM patients compared with those from normal individuals (Fig. 8). 4. Discussion The salient findings from our study revealed that knockdown of ALDH2 accentuated whereas ALDH2 overexpression or activation attenuated DOX-induced cardiac toxicity in murine hearts and cultured cardiomyocytes. Inhibition or knockdown ALDH2 was associated with upregulated autophagic protein expression and increased 4-NHE accumulation while activation or overexpression ALDH2 was linked with downregulated autophagic protein expression and reduced 4-NHE accumulation in mice or cultured cardiomyocytes. Our results collectively suggest that ALDH2 might protect against DOX-induced myocardial dysfunction and cardiomyocyte contractile dysfunction at least partly through detoxification of 4-HNE and downregulation of autophagy. Clinical findings from our study further demonstrated loss of ALDH2 in conjunction with facilitated autophagy in left ventricles from patients suffering from dilated cardiomyopathy. To the best of our knowledge, this is the first report depicting a potential role of ALDH2 in DOXinduced myocardial toxicity and contractile dysfunction in mice, and in patients with idiopathic DCM. Data from our study revealed decreased ejection fraction and fractional shortening in conjunction with enlarged LVEDD in mice following DOX challenge. These findings are reminiscent of the previous observation of enlarged LVEDD and impaired left ventricular systolic function in

mice following DOX treatment [42]. Moreover, our study revealed a close association between cardiac mechanical/geometric changes and increased myocardial autophagy induction in DOX-challenged mice, cardiomyocytes and in patients with DCM. Although a link between autophagy induction and DCM was implicated previously [43,44], the underlying mechanisms for a role of autophagy in DCM pathogenesis remain essentially elusive. In our present study, we demonstrated that overexpression or activation of ALDH2 may improve cardiac function and suppress autophagy in DOX challenged murine models. To the contrary, ALDH2 knockout significantly accentuates DOX-induced cardiac contractile and autophagic anomalies. These data have shed some lights towards consolidating a role for loss of ALDH2 and excess autophagy in the pathogenesis of doxorubicin-induced cardiac toxicity, and more importantly, possible etiology of DCM in human. As a potent stimulant for oxidative stress, DOX promotes the quinone production and redox-cycling, which may facilitate ROS-dependent lipid peroxidation [45] and production of 4-HNE, a highly reactive carbonyl lipid peroxidation end product [46]. 4-HNE is directly cardiac toxic while the formation of 4-HNE adduct in the heart has been shown to impair ATP production from mitochondria [47], contributing to impaired myocardial contractile function [41]. Moreover, 4-HNE is one of the most important mediators for lipid peroxidation-derived aldehydeinduced autophagy [48]. Our study showed that inhibition or knockdown ALDH2 was associated with 4-NHE accumulation and upregulated autophagy while activation or overexpression ALDH2 was accompanied with 4-NHE detoxification and downregulated autophagy in mice or cultured cardiomyocytes. Given that 4-HNE serves as a main substrate for ALDH2, it is plausible to speculate that ALDH2 may be primarily responsible for the governance of 4-HNE-induced autophagy and toxicity in DOX-induced cardiac toxicity. It is noteworthy that 4-HNE is one of the

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Fig. 6. Doxorubicin (DOX)-induced changes in autophagy regulatory signaling molecules in vivo. Hearts from WT and ALDH2 knockout mice were treated with or without doxorubicin (15 mg/kg, i.p., for 6 days) prior to determination of protein levels of autophagy signaling molecules. A: Representative gel blots depicting levels of PI3K Class III, Class I (p110α), LKB1, AMPK, pAMPK, Akt, pAkt and GAPDH (loading control) using specific antibodies; B: PI3K Class III; C: PI3K Class I (p110α); D: LKB1; E: AMPK; F: pAMPK; G: Akt; and H: pAkt; Mean ± SEM, n = 4 mice per group, *p b 0.05 vs. WT group, #p b 0.05 vs. WT-DOX group.

most important mediators for mitochondrial oxidative stress en route to mitochondrial injury [41,49]. Mitochondrial dysfunction may be both the trigger and result of altered autophagic response [16]. Our current study

suggested that ALDH2 reversed DOX-induced autophagy although a role of mitochondrial integrity cannot be determined in ALDH2- and DOXinduced cardiac mechanical and autophagy responses.

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Fig. 7. Effect of the autophagy inhibitor 3-MA, autophagy inducer rapamycin and the ALDH2 activator Alda-1 on doxorubicin (DOX)-induced cardiomyocyte contractile dysfunction in vitro. Isolated cardiomyocytes from WT mice were incubated with DOX (1 μM) for 4 h in the absence or presence of 3-MA (10 mM), rapamycin (5 μM) or Alda-1 (20 μM). A: Resting cell length; B: Peak shortening (normalized to resting cell length); C: Maximal velocity of shortening (+dL/dt); D: Maximal velocity of relengthening (−dL/dt); E: Time-to-peak shortening (TPS) and F: Time-to-90% relengthening (TR90). Mean ± SEM, n = 60 cells per group, *p b 0.05 vs. Control group, #p b 0.05 vs. DOX group.

Result from our study showed that both overexpression and activation of ALDH2 may effectively suppress 4-HNE accumulation and autophagy induction following DOX challenge, while ALDH2 knockout exacerbated DOX-induced contractile dysfunction and autophagy. Early evidence revealed that ALDH2 protects against myocardial ischemia through reduced apoptosis and preserved mitochondrial function along with activation of Akt and GSK3β [27]. In our hands, DOX significantly dampened Akt phosphorylation in hearts, the effect of which was exacerbated by ALDH2 knockout. Downregulation of Akt phosphorylation may promote mitochondrial injury and oxidative stress [50,51]. It is possible that 4-HNE plays a central role in the regulation of PI3K/Akt/mTOR signaling and cardiomyocyte survival [52] in our current experimental setting. Our

data noted overt 4-HNE accumulation in response to DOX challenge and subsequently autophagy induction in response to 4-HNE exposure. These findings favor a role for 4-HNE in autophagy induction. AMPK and Akt were considered as major regulators for myocardial autophagy and function [27,53,54]. mTOR serves as the key converging point between Akt and AMPK for energy metabolism, autophagy and cell survival [2,42]. Recent findings from our group indicated that ALDH2 protected heart from myocardial ischemia and reperfusion injury via regulation of AMPK-Akt-mTOR-governed autophagy [6,27]. In the current study, DOX treatment suppressed phosphorylation of Akt but not AMPK, the effect of which was augmented by ALDH2 knockout. These findings are supported by unchanged LKB1 and suppressed Class I PI3K (upstream of

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Fig. 8. Expression of autophagic proteins and ALDH2 in left ventricular tissues from normal healthy donors and patients with dilated cardiomyopathy (DCM). A: Representative gel blots depicting expression of ALDH2, Beclin-1, Atg5, LC3-II and GAPDH (loading control) using specific antibodies; B: ALDH2; C: Beclin-1; D: Atg5; and E: LC3-II. Mean ± SEM, n = 3 individuals per group, *p b 0.05 vs. Normal group.

Akt) following DOX challenge. Although our previous studies have depicted a key role for both Akt and AMPK in ALDH2-triggered autophagy inhibition under ischemia–reperfusion and ER stress [27,32,55], it appears that only Akt is involved in ALDH2-offered beneficial responses against DOX-induced myocardial dysfunction and autophagy. 4.1. Experimental limitations Several limitations of our study should be noted. First, no data were provided on the size changes of individual cardiomyocytes, thereby limiting the conclusion of cardiac remodeling at the organ level. Second, the conclusion regarding the role of ALDH2 in the pathogenesis of dilated cardiomyopathy must be interpreted with caution as doxorubicin cardiomyopathy does not truly recapitulate the clinical changes in idiopathic dilated cardiomyopathy in our patients. The data leave more unanswered questions such as whether or not ALDH2 participates in the etiology of other forms of cardiomyopathy. Third, the autophagy

data may be merely associated with changes in cardiac function. Although our in vitro study using 3-MA and rapamycin suggested a permissive role for autophagy in DOX- and Alda-1-offered cardiac responses, in depth investigation is needed to better consolidate such connection. Last, the present study failed to explain any of the downstream or parallel elements of Akt en route to autophagy regulation such as mTOR signaling and the mTOR-independent pathways which may be involved. 4.2. Clinical implication Doxorubicin induces cardiac toxicity and ultimate development of congestive heart failure [47]. Although a number of approaches have been engaged to minimize its toxic effects such as modification of doxorubicin chemical structure [56], severe cardiac toxicity remains a rather pertinent clinical issue impeding the clinical application of this antineoplastic drug [47]. Results from our current study

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have provided compelling evidence that ALDH2 may exert a unique beneficial role in the DOX-induced cardiac injury likely via detoxification of the lipid peroxidation product 4-HNE and suppression of excess autophagy. Further study is warranted to better elucidate if targeting ALDH2 might serve as a novel therapeutic avenue for the proper management of doxorubicin cardiomyopathy, DCM and possibly heart failure. Funding This work was supported in part by the National Natural Science Foundation of China (30971250), the National Basic Research Program of China (2011CB503905) and the New Century Talents Plan from Ministry of Education. Conflict of interest None declared. References [1] Stewart MJ, Malek K, Crabb DW. Distribution of messenger RNAs for aldehyde dehydrogenase 1, aldehyde dehydrogenase 2, and aldehyde dehydrogenase 5 in human tissues. J Investig Med 1996;44:42–6. [2] Zhang Y, Ren J. ALDH2 in alcoholic heart diseases: molecular mechanism and clinical implications. Pharmacol Ther 2011;132:86–95. [3] Chen CH, Budas GR, Churchill EN, Disatnik MH, Hurley TD, Mochly-Rosen D. Activation of aldehyde dehydrogenase-2 reduces ischemic damage to the heart. Science 2008;321:1493–5. [4] Doser TA, Turdi S, Thomas DP, Epstein PN, Li SY, Ren J. Transgenic overexpression of aldehyde dehydrogenase-2 rescues chronic alcohol intakeinduced myocardial hypertrophy and contractile dysfunction. Circulation 2009;119:1941–9. [5] Wang J, Wang H, Hao P, Xue L, Wei S, Zhang Y, et al. Inhibition of aldehyde dehydrogenase 2 by oxidative stress is associated with cardiac dysfunction in diabetic rats. Mol Med 2011;17:172–9. [6] Ma H, Yu L, Byra EA, Hu N, Kitagawa K, Nakayama KI, et al. Aldehyde dehydrogenase 2 knockout accentuates ethanol-induced cardiac depression: role of protein phosphatases. J Mol Cell Cardiol 2010;49:322–9. [7] Zhang Y, Babcock SA, Hu N, Maris JR, Wang H, Ren J. Mitochondrial aldehyde dehydrogenase (ALDH2) protects against streptozotocin-induced diabetic cardiomyopathy: role of GSK3beta and mitochondrial function. BMC Med 2012;10:40. [8] Sun A, Ren J. ALDH2, a novel protector against stroke? Cell Res 2013;23:874–5. [9] Guo JM, Liu AJ, Zang P, Dong WZ, Ying L, Wang W, et al. ALDH2 protects against stroke by clearing 4-HNE. Cell Res 2013;23:915–30. [10] Olson RD, Mushlin PS. Doxorubicin cardiotoxicity: analysis of prevailing hypotheses. FASEB J 1990;4:3076–86. [11] Mukhopadhyay P, Batkai S, Rajesh M, Czifra N, Harvey-White J, Hasko G, et al. Pharmacological inhibition of CB1 cannabinoid receptor protects against doxorubicininduced cardiotoxicity. J Am Coll Cardiol 2007;50:528–36. [12] Pacher P, Liaudet L, Bai P, Virag L, Mabley JG, Hasko G, et al. Activation of poly(ADPribose) polymerase contributes to development of doxorubicin-induced heart failure. J Pharmacol Exp Ther 2002;300:862–7. [13] Nemchenko A, Chiong M, Turer A, Lavandero S, Hill JA. Autophagy as a therapeutic target in cardiovascular disease. J Mol Cell Cardiol 2011;51:584–93. [14] Xie M, Morales CR, Lavandero S, Hill JA. Tuning flux: autophagy as a target of heart disease therapy. Curr Opin Cardiol 2011;26:216–22. [15] Yitzhaki S, Huang C, Liu W, Lee Y, Gustafsson AB, Mentzer Jr RM, et al. Autophagy is required for preconditioning by the adenosine A1 receptor-selective agonist CCPA. Basic Res Cardiol 2009;104:157–67. [16] De Meyer GR, De Keulenaer GW, Martinet W. Role of autophagy in heart failure associated with aging. Heart Fail Rev 2010;15:423–30. [17] Mathew R, Kongara S, Beaudoin B, Karp CM, Bray K, Degenhardt K, et al. Autophagy suppresses tumor progression by limiting chromosomal instability. Genes Dev 2007;21:1367–81. [18] Zhang YS, He L, Liu B, Li NS, Luo XJ, Hu CP, et al. A novel pathway of NADPH oxidase/ vascular peroxidase 1 in mediating oxidative injury following ischemia–reperfusion. Basic Res Cardiol 2012;107:266. [19] Zhu H, Tannous P, Johnstone JL, Kong Y, Shelton JM, Richardson JA, et al. Cardiac autophagy is a maladaptive response to hemodynamic stress. J Clin Invest 2007;117:1782–93. [20] Ceylan-Isik AF, Dong M, Zhang Y, Dong F, Turdi S, Nair S, et al. Cardiomyocytespecific deletion of endothelin receptor A rescues aging-associated cardiac hypertrophy and contractile dysfunction: role of autophagy. Basic Res Cardiol 2013;108:335. [21] Ito H, Daido S, Kanzawa T, Kondo S, Kondo Y. Radiation-induced autophagy is associated with LC3 and its inhibition sensitizes malignant glioma cells. Int J Oncol 2005;26:1401–10.

103

[22] Karantza-Wadsworth V, Patel S, Kravchuk O, Chen G, Mathew R, Jin S, et al. Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis. Genes Dev 2007;21:1621–35. [23] Kostin S, Pool L, Elsasser A, Hein S, Drexler HC, Arnon E, et al. Myocytes die by multiple mechanisms in failing human hearts. Circ Res 2003;92:715–24. [24] Tanaka Y, Guhde G, Suter A, Eskelinen EL, Hartmann D, Lullmann-Rauch R, et al. Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice. Nature 2000;406:902–6. [25] Wang X, Robbins J. Proteasomal and lysosomal protein degradation and heart disease. J Mol Cell Cardiol 2013. [26] Kassiotis C, Ballal K, Wellnitz K, Vela D, Gong M, Salazar R, et al. Markers of autophagy are downregulated in failing human heart after mechanical unloading. Circulation 2009;120:S191–7. [27] Ma H, Guo R, Yu L, Zhang Y, Ren J. Aldehyde dehydrogenase 2 (ALDH2) rescues myocardial ischaemia/reperfusion injury: role of autophagy paradox and toxic aldehyde. Eur Heart J 2011;32:1025–38. [28] Bian Y, Chen YG, Xu F, Xue L, Ji WQ, Zhang Y. The polymorphism in aldehyde dehydrogenase-2 gene is associated with elevated plasma levels of high-sensitivity C-reactive protein in the early phase of myocardial infarction. Tohoku J Exp Med 2010;221:107–12. [29] Pavan M, Ruiz VF, Silva FA, Sobreira TJ, Cravo RM, Vasconcelos M, et al. ALDH1A2 (RALDH2) genetic variation in human congenital heart disease. BMC Med Genet 2009;10:113. [30] Xu F, Chen YG, Xue L, Li RJ, Zhang H, Bian Y, et al. Role of aldehyde dehydrogenase 2 Glu504lys polymorphism in acute coronary syndrome. J Cell Mol Med 2011;15:1955–62. [31] Ge W, Guo R, Ren J. AMP-dependent kinase and autophagic flux are involved in aldehyde dehydrogenase-2-induced protection against cardiac toxicity of ethanol. Free Radic Biol Med 2011;51:1736–48. [32] Zhang B, Zhang Y, La Cour KH, Richmond KL, Wang XM, Ren J. Mitochondrial aldehyde dehydrogenase obliterates endoplasmic reticulum stress-induced cardiac contractile dysfunction via correction of autophagy. Biochim Biophys Acta 1832;2013:574–84. [33] Yalcin E, Oruc E, Cavusoglu K, Yapar K. Protective role of grape seed extract against doxorubicin-induced cardiotoxicity and genotoxicity in albino mice. J Med Food 2010;13:917–25. [34] Sano M, Minamino T, Toko H, Miyauchi H, Orimo M, Qin Y, et al. p53-Induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload. Nature 2007;446:444–8. [35] Zou Y, Zhu W, Sakamoto M, Qin Y, Akazawa H, Toko H, et al. Heat shock transcription factor 1 protects cardiomyocytes from ischemia/reperfusion injury. Circulation 2003;108:3024–30. [36] Krusche CA, Holthofer B, Hofe V, van de Sandt AM, Eshkind L, Bockamp E, et al. Desmoglein 2 mutant mice develop cardiac fibrosis and dilation. Basic Res Cardiol 2011;106:617–33. [37] Wold LE, Aberle II NS, Ren J. Doxorubicin induces cardiomyocyte dysfunction via a p38 MAP kinase-dependent oxidative stress mechanism. Cancer Detect Prev 2005;29:294–9. [38] Jay SM, Murthy AC, Hawkins JF, Wortzel JR, Steinhauser ML, Alvarez LM, et al. An engineered bivalent neuregulin protects against doxorubicin-induced cardiotoxicity with reduced proneoplastic potential. Circulation 2013;128:152–61. [39] Kobayashi S, Volden P, Timm D, Mao K, Xu X, Liang Q. Transcription factor GATA4 inhibits doxorubicin-induced autophagy and cardiomyocyte death. J Biol Chem 2010;285:793–804. [40] Yao Y, Xu X, Zhang G, Zhang Y, Qian W, Rui T. Role of HMGB1 in doxorubicininduced myocardial apoptosis and its regulation pathway. Basic Res Cardiol 2012;107:267. [41] Aberle II NS, Picklo Sr MJ, Amarnath V, Ren J. Inhibition of cardiac myocyte contraction by 4-hydroxy-trans-2-nonenal. Cardiovasc Toxicol 2004;4:21–8. [42] Zhu W, Shou W, Payne RM, Caldwell R, Field LJ. A mouse model for juvenile doxorubicin-induced cardiac dysfunction. Pediatr Res 2008;64:488–94. [43] Diwan A, Dorn II GW. Decompensation of cardiac hypertrophy: cellular mechanisms and novel therapeutic targets. Physiology (Bethesda) 2007;22:56–64. [44] Shimomura H, Terasaki F, Hayashi T, Kitaura Y, Isomura T, Suma H. Autophagic degeneration as a possible mechanism of myocardial cell death in dilated cardiomyopathy. Jpn Circ J 2001;65:965–8. [45] Keizer HG, Pinedo HM, Schuurhuis GJ, Joenje H. Doxorubicin (adriamycin): a critical review of free radical-dependent mechanisms of cytotoxicity. Pharmacol Ther 1990;47:219–31. [46] Petersen DR, Doorn JA. Reactions of 4-hydroxynonenal with proteins and cellular targets. Free Radic Biol Med 2004;37:937–45. [47] Yan LJ, Sohal RS. Mitochondrial adenine nucleotide translocase is modified oxidatively during aging. Proc Natl Acad Sci U S A 1998;95:12896–901. [48] Hill BG, Haberzettl P, Ahmed Y, Srivastava S, Bhatnagar A. Unsaturated lipid peroxidation-derived aldehydes activate autophagy in vascular smooth-muscle cells. Biochem J 2008;410:525–34. [49] Tjalkens RB, Cook LW, Petersen DR. Formation and export of the glutathione conjugate of 4-hydroxy-2, 3-E-nonenal (4-HNE) in hepatoma cells. Arch Biochem Biophys 1999;361:113–9. [50] Yan F, Wang M, Chen H, Su J, Wang X, Wang F, et al. Gambogenic acid mediated apoptosis through the mitochondrial oxidative stress and inactivation of Akt signaling pathway in human nasopharyngeal carcinoma CNE-1 cells. Eur J Pharmacol 2011;652:23–32. [51] Zhang Y, Ren J. Thapsigargin triggers cardiac contractile dysfunction via NADPH oxidase-mediated mitochondrial dysfunction: role of Akt dephosphorylation. Free Radic Biol Med 2011;51:2172–84.

104

A. Sun et al. / Journal of Molecular and Cellular Cardiology 71 (2014) 92–104

[52] Lee JH, Won YS, Park KH, Lee MK, Tachibana H, Yamada K, et al. Celastrol inhibits growth and induces apoptotic cell death in melanoma cells via the activation ROS-dependent mitochondrial pathway and the suppression of PI3K/AKT signaling. Apoptosis 2012;17:1275–86. [53] Ginion A, Auquier J, Benton CR, Mouton C, Vanoverschelde JL, Hue L, et al. Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake. Am J Physiol Heart Circ Physiol 2011;301:H469–77.

[54] Morrison A, Yan X, Tong C, Li J. Acute rosiglitazone treatment is cardioprotective against ischemia–reperfusion injury by modulating AMPK, Akt, and JNK signaling in nondiabetic mice. Am J Physiol Heart Circ Physiol 2011;301:H895–902. [55] Zhang Y, Ren J. Autophagy in ALDH2-elicited cardioprotection against ischemic heart disease: slayer or savior? Autophagy 2010;6:1212–3. [56] Turdi S, Xu P, Li Q, Shen Y, Kerram P, Ren J. Amidization of doxorubicin alleviates doxorubicin-induced contractile dysfunction and reduced survival in murine cardiomyocytes. Toxicol Lett 2008;178:197–201.