Published online before print March 30, 2009, doi: 10.1161/CIRCULATIONAHA.108.823799
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(Circulation. 2009;119:1941-1949.)
© 2009 American Heart Association, Inc.
Molecular Cardiology |
Transgenic Overexpression of Aldehyde Dehydrogenase-2 Rescues Chronic Alcohol Intake–Induced Myocardial Hypertrophy and Contractile Dysfunction
From the Center for Cardiovascular Research and Alternative Medicine (T.A.D., S.T., S.-Y.L., J.R.), Division of Kinesiology and Health (D.P.T.), University of Wyoming College of Health Sciences, Laramie, Wyo, and the Department of Pediatrics (P.N.E.), University of Louisville School of Medicine, Louisville, Ky.
Correspondence to Dr Jun Ren, University of Wyoming College of Health Sciences, Laramie, WY 82071. E-mail jren{at}uwyo.edu
Received September 13, 2007; accepted February 2, 2009.
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Abstract |
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Background— Chronic alcoholism leads to the onset and progression of alcoholic cardiomyopathy through toxic mechanisms of ethanol and its metabolite, acetaldehyde. This study examined the impact of altered acetaldehyde metabolism through systemic transgenic overexpression of aldehyde dehydrogenase-2 (ALDH2) on chronic alcohol ingestion–induced myocardial damage.
Methods and Results— ALDH2 transgenic mice were produced with the chicken β-actin promoter. Wild-type FVB and ALDH2 mice were placed on a 4% alcohol diet or a control diet for 14 weeks. Myocardial and cardiomyocyte contraction, intracellular Ca2+ handling, histology (hematoxylin and eosin, Masson trichrome), protein damage, and apoptosis were determined. Western blot was used to monitor the expression of NADPH oxidase, calcineurin, apoptosis-stimulated kinase (ASK-1), glycogen synthase kinase-3β (GSK-3β), GATA4, and cAMP-response element binding (CREB) protein. ALDH2 reduced the chronic alcohol ingestion–induced elevation in plasma and tissue acetaldehyde levels. Chronic alcohol consumption led to cardiac hypertrophy, reduced fractional shortening, cell shortening, and impaired intracellular Ca2+ homeostasis, the effect of which was alleviated by ALDH2. In addition, the ALDH2 transgene significantly attenuated chronic alcohol intake–induced myocardial fibrosis, protein carbonyl formation, apoptosis, enhanced NADPH oxidase p47phox and calcineurin expression, as well as phosphorylation of ASK-1, GSK-3β, GATA4, and CREB.
Conclusions— The present results suggest that transgenic overexpression of ALDH2 effectively antagonizes chronic alcohol intake–elicited myocardial hypertrophy and contractile defect through a mechanism that is associated, at least in part, with phosphorylation of ASK-1, GSK-3β, GATA4, and CREB. These data strongly support the notion that acetaldehyde may be an essential contributor to the chronic development of alcoholic cardiomyopathy.
Key Words: alcohol • cardiomyopathy • hypertrophy • myocytes • myocardial contraction • apoptosis
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Introduction |
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Chronic alcohol ingestion often leads to cardiovascular complications, including alcoholic cardiomyopathy, which is mainly manifested as cardiac hypertrophy and contractile dysfunction.1,2 Although several rationales have been suggested for this myopathic change after alcohol intake, such as ethanol toxicity and buildup of fatty acid ethyl esters,2,3 the precise mechanisms underscoring alcoholic cardiomyopathy remain elusive. Acetaldehyde, the first oxidized metabolite of ethanol, is far more reactive and toxic than ethanol and may contribute to alcoholic injury.3,4 Clinical evidence suggests that blood acetaldehyde levels may reach the low-millimolar range after alcohol ingestion in Asian and black populations with defective aldehyde dehydrogenase (ALDH),5,6 which makes them prone to alcoholic tissue injury. We and others have shown that acetaldehyde impairs cardiac excitation-contraction coupling, inhibits sarco(endo)plasmic reticulum Ca2+ release,7–9 and forms protein adducts.10 The acetaldehyde toxicity theory recently received convincing support from our study in which cardiac overexpression of alcohol dehydrogenase, which converts ethanol into acetaldehyde, resulted in an exacerbated cardiac hypertrophy and contractile defect after alcohol exposure.5,11,12
Clinical Perspective p 1949
To further explore the role of acetaldehyde in alcoholic cardiomyopathy, we produced a transgenic mouse line overexpressing human mitochondrial ALDH type 2 (ALDH2) to examine whether facilitated acetaldehyde detoxification affects alcohol intake–induced myocardial tissue damage and contractile function. We also examined the role of glycogen synthase kinase-3β (GSK-3β) and apoptosis signaling regulated kinase-1 (ASK-1), 2 signaling molecules essential for cardiac hypertrophy and cell survival.13,14 GSK-3β, which belongs to the serine/threonine kinase family, is inactivated by phosphorylation of serine 9 by oxidative stress during hypertrophic conditions. On the other hand, ASK-1 and the mitogen-activated protein kinase cascade may contribute to oxidative stress–elicited cardiomyocyte hypertrophy and gene reprogramming,13,14 which indicates a role of oxidative stress in GSK-3β– and ASK-1–mediated cardiomyocyte events. More recently, a role of GATA4 and the transcription factor cAMP-response element binding protein (CREB) has been revealed in GSK-3β–regulated cardiomyocyte hypertrophy and gene expression.13,15,16 Nonetheless, the role of these signaling molecules in alcohol-induced cardiac hypertrophy has not been elucidated.
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Methods |
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Generation of ALDH2 Transgenic Mice
All animal procedures were approved by the University of Wyoming Institutional Animal Care and Use Committee. The human ALDH2 gene was amplified by polymerase chain reaction from pT7-7-hpALDH2 (kindly provided by Dr Henry Weiner from Purdue University, Lafayette, Ind) with the following primers: ALDH-F (5-tcgaattctatgttgcgcgctgccgcccg) and ALDH-R (3-cacggagtcttcttgagtattcttaaggc). The amplified ALDH2 fragment was digested with EcoRI and cloned into the EcoRI site of vector pBsCAG-2 under the CAG cassette, where ALDH activity was increased with the promoter of the chicken β-actin gene, as we reported previously.17 This promoter has been widely used to produce high-level expression in the liver of transgenic mice.18 Elevated ALDH activity should reduce circulating acetaldehyde by metabolizing it to acetate. The full length of the promoter portion of the CAG-ALDH gene was sequenced to confirm that no errors were inserted. The transgene can be removed from the plasmid by digestion with KpnI and SstI, which was shown to drive expression in many mammalian cells.17,19 The ALDH2 insert was excised and separated from the plasmid by KpnI/SstI restriction digestion and agarose gel electrophoresis. The insert was purified on Qiagen-tip 20 columns (Qiagen, Valencia, Calif), followed by spin gel chromatography and filtration through 0.22-µm filters. A concentration of 1 µg/µL of the purified transgene insert DNA was microinjected into a 1-cell embryo of the inbred strain FVB. Approximately 20 to 30 microinjected embryos were implanted into each pseudopregnant female and allowed to come to term. After weaning, mice tail clips were collected for genotype of DNA insertion of ALDH2 (Figure 1). The primer pair used for ALDH2 genotyping was 5'-cat cac aac cac gtt tcc ag-3' (ALDH-F) and 5'-aca atg gca agc cct atg tc-3' (ALDH-R). Further breeding was conducted with the same background wild-type FVB. All mice were housed in a temperature-controlled room under a 12-hour-light/12-hour-dark cycle and allowed access to tap water ad libitum. Four-month-old adult male FVB and ALDH2 (F8) mice were placed on a nutritionally complete liquid diet (Shake & Pour Bio-Serv Inc, Frenchtown, NJ) for a 1-week acclimation period. The use of a liquid diet was based on the scenario that ethanol self-administration resulted in fewer nutritional deficiencies and less stress to the animals than forced-feeding regimens, intravenous administration, or aerosolized inhalation.20 On completion of the acclimation period, half of the FVB and ALDH2 mice were maintained on the regular liquid diet (without ethanol), and the remaining half began a 14-week period of isocaloric 4% (vol/vol) ethanol diet feeding. An isocaloric pair-feeding regimen was used to eliminate the possibility of nutritional deficits. Control mice were offered the same quantity of diet that ethanol-consuming mice drank the previous day. Body weight was monitored weekly.11
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Measurement of Blood Ethanol and Acetaldehyde Levels
On the last day of diet feeding, mice were euthanized under anesthesia (ketamine/xylazine 3:1, 1.32 mg/kg IP). Blood was collected and stored in sealed vials. A volume of 100 µL of plasma from each sample was put into an autosampler vial. Six microliters of n-propanol and 194 µL of H2O were then added to the vial. After a 20-minute incubation at 50°C, a 50-µL aliquot of headspace gas was removed and transferred to an Agilent 6890 gas chromatograph (Agilent Technologies, Inc, Wilmington, Del) equipped with a flame ionization detector. Ethanol, n-propanol, and other components such as acetaldehyde were separated on a 60-m VOCOL capillary column (Supelco Inc, Bellefonte, Pa) with film of 1.8-µm thickness and an inner diameter of 320 µm. The carrier gas was helium at a flow rate of 18.0 mL/min. Quantitation was achieved by calibrating peak areas against those from headspace samples of known ethanol and acetaldehyde standards.21
Echocardiographic Assessment
Cardiac geometry and function were evaluated in anesthetized (Avertin 2.5%, 10 µL/g body weight IP) mice with 2D guided M-mode echocardiography with a Sonos 5500 (Philips Medical Systems) equipped with a 15- to 16-MHz linear transducer. Left ventricular (LV) anterior and posterior wall dimensions during diastole and systole were recorded from 3 consecutive cycles in M mode by methods adopted by the American Society of Echocardiography. Fractional shortening was calculated from LV end-diastolic (EDD) and end-systolic (ESD) diameters with the equation (EDD–ESD)/EDD. Heart rates were averaged over 10 cardiac cycles.22
Isolation of Cardiomyocytes
After ketamine/xylazine sedation, hearts were removed and perfused with Krebs-Henseleit bicarbonate buffer containing (in mmol/L) 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 10 HEPES, and 11.1 glucose. Hearts were digested with collagenase D for 20 minutes. LVs were removed and minced before being filtered. Myocyte yield was 
75% and was not affected by high-fat diet or metallothionein. Only rod-shaped myocytes with clear edges were selected for mechanical and intracellular Ca2+ study.11
Cell Shortening/Relengthening
Mechanical properties of cardiomyocytes were assessed with an IonOptix soft-edge system (IonOptix, Milton, Mass). Myocytes were placed in a chamber mounted on the stage of an Olympus IX-70 microscope and superfused (2 mL/min at 25°C) with a Krebs-Henseleit bicarbonate buffer containing 1 mmol/L CaCl2. Myocytes were field stimulated at 0.5 Hz unless otherwise stated. Cell shortening and relengthening were assessed, including peak shortening (PS), time to PS, time to 90% relengthening, and maximal velocities of shortening/relengthening (±dL/dt).11 In the case of alterations of stimulus frequency from 0.1 to 5.0 Hz, the steady state contraction of the myocyte was achieved (usually after the first 5 to 6 beats) before PS was recorded.
Intracellular Ca2+ Transients
A cohort of myocytes were loaded with fura-2/AM (0.5 µmol/L) for 10 minutes, and fluorescence intensity was recorded with a dual-excitation fluorescence photomultiplier system (IonOptix). Myocytes were placed onto an Olympus IX-70 inverted microscope and imaged through a Fluor 40x oil objective. Cells were exposed to light emitted by a 75-W lamp and passed through either a 360- or a 380-nm filter while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm, and qualitative change in fura-2 fluorescence intensity was inferred from the fura-2 fluorescence intensity ratio at the 2 wavelengths (360/380). Fluorescence decay time was calculated as an indicator of intracellular Ca2+ clearing.11
Sarcoplasmic Reticulum Ca2+ ATPase Activity Measured by 45Ca2+ Uptake
Cardiomyocytes were sonicated and solubilized in a Tris-sucrose homogenization buffer that consisted of 30 mmol/L Tris-HCl, 8% sucrose, 1 mmol/L phenylmethanesulfonyl fluoride, and 2 mmol/L dithiothreitol, pH 7.1. To determine sarcoplasmic reticulum Ca2+ ATPase (SERCA)–dependent Ca2+ uptake, samples were treated with and without the SERCA inhibitor thapsigargin (10 µmol/L) for 15 minutes. The difference between the 2 readings was deemed the thapsigargin-sensitive uptake through SERCA. Uptake was initiated by the addition of an aliquot of supernatant to a solution that consisted of (in mmol/L) 100 KCl, 5 NaN3, 6 MgCl2, 0.15 EGTA, 0.12 CaCl2, 30 Tris-HCl pH 7.0, 10 oxalate, and 2 ATP, with 1 µCi of 45CaCl2, at 37°C. Aliquots of samples were injected onto glass filters on a suction manifold and washed 3 times. Filters were then removed from the manifold, placed in scintillation fluid, and counted. SERCA activity was expressed as counts per minute per milligram of protein.23
Histological Examination
After anesthesia, hearts were excised and immediately placed in 10% neutral-buffered formalin at room temperature for 24 hours after a brief rinse with PBS. The specimen were embedded in paraffin, cut in 5-µm sections, and stained with hematoxylin and eosin. Cardiomyocyte cross-sectional areas were calculated on a digital microscope (x400) with ImageJ (version 1.34S) software. Masson’s trichrome staining was used to detect fibrosis in heart sections. The percentage of fibrosis was calculated with the histogram function of the Photoshop software. Briefly, 7 random fields (6 mm2) from each section were assessed at x200 magnification for fibrosis. The fraction of the light blue–stained area normalized to the total area was used as an indicator of myocardial fibrosis while omitting fibrosis of the perivascular, epicardial, and endocardial areas from the study.24
Protein Carbonyl Assay
To assess cardiac oxidative damage, the protein carbonyl content of tissue was determined as described previously.11 Briefly, proteins were extracted and minced to prevent proteolytic degradation. Nucleic acids were eliminated by treatment of the samples with 1% streptomycin sulfate for 15 minutes, followed by a 10-minute centrifugation (11 000g). Protein was precipitated by the addition of an equal volume of 20% trichloroacetic acid to protein (0.5 mg) and then centrifuged for 1 minute. The trichloroacetic acid solution was removed and the sample resuspended in 10 mmol/L 2,4-dinitrophenylhydrazine solution. Samples were incubated at room temperature for 15 to 30 minutes. After addition of 500 µL of 20% trichloroacetic acid, samples were centrifuged for 3 minutes. The supernatant was discarded, and the pellet was washed in ethanol–ethyl acetate and allowed to incubate at room temperature for 10 minutes. The samples were centrifuged again for 3 minutes and the ethanol–ethyl acetate steps repeated 2 more times. The precipitate was resuspended in 6 mol/L guanidine solution and centrifuged for 3 minutes, and insoluble debris was removed. The maximum absorbance (360 to 390 nm) of the supernatant was read against appropriate blanks (water, 2 mol/L HCl), and the carbonyl content was calculated with the molar absorption coefficient of 22 000 mol/L–1 · cm–1.
Caspase-3 Assay
Caspase-3 is an enzyme activated during induction of apoptosis. In brief, 1 mL of PBS was added to flasks that contained human cardiac myocytes, and the monolayer was scraped and collected in a microfuge tube. The cells were centrifuged at 10 000g at 4°C for 10 minutes, and cell pellets were lysed in 100 µL of ice-cold cell lysis buffer (50 mmol/L HEPES, 0.1% CHAPS, 1 mmol/L dithiothreitol, 0.1 mmol/L EDTA, 0.1% NP40). After cells were lysed, 70 µL of reaction buffer was added to cell lysate (30 µL), followed by the addition of 20 µL of caspase-3 colorimetric substrate (Ac-DEVD-pNA) and incubation at 37°C for 1 hour, during which time the caspase in the sample was allowed to cleave the chromophore p-nitroaniline from the substrate molecule. The samples were then read with a microplate reader at 405 nm. Caspase-3 activity was expressed as picomoles of p-nitroaniline released per microgram of protein per minute.17
Western Blot Analysis
The protein was prepared as described previously.17 Samples containing equal amount of proteins were separated on 10% SDS-polyacrylamide gels in a minigel apparatus (Mini-PROTEAN II, Bio-Rad Laboratories, Hercules, Calif) and transferred to nitrocellulose membranes. The membranes were blocked with 5% milk in TBS-T buffer and were incubated overnight at 4°C with anti-ALDH2 (kindly provided by Dr Henry Weiner, Purdue University Lafayette, Ind), anti-p47phox, anti-calcineurin A, anti-ASK-1, anti-phosphorylated ASK-1 (anti-pASK-1; Ser83), anti-GSK-3β, anti-phosphorylated GSK-3β (anti-p GSK-3β; Ser9), anti-GATA4, anti-phosphorylated GATA4 (anti-pGATA4; Ser105), anti-CREB, and anti-pCREB (Ser133) antibodies. After immunoblotting, the film was scanned, and the intensity of immunoblot bands was detected with a Bio-Rad calibrated densitometer. β-Actin was used as the loading control.
Data Analysis
Data are presented as mean±SEM. Statistical significance (P<0.05) for each variable was estimated by ANOVA followed by Tukey’s post hoc analysis.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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General Features and Echocardiographic Properties of FVB and ALDH2 Mice Fed With Alcohol
Chronic alcohol feeding did not affect body, liver, or kidney weights, although the heart was significantly enlarged compared with control mice. ALDH2 did not affect body or organ weights, although it significantly alleviated alcohol-induced cardiac hypertrophy. Blood alcohol levels were significantly and equally elevated in alcohol-consuming FVB and ALDH2 mice. ALDH2 significantly reduced the chronic alcohol ingestion–induced elevation in blood acetaldehyde levels. The levels of blood alcohol and acetaldehyde were either undetectable or minimal in non–alcohol-consuming mice (Table). Analysis of cardiac tissue acetaldehyde levels further supported the notion that the ALDH2 transgene alleviated the chronic alcohol ingestion–induced increase in tissue acetaldehyde levels (Figure 1). Heart rate and LV end-systolic diameter were comparable among all groups. Although LV mass, end-diastolic diameter, and fractional shortening were no different between control groups, alcohol intake significantly increased LV end-diastolic diameter, reduced LV wall thickness, and enhanced LV mass (absolute or normalized to body weight) while reducing fractional shortening in FVB but not ALDH2 mice. These deleterious changes seen in the FVB-ethanol group indicate cardiac hypertrophy and associated dilated cardiomyopathy (Table).
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Cardiomyocyte Contractile and Intracellular Ca2+ Properties, 45Ca2+ Uptake, and Frequency Response
Consistent with our data on hypertrophied hearts in response to alcohol intake, chronic alcohol intake but not ALDH2 significantly enhanced longitudinal cross-sectional area. Moreover, chronic alcohol intake significantly reduced PS and ±dL/dt and prolonged time to 90% relengthening without affecting time to PS in FVB cardiomyocytes. Importantly, ALDH2 abolished chronic alcohol intake–induced mechanical abnormalities (Figure 2). In addition, cardiomyocytes from alcohol-fed mice displayed a significantly depressed intracellular Ca2+ rise in response to electrical stimulus (
fura-2 fluorescence intensity) and a reduced intracellular Ca2+ decay rate associated with an unchanged baseline intracellular Ca2+. The reduced intracellular Ca2+ decay was consistent with the dampened 45Ca2+ uptake, which indicates impaired SERCA activity in murine cardiomyocytes after alcohol intake. ALDH2 negated the alcohol-induced changes in fura-2 fluorescence intensity, intracellular Ca2+ decay, and 45Ca2+ uptake without eliciting any effect on intracellular Ca2+ properties in the absence of chronic alcohol intake (Figures 3A through 3D).
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Mouse hearts beat at high frequencies (>400 bpm at 37°C), with sarcoplasmic reticulum Ca2+ store being the primary determinant of the frequency-dependent response. We initially stimulated cardiomyocytes to contract at 0.5 Hz for 5 minutes to ensure achievement of steady state before altering the frequency in a stepwise manner from 0.1 to 5 Hz (300 bpm). All recordings were normalized to the PS obtained at 0.1 Hz of the same cell. Myocytes from the alcohol-fed group exhibited significantly exaggerated depression in PS at 1.0 Hz and higher. The ALDH2 transgene did not alter the pattern of PS response at any of the frequencies tested, regardless of alcohol or control diet intake (Figure 3E).
Effects of Alcohol Treatment on Myocardial Histology
To assess the impact of ALDH2 on myocardial histology after chronic alcohol ingestion, cardiomyocyte cross-sectional area and interstitial fibrosis were examined. In the hematoxylin-and-eosin–stained sections, alcohol ingestion increased cardiomyocyte transverse cross-sectional area, consistent with increased ventricular mass in FVB mice. Alcohol-induced cardiomyocyte hypertrophy was significantly attenuated by ALDH2 even though cardiomyocyte areas from the ALDH2-plus-ethanol group remained significantly greater that those of nondrinking groups. Further examination with Masson trichrome staining revealed overt myocardial fibrosis after chronic alcohol ingestion, the effect of which was significantly attenuated by the ALDH2 transgene (Figure 4).
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Effects of ALDH2 on Alcohol-Induced Apoptosis and Protein Carbonyl Formation
To examine the potential mechanism of action behind ALDH2-elicited protection against alcoholic cardiomyopathy, myocardial apoptosis and protein damage were examined in cardiac tissues from FVB and ALDH2 mice consuming control or alcohol diets. Results shown in Figure 5 indicate that caspase-3 activity and protein carbonyl formation were both significantly elevated in hearts of alcohol-fed FVB mice. Consistent with its mechanical and morphometric response, ALDH2 significantly ameliorated alcohol-induced apoptosis and protein damage. ALDH2 itself displayed minimal effects on apoptosis and protein carbonyl formation in the absence of alcohol intake, which indicates that the transgene itself is not innately harmful.
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Western Blot Analysis of p47phox, Calcineurin, and Activation of ASK-1, GSK-3β, GATA4, and CREB
To elucidate the potential mechanism(s) involved in ALDH2-elicited cardiac protection against alcohol-induced cardiac hypertrophy and contractile dysfunction, we further examined expression of the NADPH oxidase p47phox and p67phox subunits, the cardiac hypertrophic gene calcineurin, and total and phosphorylated levels of ASK-1, GSK-3β, GATA4, and CREB. As shown in Figure 6, chronic alcohol intake led to upregulated p47phox (but not p67phox; data not shown) and calcineurin A, as well as enhanced phosphorylation of ASK-1, GSK-3β, GATA4, and CREB in FVB mice. Interestingly, ALDH2 reversed alcohol-induced abnormal upregulation or activation of these proteins without eliciting any effect by itself. Total protein expression of ASK-1, GSK-3β, GATA4, and CREB was not affected by either chronic alcohol intake or the ALDH2 transgene.
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Discussion |
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The myocardial morphometric and functional observations from the present study demonstrated that the ALDH2 transgene significantly attenuated or ablated chronic alcohol intake–induced cardiac hypertrophy and contractile dysfunction. The present data further revealed that ALDH2 significantly ameliorated chronic alcohol intake–induced myocardial fibrosis, protein carbonyl formation, apoptosis, and enhanced NADPH oxidase p47phox and calcineurin expression, as well as hyperactivation of ASK-1, GSK-3β, GATA4, and CREB, which indicates a role of these signaling molecules in chronic alcohol ingestion–induced cardiac functional and morphometric abnormalities. These data strongly support the notion that acetaldehyde may be an essential player in the pathogenesis of alcoholic cardiomyopathy and suggest a therapeutic potential of ALDH2 and acetaldehyde detoxification in the management of chronic alcoholism–associated complications.
The ALDH2 transgene, along with alcohol dehydrogenase, is useful for artificial alteration of acetaldehyde and/or ethanol metabolism.5,12 The availability of both transgenes has made it possible to evaluate the role of acetaldehyde in the pathogenesis of alcoholic cardiomyopathy. Although ethanol toxicity, oxidative damage, lipid peroxidation, and altered membrane integrity have been speculated to contribute to alcohol-induced tissue injury,25,26 none of these hypotheses has been fully validated experimentally or clinically. The acetaldehyde theory has received recent attention because much of the alcohol-elicited cell damage, such as reactive oxygen species production and peroxidation of lipid, protein, and DNA, may be mimicked by acetaldehyde.5,11,25,26 Evidence from our laboratory indicated that transgene mice with cardiac overexpression of alcohol dehydrogenase manifested exaggerated cardiac hypertrophy, contractile dysfunction, oxidative stress, lipid peroxidation, and endoplasmic reticulum stress after chronic alcohol intake, which was associated with significantly elevated cardiac acetaldehyde levels.5,11,12,27 Several reactive oxygen species or stress signaling pathways, including the ethanol-inducible CYP2E1 isoform of cytochrome P-450, xanthine oxidase, and aldehyde oxidase, have been implicated in acetaldehyde-induced cellular toxicity. Metabolism of acetaldehyde through these enzymatic pathways promotes free radical generation en route to cell oxidant stress and apoptosis.28–31 Acetaldehyde may also facilitate depletion of cellular glutathione and promote protein-adduct formation between acetaldehyde and the glutathione precursor, L-cysteine, which contributes to glutathione depletion and peroxidative reaction.5,28,31 The ability of acetaldehyde to promote myocardial fibrosis, oxidative stress, and apoptosis is consistent with the present observation of enhanced Masson trichrome staining, protein carbonyl formation, and caspase-3 activity in chronic alcohol-fed FVB but not ALDH2 mice.
Results from immunoblotting analysis indicated that chronic alcohol intake–induced cardiac hypertrophy and contractile dysfunction are associated, at least in part, with upregulated NADPH oxidase p47 subunit and calcineurin A and with hyperphosphorylation of ASK-1, GSK-3β, GATA4, and CREB. The ALDH2 transgene ameliorates chronic alcohol intake–induced hyperphosphorylation of ASK-1, GSK-3β, GATA4, and CREB, which suggests a possible role of these molecules in the cytoprotection of ALDH2. Inactivation of GSK-3β by phosphorylation at serine 9 plays an essential role in the regulation of the GSK-3β downstream signaling molecules GATA4 and calcineurin, as well as in cardiac hypertrophy.13,15 In the present study, we showed that the ALDH2 transgene prevented chronic alcohol consumption–induced cardiac hypertrophy, as evidenced by heart weight/body weight ratio, LV mass, and histological examination. Acetaldehyde has been shown to trigger oxidative stress and apoptosis via activation of stress signaling, which may in turn induce myocardial hypertrophy.2,32,33 This is consistent with our observation of enhanced NADPH oxidase and ASK-1 phosphorylation after alcohol intake. The NADPH oxidase (Nox) enzymes are a particularly important source of reactive oxygen species that play a critical role in ASK-1 activation, cardiac hypertrophy, and contractile dysfunction.34 The present observation that ALDH2 reversed chronic alcohol intake–induced phosphorylation of GATA4 and CREB also indicated a role of GATA4 and CREB in the regulation of cardiac hypertrophy. With the upregulated calcineurin, inhibition of GSK-3β (by its phosphorylation) and activated GATA4 may facilitate nuclear translocation of NFAT (nuclear factor of activated T cells), thereby stimulating cardiac hypertrophy.13 Nonetheless, the precise interplay among ASK-1, GSK-3β, GATA4, and CREB is essentially unclear and warrants further investigation to elucidate the precise mechanism behind ALDH2-elicited protection against chronic alcohol intake–induced cardiac injury.
In summary, the present study provides evidence that overexpression of the ALDH2 transgene rescues chronic alcohol intake–induced cardiac hypertrophy and contractile dysfunction. The present data indicate that activation of calcineurin, ASK-1, GATA4, and CREB associated with inhibition of GSK-3β is intimately involved in acetaldehyde and ALDH2-elicted cardiac remodeling. Given that activation of the ALDH2 enzyme can confer myocardial protection against ischemic damage independent of alcohol metabolism,35 the present data with the novel ALDH2 transgenic model further suggest the potential of the ALDH2 enzyme as a therapeutic target clinically in alcoholic cardiomyopathy and other cardiac myopathic complications.
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Acknowledgments |
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The authors gratefully acknowledge Dr Feng Dong, Dr Qun Li, and Sara A. Babcock from the University of Wyoming (Laramie, Wyo) for their skillful assistance.
Sources of Funding
This work was supported in part by National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism grant 1R01 AA013412 and NIH/NCRR 5P20RR016474 (to Dr Ren).
Disclosures
None.
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Related Article:
CLINICAL PERSPECTIVE
Almost 1 of every 3 alcoholics displays some degree of heart problems, collectively known as alcoholic cardiomyopathy. The present study shows that the ALDH2 enzyme is capable of mitigating cardiac remodeling and myocardial dysfunction after chronic alcohol ingestion, possibly through facilitated acetaldehyde detoxification. Blood acetaldehyde levels are 10-fold higher in humans with defective ALDH2 (eg, Asians and blacks) than in normal individuals after alcohol ingestion. Allelic variation of ALDH genes, especially ALDH2 due to a point mutation in the active ALDH2*1 gene, significantly alters vulnerability for alcoholism and alcoholic complications; however, the jury is still out as to whether elevated acetaldehyde levels are directly involved in the origin of alcoholic cardiomyopathy or are simply the result of alcohol metabolism. The present study, which used transgenic mice with overexpression of ALDH2, provides the first evidence that facilitated acetaldehyde detoxification alone is sufficient to reverse the cardiac remodeling processes that lead to alcoholic cardiomyopathy. Results obtained in the present study support the conclusion that elevated acetaldehyde levels participate in cardiac remodeling and contractile defects, perhaps through NADPH oxidase–mediated oxidative stress and activation of hypertrophic signaling molecules. These data indicated that ALDH2 may be cardioprotective and counteract cardiac remodeling and myocardial dysfunction after chronic alcohol intake, thus providing its therapeutic potential in alcoholic and other forms of myocardial damage. Because convincing human case studies on the interaction between the ALDH2 gene polymorphism and heart function after chronic alcohol intake are lacking, caution must be exercised when evaluating the role of acetaldehyde and ALDH2 in the pathogenesis and management of alcoholic cardiomyopathy.
Circulation 2009 119: 1843-1845.
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