Author + information
- Received October 28, 2008
- Revision received March 10, 2009
- Accepted April 2, 2009
- Published online December 1, 2009.
- ↵⁎Reprint requests and correspondence:
Dr. Jun Ren, University of Wyoming College of Health Sciences, 1000 East University Avenue, Laramie, Wyoming 82071
Objectives This study was designed to evaluate the role of facilitated detoxification of acetaldehyde, the main metabolic product of ethanol, through systemic overexpression of mitochondrial aldehyde dehydrogenase-2 (ALDH2) on acute ethanol exposure-induced myocardial damage.
Background Binge drinking may exert cardiac toxicity and interfere with heart function, manifested as impaired ventricular contractility, although the underlying mechanism remains poorly defined.
Methods ALDH2 transgenic mice were produced using the chicken beta-actin promoter. Wild-type FVB (friend virus B) and ALDH2 mice were challenged with ethanol (3 g/kg, intraperitoneally), and cardiac function was assessed 24 h later using the Langendroff and cardiomyocyte edge-detection systems. Western blot analysis was used to evaluate protein phosphatase 2A and 2C (PP2A and PP2C), phosphorylation of Akt, AMP-activated protein kinase (AMPK), and the transcription factors Foxo3 (Thr32 and Ser413).
Results ALDH2 reduced ethanol-induced elevation in cardiac acetaldehyde levels. Acute ethanol challenge deteriorated myocardial and cardiomyocyte contractile function evidenced by reduction in maximal velocity of pressure development and decline (±dP/dt), left ventricular developed pressure, cell shortening, and prolonged relengthening duration, the effects of which were alleviated by ALDH2. Ethanol treatment dampened phosphorylation of Akt and AMPK associated with up-regulated PP2A and PP2C, which was abrogated by ALDH2. ALDH2 significantly attenuated ethanol-induced decrease in Akt- and AMPK-stimulated phosphorylation of Foxo3 at Thr32 and Ser413, respectively. Consistently, ALDH2 rescued ethanol-induced myocardial apoptosis, protein damage, and mitochondrial membrane potential depolarization.
Conclusions Our results suggest that ALDH2 is cardioprotective against acute ethanol toxicity, possibly through inhibition of protein phosphatases, leading to enhanced Akt and AMPK activation, and subsequently, inhibition of Foxo3, apoptosis, and mitochondrial dysfunction.
Alcoholism, a worldwide major health problem, affects approximately 10% of the adult population in the U.S. In contrast to the cardioprotective benefits of light-to-moderate alcohol consumption (up to 1 drink daily for women and 1 or 2 drinks daily for men), incidental heavy or binge drinking increases cardiovascular events and mortality (1). Similar to chronic alcohol intake, frequent binge drinking may predispose hearts to myopathic changes, including myofibrillar disruption and reduced ventricular contractility en route to the onset of alcoholic cardiomyopathy (2). Although several hypotheses have been proposed for myopathic alterations following alcohol exposure including ethanol toxicity, reactive oxygen species, and fatty acid ethyl ester accumulation (3,4), the ultimate culprit factor(s) still remain unclear. As the first metabolic product of ethanol, acetaldehyde is thought to mediate ethanol-induced cardiac toxicity (3). Evidence from our lab and others has shown that acetaldehyde interrupts cardiac excitation–contraction coupling and sarcoplasmic reticulum (SR) Ca2+release (3,5). Acetaldehyde is capable of reacting with functional proteins to form protein adducts leading to tissue injury (6). In general, aldehydes may increase the myocardial susceptibility to ischemia reperfusion injury and mitigate certain cardioprotective mechanisms such as nitric oxide (7). The “acetaldehyde toxicity” theory received some convincing support from our recent observation that overexpression of alcohol dehydrogenase (ADH), which metabolizes ethanol to acetaldehyde, accentuated alcohol-induced myocardial injury (8). Further evidence from our lab indicated a protective role of the mitochondrial isoform of aldehyde dehydrogenase (ALDH), ALDH2, in acetaldehyde or alcohol-induced tissue injury (9). Consistent with these observations, ALDH2 was recently shown to reduce ischemic injury to the heart (10), suggesting its cardioprotective role. Nonetheless, the mechanism behind ALDH2-mediated protection and whether it relates to the ALDH2-engaged breakdown of acetaldehyde remain unknown. To test our hypothesis that ALDH2 offers protection against acute ethanol toxicity through detoxification of acetaldehyde, a transgenic mouse line with overexpression of human ALDH2 was produced to examine acute ethanol exposure-elicited cardiac toxicity and the underlying mechanisms, with a special focus on AMP-activated protein kinase (AMPK) and Akt, key regulators of survival and metabolism (11,12). Given that a member of Forkhead transcription factors family (Foxo3) may serve as a suitable candidate at the convergence of AMPK and Akt signaling (13), the phosphorylation of Foxo3 at different phosphorylation sites was further evaluated. Activation of Foxo3 at Ser413 by AMPK appears to promotes stress resistance and longevity (14). On the other hand, Foxo3 activation at Thr32 by Akt shuts down Foxo3 transcriptional activity, favoring antiapoptosis, improved mitochondrial function, and insulin sensitivity (15). Therefore, the convergence of AMPK and Akt signaling at the level of Foxo3 may play a critical role in the regulation of myocardial function.
Experimental animals, acute ethanol challenge, assay for blood ethanol, and acetaldehyde
All animal procedures were approved by the University of Wyoming Institutional Animal Care and Use Committee (Laramie, Wyoming). The human ALDH2 gene was amplified by polymerase chain reaction from pT7-7-hpALDH2 (kindly provided by Dr. Henry Weiner from Purdue University, Lafayette, Indiana) using 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 using the chicken beta-actin promoter (9). The full length of the promoter portion of the CAG-ALDH gene was sequenced to confirm accuracy. The transgene can be removed from the plasmid by digestion with KpnI and BamHI. The ALDH2 insert (Fig. 1A)was excised and separated from the plasmid by KpnI/SstI restriction digestion and agarose gel electrophoresis. The insert was purified on Qiagen 20 columns (QIAGEN Inc., Valencia, California), followed by spin gel chromatography and filtration through 0.22-μm filters. A concentration of 1 μg/μl of the purified transgene insert deoxyribonucleic acid was microinjected into a 1-cell embryo of the inbred strain FVB. Around 20 to 30 microinjected embryos were implanted into each pseudopregnant female and allowed to come to term. After weaning, mouse tail clips were collected for genotyping of deoxyribonucleic acid insertion of ALDH2. Further breeding was conducted with the same background wild-type FVB. All mice were housed in a temperature-controlled room under a 12-h/12-h light/dark cycle with access to tap water ad libitum. Four-month-old adult male FVB and ALDH2 (F8) mice were selected for study. For acute ethanol challenge, mice were injected intraperitoneally with ethanol (3 g/kg) and were sacrificed under anesthesia (ketamine/xylazine: 3:1, 1.32 mg/kg, intraperitoneal) 24 h after ethanol injection.
Blood and hearts were collected in sealed vials and were stored at −80°C until analysis. For analysis, a 2-ml aliquot of the headspace gas from each vial was removed through the septum on the cap with a gas-tight syringe and transferred to a 200-μl loop injection system. 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. Following a 20-min incubation at 50°C, a 50-μl aliquot of headspace gas was removed. Plasma and heart samples transferred to a Hewlett-Packard 5890 gas chromatograph (Hewlett-Packard, Palo Alto, California) equipped with a flame ionization detector. Ethanol and acetaldehyde were separated on a 9-m VOCOL capillary column with film of 1.8-μm thickness and an inner diameter of 0.32 mm. The temperature was held at 30°C, and the carrier gas was helium at a flow rate of 1.8 ml/min. Quantitation was achieved by calibrating the gas chromatograph peak areas against those from headspace samples of known ethanol and acetaldehyde standards, over a similar concentration range as the tissue samples in the same buffer (16).
ALDH2 enzymatic activity
ALDH2 enzymatic activity was measured at 25°C in 33 mmol/l sodium pyrophosphate containing 0.8 mmol/l nicotinamide adenine dinucleotide (NAD+), 15 μmol/l propionaldehyde, and 0.1 ml of cellular extract (50 μg of protein). Propionaldehyde, the substrate of ALDH2, was oxidized in propionic acid by ALDH2, whereas NAD+was reduced to reduced form of nicotinamide-adenine dinucleotide (NADH) to quantitate the ALDH2 activity. Production of NADH was determined by spectrophotometric absorbance at 340 nm. ALDH2 activity was expressed as nanomoles NADH/minute per milligram protein. An extinction coefficient of 6.22/mmol per cm for NADH was used for the calculation of reaction rates (17).
Protein carbonyl formation
Protein was precipitated by adding an equal volume of 20% trichloroacetic acid and centrifuged for 1 min. The sample was resuspended in 10 mmol/l 2,4-dinitrophenylhydrazine (2,4-DNPH) solution for 15 to 30 min at room temperature before 20% trichloroacetic acid was added and samples were centrifuged for 3 min. The precipitate was resuspended in 6 mol/l guanidine solution. The maximum absorbance (360 to 390 nm) was read against appropriate blanks (2 mol/l HCl), and the carbonyl content was calculated using the formula: absorption at 360 nm × 45.45 nmol/protein content (mg) (16).
Mouse heart perfusion
Mouse hearts were retrogradely perfused with a Krebs-Henseleit buffer containing 7 mmol/l glucose, 0.4 mmol/l oleate, 1% BSA, and a low fasting concentration of insulin (10 μU/ml). Hearts were perfused at a constant flow of 4 ml/min (equal to an aortic pressure of 80 cm H2O) at baseline for 60 min. A fluid-filled latex balloon connected to a solid-state pressure transducer was inserted into the left ventricle through a left atriotomy to measure pressure. Left ventricular developed pressure (LVDP), maximal velocity of pressure development and decline (±dP/dt), and heart rate were recorded using a digital acquisition system at a balloon volume that resulted in a baseline LV end-diastolic pressure of 5 mm Hg (18).
After ketamine/xylazine sedation, hearts were removed and perfused with Krebs-Henseleit buffer containing (in mmol/l): 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 10 hydroxyethnyl piperazine ethanesulfonic acid (HEPES), and 11.1 glucose. Hearts were digested with 223 U/ml collagenase D for 20 min. Left ventricles were removed and minced before being filtered. Myocyte yield was ∼75%, which was not affected by either acute ethanol challenge or ALDH2 transgene. Only rod-shaped myocytes with clear edges were selected for mechanical study (16).
Cell shortening and relengthening
Mechanical properties of cardiomyocytes were evaluated utilizing a SoftEdge MyoCam system (IonOptix Corporation, Milton, Massachusetts) (16). Briefly, cardiomyocytes were visualized under an inverted microscope (Olympus, IX-70, Olympus Optical Co., Tokyo, Japan) and were stimulated with a voltage frequency of 0.5 Hz. The myocyte being observed was shown on a computer monitor using an IonOptix MyoCam camera. IonOptix SoftEdge software was utilized to capture cell shortening and relengthening changes. The indexes considered were peak shortening amplitude (PS), time-to-peak shortening (TPS), time-to-90% relengthening (TR90), and maximal velocity of shortening/relengthening (±dL/dt). In the case of stimulus alternation from 0.1 to 5.0 Hz, the steady-state contraction of myocytes was achieved (usually after the first 5 to 6 beats) before PS was recorded.
Ventricular tissues were homogenized and sonicated in a lysis buffer containing 20 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 1 mmol/l ethylenediaminetetraacetic acid (EDTA), 1 mmol/l ethyleneglycoltetraacetic acid (EGTA), 1% Triton, 0.1% SDS, and 1% protease inhibitor cocktail. Equal amounts (30 μg) of proteins were separated on 10% or 15% SDS-polyacrylamide gels in a minigel apparatus (Mini-PROTEAN II, Bio-Rad Laboratories Inc., Hercules, California) and were then transferred electrophoretically to nitrocellulose membranes. The membranes were blocked with 5% milk in Tris-buffered saline before overnight incubation at 4°C with anti-ALDH2 (1:1,000), anti-Akt (1:1,000), anti-pAkt (Thr308, 1:1,000), anti-AMPK (1:1,000), anti-pAMPK (Thr172, 1:1,000), anti-Foxo3 (1:1,000), anti-pFoxo3 (Ser413, Thr32, 1:1,000), anti-PP2C (1:1,000), and anti-PP2A (1:1,000) antibodies. Membranes were then incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000). After immunoblotting, films were scanned and the intensity of immunoblot bands was detected with a Bio-Rad Calibrated Densitometer (8).
Caspase-3 is an enzyme activated during induction of apoptosis. Caspase-3 activity was determined according to published method (9). In brief, myocytes were lysed in 100 μl of ice-cold cell lysis buffer (50 mmol/l HEPES, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1 mmol/l dithiothreitol, 0.1 mmol/l EDTA, 0.1% NP40). Following cell lysis, 70 μl of reaction buffer and 20 μl of caspase-3 colorimetric substrate (Ac-DEVD-p-nitroanilide) were added to cell lysate and incubated for 1 h at 37°C, during which time, caspase enzyme in the sample was allowed to cleave the chromophore pNA from its substrate molecule. Absorbency was detected at 405 nm, with caspase-3 activity being proportional to the color reaction. Caspase-3 activity was expressed as picomoles of p-nitroanilide released per micrograms of protein per minute.
Measurement of mitochondrial membrane potential (ΔΨm)
Cardiomyocytes were suspended in HEPES-saline buffer, and ΔΨm was detected as described (19). Briefly, following a 10-min pre-incubation with 5 μmol/l JC-1 at 37°C, cells were rinsed twice using the HS buffer free of JC-1. Fluorescence of each sample was read at excitation wavelength of 490 nm and emission wavelength of 530 and 590 nm using a spectrofluorimeter (Spectra MaxGeminiXS, Spectra Max, Atlanta, Georgia) at an interval of 10 s. Results in fluorescence intensity were expressed as the 590- to 530-nm emission ratio. The mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 μmol/l) was used as a positive control for ΔΨm measurement.
Data were expressed as mean ± standard error of the mean (SEM). Statistical significance (p < 0.05) for each variable was estimated by analysis of variance followed by Tukey's test for post-hoc analysis.
General features and whole-heart function of FVB and ALDH2 mice
ALDH2 transgene itself did not affect body and organ weights. Acute ethanol injection elicited comparable elevation in blood alcohol levels and cardiac acetaldehyde levels, which were minimal in nonethanol-treated mice, without affecting body and organ weights. Although blood alcohol levels were equally elevated in both FVB and ALDH2 mice at 1 or 6 h following ethanol injection, cardiac acetaldehyde levels were significantly lower in ALDH2 mice following ethanol challenge, validating this transgenic model of facilitated acetaldehyde detoxification. Protein carbonyl formation, an indicator of protein oxidation and protein damage, was significantly elevated in response to acute ethanol challenge, the effect of which was partially inhibited by the ALDH2 transgene. Assessment of whole-heart function including LVDP and maximal velocity of pressure development and decline (±dP/dt) revealed a significant decline in response to acute ethanol challenge, the effect of which was attenuated by ALDH2. No difference was noted in ex vivo heart rate among all mouse groups tested (Table 1).Transgenic overexpression of ALDH2 significantly enhanced cardiac ALDH2 expression and its enzymatic activity. Acute ethanol treatment failed to alter ALDH2 protein expression although it significantly promoted ALDH2 enzymatic activity equally in both FVB and ALDH2 mice (Figs. 1B and 1C).
Effect of acute ethanol exposure on cardiomyocyte mechanics in FVB and ALDH2 mice
Our further assessment of cardiomyocyte mechanics revealed that acute ethanol treatment significantly depressed peak shortening and ±dL/dt as well as prolonged TR90without affecting TPS. Although the ALDH2 transgene itself did not affect these mechanical indexes, it significantly attenuated acute ethanol exposure-induced cardiomyocyte mechanical abnormalities (Fig. 2).To evaluate the potential contribution of SR in ethanol and/or ALDH2-elicited cardiac contractile responses, cardiomyocytes from FVB or ALDH2 mice treated with or without ethanol were paced at higher stimulating frequencies to examine the SR Ca2+handling capacity. The cells were initially stimulated to contract at 0.5 Hz for 5 min to ensure a steady state prior to raising the stimulating frequency to 5.0 Hz. Figure 3exhibits a comparable negative staircase in PS with increased stimulus frequency between FVB and ALDH2 mice in the absence of ethanol exposure. Acute ethanol exposure exaggerated high stimulus frequency-elicited depression in PS, the effect of which was significantly attenuated by ALDH2.
Effect of ALDH2 on ethanol-induced change in Akt, AMPK, and Foxo signaling
To examine the potential signaling pathways involved in ethanol and/or ALDH2-elicited cardiac mechanical response, we examined levels of the key cardiac surviving factor Akt and the cardiac energy fuel AMPK. Our results indicated that acute ethanol treatment markedly decreased phosphorylation of both Akt and AMPK in FVB mice, which was abrogated by ALDH2. These data suggest that ALDH2 may compensate for the loss of activation in Akt and AMPK in response to acute ethanol treatment. Consistently, our data further revealed reduction of the Akt-engaged phosphorylation of Foxo3 at Thr32 and the AMPK-activated Foxo3 phosphorylation at Ser413 following ethanol treatment. ALDH2 restored acute ethanol exposure-depressed Foxo3 phosphorylation at both sites (Fig. 4).These results suggest involvement of the Akt-Foxo3 (Thr32) and AMPK-Foxo3 (Ser413) cascades in ethanol and/or ALDH2-induced cardiac responses.
Effect of ALDH2 on ethanol-induced changes in protein phosphatases
Recent evidence has indicated Akt is dephosphorylated by protein phosphatase 2A (PP2A), whereas AMPK is dephosphorylated by protein phosphatase 2C (PP2C) (15,20,21). To examine the possible mechanisms behind ethanol-induced reduction in the phosphorylation of Akt and AMPK, expression of PP2A and PP2C was evaluated in myocardium from FVB and ALDH2 mice with or without acute ethanol exposure. Our result shown in Figure 5indicated that ethanol treatment significantly up-regulated the levels of PP2A and PP2C in FVB but not ALDH2 mice, indicating a likely role of up-regulated PP2A and PP2C proteins in the ethanol-induced loss of phosphorylation in Akt and AMPK, respectively.
Effect of ALDH2 on ethanol-induced cardiomyocyte apoptosis and mitochondrial damage
Apoptosis, a key element for a variety of pathological heart conditions, contributes to alcoholic heart injury (12). Our current data suggested a possible role of Akt-Foxo3 (Thr32) and AMPK-Foxo3 (Ser413) pathways in ethanol- and/or ALDH2-elicited response. Considering the close tie among Akt, AMPK, and Foxo3 in cardiomyocyte survival and function (11,12,22), we further examined apoptosis in myocardium following ethanol treatment. Results shown in Figure 6Arevealed that caspase-3 activity was significantly elevated in cardiomyocytes following acute ethanol challenge, the effect of which was significantly attenuated by ALDH2.
Given that mitochondrial function is essential to cardiomyocyte viability and function (4,23), the cationic lipophilic probe JC-1 was employed to monitor ΔΨm in response to acute ethanol treatment. The dynamic change of ΔΨm was displayed by change in the ratio between red (aggregated JC-1) and green (monomeric form of JC-1) fluorescence. Quantitative analysis showed a significant reduction in the ratio between red and green fluorescence in response to a 30-min ethanol treatment, indicating a fall in ΔΨm and mitochondrial damage. Interestingly, the ethanol-induced fall in ΔΨm was abrogated by ALDH2. The ALDH2 transgene itself did not exert any significant effect on ΔΨm (Fig. 6B).
Our study was designed based on the hypothesis that acetaldehyde is the ultimate culprit toxin for ethanol-induced toxicity. To test this hypothesis, a transgenic mouse line was generated with systemic overexpression of ALDH2 to facilitate the detoxification of acetaldehyde. Our major findings revealed that ALDH2 reduced cardiac acetaldehyde levels and lessened acute ethanol-induced myocardial contractile dysfunction, apoptosis, and protein damage. Our data further indicated that ALDH2 reconciled the acute ethanol-induced dephosphorylation of Akt and AMPK possibly associated with an up-regulation of protein phosphatases (PP2A and PP2C). Meanwhile, our study revealed a cross-talk between Akt and AMPK signaling pathways at the converging point of Foxo3, leading to inactivation (phosphorylation) of Forkhead transcriptional factors and inhibition of myocardial apoptosis, protein, and mitochondrial damage in the ethanol-treated ALDH2 mice. Taken together, this study demonstrated that ALDH2 may be protective against acute ethanol-induced cardiac toxicity through inhibition of protein phosphatases, preserved phosphorylation of Akt and AMPK on Foxo3, and reduced apoptosis, protein, and mitochondrial damage.
Low-to-moderate intake of alcohol is associated with a better health outcome than less frequent consumption. Binge drinking, even among otherwise light drinkers, contributes to cardiovascular events, myocardial abnormalities, and mortality (1,2). Ethanol-induced cardiac damage is evident if acute alcohol consumption exceeds 90 to 100 g/day in humans (2), which can be transpired to a dosage of ∼1.5 g/kg for an average adult weighing 70 kg. Therefore, the dosage of ethanol used in our study (3 g/kg) closely resembles a state of heavy ethanol consumption, given that rodents are more resist than humans to ethanol intoxication. The principal indicator of myopathic alteration following ethanol exposure is characterized by compromised myocardial contractility (2,24). This is supported by results from our current study in that acute ethanol exposure triggered deteriorated in heart contractile function, as evidenced by reduced ±dP/dt and LVDP, prolonged TR90, and depressed cell shortening and ±dL/dt associated with normal TPS. Not surprisingly, little geometric alteration was observed in our acute setting of ethanol treatment. Several rationales may be derived toward the acute ethanol exposure-induced cardiac toxicity may have multiple causes, including lipid peroxidation, oxidative damage (3), acetaldehyde oxidation (5), and altered membrane properties (25). Among which, acetaldehyde may be the top candidate for the rapid onset of cardiac toxicity responsible for overt myocardial injury within 24 h. As the major metabolite of ethanol, acetaldehyde is capable of generating free radicals via aldehyde oxidase and/or xanthine oxidase-associated oxidation (26). In our earlier study, we reported greater levels of lipid peroxidation and protein carbonyls associated with dampened intercellular Ca2+release and SR Ca2+load in myocardium from cardiac-specific ADH transgenic mice (16). More recently, we demonstrated that elevated cardiac acetaldehyde exposure via ADH overexpression exacerbates alcohol-induced myocardial dysfunction, hypertrophy, insulin insensitivity, and endoplasmic reticulum (ER) stress (8). Although our previous studies demonstrated overexpression of ALDH2 in cell culture alleviates acetaldehyde-induced cell injury in human umbilical vein endothelial cells and human cardiac cells (9,17), little knowledge is available with regards to the effect of ALDH2 on acute ethanol toxicity on the hearts. Data from our current study revealed for the first time, to our knowledge, that ALDH2 counteracts cardiac acetaldehyde exposure and protects myocardial function against acute ethanol toxicity. These results have convincingly validated the notion that acetaldehyde may be a critical player in acute ethanol toxicity in the hearts, favoring a cardioprotective role of ALDH2. Pharmacological elevation of ALDH2 activity was previously shown to reduce the ischemic heart damage (10). Interestingly, our data revealed overtly elevated ALDH activity in response to acute ethanol challenge, representing the likelihood of a compensatory protective response against harmful insults.
In hearts, both AMPK and Akt are deemed key regulators of myocardial function (11,12). Nonetheless, possible cross-talk between the 2 under physiological or pathophysiological state is still unclear. Our earlier study indicated that alcohol intake-induced cardiomyocyte contractile dysfunction is associated with a reduced Akt activity (27), although little is known for AMPK. Data from our current study revealed dampened phosphorylation of both Akt and AMPK in conjunction with compromised cardiac function in response to acute ethanol treatment. Akt and AMPK are both essential for cardiac survival and energy fuel, as well as contractile function (11,12,27). Our observation that ALDH2 compensates for the loss of activation in Akt and AMPK in response to ethanol treatment suggests a possible role of these signaling molecules in the ALDH2-induced cardioprotection against acute ethanol toxicity. It was recently shown that Akt and AMPK are dephosphorylated by PP2A (15,20) and PP2C (28), respectively. Our current study detected significantly up-regulated PP2A and PP2C proteins in FVB mice following acute ethanol treatment, suggesting a role of PP2A and PP2C in the reduced phosphorylation of Akt and AMPK, respectively. The ability of ALDH2 to alleviate the acute ethanol exposure-induced elevation in these 2 protein phosphatases provides further evidence for the regulation of PP2A and PP2C in Akt and AMPK signaling, respectively, under our current experimental setting.
Foxo3 has been considered a converging point for Akt and AMPK signaling pathways (13). AMPK phosphorylation of Foxo3 at the site of Ser413 enhances the stress resistance and longevity (13,14). On the other hand, Akt phosphorylation of Foxo3 at Thr32 inhibits Foxo3 transcriptional activity, promoting antiapoptosis and insulin sensitivity (15). Therefore, Akt and AMPK signaling may elicit transcriptional regulation via Foxo3 to allow organismal adaptation to physiological or pathophysiological changes. The convergence of the 2 pathways at the level of Foxo3 may play a critical role for the cross-talk between Akt and AMPK. In the present study, acute ethanol exposure-induced loss of phosphorylation of Akt and AMPK was restored in ALDH2 mice. These data favor Foxo3 as a converging point between Akt and AMPK pathways following ethanol exposure. The ALDH2 enzyme-induced protection on both Akt-Foxo3 (Thr32) and AMPK-Foxo3 (Ser413) pathways is likely a secondary effect of facilitated detoxification of acetaldehyde following ethanol exposure. Foxo transcriptional factors are known regulators for cell cycle and apoptosis, especially for Akt and AMPK-mediated antiapoptotic properties (22). Our data revealed enhanced caspase-3 activity, carbonyl formation, and mitochondrial damage in FVB but not ALDH2 mice following acute ethanol challenge. These data suggest a possible role of Akt-Foxo and AMPK-Foxo in the ethanol-induced apoptosis and mitochondrial dysfunction as well as the beneficial role of ALDH2. However, given the nature of associate rather than causal relationship of our data in cell signaling and cell injury, further study is warranted to better elucidate the role of Akt, AMPK, and Foxo3 in ALDH2-elicited cardioprotection against ethanol toxicity.
The present study has provided convincing evidence that ALDH2 attenuated acute ethanol exposure-induced myocardial dysfunction, apoptosis, and protein and mitochondrial damage, possibly through inhibition of protein phosphatases, leading to preserved phosphorylation of Akt and AMPK on Foxo3. Further study is warranted to unveil the impact of ALDH2 on the more clinically relevant condition of chronic alcohol ingestion-induced onset and progression of alcoholic cardiomyopathy.
The authors wish to thank Dr. Paul N. Epstein from the University of Louisville for production of the ALDH2 transgenic line.
This work was supported by NIAAA 1R01 AA013412, NCRR P20RR016474, and the National Science Foundation of China (#30728023).
- Abbreviations and Acronyms
- maximal velocity of shortening/relengthening
- maximal velocity of pressure development and decline
- alcohol dehydrogenase
- aldehyde dehydrogenase
- AMP-activated protein kinase
- left ventricular developed pressure
- peak shortening
- sarcoplasmic reticulum
- time-to-90% peak shortening
- time-to-90% relengthening
- mitochondrial membrane potential
- Received October 28, 2008.
- Revision received March 10, 2009.
- Accepted April 2, 2009.
- American College of Cardiology Foundation
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