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J Appl Physiol 99: 2246-2254, 2005. First published August 18, 2005; doi:10.1152/japplphysiol.00750.2005
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Cardiac overexpression of catalase antagonizes ADH-associated contractile depression and stress signaling after acute ethanol exposure in murine myocytes

Xiaochun Zhang,1 Feng Dong,1 Qun Li,1 Anthony J. Borgerding,2 Aaron L. Klein,1 and Jun Ren1

1Division of Pharmaceutical Sciences & Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, Wyoming; and 2Department of Chemistry, University of St. Thomas, St. Paul, Minnesota

Submitted 24 June 2005 ; accepted in final form 11 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Alcohol dehydrogenase (ADH), which oxidizes ethanol into acetaldehyde, exacerbates ethanol-induced cardiac depression, although the mechanism of action remains unclear. This study was designed to examine the impact of antioxidant catalase (CAT) on cardiac contractile response to ethanol and activation of stress signaling. ADH-CAT double transgenic mice were generated by crossing CAT and ADH lines. Mechanical, intracellular Ca2+ properties and reactive oxygen species generation were measured in ventricular myocytes. ADH-CAT, ADH, CAT and wild-type FVB myocytes exhibited similar mechanical and intracellular Ca2+ properties. ADH or ADH-CAT myocytes had higher acetaldehyde-producing ability. Ethanol (80–640 mg/dl) suppressed FVB cell shortening and intracellular Ca2+ transients with maximal inhibitions of 43.5 and 45.2%, respectively. Ethanol-induced depression on cell shortening and intracellular Ca2+ was augmented in ADH group with maximal inhibitions of 66.8 and 69.6%, respectively. Interestingly, myocytes from CAT-ADH mice displayed normal ethanol response with maximal inhibitions of 46.0 and 47.2% for cell shortening and intracellular Ca2+, respectively. CAT transgene lessened ethanol-induced inhibition on cell shortening (maximal inhibition of 30.3%) but not intracellular Ca2+. ADH amplified ethanol-induced reactive oxygen species generation, which was nullified by the CAT transgene. Western blot analysis showed that ethanol reduced ERK phosphorylation and enhanced JNK phosphorylation without affecting p38 phosphorylation. The ethanol-induced changes in phosphorylation of ERK and JNK were amplified by ADH. CAT transgene itself did not affect ethanol-induced response in ERK and JNK phosphorylation, but it cancelled ADH-induced effects. These data suggest that antioxidant CAT may effectively antagonize ADH-induced enhanced cardiac depression in response to ethanol.

myocyte; shortening; intracellular Ca2+ transient

ALCOHOLISM LEADS TO heart muscle damage, namely alcoholic cardiomyopathy, and increased cardiac mortality (8, 31). Alcoholic cardiomyopathy is usually featured by cardiac hypertrophy, disrupted myocardial ultrastructure, decreased ejection fraction, reduced myocardial contractility, and elevated risk of stroke and hypertension (19, 31). Several theories have been speculated to be responsible for pathogenesis of alcoholic cardiomyopathy, including toxicity of alcohol, its metabolite acetaldehyde and fatty acid ethyl esters, as well as accumulation of reactive oxygen species (ROS) and/or reduced antioxidant defense (20, 24, 31), suggesting a likelihood role of the alcohol metabolite acetaldehyde and oxidative stress in alcohol-initiated heart diseases in populations with chronic alcohol consumption. This is supported with the notion that free radical generation and oxidative stress following alcohol ingestion appear to be essential to the toxicity of ethanol and acetaldehyde (10, 31). Ethanol-induced accumulation of free radicals and oxidative stress is directly associated with its metabolic pathways, including alcohol dehydrogenase (ADH), microsomal ethanol oxidation system, and catalase, which may produce superoxide anion 1-hydroxyethyl radical and lessen the enzymatic/nonenzymatic antioxidant defense systems (10, 31). However, exactly how ethanol produces cytotoxicity and mediates cardiomyopathy through its metabolic intermediate(s) remains poorly understood.

The "acetaldehyde toxicity" theory of alcohol cardiomyopathy received convincing support from our laboratories recent studies using a transgenic mouse model of cardiac-specific overexpression of ADH (6, 7, 12). Through elevated cardiac expression of ADH, the enzyme that hydrolyzes ethanol into acetaldehyde, the progression and severity of cardiomyopathy following alcohol consumption or ethanol exposure become significantly "accelerated" both functionally and morphologically (6, 7, 12, 15). This accelerated alcoholic cardiomyopathy is apparently associated with enhanced cardiac acetaldehyde levels (7, 12) and seems to be consistent with the observation that acetaldehyde directly impairs cardiac excitation-contraction coupling and inhibits sarco(endo)plasmic reticulum Ca2+ release function (1, 21, 22). Nevertheless, the mechanism through which facilitated alcoholic myopathic alteration develops has not been fully unveiled. The aim of this study was to investigate the impact of the antioxidant catalase (CAT) on ADH-induced amplification of ethanol-elicited depression of cardiac function to examine the role of antioxidant defense in ADH-induced accelerated cardiomyopathy. We generated a novel double transgenic model with cardiac-specific overexpression of both ADH and CAT genes. Myocyte shortening, intracellular Ca2+ homeostasis, and mitogen-activated protein (MAP) kinase stress signals including extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase following acute ethanol exposure were evaluated in hearts from wild-type FVB, catalase, ADH, and ADH-CAT double transgenic mice. Activation of ERK, JNK, and p38 MAP kinases is essential in the onset of morphological and mechanical dysfunctions of the hearts with characteristic gene expression alteration including elevated transcription of atrial natriuretic factor, {beta}-myosin heavy chain (MHC), {alpha}-skeletal actin, integrins and tubulin, and reduced expression of phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPase. It has been demonstrated that ethanol, acetaldehyde, and other reactive aldehydes are important inducers of the MAP kinase signaling cascade, which play critical roles in the progression of alcoholic cardiomyopathy (3, 28, 31).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Generation and identification of cardiac ADH-CAT double transgenic mice.   All animal procedures were approved by the Animal Care and Use Committees at the University of North Dakota (Grand Forks, ND) and the University of Wyoming (Laramie, WY). Briefly, either ADH or CAT transgene was constructed to produce transgenic mice with cardiac-specific overexpression of either ADH or CAT as characterized earlier (7, 13, 15, 30). Mouse {alpha}-MHC promoter was used to drive cardiac-specific expression. cDNA for either murine class I ADH, which converts ethanol to acetaldehyde, or rat catalase, which converts H2O2 into O2 and H2O, was inserted behind the MHC promoter (13). White albino FVB mice were used as the background (26). For ADH line, a second transgene containing a cDNA for the enzyme tyrosinase was co-injected with ADH, resulting in coat color (light gray) pigmentation for identification of ADH-positive (~40-fold increase in ADH expression) transgene (18). On the other hand, identification of cardiac-specific CAT transgenic mice was performed using PCR technique as described previously (~60-fold increase in CAT expression) (30). A primer pair derived from MHC promoter and rat CAT cDNA was used with the reverse sequence of AAT ATC GTG GGT GAC CTC AA and the forward sequence of CAG ATG AAG CAG TGG AAG GA. The ADH-CAT double transgenic mice were generated by crossing ADH and CAT transgenic lines followed by PCR identification in light-gray colored offspring (ADH positive) (Fig. 1). All mice were maintained with a 12:12-h light-dark cycle with free access to tap water and rodent chow. Male mice of ~4 mo of age were used for experimentation. For acute ethanol challenge experiment, mice were injected intraperitoneally with ethanol (3 g/kg body wt) and were killed 30 min later for cardiac tissue collection.



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  		Fig. 1. Identification of catalase-overexpressed transgenic mice. Genomic DNA was isolated from 2-cm tail clips from 1-mo-old mice. Catalase gene was identified by polymerase chain reaction using a primer pair derived from the myosin heavy chain (MHC) promoter and rat catalase cDNA.

 
Cell isolation procedures.   After ketamine/xylazine sedation, mouse hearts were removed and perfused with Krebs-Henseleit bicarbonate buffer containing (in mM) 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 10 HEPES, and 11.1 glucose, with 5% CO2-95% O2. Hearts were subsequently digested with a Krebs-Henseleit bicarbonate buffer containing 223 U/ml collagenase D (Boehringer Mannheim, Indianapolis, IN) for 20 min. After perfusion, left ventricles were removed and minced before being filtered. Extracellular Ca2+ was slowly added back to 1.25 mM. Myocytes with obvious sarcolemmal blebs or spontaneous contractions were not used (30). Myocytes were used within 6 h of isolation.

Assessment of the acetaldehyde production after acute ethanol challenge.   Isolated ventricular myocytes (200,000 cells/ml) from FVB, ADH, catalase, or ADH-CAT mice were exposed to 240 mg/dl ethanol for 15 min in a sealed vial before the reaction was terminated by 4-methylpyrazole (1 mM). Vials were then stored at –80°C until analysis. Immediately before analysis, the samples were warmed to 25°C. Two milliliters 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 on a Hewlett-Packard 5890 gas chromatograph equipped with a flame ionization detector. Acetaldehyde and other components were separated on a 9-m VOCOL capillary column (Supelco) with a 1.8-µm film thickness and an inner diameter of 0.32 mm. The temperature was held isothermally at 30°C, and the carrier gas was helium at a flow rate of 1.8 ml/min. Under these conditions, separation of acetaldehyde from ethanol and other compounds was complete in 1 min. Quantitation was achieved by calibrating the gas chromatography areas against those from headspace samples of known acetaldehyde standards over a similar concentration range as the cell samples in the same buffer (7).

Cell shortening/relengthening.   Mechanical properties of ventricular myocytes were assessed using an IonOptix MyoCam video-based edge-detection system (IonOptix, Milton, MA) as described previously (30). In brief, cells were placed in a chamber mounted on the stage of an inverted microscope (Olympus IX-70) and superfused (~2 ml/min at 25°C) with a buffer containing (in mM) 131 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 mM glucose, 10 HEPES, at pH 7.4. The cells were field stimulated to contract at a frequency of 0.5 Hz. Changes in cell length during shortening and relengthening were captured and converted to a digital signal. The myocyte being studied was rapidly scanned with a camera at 120 Hz to ensure recording with good fidelity. Cell shortening and relengthening were assessed using the following indexes: peak shortening, time to peak shortening (TPS) and time to 90% relengthening (TR90), and ±dL/dt.

Intracellular Ca2+ fluorescence measurement.   Myocytes were loaded with fura 2-AM (0.5 µM) for 10 min at 25°C, and fluorescence measurements were recorded with a dual-excitation fluorescence photomultiplier system (Ionoptix) as previously described (30). Myocytes were imaged through an Olympus IX-70 Fluor oil objective. Cells were exposed to light emitted by a 75-W lamp and passed through either a 360- or a 380-nm filter (bandwidths were ±15 nm), while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after first illuminating the cells at 360 nm for 0.5 s then at 380 nm for the duration of the recording protocol (333-Hz sampling rate). The 360 excitation scan was repeated at the end of the protocol, and qualitative changes in intracellular Ca2+ concentration was inferred from the ratio of the fura 2 fluorescence intensity (FFI) at the two wavelengths.

Intracellular fluorescence measurement of ROS.   The membrane-permeable probe 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (Molecular Probes, Eugene, OR) enters the cells and produces a fluorescent signal after intracellular oxidation by ROS such as H2O2. Intracellular ROS were monitored by changes in fluorescence intensity resulting from intracellular probe oxidation. After a 3-min acute ethanol (240 mg/dl) treatment, cardiac myocytes from FVB, ADH, CAT, and CAT-ADH mice were loaded with 10 µM DCF for 30 min at 37°C before being washed with PBS buffer. Cells were sampled randomly using an Olympus BX-51 microscope equipped with an Olympus MagnaFire SP digital camera and ImagePro image analysis software (Media Cybernetics, Silver Spring, MD). Fluorescence was calibrated with InSpeck microspheres (Molecular Probes). More than 150 cells per data point were evaluated using the grid-crossing method for cell selection in >15 visual fields (5).

Western blot analysis of ERK, pERK, JNK, p-JNK, p38, and pp38.   The total protein was prepared as described previously (30). In brief, left ventricles were rapidly removed and homogenized in a lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 0.1% SDS, and 1% protease inhibitor cocktail. Samples were then sonicated for 15 s and centrifuged at 12,000 g for 20 min at 4°C. The protein concentration of the supernatant was evaluated using the protein assay reagent (Bio-Rad, Hercules, CA). Equal-amount (50 µg protein/lane) protein and prestained molecular weight marker (GIBCO, Gaithersburg, MD) were loaded onto 15% SDS-polyacrylamide gels in a minigel apparatus (Mini-PROTEAN II, Bio-Rad), separated, and transferred to nitrocellulose membranes (0.2-µm pore size, Bio-Rad). Membranes were incubated for 1 h in a blocking solution containing 5% nonfat milk in TBS before being washed in TBS and incubated overnight at 4°C with anti-ERK (1:1,000), anti-phospho-ERK (pERK; 1:1,000), anti-JNK (1:1,000), anti-phospho-JNK (pJNK, 1:1,000), anti-p38 (1:1,000), and anti-phospho-p38 (pp38, 1:1,000) antibodies. Anti-ERK and anti-pERK antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other antibodies were obtained from Cell Signaling Technology (Beverly, MA). After incubation with the primary antibodies, blots were incubated with either anti-mouse or anti-rabbit IgG horseradish peroxidase-linked antibodies at a dilution of 1:5,000 for 60 min at room temperature. Immunoreactive bands were detected using the Super Signal West Dura Extended Duration Substrate (Pierce, Milwaukee, WI). The intensity of bands was measured with a scanning densitometer (model GS-800; Bio-Rad) coupled with Bio-Rad personal computer analysis software.

Experimental protocols.   For in vitro ethanol exposure, myocytes (fura 2 loaded or nonloaded) were first allowed to contract at a stimulation frequency of 0.5 Hz for 5 min to ensure steady state (myocytes with rundown >10% were not studied further) before perfusing with ethanol (80–640 mg/dl) containing contractile buffer. A 3-min interval was allowed between the ethanol doses. Our previous experience indicated that ethanol-induced inhibition of cell shortening remains stable for up to 30 min (7, 23). In ROS generation study, myocytes were loaded with 10 µM dichlorofluorescein for 30 min at 37°C after the 3-min acute ethanol (240 mg/dl) exposure. For in vivo ethanol exposure, mice were injected intraperitoneally with ethanol (3 g/kg). Cardiac tissues were collected 30 min later for gel electrophoresis assessment.

Data analysis.   Data were reported as means ± SE. All data were statistically evaluated using a 2 x 2 ANOVA design where one factor was transgene and the other ethanol concentration. Each index was assessed in separate ANOVAs. If the interaction term (transgene x ethanol concentration) reached statistical significance (P < 0.05), then a Tukey's multiple comparison test was used to avoid inflated group {alpha} effects and to identify which groups were statistically different.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
General features of FVB wild-type, ADH, catalase, and ADH-CAT transgenic mice and left ventricular myocytes from each group.   As shown in Table 1, overexpression of ADH, catalase, or both did not elicit any notable effect on body, heart, liver, and kidney weights or organ size (when normalized to respective body weight) compared with those of the FVB wild-type littermates. Baseline mechanical and intracellular Ca2+ properties of left-ventricular myocytes were essentially similar in left-ventricular myocytes from FVB, ADH, catalase, and ADH-CAT transgenic mice, with the only exception of significantly shorter resting myocyte length from the CAT mice (Table 2), suggesting that the ADH or CAT transgenes themselves were unlikely innately harmful to intrinsic ventricular contractile function. The ability of ventricular myocytes to oxidize ethanol (240 mg/dl) into acetaldehyde measured by gas chromatography and flame ionization detection was significantly higher in myocytes from ADH and ADH-CAT groups compared with FVB and CAT groups (Fig. 2), validating the identity of transgenic overexpression of ADH enzyme in both groups (increases ADH activity by ~40 times) (7, 15). No difference was observed between ventricular myocytes from ADH and ADH-CAT double transgenic groups in their ability to convert ethanol into acetaldehyde (Fig. 2).


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  		Table 1. General features of FVB, ADH, CAT, and ADH-CAT mice

 

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  		Table 2. Baseline properties of isolated ventricular myocytes from FVB, ADH, CAT, and ADH-CAT mice

 


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  		Fig. 2. Acetaldehyde production in cardiomyocytes from FVB, alcohol dehydrogenase (ADH), catalse (CAT), or ADH-CAT mice. Cardiomyocytes were exposed to ethanol (240 mg/dl) for 15 min before levels of acetaldehyde were determined using gas chromatography. Values are means ± SE; n = 6–7 mice per group. *P < 0.05 vs. FVB.

 
Acute effects of ethanol on cell shortening and intracellular Ca2+ transients.   To determine whether ADH and CAT transgenes affect ethanol-induced depression of cardiac myocyte shortening, concentration-dependent responses were constructed for ethanol (0–640 mg/dl) in myocytes from FVB wild-type, ADH, catalase, and ADH-CAT mice. Acute exposure of ethanol did not affect the resting cell length (data not shown); however, it significantly depressed cell-shortening amplitude in all mouse groups. Consistent with our laboratory's previous reports (6, 7), ADH transgene significantly augmented the degree of ethanol-induced inhibition in cell shortening. The maximal inhibitions of myocyte shortening in response to ethanol were 43.5 and 66.8% in FVB and ADH groups, respectively. The threshold of ethanol-induced inhibition was reduced from 80–120 mg/dl in FVB myocytes to <80 mg/dl in ADH myocytes. CAT transgene itself did not alter ethanol-induced inhibition of cell shortening (with a maximal inhibition of 30.3% and a threshold of inhibition between 80 and 120 mg/dl). Interestingly, CAT alleviated ADH transgene-augmented suppression of cell shortening in response to ethanol exposure. The maximal inhibition of ethanol-induced cell shortening was 46.0% with a threshold between 80 and 120 mg/dl. The ethanol-induced inhibition of cell shortening in ADH myocytes was significantly greater than all other groups at doses of >80 mg/dl (Fig. 3A). Acute ethanol exposure also prolonged duration of shortening (TPS) and relengthening (TR90) and inhibited ±dL/dt. Interestingly, ventricular myocytes from ADH mice were more sensitive to ethanol-induced effect on TPS, TR90, and ±dL/dt with a much lower threshold of effectiveness (Table 3). Consistent with its response on peak shortening, ethanol depressed the electrically stimulated rise in intracellular Ca2+ FFI in a concentration-dependent manner (80–640 mg/dl) in FVB myocytes, with a threshold between 80 and 120 mg/dl. CAT transgene did not affect the pattern of response for ethanol, whereas ADH transgene exacerbated ethanol-induced depression of FFI with a threshold of <80 mg/dl. Interestingly, CAT transgene nullified ADH transgene-elicited augmentation of ethanol-induced inhibition of FFI (Fig. 3B). These results strongly indicated that the antioxidant CAT may abolish ADH transgene-amplified cardiac depression in response to acute ethanol exposure.



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  		Fig. 3. Effect of acute ethanol exposure on cell shortening (A) and intracellular Ca2+ transient fura-2 fluorescent intensity (FFI; B) in isolated myocytes from FVB, ADH, CAT, and ADH-CAT mice. Graph shows concentration-dependent response of ethanol (0–640 mg/dl) on peak cell shortening (PS) and FFI. Both peak shortening and FFI are expressed as percent change from respective baseline (0 ethanol) value. Valus are means ± SE; cell number is given in parenthesis. *P < 0.05 vs. baseline. #P < 0.05 vs. all other groups at the same concentration.

 

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  		Table 3. Effect of ethanol on myocyte shortening and relengthening indexes in FVB, ADH, CAT, and ADH-CAT mice

 
Acute effect of ethanol on ROS generation.   Figure 4 reveals that a 3-min acute incubation of ethanol (240 mg/dl) with myocytes significantly increased intracellular ROS generation measured by 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate fluorescent intensity in all four myocyte groups tested. Interestingly, myocytes obtained from ADH mice displayed significantly enhanced ROS accumulation in response to ethanol exposure compared with FVB group with ethanol exposure, suggesting that facilitation of acetaldehyde production may exacerbate ethanol-induced cellular toxicity. CAT effectively antagonized ethanol-induced ROS generation and was able to nullify ADH transgene-induced exacerbation in ROS generation in response to ethanol exposure. These data indicated that the antioxidant CAT may eliminate ADH transgene-amplified cardiac depression in response to acute ethanol exposure via a mechanism related to reduction in ROS generation.



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  		Fig. 4. Effect of acute ethanol exposure (240 mg/dl) on intracellular reactive oxygen species (ROS) production using dichlorofluorescein (DCF) fluorescence detection in left ventricular myocytes from FVB, ADH, CAT, and ADH-CAT mice. Values are means ± SE; n = 15 visual fields from 2–4 mice per group. *P < 0.05 vs. FVB-control (without ethanol treatment). #P < 0.05 vs. FVB-ethanol group.

 
Protein expression of stress-signaling molecules under acute ethanol exposure.   Ethanol or its metabolite acetaldehyde has been demonstrated to exert its cytotoxic effect through one or more of the three distinct but parallel MAP kinase cascades, namely ERK, JNK, and p38 MAP kinase (31). To examine possible involvement of these pathways, all four groups of mice (FVB, ADH, catalase, and ADH-catalase) were injected intraperitoneally with ethanol (3 g/kg) before cardiac tissues were collected 30 min later for gel electrophoresis. Our data revealed that total ERK or total JNK protein expression was not affected by ethanol treatment among all groups tested (including an ethanol-untreated FVB control group). However, phosphorylation of ERK and JNK was significantly reduced and enhanced, respectively, by ethanol treatment. The ethanol-induced response on phosphorylation of ERK and JNK was significantly amplified by the ADH transgene. Interestingly, the ADH transgene-elicited augmentation of ethanol response (depressed ERK phosphorylation and enhanced JNK phosphorylation) was blunted by the CAT transgene. CAT transgene itself did not affect pattern of ERK or JNK phosphorylation in response to ethanol. Although the extent of ethanol-induced reduction in ERK phosphorylation was significantly less in the CAT group compared with FVB group, the ratio of phosphorylated ERK to total ERK was similar between the two groups (Figs. 5 and 6). Finally, ethanol treatment did not affect the total and phosphorylated p38 MAP kinase. Neither ADH nor CAT transgene affected the nonresponse of ethanol on p38 MAP kinase expression and p38 MAP kinase phosphorylation (Fig. 7).



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  		Fig. 5. Effect of acute ethanol treatment (3 g/kg ip for 30 min) on expression of total extracellular-regulated kinase (ERK) and phosphorylated ERK (pERK) in left ventricles from FVB, ADH, CAT, and ADH-CAT mice. A: representative Western gel blots depicting ERK and pERK expression using anti-ERK and anti-pERK antibodies. B: total ERK expression. C: pERK expression. D: ratio of pERK to total ERK. Values are means ± SE; n = 4–8 mice per group. *P < 0.05 vs. FVB control (without ethanol treatment). #P < 0.05 vs. all other groups.

 


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  		Fig. 6. Effect of acute ethanol treatment (3 g/kg ip for 30 min) on expression of total JNK and phosphorylated JNK (pJNK) in left ventricles from FVB, ADH, CAT, and ADH-CAT mice. A: representative Western gel blots depicting JNK and pJNK expression using anti-JNK and anti-pJNK antibodies. B: total JNK expression. C: pJNK expression. D: ratio of pJNK to total JNK. Values are means ± SE; n = 4–8 mice per group. *P < 0.05 vs. FVB control (without ethanol treatment). #P < 0.05 vs. FVB ethanol (FVB+ETOH) and CAT-ethanol (CAT-ETOH) groups.

 


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  		Fig. 7. Effect of acute ethanol treatment (3 g/kg ip for 30 min) on expression of total p38 MAPK and phosphorylated p38 MAPK (pp38) in left ventricles from FVB, ADH, CAT, and ADH-CAT mice. A: representative Western gel blots depicting p38 and pp38 expression using anti-p38 and anti-pp38 antibodies. B: total p38 expression. C: pp38 expression. D: ratio of pp38 to total p38. Values are means ± SE; n = 4–8 mice per group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The major finding of this study was that antioxidant CAT counteracts the ADH gene-elicited augmentation of ethanol-induced cardiac contractile depression, ROS generation, and alteration of stress signaling including ERK and JNK. Our laboratory's earlier study has indicated the significant role of acetaldehyde as an ultimate candidate toxin for alcoholic cardiomyopathy (6, 7, 12, 31). Data from our present study revealed that cardiac-specific overexpression of ADH-amplified cardiac abnormalities (myocyte contraction and intracellular Ca2+ handing) is likely due to enhanced acetaldehyde-mediated oxidative stress and/or reduction in antioxidant defense. Consistent with its effect on cell shortening, intracellular Ca2+ transient, and ROS generation, ADH transgene sensitized myocardium to ethanol-induced response in stress signaling activation, which was also antagonized by the CAT transgene. These results suggested that the ADH enzyme may be a significant regulator in ethanol-induced cardiac depression by way of acetaldehyde production, alteration of redox state, and activation of stress signaling molecules.

As stated earlier, alcoholic cardiomyopathy usually displays cardiomegaly, disruptions of myofibrillary architecture, reduced myocardial contractility, and decreased ejection volume (19, 24). Among all these cardiac anomalies, impaired contractile function and interrupted intracellular Ca2+ handling are perhaps the most significant mechanisms leading to compromised myocardial function following ethanol exposure (4, 9, 27). Evidence from our laboratory as well as others has indicated that the main metabolic product acetaldehyde plays a crucial role in disrupted intracellular Ca2+ handling and cardiac excitation-contraction coupling (7, 17, 22, 31), consistent with the observation that enhanced cardiac acetaldehyde exposure is directly linked to exacerbated cardiac contractile dysfunction in ADH transgenic mice (12). Acetaldehyde exerts a myogenic negative myocardial inotropic effect (1, 31). Data from our present study revealed that ventricular myocytes from both ADH and ADH-CAT mice are capable of producing equally high levels of acetaldehyde from ethanol oxidization. However, the myocyte contraction and intracellular Ca2+ transient properties were drastically different in these two groups, with myocytes from the ADH-CAT group showing a great degree of protection against acute ethanol-initiated insults. Because CAT is a potent antioxidant that elicits antioxidant protection against ethanol-induced cardiac contractile dysfunction (30), it is natural to speculate that the catalase-elicited protection against ADH-augmented cardiac contraction dysfunction in response to ethanol is due to antioxidant defense from catalase. This is supported by the observation that alcohol intake directly contributes to reduction of the CAT enzymatic activity (11). This notion received further support from our ERK and JNK phosphorylation study. ERK, JNK, and p38 MAP kinases have been shown to participate in acetaldehyde-induced cell toxicity (2, 25), consistent with the concept that activation of MAP kinase family serves as a significant signaling mechanism for chronic alcohol ingestion-induced oxidative injury (29). Acetaldehyde, as the highly reactive product from the oxidative metabolism of ethanol, is a critical mediator of ethanol-induced apoptosis via the activation of MAP kinase signaling pathway (14, 25). This scenario should explain ADH-induced augmented response in ERK and JNK phosphorylation. The discrepant phosphorylation between ERK and JNK observed in our study may be due to pro- vs. anti-apoptotic properties of these two stress-signaling molecules. It is possible that acetaldehyde may selectively activate certain but not all family of the MAP kinase in different experimental settings. Further study is warranted to elucidate the stress signaling mechanisms involved in the ADH enzyme or acetaldehyde-induced cell toxicity.

In our present study, we found that ethanol (at 240 and 640 mg/dl) prolonged the duration of shortening (TPS) and relengthening (TR90) in conjunction with depression of peak shortening, ±dL/dt in myocytes from FVB mice. This is consistent with our laboratory's earlier study (7). However, our recent study using both FVB and CAT mice revealed that ethanol (80–640 mg/dl) prolonged relengthening duration but not that of shortening in myocytes from both FVB and CAT mice (30). The rationale responsible for this apparent discrepancy in duration of cell shortening is unknown at this time. However, it may be speculated that subtle differences in the age and sex of FVB mice. Male FVB mice at the age of 4 mo were used in the present study compared with 5-mo-old mice of both genders used in an earlier study (30). In addition, employment of a different batch of isolation enzyme (collagenase D) due to the availability from the vender may also contribute to viability of left ventricular myocytes. Other mechanisms not identified here may be also involved.

In summary, our present study provides evidence that antioxidant enzyme CAT may protect against ADH enzyme-elicited augmentation of cardiac contractile and intracellular Ca2+ dysfunction as well as ROS generation in response to ethanol exposure, supporting a role of oxidative stress in ADH-/acetaldehyde-associated myopathic alteration following ethanol exposure. Oxidant balance is crucial in maintaining normal cardiac contractile performance (10, 16). However, the antioxidant reserves become inadequate under alcoholism such as CAT (11). Future work with alcoholism and the assessment of cardiac contractile protein function using ADH and other antioxidant transgenic mice should provide in-depth knowledge for the precise nature of acetaldehyde and oxidative stress in the development of alcoholic cardiomyopathy.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This research was supported in part by National Institutes of Health Grants R15 AA-13575-01, RR-16474, and P20 RR-15640.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors are grateful to Bonnie H. Zhao, Faye L. Lopaz, and Karissa H. LaCour for technical assistance and data analysis. The ADH and CAT founder mice were kindly provided by Paul N. Epstein from University of Louisville, Louisville, KY.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Ren, Division of Pharmaceutical Sciences & Center for Cardiovascular Research and Alternative Medicine, Univ. of Wyoming, Laramie, WY 82071 (e-mail: jren{at}uwyo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Brown RA, Jefferson L, Sudan N, Lloyd TC, and Ren J. Acetaldehyde depresses myocardial contraction and cardiac myocyte shortening in spontaneously hypertensive rats: role of intracellular Ca2+. Cell Mol Biol (Oxf) 45: 453–465, 1999.
  2. Cao Q, Mak KM, and Lieber CS. Dilinoleoylphosphatidylcholine decreases acetaldehyde-induced TNF-{alpha} generation in Kupffer cells of ethanol-fed rats. Biochem Biophys Res Commun 299: 459–464, 2002.[CrossRef][ISI][Medline]
  3. Che W, Asahi M, Takahashi M, Kaneto H, Okado A, Higashiyama S, and Taniguchi N. Selective induction of heparin-binding epidermal growth factor-like growth factor by methylglyoxal and 3-deoxyglucosone in rat aortic smooth muscle cells. The involvement of reactive oxygen species formation and a possible implication for atherogenesis in diabetes. J Biol Chem 272: 18453–18459, 1997.[Abstract/Free Full Text]
  4. Danziger RS, Sakai M, Capogrossi MC, Spurgeon HA, Hansford RG, and Lakatta EG. Ethanol acutely and reversibly suppresses excitation-contraction coupling in cardiac myocytes. Circ Res 68: 1660–1668, 1991.[Abstract/Free Full Text]
  5. Dong F, Zhang X, Li SY, Zhang Z, Ren Q, Culver B, and Ren J. Possible involvement of NADPH oxidase and JNK in homocysteine-induced oxidative stress and apoptosis in human umbilical vein endothelial cells. Cardiovasc Toxicol 5: 9–20, 2005.[Medline]
  6. Duan J, Esberg LB, Ye G, Borgerding AJ, Ren BH, Aberle NS, Epstein PN, and Ren J. Influence of gender on ethanol-induced ventricular myocyte contractile depression in transgenic mice with cardiac overexpression of alcohol dehydrogenase. Comp Biochem Physiol A Mol Integr Physiol 134: 607–614, 2003.[CrossRef][Medline]
  7. Duan J, McFadden GE, Borgerding AJ, Norby FL, Ren BH, Ye G, Epstein PN, and Ren J. Overexpression of alcohol dehydrogenase exacerbates ethanol-induced contractile defect in cardiac myocytes. Am J Physiol Heart Circ Physiol 282: H1216–H1222, 2002.[Abstract/Free Full Text]
  8. Fernandez-Sola J, Estruch R, Grau JM, Pare JC, Rubin E, and Urbano-Marquez A. The relation of alcoholic myopathy to cardiomyopathy. Ann Intern Med 120: 529–536, 1994.[Abstract/Free Full Text]
  9. Guarnieri T and Lakatta EG. Mechanism of myocardial contractile depression by clinical concentrations of ethanol. A study in ferret papillary muscles. J Clin Invest 85: 1462–1467, 1990.[ISI][Medline]
  10. Guerri C, Montoliu C, and Renau-Piqueras J. Involvement of free radical mechanism in the toxic effects of alcohol: implications for fetal alcohol syndrome. Adv Exp Med Biol 366: 291–305, 1994.[Medline]
  11. Gutierrez-Ruiz MC, Gomez Quiroz LE, Hernandez E, Bucio L, Souza V, Llorente L, and Kershenobich D. Cytokine response and oxidative stress produced by ethanol, acetaldehyde and endotoxin treatment in HepG2 cells. Isr Med Assoc J 3: 131–136, 2001.[Medline]
  12. Hintz KK, Relling DP, Saari JT, Borgerding AJ, Duan J, Ren BH, Kato K, Epstein PN, and Ren J. Cardiac overexpression of alcohol dehydrogenase exacerbates cardiac contractile dysfunction, lipid peroxidation, and protein damage after chronic ethanol ingestion. Alcohol Clin Exp Res 27: 1090–1098, 2003.[CrossRef][ISI][Medline]
  13. Kang YJ, Chen Y, and Epstein PN. Suppression of doxorubicin cardiotoxicity by overexpression of catalase in the heart of transgenic mice. J Biol Chem 271: 12610–12616, 1996.[Abstract/Free Full Text]
  14. Lee YJ, Aroor AR, and Shukla SD. Temporal activation of p42/44 mitogen-activated protein kinase and c-Jun N-terminal kinase by acetaldehyde in rat hepatocytes and its loss after chronic ethanol exposure. J Pharmacol Exp Ther 301: 908–914, 2002.[Abstract/Free Full Text]
  15. Liang Q, Carlson EC, Borgerding AJ, and Epstein PN. A transgenic model of acetaldehyde overproduction accelerates alcohol cardiomyopathy. J Pharmacol Exp Ther 291: 766–772, 1999.[Abstract/Free Full Text]
  16. McDonough KH. The role of alcohol in the oxidant antioxidant balance in heart. Front Biosci 4: 601–606, 1999.
  17. Morales JA, Ram JL, Song J, and Brown RA. Acetaldehyde inhibits current through voltage-dependent calcium channels. Toxicol Appl Pharmacol 143: 70–74, 1997.[CrossRef][ISI][Medline]
  18. Overbeek PA, Aguilar-Cordova E, Hanten G, Schaffner DL, Patel P, Lebovitz RM, and Lieberman MW. Coinjection strategy for visual identification of transgenic mice. Transgenic Res 1: 31–37, 1991.[CrossRef][Medline]
  19. Patel VB, Why HJ, Richardson PJ, and Preedy VR. The effects of alcohol on the heart. Adverse Drug React Toxicol Rev 16: 15–43, 1997.[ISI][Medline]
  20. Preedy VR, Patel VB, Reilly ME, Richardson PJ, Falkous G, and Mantle D. Oxidants, antioxidants and alcohol: implications for skeletal and cardiac muscle. Front Biosci 4: 58–66, 1999.
  21. Ren J and Brown RA. Influence of chronic alcohol ingestion on acetaldehyde-induced depression of rat cardiac contractile function. Alcohol Alcohol 35: 554–560, 2000.[Abstract/Free Full Text]
  22. Ren J, Davidoff AJ, and Brown RA. Acetaldehyde depresses shortening and intracellular Ca2+ transients in adult rat ventricular myocytes. Cell Mol Biol 43: 825–834, 1997.[ISI][Medline]
  23. Ren J, Wold LE, Natavio M, Ren BH, Hannigan JH, and Brown RA. Influence of prenatal alcohol exposure on myocardial contractile function in adult rat hearts: role of intracellular calcium and apoptosis. Alcohol Alcohol 37: 30–37, 2002.[Abstract/Free Full Text]
  24. Richardson PJ, Patel VB, and Preedy VR. Alcohol and the myocardium. Novartis Found Symp 216: 35–45, 1998.[Medline]
  25. Svegliati-Baroni G, Ridolfi F, Di Sario A, Saccomanno S, Bendia E, Benedetti A, and Greenwel P. Intracellular signaling pathways involved in acetaldehyde-induced collagen and fibronectin gene expression in human hepatic stellate cells. Hepatology 33: 1130–1140, 2001.[CrossRef][ISI][Medline]
  26. Taketo M, Schroeder AC, Mobraaten LE, Gunning KB, Hanten G, Fox RR, Roderick TH, Stewart CL, Lilly F, and Hansen CT. FVB/N: an inbred mouse strain preferable for transgenic analyses. Proc Natl Acad Sci USA 88: 2065–2069, 1991.[Abstract/Free Full Text]
  27. Thomas AP, Rozanski DJ, Renard DC, and Rubin E. Effects of ethanol on the contractile function of the heart: a review. Alcohol Clin Exp Res 18: 121–131, 1994.[CrossRef][ISI][Medline]
  28. Uchida K, Shiraishi M, Naito Y, Torii Y, Nakamura Y, and Osawa T. Activation of stress signaling pathways by the end product of lipid peroxidation. 4-hydroxy-2-nonenal is a potential inducer of intracellular peroxide production. J Biol Chem 274: 2234–2242, 1999.[Abstract/Free Full Text]
  29. Weng Y and Shukla SD. Ethanol alters angiotensin II stimulated mitogen activated protein kinase in hepatocytes: agonist selectivity and ethanol metabolic independence. Eur J Pharmacol 398: 323–331, 2000.[CrossRef][ISI][Medline]
  30. Zhang X, Klein AL, Alberle NS, Norby FL, Ren BH, Duan J, and Ren J. Cardiac-specific overexpression of catalase rescues ventricular myocytes from ethanol-induced cardiac contractile defect. J Mol Cell Cardiol 35: 645–652, 2003.[CrossRef][ISI][Medline]
  31. Zhang X, Li SY, Brown RA, and Ren J. Ethanol and acetaldehyde in alcoholic cardiomyopathy: from bad to ugly en route to oxidative stress. Alcohol 32: 175–186, 2004.[CrossRef][ISI][Medline]



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T. Oba, Y. Maeno, M. Nagao, N. Sakuma, and T. Murayama
Cellular redox state protects acetaldehyde-induced alteration in cardiomyocyte function by modifying Ca2+ release from sarcoplasmic reticulum
Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H121 - H133.
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