Alcoholic cardiomyopathy is characterized by impaired ventricular function although its toxic mechanism is unclear. This study examined the impact of cardiac overexpression of alcohol dehydrogenase (ADH), which oxidizes ethanol into acetaldehyde (ACA), on ethanol-induced cardiac contractile defect. Mechanical and intracellular Ca2+properties were evaluated in ventricular myocytes from ADH transgenic and wild-type (FVB) mice. ACA production was assessed by gas chromatography. ADH myocytes exhibited similar mechanical properties but a higher efficiency to convert ACA compared with FVB myocytes. Acute exposure to ethanol depressed cell shortening and intracellular Ca2+ in the FVB group with maximal inhibitions of 23.3% and 23.4%, respectively. Strikingly, the ethanol-induced depression on cell shortening and intracellular Ca2+ was significantly augmented in the ADH group, with maximal inhibitions of 43.7% and 40.6%, respectively. Pretreatment with the ADH inhibitor 4-methylpyrazole (4-MP) or the aldehyde dehydrogenase inhibitor cyanamide prevented or augmented the ethanol-induced inhibition, respectively, in the ADH but not the FVB group. The ADH transgene also substantiated the ethanol-induced inhibition of maximal velocity of shortening/relengthening and unmasked an ethanol-induced prolongation of the duration of shortening/relengthening, which was abolished by 4-MP. These data suggest that elevated cardiac ACA exposure due to enhanced ADH expression may play an important role in the development of alcoholic cardiomyopathy.
- intracellular calcium ion transient
chronic alcoholism causes heart muscle damage leading to the onset of alcoholic cardiomyopathy, which accounts for ∼33% of dilated cardiomyopathy (6). The incidence of cardiomyopathy is increased up to 50% in patients with chronic alcoholism, and a high proportion of these patients die from cardiac disease. Alcoholic cardiomyopathy is manifested by cardiomegaly, disruptions of the myofibrillary architecture, reduced myocardial contractility, decreased ejection volumes, and enhanced risk of stroke and hypertension (21, 27). Clinical studies indicate that alcoholic cardiomyopathy can be reversed by abstinence from alcohol before its progression beyond a certain, as yet poorly defined, point of severity. Diagnosis of alcoholic cardiomyopathy is often made on the basis of deteriorating cardiac function, increased heart size, and a history of alcohol abuse (31). Although several hypotheses have been postulated for the development of alcoholic cardiomyopathy, including direct and indirect cardiotoxicity of alcohol (22) and accumulation of fatty acid ethyl esters (13), no hypothesis has received convincing and consistent clinical and experimental support to be fully validated.
Acetaldehyde (ACA), the first oxidized metabolic product of ethanol, has been considered a favorable candidate toxin for the pathogenesis of alcoholic cardiomyopathy because it concentrates in the heart (5) and is far more reactive than ethanol. Elevated levels of circulating ACA and antibodies to cardiac ACA-protein adducts have been found in alcoholics with alcoholic cardiomyopathy (10, 12,18). Our laboratories have shown (1, 24, 25, 26) that ACA may directly impair cardiac excitation-contraction (E-C) coupling and inhibit sarco(endo)plasmic reticulum (SR) Ca2+release function. However, the advancement of the ACA toxicity theory has been stalled over the past years largely because of the lack of suitable methods for experimentally altering blood ACA and the technical constraints of ACA manipulation such as low boiling point and high chemical reactivity. Normally, ACA metabolizes quite rapidly (∼5 times faster than ethanol) to keep a low blood level (<0.5 μM) after a 0.5 g/kg ethanol intake. Higher ACA levels (∼2 μM) may be seen in alcoholics given the same dose of ethanol, probably because of hepatic inhibition of aldehyde dehydrogenase (ALDH) activity (14). Furthermore, blood ACA levels may reach 30–500 μM after ethanol intoxication in certain populations such as Asians and African Americans who carry a mutation of the low-K m ALDH (32, 34), making them a theoretically ideal cohort for testing the role of ACA. However, the intolerance among these populations to ethanol ingestion makes it practically impossible to carry out this analysis. Earlier experiments using metabolic inhibitors to alter ACA level (e.g., the ALDH inhibitor cyanamide) revealed the inhibitors to be nonspecific, ineffective, toxic, and difficult to maintain chronically in experimental animals (11, 23, 33). To overcome this lack of a suitable model or method to chronically alter the ACA level in vivo for a definitive test of ACA, our group recently developed a transgenic mouse model to overexpress alcohol dehydrogenase (ADH) specifically in the heart. Our results indicated that elevated cardiac ACA exposure due to ADH overexpression increased the severity of alcoholic cardiomyopathy functionally and morphologically at the whole heart level (15). However, whether these elevated ACA levels directly facilitate the development of alcoholic cardiomyopathy or indirectly impair cardiac function through interstitial fibrosis is unknown.
The aim of the present study, therefore, was to investigate the direct influence of elevated cardiac ACA exposure on cell shortening and intracellular Ca2+ homeostasis in isolated ventricular myocytes from ADH transgenic mice and age-matched wild-type controls (FVB mice). Direct measurement of myocyte mechanics and intracellular Ca2+ transient on a beat-to-beat basis should provide fundamental information on cardiac E-C coupling and has been proven to be a valid physiological technique to assess the heart function under pathophysiological conditions such as chronic alcoholism.
MATERIALS AND METHODS
Development of ADH transgenic mice.
All animal procedures were approved by the University of North Dakota School of Medicine Institutional Animal Care and Use Committee. Briefly, the MyADH transgene was constructed to produce local overexpression of ADH in the heart. This gene includes the mouse α-myosin heavy chain (MHC) promoter to drive cardiac-specific expression. The cDNA for murine class I ADH (4) was inserted behind the promoter. This cDNA was chosen because class I ADH is the most efficient in the oxidation of ethanol. FVB mice obtained from the University of North Dakota Biomedical Research Center were used to produce transgenic lines containing the MyADH transgene (28). Standard procedures were used for producing transgenic animals. A second transgene containing a cDNA for the enzyme tyrosinase was coinjected with MyADH. The enzyme tyrosinase produces coat color pigmentation in albino mice (16) and was used to conveniently identify transgenic animals (15).
Assessment of ACA production after acute ethanol challenge.
Isolated ventricular myocytes (20,000 cells/ml) from either FVB or ADH groups were exposed to 80, 120, 240, and 640 mg/dl ethanol for 15 min in a sealed vial before the reaction was terminated by 4-methylpyrazole (4-MP; 1 mM). Vials were then stored at −80°C until being analyzed. 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. ACA 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 ACA from ethanol and other compounds was completed in 1 min. Quantitation was achieved by calibrating the gas chromatographic peak areas against those from headspace samples of known ACA standards, over a similar concentration range as the cell samples, in the same buffer (15).
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 223 U/ml collagenase D (Boehringer Mannheim, Indianapolis, IN) for 20 min. After perfusion, ventricles were removed and minced before being filtered. Extracellular Ca2+ was slowly added back to reach 1.25 mM. Myocytes with obvious sarcolemmal blebs or spontaneous contractions were not used (24).
24). 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, and 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 digital signal before being analyzed with pCLAMP software. The myocyte being studied was scanned rapidly with a camera at 120 Hz to ensure recording with good fidelity. Cell shortening and relengthening were assessed with the following indexes: peak shortening (PS), time to PS (TPS) and time to 90% relengthening (TR90), and maximal velocities of shortening and relengthening (±dL/dt). Steady-state contraction of the myocyte was achieved before application of ethanol.
Intracellular 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 (24). Myocytes were imaged through an Olympus IX-70 fluor ×40 oil objectives. 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 and then at 380 nm for the duration of the recording protocol (333-Hz sampling rate). The 360-nm excitation scan was repeated at the end of the protocol, and qualitative changes in intracellular Ca2+level were inferred from the ratio of the fura fluorescence intensity (FFI) at the two wavelengths. Fluorescence decay time (τ) was also measured as an indication of the intracellular Ca2+clearing rate.
For each experimental series, data are reported as means ± SE. Differences in means between groups were assessed with the Student'st-test, whereas within-group comparisons between mean values were calculated by repeated-measures ANOVA. When an overall significance was determined, a Dunnett's post hoc analysis was incorporated. A P value <0.05 was considered significant.
General features of FVB wild-type and ADH transgenic mice and left ventricular myocytes.
As shown in Table 1, transgenic ADH overexpression did not elicit any notable effect on body, heart, liver, and kidney weights compared with those of the FVB wild-type littermates. The ability of freshly isolated ventricular myocytes to oxidize ethanol into ACA was measured by gas chromatography and flame ionization detection. The results are shown in Fig.1. After a 15-min acute challenge of ethanol, the ventricular myocytes from the ADH group generated a significantly larger amount of ACA compared with the FVB group, validating the transgenic elevation of cardiac ADH activity (by ∼40 times) (15). The baseline mechanical and intracellular Ca2+ properties of left ventricular myocytes were also similar in left ventricular myocytes isolated from both FVB and ADH animals (Table 2), suggesting that the ADH transgene itself was not innately harmful to the ventricular mechanical function.
Acute effects of ethanol on cell shortening.
To determine whether local overexpression of ADH affects the ethanol-induced depression on cardiac myocyte shortening, concentration-dependent response was determined for ethanol (0–640 mg/dl). The concentration range of ethanol was chosen largely based on the national legal driving limit (100 mg/dl), the fact that 90 mg/dl is a moderate ethanol intake dose, the LD50 of ethanol in rodents (∼1,000 mg/dl), and our previous experience with the acute cardiac depressant effect of ethanol (26). Representative traces depicting the effect of ethanol on cell shortening in myocytes from the FVB and ADH groups are shown in Fig.2 A. Acute exposure to ethanol (240 mg/dl) caused an appreciable decrease in the extent of cell shortening (PS) in both FVB (∼19%) and ADH (∼52%) mice. The concentration-dependent response further indicated that ethanol induced a stepwise depression of PS in both cell types. Interestingly, ventricular myocytes from the ADH group were significantly more sensitive to ethanol-induced inhibition compared with those from the FVB wild-type group (Fig. 2 B). The ethanol-induced attenuation of PS in FVB myocytes was associated with depressed maximal velocities of shortening/relengthening (±dL/dt, threshold between 80 and 120 mg/dl) and unaltered duration (TPS and TR90). Consistent with its augmented response on PS, the ADH transgene exacerbated the ethanol-induced depression of ±dL/dt with a threshold <80 mg/dl. In addition, ADH unmasked a prolongation in the duration of both shortening (TPS) and relengthening (TR90) (Table3). These results convincingly demonstrated an augmented cardiac depression in response to acute ethanol exposure.
Effects of ethanol on cell shortening in the presence of ADH or ALDH inhibitor.
To evaluate whether the augmented ethanol-induced depression of PS in ADH myocytes was due to elevated cardiac exposure of ACA, the cell shortening response to acute ethanol administration was reexamined in the presence of either the ADH inhibitor 4-MP (25 μM) or the ALDH inhibitor cyanamide (25 μM). 4-MP blocks the conversion from ethanol to ACA, whereas cyanamide prevents ACA breakdown, leading to accumulation of ACA. 4-MP or cyanamide alone had no effect on PS in the FVB myocytes (4-MP −2.82 ± 7.85%, cyanamide −1.13 ± 4.34%; n = 8) or the ADH myocytes (4-MP −4.51 ± 8.31%, cyanamide −10.12 ± 11.02%; n = 8). Figure 2, B and C, shows that 4-MP or cyanamide did not significantly alter the ethanol-induced depression of PS in myocytes from FVB mouse hearts, indicating the cardiodepressive property of ethanol by itself. However, 4-MP and cyanamide attenuated or exacerbated, respectively, the ethanol-induced cardiac depression in myocytes from ADH mouse hearts. Consistent with their response on PS, 4-MP and cyanamide attenuated or exacerbated, respectively, the ethanol-induced responses on duration or maximal velocities of shortening and relengthening (data not shown). These results confirmed that enhanced cardiac depression in ADH myocytes was likely due to the elevated ACA levels triggered by the ADH transgene.
Acute effects of ethanol on intracellular Ca2+ transients: influence of 4-MP and cyanamide.
To determine whether the disparate ethanol-induced depression of PS between the FVB and the ADH myocytes was due to changes in intracellular Ca2+ concentration ([Ca2+]i), we used the fluorescent dye fura 2 to estimate [Ca2+]i in the myocytes from both groups. Representative tracings depicting ethanol-induced inhibition of intracellular Ca2+ transient in both FVB and ADH groups are shown in Fig. 3 A. Consistent with its effect on cell shortening, ethanol exerted an enhanced concentration-dependent depression in intracellular Ca2+transients recorded from the ADH group compared with the FVB group (Fig. 3 B), suggesting that the disparate response in PS was likely due to a difference in the intracellular Ca2+transients between the FVB and ADH myocytes. Consistent with the observation in cell shortening, the ADH inhibitor 4-MP abolished the ADH transgene-induced augmentation of ethanol-induced depression in intracellular Ca2+ transients. On the other hand, the ALDH inhibitor cyanamide exacerbated depression induced by higher concentrations of ethanol in intracellular Ca2+ transients in myocytes from ADH but not FVB mouse hearts (Fig.3 C). Collectively, these data confirmed an enhanced contribution of ACA to depression of intracellular Ca2+handling in myocytes from the ADH mouse hearts. Finally, neither resting FFI nor τ was affected by ethanol (data not shown).
The basis of this study stems from the hypothesis that ACA is responsible, at least in part, for ethanol-induced cardiac contractile depression and may be considered as an ultimate candidate toxin for alcoholic cardiomyopathy after chronic ethanol ingestion. Our major finding revealed that overexpression of ADH specifically in the hearts is likely to lead to enhanced cardiac ACA exposure and augmented ethanol-induced depression in cardiac contractile function and intracellular Ca2+ recruitment. The augmentation of ethanol-induced cardiac depression was prevented by the ADH inhibitor 4-MP, or even further substantiated by the ALDH inhibitor cyanamide, although only in ADH (not FVB) myocytes. Consistent with its effect on PS, ADH transgene sensitized the mouse myocytes to ethanol-induced effects on most other mechanical properties including ±dL/dt, TPS, and TR90. These results suggested that, although ACA may not be a significant factor in acute ethanol-induced cardiac depression under normal conditions, elevated ACA levels surely can deteriorate cardiac contractile function once it exceeds certain points.
The hallmarks of alcoholic cardiomyopathy are altered cardiac contractile function and impaired intracellular Ca2+mobilization such as decreased SR Ca2+ function, which may underlie the altered cardiac mechanics (2, 3, 8, 24, 29,30). It is postulated that inhibition by ethanol of Ca2+ regulatory proteins such as Ca2+ pumps and channels may play a role in the decreased cytosolic Ca2+concentration. However, evidence indicated that the chronic alcoholism-associated changes in myocardial contractility may not result from altered Ca2+ handling but rather from changes of the myofilament Ca2+ sensitivity (7). Whether ACA itself affects these proteins or the myofilament Ca2+ sensitivity is not fully clear and deserves further study.
ACA has been shown to exert biphasic chronotropic and inotropic effects in hearts. The positive effect was observed at low doses because of β-adrenergic activation. The negative inotropic effects are believed to be myogenic, independent of cholinergic or purinergic mechanisms (1). Data from the current study favor the cardiac depressive action induced by elevated ACA levels, although it is not known at this time whether the trace ACA amount produced in FVB myocytes plays a role in the less depressed cardiac contraction compared with that of ADH myocytes. The potential mechanisms involved in the augmented cardiac depression in the ADH myocytes, likely attributed to ACA, are largely unknown. It may be postulated that ACA impairs the cardiac E-C coupling through one or more of the following mechanisms. 1) ACA is known to disrupt cellular functions by producing ACA-protein adducts even at very low concentration. Patients with alcoholic cardiomyopathy possess circulating antibodies to cardiac ACA-protein adducts (10). Protein adducts are polypeptides bound with other molecules (usually reactive biochemicals), which either render the protein inoperative or immunogenic. The formation of protein adducts may contribute to the pathogenesis of cardiomyopathy by provoking an immunologically based reaction, by inactivating functional proteins (e.g., those involved in cardiac E-C coupling), or by causing the adducted proteins to be selectively targeted for more rapid degradation. 2). Several alcohol-induced detrimental actions in the heart have been attributed to the alcohol-induced changes in the oxidant-antioxidant balance (16). Oxidant balance is crucial in maintaining normal cardiac contractile performance. However, the antioxidant reserves become inadequate under pathological situations such as alcoholism. ACA was shown to directly enhance free radical generation by its oxidation via aldehyde oxidase and/or xanthine oxidase with concurrent accumulation of superoxide anions (9). It has been suggested that the metabolism of ACA through xanthine oxidase plays an important role in the production of oxidative stress in the heart and may be one of the mechanisms mediating cardiac pathology in alcoholism (19).3). Evidence has shown that ACA may directly attenuate intracellular Ca2+ mobilization and inhibit membrane voltage-dependent Ca2+ channels (1, 17, 25).
In summary, the present study provides convincing evidence that elevated cardiac ACA exposure substantiated acute ethanol-induced cardiac depression in isolated ventricular myocytes, supporting a role of ACA in the development of alcoholic cardiomyopathy. Future work with chronic alcohol consumption and the assessment of cardiac contractile protein function with this transgenic mouse line is warranted to better understand the precise nature of ACA in the development of alcoholic cardiomyopathy.
The authors are grateful to Kadon K. Hintz and the Center for Biomedical Research at University of North Dakota School of Medicine for skillful assistance.
First published December 13, 2001;10.1152/ajpheart.00780.2001
This research was supported in part by grants from the American Heart Association and the American Diabetes Association and the University of North Dakota New Faculty Scholar Award.
Address for reprint requests and other correspondence: J. Ren, Dept. of Pharmacology, Physiology, and Therapeutics, Univ. of North Dakota School of Medicine, 501 N. Columbia Rd., Grand Forks, ND 58203 (E-mail:).
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.
- Copyright © 2002 the American Physiological Society