Department of Pharmacology, Physiology, and Therapeutics, University of North Dakota School of Medicine, 501 N. Columbia Road, Grand Forks, ND 58203 and
1 Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA
Received 27 March 2000; in revised form 23 May 2000; accepted 29 May 2000
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ABSTRACT |
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INTRODUCTION |
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Acetaldehyde (ACA), the major metabolite of ethanol which is found highly concentrated in the heart (Espinet and Argiles, 1984), has been shown to exert biphasic chronotropic and inotropic cardiac effects (Stratton et al., 1981
; Brown and Carpentier, 1989
; Savage et al., 1995
). The positive component including increases in heart rate, cardiac output, coronary blood flow and left ventricular pressure, was often observed at low concentrations and may be associated with stimulation of the ß-adrenergic system. Although the negative chronotropic and inotropic effects on myocardial tissue have not been precisely determined, ACA has been shown to elicit myocardial depression independently of cholinergic or purinergic mechanisms (Savage et al., 1995
; Brown and Savage, 1996
). Our further study confirmed that ACA-induced cardiac depression is likely to be due to its direct inhibition of single ventricular myocyte contraction (Ren et al., 1997
; Brown et al., 1999
). These data suggest that ACA may play a role in the action of ethanol on cardiovascular dysfunction.
Recently, we reported that ethanol-induced myocardial depression is substantiated by chronic ethanol ingestion (Brown et al., 1998). To determine the action of ACA following chronic ethanol ingestion, the present study examined the impact of chronic ethanol ingestion on ACA-induced cardiac contractile depression using isolated papillary muscles and cardiac myocytes isolated from left ventricles from control animals and those chronically fed with ethanol.
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MATERIALS AND METHODS |
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Ventricular papillary muscle isolation and measurement of isometric tension
Once the animals were sedated, hearts were rapidly excised and immersed in oxygenated Tyrode's solution at 37°C. Left ventricular papillary muscles were dissected and mounted vertically in a temperature-controlled bath superfused with oxygenated Tyrode's solution (mM: KCl 5.4, NaCl 136.9, NaHCO3 11.9, MgCl2 0.50, CaC12 2.70, NaH2PO4 0.45 and glucose 5.6, pH 7.4) at 30°C. Preparations were allowed to equilibrate in Tyrode's solution for 90 min while electrically driven by a Grass stimulator (S-88) at 0.5 Hz, to establish baseline isometric tension development. Lengthtension curves were constructed for each preparation and the peak tension development (PTD) was recorded at ~90% of Lmax using a force transducer (Grass, FT 03). Signals were amplified, differentiated and displayed on a chart recorder (Grass-79). PTD was normalized to respective control values and presented as a percentage change to minimize inter-muscle variance. The following parameters were measured: PTD, time-to-PTD (TPT); time-to-90% relaxation (RT90) and the maximum velocities of tension developed and decline (±VT) (Brown et al., 1998).
Cell isolation procedures
Ventricular myocytes were enzymatically dispersed from the rat heart as described previously (Ren et al., 1998). Briefly, hearts were rapidly removed and perfused (at 37°C) with KrebsHenseleit bicarbonate (KHB) buffer containing (in mM): 118 NaCl, 4.7 KCl, 1.25 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 10 N-[2-hydro-ethyl]-piperazine-N'-[2-ethanesulphonic acid] (HEPES) and 11.1 glucose. Hearts were digested with Ca2+-free KHB containing 223 U/ml collagenase (Worthington Biochemical Corp., Freehold, NJ, USA) and 0.1 mg/ml hyaluronidase (Sigma Chemical, St Louis, MO, USA). After perfusion, ventricles were removed, minced and filtered through a nylon mesh (300 µm). Cells were initially washed with Ca2+-free KHB buffer to remove remnant enzyme and extracellular Ca2+ was added incrementally back to the cells, up to 1.25 mM. Cells were not used if they had any obvious sarcolemmal blebs or spontaneous contractions.
Cell shortening/relengthening
Mechanical properties of ventricular myocytes were assessed using a video-based edge-detection system (IonOptix, Milton, MA, USA) as described (Ren et al., 1998). In brief, cells were placed in a chamber mounted on the stage of an inverted microscope (Olympus X-70) and superfused (~2 ml/min at 37°C) with a buffer containing (in mM): 131 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, at pH 7.4. The cells were field-stimulated to contract at a frequency of 0.5 Hz. Changes in cell length during shortening and relengthening were captured and converted to digital signals before being analysed with pClamp software. 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 indices: peak shortening (PS), time-to-PS (TPS) and time-to-90% relengthening (TR90), maximal velocities of shortening and relengthening (± dL/dt). Steady state contraction of myocyte was achieved before application of ACA (Aldrich, Milwaukee, WI, USA).
Intracellular fluorescence measurement
Myocytes were loaded with fura-2/AM (0.5 µM) for 10 min at 30°C, and fluorescence measurements were recorded with a dual-excitation fluorescence photomultiplier system (Ionoptix) as previously described (Ren et al., 1998). Myocytes were imaged through an Olympus X-70 Fluor x40 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 480520 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 ([Ca2+]i) were inferred from the ratio of the fluorescence intensity at the two wavelengths.
Data analysis
For each experimental series, data are reported as mean ± SEM. Differences between group means were assessed using Student's t-test, whereas within-group comparisons between mean values were calculated by repeated measures analysis of variance (ANOVA). When an overall significance was determined, Dunnett's post-hoc analysis was incorporated. P < 0.05 was considered significant.
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RESULTS |
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DISCUSSION |
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Altered contractile function has been reported to be associated with chronic ethanol exposure (Thomas et al., 1994). However, the mechanisms responsible for the altered tension development and velocity of contraction and relaxation are not well known. Consistent with this study, several laboratories have shown depressed cardiac contraction following chronic ethanol exposure (Kino et al., 1981
; Tepper et al., 1986
; Capasso et al., 1991
; Figueredo et al., 1998
). However, conflicting results regarding the impact of chronic ethanol ingestion on velocity of contraction and relaxation have included increased (Tepper et al., 1986
), no change or decreased parameters (Kino et al., 1981
; Capasso et al., 1991
, 1992
). Such discrepancies may be related to multiple factors such as animal models, dosages and duration of exposure to ethanol.
It has been reported that impaired intracellular Ca2+ handling, such as decreased sarcoplasmic reticulum Ca2+ uptake and binding, may be mainly responsible for altered cardiac contraction following chronic ethanol exposure (Sarma et al., 1976; Kino et al., 1981
; Segel et al., 1981
; Guarnieri and Lakatta, 1990
; Danziger et al., 1991
; Thomas et al., 1994
). In the present study, despite comparable myocyte mechanical parameters with chronic ethanol exposure, diastolic Ca2+ level was found to be reduced, but associated with a normal intracellular Ca2+ decay rate in the ethanol-consuming group. Acute ethanol exposure has been reported to reduce the peak amplitude of cytosolic Ca2+ increase without affecting basal (diastolic) Ca2+ concentration (Thomas et al., 1994
). It is possible that inhibition by ethanol of certain Ca2+ regulatory proteins, such as Ca2+ pumps and channels, may play a role in the decreased cytosolic Ca2+ concentration. However, recent evidence has suggested that chronic ethanol-associated changes in myocardial contractility do not result from altered Ca2+ handling, but, rather, from changes at the level of the myofilament that do not involve myosin heavy chain isoform shifts (Figueredo et al., 1998
).
ACA is the major metabolic product of ethanol metabolism and has been shown to bind to proteins to form adducts. The role of ACA in cardiac function is still somewhat controversial. Long-term ethanol consumption leads to accumulation of ACA and increased reaction with protein-bound Amadori products (Thiele et al., 1996). The formation of ACAprotein adducts effectively removes Amadori products, the precursors to advanced glycation endproduct which leads to the development of diabetes- and age-related cardiovascular disease. This ACAprotein adduct has been considered the possible mechanism for the so-called French paradox, where the cardioprotection is conferred by moderate ethanol ingestion (Al-Abed et al., 1999
). In contrast, although one report indicates that ACA has no influence on the function of the cardiovascular system after the ingestion of ethanol (Pawlak et al., 1993
), accumulating evidence has also shown that ACA depresses myocardial contraction, attenuates intracellular Ca2+ mobilization and inhibits membrane voltage-dependent Ca2+ channels (Savage et al., 1995
; Morales et al., 1997
; Ren et al., 1997
; Brown et al., 1999
). Transgenic over-expression of alcohol dehydrogenase to elevate cardiac exposure to ACA displayed ultrastructural as well as functional damage in the myocardium consistent with chronic ethanol ingestion-induced cardiomyopathy (Liang et al., 1999
). The ACA-induced myocardial depression may play a role in ethanol-induced myocardial depression in both human and experimental animals.
In this study, we observed a disparate response to ACA at the papillary muscle, but not the myocyte, level. Although the mechanism responsible for such difference is unknown, the non-myocyte component is speculated to play a role. Chronic ethanol ingestion is associated with a reduction in the amount of contractile proteins and an adaptive increase in fibrillary protein synthesis, which may be responsible for the increased myocardial stiffness and fibrosis (Rajiyah et al., 1996).
Despite prolonged exposure to heavy ethanol, cardiac contractile changes are relatively modest, and the majority of alcoholics demonstrate subclinical contractile abnormalities. Future work will be focused on whether blood ACA concentrations obtained from metabolism of ethanol at commonly acceptable doses are sufficient to impose overt cardiac effect, and if so, the mechanism of action to altered contractile function.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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