Department of Pharmacology, Physiology, and Therapeutics, University of North Dakota School of Medicine, Grand Forks, ND 58203,
1 Departments of Physiology,
2 Obstetrics & Gynecology, Wayne State University School of Medicine, Detroit, MI 48201 and
3 Department of Biology, Morgan State University, Baltimore, MD 21251, USA
Received 23 April 2001; in revised form 6 July 2001; accepted 8 August 2001
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ABSTRACT |
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INTRODUCTION |
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We utilized SpragueDawley rats fed an ethanol liquid diet during embryogenesis as a model for FAS (Hannigan and Abel, 1996). One of the more consistent findings in both the ethanol-exposed rodent fetus and neonate was decreased body weight combined with parallel decreases in heart, liver and kidney growth (Hannigan, 1996
; Stratton et al., 1996
). The aim of the present study was to examine the teratogenic effects of ethanol on cardiac contractile function in offspring exposed to ethanol in utero. The isolated papillary muscle has been employed extensively over the past three decades in the evaluation of myocardial contraction, transmembrane electrical events, contractile protein enzyme activities, and morphological parameters. The papillary muscle is particularly ideal for the assessment of myocardial contractility due to the fact that it is an extension of the endomyocardium with all the fibres lined up in parallel with the long axis of the muscle. Its small size also allows better diffusion of nutrients and oxygen to the innermost region, compared to the whole heart, thus making it available for a longer period of time. The myocyte versus non-myocyte composition has also been found to be similar between papillary muscle and ventricular wall, and the muscle mechanical response to inotropic and chronic agents is representative of the whole heart (Capasso, 1997
). However, due to the presence of heterogeneous cell types and nerve terminals, the results obtained using papillary muscles may not accurately represent functional changes at the single myocyte level (Ren and Brown, 2000
). Mechanical function may be affected by non-myocyte factors, such as the coronary vasculature and/ or interstitial connective tissue. For example, alterations in contractile performance under ethanol exposure may be simply due to enhanced interstitial fibrosis, but not reduced function of individual myocytes. Therefore, both left-ventricular papillary muscles and ventricular myocytes were isolated from 10-week-old offspring exposed to ethanol in utero and age-matched, non-exposed controls, and used for cardiac contractile function assessment. As ethanol is capable of augmenting programmed cell death (apoptosis) in cardiomyocytes, contributing to the development of several types of cardiomyopathies (Chen et al., 2000
), the activation of apoptotic signalling was also evaluated in isolated ventricular myocytes.
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MATERIALS AND METHODS |
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Ventricular papillary muscle isolation and measurement of force-generating capacity
The rats were anaesthetized with a ketamine/xylazine solution (3:1, 1.32 mg/kg intraperitoneally) and the hearts were rapidly excised and immersed in oxygenated Tyrode's solution (mM: KCl 5.4, NaCl 136.9, NaHCO3 11.9, MgCl2 0.50, CaCl2 2.70, NaH2PO4 0.45 and glucose 5.6, pH 7.4) at 37°C. Left-ventricular papillary muscles were dissected and mounted vertically in a temperature-controlled bath superfused with oxygenated Tyrode's solution at 30°C. Preparations were allowed to equilibrate for 60 min while being electrically stimulated by a Grass stimulator (S-88) at 0.5 Hz, to establish baseline force (isometric tension). Lengthforce curves were constructed for each preparation and the peak tension development (PTD), which indicates the myocardial force-generating capacity, 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). The following parameters were measured: PTD, time-to-PTD (TPT); time-to-90% relaxation (RT90) and the maximum velocities of contraction and relaxation (±VT) (Brown et al., 1998
). The papillary muscle is an extension of the endomyocardium and exhibits inotropic and chronic responses similar to the whole heart (Capasso, 1997
).
Isolation of ventricular myocytes
Single ventricular myocytes were isolated as described (Ren and Brown, 2000). Briefly, hearts were rapidly removed and perfused (at 37°C) with oxygenated KrebsHenseleit bicarbonate (KHB) buffer, pH 7.4. Hearts were subsequently perfused with a nominally Ca2+-free KHB buffer for 23 min followed by a 20 min perfusion with Ca2+-free KHB containing 223 U/ml of collagenase (Worthington Biochemical Corp., Freehold, NJ, USA) and 0.1 mg/ml hyaluronidase (Sigma Chemical, St Louis, MO, USA). After perfusion, the left ventricle was removed, minced and further digested with trypsin (Sigma) before being filtered through a nylon mesh (300 µm) and collected by centrifugation. Cells were initially washed with Ca2+-free KHB buffer to remove remnant enzyme and extracellular Ca2+ was added incrementally back to 1.25 mM.
Myocyte shortening and re-lengthening
Mechanical properties of ventricular myocytes were assessed by a video-based edge-detection system. Coverslips with cells attached were placed in a chamber mounted on the stage of an inverted microscope and superfused (at 30°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 at a frequency of 0.5 Hz. Cell shortening and re-lengthening were assessed using the following indices: peak shortening (PS), time-to-90% peak shortening (TPS) and time-to-90% re-lengthening (TR90), maximal velocities of shortening (+dL/dt) and re-lengthening (dL/dt) (Ren and Brown, 2000). The use of isolated ventricular myocytes should allow us to differentiate the contractile response contributed by the myocyte component from that of other heterogeneous cell types, including nerve terminals.
Intracellular Ca2+fluorescence measurement
Isolated ventricular myocytes were loaded with fura-2/AM (0.5 µM) for 10 min and fluorescence measurements were recorded with a dual-excitation fluorescence photomultiplier system (Ionoptix Corp., Milton, MA, USA) as described (Ren and Brown, 2000). Myocytes were plated on glass coverslips on an Olympus IX-70 inverted microscope and imaged through a 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 480 and 520 nm after first illuminating 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 nm 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.
Caspase-3 activation assay
Capase-3 is an enzyme activated during induction of apoptosis. Isolated ventricular myocytes were plated on 100-mm Petri dishes. Caspase-3 activity was determined using the colorimetric kit purchased from R & D Systems (Minneapolis, MN, USA). Myocytes were harvested and washed once with phosphate-buffered saline. After the cells were lysed, reaction buffer was added to the myocytes followed by the additional 5 µl of Caspase-3 colorimetric substrate (DEVD-pNA) and incubated in a 96-well plate for 4 h at 37°C in a CO2 incubator. The plate was then read with a microplate reader at 405 nm.
Data analysis
Data are reported as means ± SEM. Statistical significance (P < 0.05) was estimated by analysis of variance (ANOVA) or t-test, where appropriate. When ANOVA showed overall significance, a Dunnett's post hoc analysis was incorporated.
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RESULTS |
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Effects of ethanol on PTD in papillary muscle from the control and prenatal ethanol groups
To determine the influence of experience with ethanol in utero on postnatal ethanol-induced cardiac contractile response, an ethanol challenge ranging from 80 to 640 mg/dl was administered acutely to the myocardium. A concentration-dependent negative inotropic effect was observed in both the ethanol and control groups, when PTD was expressed in absolute value or normalized to muscle weight or to respective baseline values (Fig. 4). The maximal inhibition achieved at 640 mg/dl was comparable in the control (52%) and ethanol (53%) groups. The concentration where ethanol caused 50% of the maximal effect (EC50) was also identical in both groups (
267 mg/dl). The negative inotropic effect of ethanol was reversible upon wash-out (started 10 min after 640 mg/dl ethanol application) in both groups. The discrepancy in the pattern of ethanol response between Fig. 4A/B and Fig.4C
may be due to the different baseline PTD (without ethanol). The identical percentage inhibition in PTD by ethanol between the control and prenatal ethanol exposure groups suggests that postnatal ethanol-induced cardiac depression was unlikely to be affected by experience with ethanol in utero.
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DISCUSSION |
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Altered cardiac contraction is directly associated with chronic ethanol exposure (Thomas et al., 1994). Several laboratories have shown depressed cardiac contraction following postnatal ethanol exposure (Capasso et al., 1991
; Figueredo et al., 1998
; Ren and Brown, 2000
). It has been speculated that impaired intracellular Ca2+ handling, such as decreased SR Ca2+ uptake and binding, may be mainly responsible for altered cardiac contraction following postnatal chronic ethanol exposure (Segel et al., 1981
; Guarnieri and Lakatta, 1990
; Danziger et al., 1991
; Thomas et al., 1994
; Brown et al., 1998
). It is likely that the decreased cytosolic Ca2+ concentration may be a result of an inhibitory effect of ethanol on Ca2+ regulatory proteins, such as Ca2+ pumps and channels. Recent evidence also suggests that postnatal ethanol-associated changes in myocardial contractility do not result from altered Ca2+ handling, but, rather, from changes of myofilaments that do not involve myosin heavy chain isoform shifts (Figueredo et al., 1998
). Therefore, chronic ethanol ingestion-induced contractile changes are not due to altered Ca2+ handling by the Ca2+ regulatory proteins or organelles, such as SR. It is worth noting that stress may be needed to reveal impaired contractile reserve in hearts from chronic ethanol consumption (Segel et al., 1981
). Results from the current study suggest that the impact of prenatal ethanol exposure on cardiac contractile machineries (e.g. SR function and myofilament Ca2+ responsiveness) might be substantially different from that of postnatal ethanol exposure.
The use of alcohol during pregnancy, even in the course of the so-called social drinking, usual drinking and binge drinking can induce heart defects in the offspring. Cardiac anomalies in FAS are mainly composed of ventricular and atrial septal defects, and histological or structural changes, such as derangement of the myofibrils (Loser et al., 1992). Ethanol induces teratogenic alterations in the development of cardiomyocytes during embryogenesis, contributing to cardiac dysmorphism and immature cardiac morphology or function. The major morphological anomalies are multinucleation and alteration in the ultrastructural organization of myofilaments (Adickes et al., 1990
, 1993
). These observations were confirmed in both human infants and rat pups exposed to ethanol in utero, and therefore may help us to understand the teratogenic manifestations of ethanol in embryogenesis and organogenesis. We observed reduced heart weight, although not the papillary muscle weight/cross-sectional area or myocyte length, in offspring exposed to ethanol in utero. This is consistent with a teratogenic effect of ethanol and may contribute to the altered myocardial contractility in the ethanol group. Another potential explanation of depressed cardiac contractility may be that ethanol reduces specific cellular contents of actin and myosin in cardiac myocytes (Ni et al., 1992
). These decreases in cytoskeletal and contractile proteins may contribute directly to the morphological abnormalities and depressed ventricular function. The elevated intracellular Ca2+ levels in myocytes from the ethanol group indicate potential Ca2+ overloading or up-regulation of sarcolemmal Ca2+ channels and the propensity for developing cardiomyopathy. Further study is warranted to investigate the mechanism of the increased intracellular Ca2+ levels in myocytes following prenatal ethanol exposure. Consistent with an earlier experimental finding (De Vito et al., 2000
), we found that prenatal ethanol exposure promotes cardiac myocyte apoptosis, which could contribute to the depressed cardiac contractile function, i.e. ventricular pumping function.
Our current observations revealed a disparate contractile response to prenatal ethanol exposure at the papillary muscle and ventricular myocyte levels. Although the mechanism responsible for such difference is unknown, the non-myocyte component may play a role. Ethanol is known to increase myocardial stiffness and fibrosis (Rajiyah et al., 1996), leading to depressed myocardial contractility. However, other factors, such as different methods of recording (isometric for papillary muscle, but isotonic for myocytes) should not be excluded. In addition to the above-mentioned potential factors, direct suppression of anaesthetics on cardiac contraction may also play a role, although, in our experience, using ketamine and xylazine for tissue harvesting does not affect any of the later measures of cardiac function. Finally, it is worth pointing out that altered postnatal cardiac function under FAS may be most marked in the early postnatal period. Recovery of some aspects of cardiac function has been noted with postnatal maturation (Staley and Tobin, 1992
).
FAS occurs at the adverse end of the continuum of alcohol effects, the progression of its symptoms can begin at any point in the course of prenatal development. In this study, prenatal ethanol exposure was achieved by feeding pregnant rats with an ethanol liquid diet during GD8 and GD20. Earlier studies exposed female rats to ethanol for at least 30 days before mating and throughout the entire course of pregnancy (Chernoff, 1977). FAS is commonly identified in children born to women chronically alcoholic (Stratton et al., 1996
). However, the relationship of chronic alcoholic to the incidence of FAS has been vaguely defined. It is suggested that high ethanol levels, even transient, at a critical embryonic development stage, may be more detrimental than the entire duration of maternal intoxication (Hannigan and Abel, 1996
). The embryonic stage is the early period between weeks 2 and 8 after conception marked by cell development. These cells later differentiate to produce tissues and organs. Maternal ethanol exposure during the embryonic period, which often results in structural irregularities, should provide sufficient information regarding the teratogen, ethanol, on cardiac contractile function.
The mechanism of FAS is still not clear. How a single compound like ethanol can cause a diverse range of cellular/ biochemical events is puzzling. It makes intuitive sense that ethanol or its metabolites may interrupt a few essential cellular processes, such as membrane integrity and energy production, that are key to cellular order. Such interruption might then trigger a cascade of secondary events manifested in FAS (Abel and Hannigan, 1995). Ethanol-induced oxidative stress and the main metabolite of ethanol, acetaldehyde, are believed to contribute to ethanol-related cardiac contractile dysfunction and cardiovascular defects in FAS (Ren et al., 1997
; Henderson et al., 1999
). The fetus/embryo is exquisitely sensitive to oxidative stress, leading to a spectrum of responses ranging from structural malformations to embryonic death. Ethanol or acetaldehyde may lead to lipid peroxidation and the intermediate radicals formed in the peroxidation process are known to adversely affect a variety of cellular functions in cell growth, including cytoskeletal disruption (Smith et al., 1992
; VanWinkle et al., 1994
), mitochondrial dysfunction (Kristal et al., 1994
), and alteration of membrane protein receptors and subsequent signal transduction (Henderson et al., 1991
; Hoek et al., 1992
).
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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