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INFLUENCE OF PRENATAL ALCOHOL EXPOSURE ON MYOCARDIAL CONTRACTILE FUNCTION IN ADULT RAT HEARTS: ROLE OF INTRACELLULAR CALCIUM AND APOPTOSIS

INFLUENCE OF PRENATAL ALCOHOL EXPOSURE ON MYOCARDIAL CONTRACTILE FUNCTION IN ADULT RAT HEARTS:... Abstract — To assess the teratogenic action of ethanol on cardiac contractile function in offspring exposed to ethanol in utero, pregnant Sprague–Dawley rats were fed with ethanol during gestation. Left-ventricular papillary muscles and myocytes were isolated from the offspring of the ethanol-ingesting and control pregnant rats. Mechanical parameters measured were peak tension development (PTD, indicating the myocardial force-generating capacity), peak cell shortening (PS), time-to-PTD/PS (TPT/TPS), time-to-90% relaxation/re-lengthening (RT90/TR90), and maximal velocities of contraction/shortening and relaxation/re-lengthening (±VT and ±dL/dt). Intracellular Ca2+ levels and apoptosis were evaluated with fura-2 fluorescent dye and Caspase-3 activation assay, respectively. Offspring of the ethanol group displayed decreased heart weight associated with comparable body, liver and kidney weight, and papillary muscle weight/size, compared to the control group. However, prenatal ethanol exposure depressed myocardial PTD and ±VT. The myocardium from the ethanol group also exhibited slightly but significantly shortened TPT, accompanied with normal RT90. Muscles from both groups exhibited comparable responses to post-rest potentiation, increasing extracellular Ca2+ concentration, noradrenaline and acute ethanol challenge. Ventricular myocytes from both the control and ethanol groups possessed similar PS, TPS, TR90 and ±dL/dt. Both resting and peak intracellular Ca2+ levels were elevated in myocytes from the ethanol group. Additionally, acute ethanol application depressed caffeine-induced intracellular Ca2+ rise in myocytes from both groups. Myocytes from the ethanol group displayed an enhanced Caspase-3 activation, compared to control myocytes. These results suggest that prenatal ethanol exposure alters myocardial contractile function and may contribute to the development of postnatal cardiac dysfunction through, in part, increased intracellular Ca2+ loading and apoptosis. INTRODUCTION A major cause of birth defects is maternal consumption of alcohol. Prenatal alcohol exposure exerts a triad of defining teratogenic effects on the developing fetus, mainly manifest by growth retardation, abnormal facial features and central nervous system (CNS) damage, collectively known as the fetal alcohol syndrome (FAS) (Stratton et al., 1996). FAS is characterized by dysgenesis of many organ systems, such as the CNS, gastrointestinal tract, and the cardiovascular system. Defects in the cardiovascular system appear in up to 50% of children diagnosed with FAS. The detrimental effects of a teratogen on an organ are believed to be greatest during the 2nd to 8th weeks of gestation in human, known as the embryonic period, when major organ systems are growing and forming rapidly. Studies have shown that cardiac myocytes exposed to ethanol during embryogenesis did not mature morphologically or functionally (Adickes et al., 1990). Depressed growth of cardiac myocytes, reduction and delay in the development of myosin and actin, alteration in Ca2+ transport, mitochondrial function, sarcoplasmic reticulum (SR) Ca2+ uptake/binding as well as intracellular Ca2+ homeostasis have been reported in several animal models of FAS (Ni et al., 1992; Staley and Tobin, 1992; Fuseler, 1993; Syslak et al., 1994; Altura et al., 1996). We utilized Sprague–Dawley 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. MATERIALS AND METHODS Prenatal ethanol exposure animal model The experimental protocol used in this study was approved by Animal Investigation Committees from Wayne State University and the University of North Dakota. Nulliparous female Sprague–Dawley rats (Charles River Farms, Portage, IL, USA) were mated with males overnight and the presence of a plug the next morning indicated successful mating and was designated gestational day 1 (GD1). Beginning on GD8, pregnant rats were randomly divided into two groups and fed with a liquid diet with or without ethanol (6 g/kg/day) through GD20. This alcohol administration regime produced blood-ethanol concentrations of 260 mg/dl (Hannigan and Abel, 1996). Male offspring of the ethanol and control groups were housed in a temperature-controlled room under a 12-h light/ 12-h dark illumination cycle, and allowed access to standard rat chow and tap water ad libitum. The rats were used for study at 10–12 weeks of age. 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). Length–force 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 Krebs–Henseleit bicarbonate (KHB) buffer, pH 7.4. Hearts were subsequently perfused with a nominally Ca2+-free KHB buffer for 2–3 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 ×40 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. RESULTS General features of control and prenatal ethanol-exposed offspring The effects of prenatal ethanol exposure on offspring's body, heart, liver and kidney weights, and blood pressure are shown in Table 1. Prenatal ethanol exposure led to reduced heart weight associated with normal body, liver and kidney weights and blood pressure, compared to offspring from the non-prenatal ethanol exposure control group. Baseline mechanical properties of papillary muscles Effects of prenatal ethanol exposure on baseline mechanical properties of left-ventricular papillary muscles are shown in Table 2. Papillary muscles from prenatally ethanol-exposed animals exhibited similar muscle weight and cross-sectional area, although the muscle was significantly wider when compared to myocardium from control animals. PTD was significantly smaller in myocardium from the ethanol, compared to the control group. PTD normalized to muscle weight (g/g) or cross-sectional area (g/mm2) to reduce the inter-muscle variation also exhibited the same pattern, i.e. reduced force-generating capacity in myocardium from ethanol group. The ethanol-induced depression in PTD was associated with significant reduction in the maximal velocity of contraction (+VT) and relaxation (–VT). The myocardium from prenatally ethanol-exposed animals also exhibited slightly, but significantly, shortened contraction (TPT) accompanied with normal relaxation (RT90) duration. It is also noteworthy that three out of 16 papillary muscles displayed anatomical variation (such as bifurcations) and were unable to generate any recognizable force. These were, however, not included in the present results. Baseline mechanical properties of isolated ventricular myocytes The effects of prenatal ethanol exposure on baseline mechanical properties of ventricular myocytes are shown in Table 3. Cells from both the control and ethanol groups exhibited comparable resting cell length, cell shortening amplitude, the duration and velocity of both shortening and re-lengthening. However, the resting as well as peak intracellular Ca2+ levels (in response to electrical stimuli) were significantly elevated in myocytes from pups with prenatal ethanol exposure, compared to the control group. These data suggest that myocyte mechanical function is unlikely to be significantly affected by prenatal ethanol exposure, indicating a potential contribution from non-myocyte components to the reduced myocardial contraction at the multicellular level due to prenatal ethanol exposure. Post-rest potentiation response of papillary muscles from control and ethanol rat hearts To examine the potential mechanism of action in the depressed cardiac contractility following prenatal ethanol exposure, the post-rest potentiation test was conducted 10 and 60 s after cessation of stimulus after the myocardium had established steady-state contraction. The first five traces after resuming stimulation were recorded, to evaluate the ability of the myocardium to replenish SR intracellular Ca2+ pools. Results depicted in Fig. 1 show a similar responsiveness in myocardium from both the ethanol and control groups after a 10- or 60-s rest. These results suggest a comparable intracellular Ca2+ uptake capacity in myocardium between the ethanol and control groups. Effect of stimulation frequency in papillary muscle on myocardial contraction Rat hearts normally contract at very high rates (300 beats/ min), whereas our baseline studies were conducted at a 0.5-Hz stimulation rate (30 beats/min). To assess possible derangement of cardiac contractile function, the stimulus frequency was increased up to 5 Hz (300 beats/min). Papillary muscle was initially stimulated to contract at 0.5 Hz for 15 min to ensure steady state before commencing the frequency study. Figure 2 shows decreases of PTD with increasing stimulus frequency that are comparable in both groups, although the baseline PTD (either the absolute value or normalized to muscle weight) was significantly less in the ethanol group. Changes in the stimulating frequency from 0.01 to 5 Hz did not affect TPT and RT90 (data not shown). These data suggest that intracellular Ca2+ storage and release are likely to be preserved in rat hearts exposed to ethanol in utero. Cardiac contractile response to changes in extracellular Ca2+concentration in papillary muscle The cardiac contractile responses to changes in extracellular Ca2+ concentration (0.5–10 mM) in papillary muscle from ethanol and control groups are shown in Fig. 3. When extracellular Ca2+ concentration was lowered from 2.7 to 0.5 mM, PTD was reduced similarly (∼35%) in both groups. Increases in extracellular Ca2+ concentration induced comparable positive contractile responses in papillary muscles from both groups, indicating that the myofilament Ca2+ responsiveness is not affected by prenatal ethanol exposure (Fig. 3). Effect of noradrenaline on myocardial contraction in papillary muscle To examine if adrenergic responsiveness plays a role in reduced myocardial contraction following prenatal ethanol exposure, the effect of the adrenergic agonist noradrenaline was studied in myocardium from both the ethanol and control groups. Results shown in Fig. 3 indicate a similar responsiveness to noradrenaline in papillary muscle from the ethanol and control groups. Not surprisingly, myocardium from the ethanol group developed less force (either absolute value or normalized to muscle weight). These data indicated that the adrenergic system does not play a significant role in the diminished cardiac contractility following prenatal ethanol exposure. 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. Effect of acute ethanol application on caffeine-induced intracellular Ca2+release in ventricular myocytes Caffeine triggers release of Ca2+ from the sarcoplasmic reticulum (SR), the major pool of Ca2+ available to contractile proteins in rat cardiac muscle. To evaluate the SR function after prenatal ethanol exposure, caffeine-induced intracellular Ca2+ transients in fura-2-loaded ventricular myocytes were studied in the absence and presence of acutely administered ethanol. Multiple applications of caffeine were given at 10-min intervals to ensure steady state. Fig. 5 shows that acute application of ethanol (240 and 640 mg/dl) reduced the caffeine (10 mM)-induced increase of intracellular Ca2+ transients by similar margins in both the control and ethanol groups. This result indicates that SR responds normally to acute ethanol exposure in adult offspring heart after prenatal ethanol exposure, consistent with a previous report (Staley and Tobin, 1992). Effect of prenatal ethanol exposure on caspase-3 activation in ventricular myocytes Caspase-3 plays a critical role in apoptotic signalling (Telford et al., 1994); induction of Bax gene expression may lead to the activation of Caspase-3. Results shown in Fig. 6 indicate that Caspase-3 activation was significantly increased in ventricular myocytes freshly isolated from prenatally ethanol-exposed offspring, compared to those of the control group, indicating that cardiac myocyte apoptosis may be enhanced by prenatal ethanol exposure. DISCUSSION The major findings of the present study are decreased heart weight, compromised myocardial contractility in papillary muscles, normal myocyte contractile function, and elevated intracellular Ca2+ concentration and myocyte apoptosis from rat offspring that were exposed to ethanol in utero. Specifically, left-ventricular papillary muscles showed depressed PTD, ±VT, shortened TPT and normal RT90 in offspring exposed to ethanol in utero. No obvious differences were observed in post-rest potentiation, force–frequency relationship, or responses to increases in extracellular Ca2+ or to noradrenaline in papillary muscles between the ethanol and control groups. These data, along with the normal mechanical and SR function in cardiac myocytes, suggest that reduced myocardial systolic function (contractility) at the multicellular level is unlikely to be due to alterations in individual myocytes or intracellular Ca2+ mechanisms attributed to SR, myofilament Ca2+ sensitivity, or adrenergic responsiveness. Our data further revealed that prenatal ethanol exposure promotes postnatal myocyte apoptosis. Although FAS-associated cardiac defects, either morphologically or functionally, have been described (Loser et al., 1992), the current study is, to our knowledge, the first report showing myocardial contractile response at both tissue and cellular levels. 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). Table 1. General features of control and prenatal ethanol exposure offspring Parameter . Control (n = 13) . Ethanol (n = 13) . Values are means ± SEM of the numbers of animals (n). *P < 0.05 versus control. BW, body weight; HW, heart weight; KW, kidney weight; BP, blood pressure. Body weight (g) 482.6 ± 14.8 426.9 ± 29.3 Heart weight (mg) 1510 ± 57 1328 ± 45* HW/BW (mg/g) 3.02 ± 0.09 3.14 ± 0.21 Liver weight (g) 17.7 ± 0.9 17.0 ± 1.3 LW/BW (mg/g) 36.5 ± 1.2 40.7 ± 2.6 Kidney weight (g) 3.78 ± 0.18 3.45 ± 0.21 KW/BW (mg/g) 7.82 ± 0.28 8.18 ± 0.28 Systolic BP (mmHg) 132.0 ± 2.1 132.3 ± 1.4 Parameter . Control (n = 13) . Ethanol (n = 13) . Values are means ± SEM of the numbers of animals (n). *P < 0.05 versus control. BW, body weight; HW, heart weight; KW, kidney weight; BP, blood pressure. Body weight (g) 482.6 ± 14.8 426.9 ± 29.3 Heart weight (mg) 1510 ± 57 1328 ± 45* HW/BW (mg/g) 3.02 ± 0.09 3.14 ± 0.21 Liver weight (g) 17.7 ± 0.9 17.0 ± 1.3 LW/BW (mg/g) 36.5 ± 1.2 40.7 ± 2.6 Kidney weight (g) 3.78 ± 0.18 3.45 ± 0.21 KW/BW (mg/g) 7.82 ± 0.28 8.18 ± 0.28 Systolic BP (mmHg) 132.0 ± 2.1 132.3 ± 1.4 Open in new tab Table 1. General features of control and prenatal ethanol exposure offspring Parameter . Control (n = 13) . Ethanol (n = 13) . Values are means ± SEM of the numbers of animals (n). *P < 0.05 versus control. BW, body weight; HW, heart weight; KW, kidney weight; BP, blood pressure. Body weight (g) 482.6 ± 14.8 426.9 ± 29.3 Heart weight (mg) 1510 ± 57 1328 ± 45* HW/BW (mg/g) 3.02 ± 0.09 3.14 ± 0.21 Liver weight (g) 17.7 ± 0.9 17.0 ± 1.3 LW/BW (mg/g) 36.5 ± 1.2 40.7 ± 2.6 Kidney weight (g) 3.78 ± 0.18 3.45 ± 0.21 KW/BW (mg/g) 7.82 ± 0.28 8.18 ± 0.28 Systolic BP (mmHg) 132.0 ± 2.1 132.3 ± 1.4 Parameter . Control (n = 13) . Ethanol (n = 13) . Values are means ± SEM of the numbers of animals (n). *P < 0.05 versus control. BW, body weight; HW, heart weight; KW, kidney weight; BP, blood pressure. Body weight (g) 482.6 ± 14.8 426.9 ± 29.3 Heart weight (mg) 1510 ± 57 1328 ± 45* HW/BW (mg/g) 3.02 ± 0.09 3.14 ± 0.21 Liver weight (g) 17.7 ± 0.9 17.0 ± 1.3 LW/BW (mg/g) 36.5 ± 1.2 40.7 ± 2.6 Kidney weight (g) 3.78 ± 0.18 3.45 ± 0.21 KW/BW (mg/g) 7.82 ± 0.28 8.18 ± 0.28 Systolic BP (mmHg) 132.0 ± 2.1 132.3 ± 1.4 Open in new tab Table 2. Baseline mechanical characteristics of papillary muscles Parameter . Control (n = 16) . Ethanol (n = 16) . PTD, peak tension development (indicates the myocardial force-generating capacity); PTD/weight, peak force-generating capacity normalized to muscle weight; ±VT = maximum velocities of contraction and relaxation; TPT, time-to-PTD; RT90, time-to-90% relaxation. *P < 0.05 versus control. n = number of muscles. an = 13 (three muscles possessed anatomical defects and failed to generate recognizable force). Muscle weight (mg) 15.25 ± 0.96 14.89 ± 1.03 Muscle length (mm) 9.22 ± 0.39 8.67 ± 0.51 Muscle width (mm) 1.30 ± 0.08 1.57 ± 0.10* Muscle cross-sectional area (mm2) 12.04 ± 0.94 13.10 ± 0.71 PTD (g) 1.38 ± 0.20 0.94 ± 0.12*,a PTD/weight (g/g) 103.7 ± 21.6 63.4 ± 9.36*,a PTD/cross-sectional area (g/mm2) 0.116 ± 0.016 0.076 ± 0.010*,a +VT (g/s) 18.7 ± 2.6 13.87 ± 1.8*,a –VT (g/s) –12.3 ± 1.7 –8.99 ± 1.0*,a TPT (ms) 109.3 ± 2.3 103.4 ± 2.1*,a RT90 (ms) 134.6 ± 4.0 130.2 ± 3.7a Parameter . Control (n = 16) . Ethanol (n = 16) . PTD, peak tension development (indicates the myocardial force-generating capacity); PTD/weight, peak force-generating capacity normalized to muscle weight; ±VT = maximum velocities of contraction and relaxation; TPT, time-to-PTD; RT90, time-to-90% relaxation. *P < 0.05 versus control. n = number of muscles. an = 13 (three muscles possessed anatomical defects and failed to generate recognizable force). Muscle weight (mg) 15.25 ± 0.96 14.89 ± 1.03 Muscle length (mm) 9.22 ± 0.39 8.67 ± 0.51 Muscle width (mm) 1.30 ± 0.08 1.57 ± 0.10* Muscle cross-sectional area (mm2) 12.04 ± 0.94 13.10 ± 0.71 PTD (g) 1.38 ± 0.20 0.94 ± 0.12*,a PTD/weight (g/g) 103.7 ± 21.6 63.4 ± 9.36*,a PTD/cross-sectional area (g/mm2) 0.116 ± 0.016 0.076 ± 0.010*,a +VT (g/s) 18.7 ± 2.6 13.87 ± 1.8*,a –VT (g/s) –12.3 ± 1.7 –8.99 ± 1.0*,a TPT (ms) 109.3 ± 2.3 103.4 ± 2.1*,a RT90 (ms) 134.6 ± 4.0 130.2 ± 3.7a Open in new tab Table 2. Baseline mechanical characteristics of papillary muscles Parameter . Control (n = 16) . Ethanol (n = 16) . PTD, peak tension development (indicates the myocardial force-generating capacity); PTD/weight, peak force-generating capacity normalized to muscle weight; ±VT = maximum velocities of contraction and relaxation; TPT, time-to-PTD; RT90, time-to-90% relaxation. *P < 0.05 versus control. n = number of muscles. an = 13 (three muscles possessed anatomical defects and failed to generate recognizable force). Muscle weight (mg) 15.25 ± 0.96 14.89 ± 1.03 Muscle length (mm) 9.22 ± 0.39 8.67 ± 0.51 Muscle width (mm) 1.30 ± 0.08 1.57 ± 0.10* Muscle cross-sectional area (mm2) 12.04 ± 0.94 13.10 ± 0.71 PTD (g) 1.38 ± 0.20 0.94 ± 0.12*,a PTD/weight (g/g) 103.7 ± 21.6 63.4 ± 9.36*,a PTD/cross-sectional area (g/mm2) 0.116 ± 0.016 0.076 ± 0.010*,a +VT (g/s) 18.7 ± 2.6 13.87 ± 1.8*,a –VT (g/s) –12.3 ± 1.7 –8.99 ± 1.0*,a TPT (ms) 109.3 ± 2.3 103.4 ± 2.1*,a RT90 (ms) 134.6 ± 4.0 130.2 ± 3.7a Parameter . Control (n = 16) . Ethanol (n = 16) . PTD, peak tension development (indicates the myocardial force-generating capacity); PTD/weight, peak force-generating capacity normalized to muscle weight; ±VT = maximum velocities of contraction and relaxation; TPT, time-to-PTD; RT90, time-to-90% relaxation. *P < 0.05 versus control. n = number of muscles. an = 13 (three muscles possessed anatomical defects and failed to generate recognizable force). Muscle weight (mg) 15.25 ± 0.96 14.89 ± 1.03 Muscle length (mm) 9.22 ± 0.39 8.67 ± 0.51 Muscle width (mm) 1.30 ± 0.08 1.57 ± 0.10* Muscle cross-sectional area (mm2) 12.04 ± 0.94 13.10 ± 0.71 PTD (g) 1.38 ± 0.20 0.94 ± 0.12*,a PTD/weight (g/g) 103.7 ± 21.6 63.4 ± 9.36*,a PTD/cross-sectional area (g/mm2) 0.116 ± 0.016 0.076 ± 0.010*,a +VT (g/s) 18.7 ± 2.6 13.87 ± 1.8*,a –VT (g/s) –12.3 ± 1.7 –8.99 ± 1.0*,a TPT (ms) 109.3 ± 2.3 103.4 ± 2.1*,a RT90 (ms) 134.6 ± 4.0 130.2 ± 3.7a Open in new tab Table 3. Baseline mechanical and intracellular characteristics of ventricular myocytes Parameter . Control (n = 46) . Ethanol (n = 46) . ±dL/dt, maximum velocities of myocyte shortening and re-lengthening; TPS, time-to-peak shortening; TR90, time-to-90% re-lengthening. *P < 0.05 versus control. n = number of myocytes. Cell length (CL, μm) 158.0 ± 4.9 148.5 ± 5.0 Peak shortening (% CL) 7.19 ± 0.57 8.13 ± 0.53 +dL/dt (μm/s) 110.8 ± 14.4 104.4 ± 7.6 –dL/dt (μm/s) –72.0 ± 6.9 –83.5 ± 8.1 TPS (ms) 175.2 ± 4.3 184.3 ± 9.5 TR90 (ms) 323.3 ± 16.3 326.1 ± 21.4 Resting [Ca2+]i (360/380) 0.955 ± 0.014 1.022 ± 0.007* Peak [Ca2+]i (360/380) 1.000 ± 0.017 1.075 ± 0.008* Parameter . Control (n = 46) . Ethanol (n = 46) . ±dL/dt, maximum velocities of myocyte shortening and re-lengthening; TPS, time-to-peak shortening; TR90, time-to-90% re-lengthening. *P < 0.05 versus control. n = number of myocytes. Cell length (CL, μm) 158.0 ± 4.9 148.5 ± 5.0 Peak shortening (% CL) 7.19 ± 0.57 8.13 ± 0.53 +dL/dt (μm/s) 110.8 ± 14.4 104.4 ± 7.6 –dL/dt (μm/s) –72.0 ± 6.9 –83.5 ± 8.1 TPS (ms) 175.2 ± 4.3 184.3 ± 9.5 TR90 (ms) 323.3 ± 16.3 326.1 ± 21.4 Resting [Ca2+]i (360/380) 0.955 ± 0.014 1.022 ± 0.007* Peak [Ca2+]i (360/380) 1.000 ± 0.017 1.075 ± 0.008* Open in new tab Table 3. Baseline mechanical and intracellular characteristics of ventricular myocytes Parameter . Control (n = 46) . Ethanol (n = 46) . ±dL/dt, maximum velocities of myocyte shortening and re-lengthening; TPS, time-to-peak shortening; TR90, time-to-90% re-lengthening. *P < 0.05 versus control. n = number of myocytes. Cell length (CL, μm) 158.0 ± 4.9 148.5 ± 5.0 Peak shortening (% CL) 7.19 ± 0.57 8.13 ± 0.53 +dL/dt (μm/s) 110.8 ± 14.4 104.4 ± 7.6 –dL/dt (μm/s) –72.0 ± 6.9 –83.5 ± 8.1 TPS (ms) 175.2 ± 4.3 184.3 ± 9.5 TR90 (ms) 323.3 ± 16.3 326.1 ± 21.4 Resting [Ca2+]i (360/380) 0.955 ± 0.014 1.022 ± 0.007* Peak [Ca2+]i (360/380) 1.000 ± 0.017 1.075 ± 0.008* Parameter . Control (n = 46) . Ethanol (n = 46) . ±dL/dt, maximum velocities of myocyte shortening and re-lengthening; TPS, time-to-peak shortening; TR90, time-to-90% re-lengthening. *P < 0.05 versus control. n = number of myocytes. Cell length (CL, μm) 158.0 ± 4.9 148.5 ± 5.0 Peak shortening (% CL) 7.19 ± 0.57 8.13 ± 0.53 +dL/dt (μm/s) 110.8 ± 14.4 104.4 ± 7.6 –dL/dt (μm/s) –72.0 ± 6.9 –83.5 ± 8.1 TPS (ms) 175.2 ± 4.3 184.3 ± 9.5 TR90 (ms) 323.3 ± 16.3 326.1 ± 21.4 Resting [Ca2+]i (360/380) 0.955 ± 0.014 1.022 ± 0.007* Peak [Ca2+]i (360/380) 1.000 ± 0.017 1.075 ± 0.008* Open in new tab Fig. 1. Open in new tabDownload slide Post-rest potentiation trials in papillary muscle from control and prenatal ethanol groups. Trials were performed after the stimulus was paused for 10 s (A) and 60 s (B) PTD (peak tension development, which indicates the myocardial force-generating capacity) of each of the first five traces (denoted as trace numbers 1–5) was taken after resuming the electrical stimulus and was normalized to the steady-state PTD value before the cessation of the stimulus. Values are means ± SEM of the numbers (bars) given in parentheses. Fig. 1. Open in new tabDownload slide Post-rest potentiation trials in papillary muscle from control and prenatal ethanol groups. Trials were performed after the stimulus was paused for 10 s (A) and 60 s (B) PTD (peak tension development, which indicates the myocardial force-generating capacity) of each of the first five traces (denoted as trace numbers 1–5) was taken after resuming the electrical stimulus and was normalized to the steady-state PTD value before the cessation of the stimulus. Values are means ± SEM of the numbers (bars) given in parentheses. Fig. 2. Open in new tabDownload slide Peak tension development (PTD) of papillary muscle from control and prenatal ethanol groups at different stimulus frequencies (0.01–5 Hz). PTD was expressed as absolute value (g, panel A), normalized value to respective muscle weight (g/g, panel B) or percentage change from the respective control (0.5 Hz) value (%, panel C). Values are means ± SEM of the numbers (bars) given in parentheses. Control frequency was at 0.5 Hz. Con, control; Rec, recovery. Fig. 2. Open in new tabDownload slide Peak tension development (PTD) of papillary muscle from control and prenatal ethanol groups at different stimulus frequencies (0.01–5 Hz). PTD was expressed as absolute value (g, panel A), normalized value to respective muscle weight (g/g, panel B) or percentage change from the respective control (0.5 Hz) value (%, panel C). Values are means ± SEM of the numbers (bars) given in parentheses. Control frequency was at 0.5 Hz. Con, control; Rec, recovery. Fig. 3. Open in new tabDownload slide Dose-dependent response of extracellular Ca2+ and noradrenaline on myocardial contraction in papillary muscles. Muscles were isolated from control (filled circles) and prenatal ethanol (filled triangles) rat hearts. Peak tension development (PTD) was expressed as absolute value (g, panel A), normalized value to respective muscle weight (g/g, panel B) or percentage change from the respective control (%, panel C). Values are means ± SEM of the numbers (bars) given in parentheses. Control and recovery extracellular Ca2+ concentration was 2.7 mM. Fig. 3. Open in new tabDownload slide Dose-dependent response of extracellular Ca2+ and noradrenaline on myocardial contraction in papillary muscles. Muscles were isolated from control (filled circles) and prenatal ethanol (filled triangles) rat hearts. Peak tension development (PTD) was expressed as absolute value (g, panel A), normalized value to respective muscle weight (g/g, panel B) or percentage change from the respective control (%, panel C). Values are means ± SEM of the numbers (bars) given in parentheses. Control and recovery extracellular Ca2+ concentration was 2.7 mM. Fig. 4. Open in new tabDownload slide Effect of acute ethanol exposure on myocardial contraction in papillary muscles. Muscles were isolated from control (filled circles) and prenatal ethanol (filled triangles) rat hearts. Peak tension development (PTD) was expressed as absolute value (g, panel A), normalized value to respective muscle weight (g/g, panel B) or percentage change from the respective control (%, panel C). Values are means ± SEM of the numbers (bars) given in parentheses. Fig. 4. Open in new tabDownload slide Effect of acute ethanol exposure on myocardial contraction in papillary muscles. Muscles were isolated from control (filled circles) and prenatal ethanol (filled triangles) rat hearts. Peak tension development (PTD) was expressed as absolute value (g, panel A), normalized value to respective muscle weight (g/g, panel B) or percentage change from the respective control (%, panel C). Values are means ± SEM of the numbers (bars) given in parentheses. Fig. 5. Open in new tabDownload slide Effect of acute ethanol exposure (240 and 640 mg/dl) on caffeine (10 mM)-induced increase in intracellular Ca2+ transients in ventricular myocytes. Muscles were isolated from control (open bar) and prenatal ethanol exposure (filled bar) groups. A 10-min interval was given between 1st and 2nd caffeine puff in the control solution to reach complete recovery. Myocytes were then switched to an ethanol-containing solution for 10 min before another caffeine puff was applied. Values are means ± SEM of 12 cells/group, *P < 0.05 versus baseline. SR, sarcoplasmic reticulum. Fig. 5. Open in new tabDownload slide Effect of acute ethanol exposure (240 and 640 mg/dl) on caffeine (10 mM)-induced increase in intracellular Ca2+ transients in ventricular myocytes. Muscles were isolated from control (open bar) and prenatal ethanol exposure (filled bar) groups. A 10-min interval was given between 1st and 2nd caffeine puff in the control solution to reach complete recovery. Myocytes were then switched to an ethanol-containing solution for 10 min before another caffeine puff was applied. Values are means ± SEM of 12 cells/group, *P < 0.05 versus baseline. SR, sarcoplasmic reticulum. Fig. 6. Open in new tabDownload slide Effect of prenatal ethanol exposure on Caspase-3 activation in fresh isolated ventricular myocytes. Myocytes were from control (open bar) and prenatal ethanol exposure (filled bar) rat hearts. The cell lysates were tested for Caspase-3 activation by addition of Caspase-3 colorimetric substrate, DEVD-pNA. The incubation time was 4 h. *P < 0.05 versus control group. Values are means ± SEM for n = 5. O.D., optical density. Fig. 6. Open in new tabDownload slide Effect of prenatal ethanol exposure on Caspase-3 activation in fresh isolated ventricular myocytes. Myocytes were from control (open bar) and prenatal ethanol exposure (filled bar) rat hearts. The cell lysates were tested for Caspase-3 activation by addition of Caspase-3 colorimetric substrate, DEVD-pNA. The incubation time was 4 h. *P < 0.05 versus control group. Values are means ± SEM for n = 5. O.D., optical density. * Author to whom correspondence should be addressed. The authors wish to acknowledge Karl Ilg, Kadon Hintz and Faye Norby for their skilful technical assistance. This research was supported in part by grants from NIH MH47181, GM08167 to R.A.B., NIH AA07606-06 and AA12015 to J.H.H., and University of North Dakota Faculty Research Committee, North Dakota Experimental Program to Stimulate Competitive Research and American Heart Association Northland Affiliate (9960204Z) to J.R. REFERENCES Abel, E. L. and Hannigan, J. H. ( 1995 ) Maternal risk factors in fetal alcohol syndrome: provocative and permissive influences. Neurotoxicology and Teratology 71 , 445 –462. Adickes, E. D., Mollner, T. J. and Lockwood, S. K. ( 1990 ) Ethanol induced morphologic alterations during growth and maturation of cardiac myocytes. Alcoholism: Clinical and Experimental Research 14 , 827 –831. Adickes, E. D., Mollner, T. J. and Makoid, M. C. 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INFLUENCE OF PRENATAL ALCOHOL EXPOSURE ON MYOCARDIAL CONTRACTILE FUNCTION IN ADULT RAT HEARTS: ROLE OF INTRACELLULAR CALCIUM AND APOPTOSIS

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Oxford University Press
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© 2002 Medical Council on Alcohol
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10.1093/alcalc/37.1.30
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Abstract

Abstract — To assess the teratogenic action of ethanol on cardiac contractile function in offspring exposed to ethanol in utero, pregnant Sprague–Dawley rats were fed with ethanol during gestation. Left-ventricular papillary muscles and myocytes were isolated from the offspring of the ethanol-ingesting and control pregnant rats. Mechanical parameters measured were peak tension development (PTD, indicating the myocardial force-generating capacity), peak cell shortening (PS), time-to-PTD/PS (TPT/TPS), time-to-90% relaxation/re-lengthening (RT90/TR90), and maximal velocities of contraction/shortening and relaxation/re-lengthening (±VT and ±dL/dt). Intracellular Ca2+ levels and apoptosis were evaluated with fura-2 fluorescent dye and Caspase-3 activation assay, respectively. Offspring of the ethanol group displayed decreased heart weight associated with comparable body, liver and kidney weight, and papillary muscle weight/size, compared to the control group. However, prenatal ethanol exposure depressed myocardial PTD and ±VT. The myocardium from the ethanol group also exhibited slightly but significantly shortened TPT, accompanied with normal RT90. Muscles from both groups exhibited comparable responses to post-rest potentiation, increasing extracellular Ca2+ concentration, noradrenaline and acute ethanol challenge. Ventricular myocytes from both the control and ethanol groups possessed similar PS, TPS, TR90 and ±dL/dt. Both resting and peak intracellular Ca2+ levels were elevated in myocytes from the ethanol group. Additionally, acute ethanol application depressed caffeine-induced intracellular Ca2+ rise in myocytes from both groups. Myocytes from the ethanol group displayed an enhanced Caspase-3 activation, compared to control myocytes. These results suggest that prenatal ethanol exposure alters myocardial contractile function and may contribute to the development of postnatal cardiac dysfunction through, in part, increased intracellular Ca2+ loading and apoptosis. INTRODUCTION A major cause of birth defects is maternal consumption of alcohol. Prenatal alcohol exposure exerts a triad of defining teratogenic effects on the developing fetus, mainly manifest by growth retardation, abnormal facial features and central nervous system (CNS) damage, collectively known as the fetal alcohol syndrome (FAS) (Stratton et al., 1996). FAS is characterized by dysgenesis of many organ systems, such as the CNS, gastrointestinal tract, and the cardiovascular system. Defects in the cardiovascular system appear in up to 50% of children diagnosed with FAS. The detrimental effects of a teratogen on an organ are believed to be greatest during the 2nd to 8th weeks of gestation in human, known as the embryonic period, when major organ systems are growing and forming rapidly. Studies have shown that cardiac myocytes exposed to ethanol during embryogenesis did not mature morphologically or functionally (Adickes et al., 1990). Depressed growth of cardiac myocytes, reduction and delay in the development of myosin and actin, alteration in Ca2+ transport, mitochondrial function, sarcoplasmic reticulum (SR) Ca2+ uptake/binding as well as intracellular Ca2+ homeostasis have been reported in several animal models of FAS (Ni et al., 1992; Staley and Tobin, 1992; Fuseler, 1993; Syslak et al., 1994; Altura et al., 1996). We utilized Sprague–Dawley 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. MATERIALS AND METHODS Prenatal ethanol exposure animal model The experimental protocol used in this study was approved by Animal Investigation Committees from Wayne State University and the University of North Dakota. Nulliparous female Sprague–Dawley rats (Charles River Farms, Portage, IL, USA) were mated with males overnight and the presence of a plug the next morning indicated successful mating and was designated gestational day 1 (GD1). Beginning on GD8, pregnant rats were randomly divided into two groups and fed with a liquid diet with or without ethanol (6 g/kg/day) through GD20. This alcohol administration regime produced blood-ethanol concentrations of 260 mg/dl (Hannigan and Abel, 1996). Male offspring of the ethanol and control groups were housed in a temperature-controlled room under a 12-h light/ 12-h dark illumination cycle, and allowed access to standard rat chow and tap water ad libitum. The rats were used for study at 10–12 weeks of age. 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). Length–force 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 Krebs–Henseleit bicarbonate (KHB) buffer, pH 7.4. Hearts were subsequently perfused with a nominally Ca2+-free KHB buffer for 2–3 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 ×40 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. RESULTS General features of control and prenatal ethanol-exposed offspring The effects of prenatal ethanol exposure on offspring's body, heart, liver and kidney weights, and blood pressure are shown in Table 1. Prenatal ethanol exposure led to reduced heart weight associated with normal body, liver and kidney weights and blood pressure, compared to offspring from the non-prenatal ethanol exposure control group. Baseline mechanical properties of papillary muscles Effects of prenatal ethanol exposure on baseline mechanical properties of left-ventricular papillary muscles are shown in Table 2. Papillary muscles from prenatally ethanol-exposed animals exhibited similar muscle weight and cross-sectional area, although the muscle was significantly wider when compared to myocardium from control animals. PTD was significantly smaller in myocardium from the ethanol, compared to the control group. PTD normalized to muscle weight (g/g) or cross-sectional area (g/mm2) to reduce the inter-muscle variation also exhibited the same pattern, i.e. reduced force-generating capacity in myocardium from ethanol group. The ethanol-induced depression in PTD was associated with significant reduction in the maximal velocity of contraction (+VT) and relaxation (–VT). The myocardium from prenatally ethanol-exposed animals also exhibited slightly, but significantly, shortened contraction (TPT) accompanied with normal relaxation (RT90) duration. It is also noteworthy that three out of 16 papillary muscles displayed anatomical variation (such as bifurcations) and were unable to generate any recognizable force. These were, however, not included in the present results. Baseline mechanical properties of isolated ventricular myocytes The effects of prenatal ethanol exposure on baseline mechanical properties of ventricular myocytes are shown in Table 3. Cells from both the control and ethanol groups exhibited comparable resting cell length, cell shortening amplitude, the duration and velocity of both shortening and re-lengthening. However, the resting as well as peak intracellular Ca2+ levels (in response to electrical stimuli) were significantly elevated in myocytes from pups with prenatal ethanol exposure, compared to the control group. These data suggest that myocyte mechanical function is unlikely to be significantly affected by prenatal ethanol exposure, indicating a potential contribution from non-myocyte components to the reduced myocardial contraction at the multicellular level due to prenatal ethanol exposure. Post-rest potentiation response of papillary muscles from control and ethanol rat hearts To examine the potential mechanism of action in the depressed cardiac contractility following prenatal ethanol exposure, the post-rest potentiation test was conducted 10 and 60 s after cessation of stimulus after the myocardium had established steady-state contraction. The first five traces after resuming stimulation were recorded, to evaluate the ability of the myocardium to replenish SR intracellular Ca2+ pools. Results depicted in Fig. 1 show a similar responsiveness in myocardium from both the ethanol and control groups after a 10- or 60-s rest. These results suggest a comparable intracellular Ca2+ uptake capacity in myocardium between the ethanol and control groups. Effect of stimulation frequency in papillary muscle on myocardial contraction Rat hearts normally contract at very high rates (300 beats/ min), whereas our baseline studies were conducted at a 0.5-Hz stimulation rate (30 beats/min). To assess possible derangement of cardiac contractile function, the stimulus frequency was increased up to 5 Hz (300 beats/min). Papillary muscle was initially stimulated to contract at 0.5 Hz for 15 min to ensure steady state before commencing the frequency study. Figure 2 shows decreases of PTD with increasing stimulus frequency that are comparable in both groups, although the baseline PTD (either the absolute value or normalized to muscle weight) was significantly less in the ethanol group. Changes in the stimulating frequency from 0.01 to 5 Hz did not affect TPT and RT90 (data not shown). These data suggest that intracellular Ca2+ storage and release are likely to be preserved in rat hearts exposed to ethanol in utero. Cardiac contractile response to changes in extracellular Ca2+concentration in papillary muscle The cardiac contractile responses to changes in extracellular Ca2+ concentration (0.5–10 mM) in papillary muscle from ethanol and control groups are shown in Fig. 3. When extracellular Ca2+ concentration was lowered from 2.7 to 0.5 mM, PTD was reduced similarly (∼35%) in both groups. Increases in extracellular Ca2+ concentration induced comparable positive contractile responses in papillary muscles from both groups, indicating that the myofilament Ca2+ responsiveness is not affected by prenatal ethanol exposure (Fig. 3). Effect of noradrenaline on myocardial contraction in papillary muscle To examine if adrenergic responsiveness plays a role in reduced myocardial contraction following prenatal ethanol exposure, the effect of the adrenergic agonist noradrenaline was studied in myocardium from both the ethanol and control groups. Results shown in Fig. 3 indicate a similar responsiveness to noradrenaline in papillary muscle from the ethanol and control groups. Not surprisingly, myocardium from the ethanol group developed less force (either absolute value or normalized to muscle weight). These data indicated that the adrenergic system does not play a significant role in the diminished cardiac contractility following prenatal ethanol exposure. 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. Effect of acute ethanol application on caffeine-induced intracellular Ca2+release in ventricular myocytes Caffeine triggers release of Ca2+ from the sarcoplasmic reticulum (SR), the major pool of Ca2+ available to contractile proteins in rat cardiac muscle. To evaluate the SR function after prenatal ethanol exposure, caffeine-induced intracellular Ca2+ transients in fura-2-loaded ventricular myocytes were studied in the absence and presence of acutely administered ethanol. Multiple applications of caffeine were given at 10-min intervals to ensure steady state. Fig. 5 shows that acute application of ethanol (240 and 640 mg/dl) reduced the caffeine (10 mM)-induced increase of intracellular Ca2+ transients by similar margins in both the control and ethanol groups. This result indicates that SR responds normally to acute ethanol exposure in adult offspring heart after prenatal ethanol exposure, consistent with a previous report (Staley and Tobin, 1992). Effect of prenatal ethanol exposure on caspase-3 activation in ventricular myocytes Caspase-3 plays a critical role in apoptotic signalling (Telford et al., 1994); induction of Bax gene expression may lead to the activation of Caspase-3. Results shown in Fig. 6 indicate that Caspase-3 activation was significantly increased in ventricular myocytes freshly isolated from prenatally ethanol-exposed offspring, compared to those of the control group, indicating that cardiac myocyte apoptosis may be enhanced by prenatal ethanol exposure. DISCUSSION The major findings of the present study are decreased heart weight, compromised myocardial contractility in papillary muscles, normal myocyte contractile function, and elevated intracellular Ca2+ concentration and myocyte apoptosis from rat offspring that were exposed to ethanol in utero. Specifically, left-ventricular papillary muscles showed depressed PTD, ±VT, shortened TPT and normal RT90 in offspring exposed to ethanol in utero. No obvious differences were observed in post-rest potentiation, force–frequency relationship, or responses to increases in extracellular Ca2+ or to noradrenaline in papillary muscles between the ethanol and control groups. These data, along with the normal mechanical and SR function in cardiac myocytes, suggest that reduced myocardial systolic function (contractility) at the multicellular level is unlikely to be due to alterations in individual myocytes or intracellular Ca2+ mechanisms attributed to SR, myofilament Ca2+ sensitivity, or adrenergic responsiveness. Our data further revealed that prenatal ethanol exposure promotes postnatal myocyte apoptosis. Although FAS-associated cardiac defects, either morphologically or functionally, have been described (Loser et al., 1992), the current study is, to our knowledge, the first report showing myocardial contractile response at both tissue and cellular levels. 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). Table 1. General features of control and prenatal ethanol exposure offspring Parameter . Control (n = 13) . Ethanol (n = 13) . Values are means ± SEM of the numbers of animals (n). *P < 0.05 versus control. BW, body weight; HW, heart weight; KW, kidney weight; BP, blood pressure. Body weight (g) 482.6 ± 14.8 426.9 ± 29.3 Heart weight (mg) 1510 ± 57 1328 ± 45* HW/BW (mg/g) 3.02 ± 0.09 3.14 ± 0.21 Liver weight (g) 17.7 ± 0.9 17.0 ± 1.3 LW/BW (mg/g) 36.5 ± 1.2 40.7 ± 2.6 Kidney weight (g) 3.78 ± 0.18 3.45 ± 0.21 KW/BW (mg/g) 7.82 ± 0.28 8.18 ± 0.28 Systolic BP (mmHg) 132.0 ± 2.1 132.3 ± 1.4 Parameter . Control (n = 13) . Ethanol (n = 13) . Values are means ± SEM of the numbers of animals (n). *P < 0.05 versus control. BW, body weight; HW, heart weight; KW, kidney weight; BP, blood pressure. Body weight (g) 482.6 ± 14.8 426.9 ± 29.3 Heart weight (mg) 1510 ± 57 1328 ± 45* HW/BW (mg/g) 3.02 ± 0.09 3.14 ± 0.21 Liver weight (g) 17.7 ± 0.9 17.0 ± 1.3 LW/BW (mg/g) 36.5 ± 1.2 40.7 ± 2.6 Kidney weight (g) 3.78 ± 0.18 3.45 ± 0.21 KW/BW (mg/g) 7.82 ± 0.28 8.18 ± 0.28 Systolic BP (mmHg) 132.0 ± 2.1 132.3 ± 1.4 Open in new tab Table 1. General features of control and prenatal ethanol exposure offspring Parameter . Control (n = 13) . Ethanol (n = 13) . Values are means ± SEM of the numbers of animals (n). *P < 0.05 versus control. BW, body weight; HW, heart weight; KW, kidney weight; BP, blood pressure. Body weight (g) 482.6 ± 14.8 426.9 ± 29.3 Heart weight (mg) 1510 ± 57 1328 ± 45* HW/BW (mg/g) 3.02 ± 0.09 3.14 ± 0.21 Liver weight (g) 17.7 ± 0.9 17.0 ± 1.3 LW/BW (mg/g) 36.5 ± 1.2 40.7 ± 2.6 Kidney weight (g) 3.78 ± 0.18 3.45 ± 0.21 KW/BW (mg/g) 7.82 ± 0.28 8.18 ± 0.28 Systolic BP (mmHg) 132.0 ± 2.1 132.3 ± 1.4 Parameter . Control (n = 13) . Ethanol (n = 13) . Values are means ± SEM of the numbers of animals (n). *P < 0.05 versus control. BW, body weight; HW, heart weight; KW, kidney weight; BP, blood pressure. Body weight (g) 482.6 ± 14.8 426.9 ± 29.3 Heart weight (mg) 1510 ± 57 1328 ± 45* HW/BW (mg/g) 3.02 ± 0.09 3.14 ± 0.21 Liver weight (g) 17.7 ± 0.9 17.0 ± 1.3 LW/BW (mg/g) 36.5 ± 1.2 40.7 ± 2.6 Kidney weight (g) 3.78 ± 0.18 3.45 ± 0.21 KW/BW (mg/g) 7.82 ± 0.28 8.18 ± 0.28 Systolic BP (mmHg) 132.0 ± 2.1 132.3 ± 1.4 Open in new tab Table 2. Baseline mechanical characteristics of papillary muscles Parameter . Control (n = 16) . Ethanol (n = 16) . PTD, peak tension development (indicates the myocardial force-generating capacity); PTD/weight, peak force-generating capacity normalized to muscle weight; ±VT = maximum velocities of contraction and relaxation; TPT, time-to-PTD; RT90, time-to-90% relaxation. *P < 0.05 versus control. n = number of muscles. an = 13 (three muscles possessed anatomical defects and failed to generate recognizable force). Muscle weight (mg) 15.25 ± 0.96 14.89 ± 1.03 Muscle length (mm) 9.22 ± 0.39 8.67 ± 0.51 Muscle width (mm) 1.30 ± 0.08 1.57 ± 0.10* Muscle cross-sectional area (mm2) 12.04 ± 0.94 13.10 ± 0.71 PTD (g) 1.38 ± 0.20 0.94 ± 0.12*,a PTD/weight (g/g) 103.7 ± 21.6 63.4 ± 9.36*,a PTD/cross-sectional area (g/mm2) 0.116 ± 0.016 0.076 ± 0.010*,a +VT (g/s) 18.7 ± 2.6 13.87 ± 1.8*,a –VT (g/s) –12.3 ± 1.7 –8.99 ± 1.0*,a TPT (ms) 109.3 ± 2.3 103.4 ± 2.1*,a RT90 (ms) 134.6 ± 4.0 130.2 ± 3.7a Parameter . Control (n = 16) . Ethanol (n = 16) . PTD, peak tension development (indicates the myocardial force-generating capacity); PTD/weight, peak force-generating capacity normalized to muscle weight; ±VT = maximum velocities of contraction and relaxation; TPT, time-to-PTD; RT90, time-to-90% relaxation. *P < 0.05 versus control. n = number of muscles. an = 13 (three muscles possessed anatomical defects and failed to generate recognizable force). Muscle weight (mg) 15.25 ± 0.96 14.89 ± 1.03 Muscle length (mm) 9.22 ± 0.39 8.67 ± 0.51 Muscle width (mm) 1.30 ± 0.08 1.57 ± 0.10* Muscle cross-sectional area (mm2) 12.04 ± 0.94 13.10 ± 0.71 PTD (g) 1.38 ± 0.20 0.94 ± 0.12*,a PTD/weight (g/g) 103.7 ± 21.6 63.4 ± 9.36*,a PTD/cross-sectional area (g/mm2) 0.116 ± 0.016 0.076 ± 0.010*,a +VT (g/s) 18.7 ± 2.6 13.87 ± 1.8*,a –VT (g/s) –12.3 ± 1.7 –8.99 ± 1.0*,a TPT (ms) 109.3 ± 2.3 103.4 ± 2.1*,a RT90 (ms) 134.6 ± 4.0 130.2 ± 3.7a Open in new tab Table 2. Baseline mechanical characteristics of papillary muscles Parameter . Control (n = 16) . Ethanol (n = 16) . PTD, peak tension development (indicates the myocardial force-generating capacity); PTD/weight, peak force-generating capacity normalized to muscle weight; ±VT = maximum velocities of contraction and relaxation; TPT, time-to-PTD; RT90, time-to-90% relaxation. *P < 0.05 versus control. n = number of muscles. an = 13 (three muscles possessed anatomical defects and failed to generate recognizable force). Muscle weight (mg) 15.25 ± 0.96 14.89 ± 1.03 Muscle length (mm) 9.22 ± 0.39 8.67 ± 0.51 Muscle width (mm) 1.30 ± 0.08 1.57 ± 0.10* Muscle cross-sectional area (mm2) 12.04 ± 0.94 13.10 ± 0.71 PTD (g) 1.38 ± 0.20 0.94 ± 0.12*,a PTD/weight (g/g) 103.7 ± 21.6 63.4 ± 9.36*,a PTD/cross-sectional area (g/mm2) 0.116 ± 0.016 0.076 ± 0.010*,a +VT (g/s) 18.7 ± 2.6 13.87 ± 1.8*,a –VT (g/s) –12.3 ± 1.7 –8.99 ± 1.0*,a TPT (ms) 109.3 ± 2.3 103.4 ± 2.1*,a RT90 (ms) 134.6 ± 4.0 130.2 ± 3.7a Parameter . Control (n = 16) . Ethanol (n = 16) . PTD, peak tension development (indicates the myocardial force-generating capacity); PTD/weight, peak force-generating capacity normalized to muscle weight; ±VT = maximum velocities of contraction and relaxation; TPT, time-to-PTD; RT90, time-to-90% relaxation. *P < 0.05 versus control. n = number of muscles. an = 13 (three muscles possessed anatomical defects and failed to generate recognizable force). Muscle weight (mg) 15.25 ± 0.96 14.89 ± 1.03 Muscle length (mm) 9.22 ± 0.39 8.67 ± 0.51 Muscle width (mm) 1.30 ± 0.08 1.57 ± 0.10* Muscle cross-sectional area (mm2) 12.04 ± 0.94 13.10 ± 0.71 PTD (g) 1.38 ± 0.20 0.94 ± 0.12*,a PTD/weight (g/g) 103.7 ± 21.6 63.4 ± 9.36*,a PTD/cross-sectional area (g/mm2) 0.116 ± 0.016 0.076 ± 0.010*,a +VT (g/s) 18.7 ± 2.6 13.87 ± 1.8*,a –VT (g/s) –12.3 ± 1.7 –8.99 ± 1.0*,a TPT (ms) 109.3 ± 2.3 103.4 ± 2.1*,a RT90 (ms) 134.6 ± 4.0 130.2 ± 3.7a Open in new tab Table 3. Baseline mechanical and intracellular characteristics of ventricular myocytes Parameter . Control (n = 46) . Ethanol (n = 46) . ±dL/dt, maximum velocities of myocyte shortening and re-lengthening; TPS, time-to-peak shortening; TR90, time-to-90% re-lengthening. *P < 0.05 versus control. n = number of myocytes. Cell length (CL, μm) 158.0 ± 4.9 148.5 ± 5.0 Peak shortening (% CL) 7.19 ± 0.57 8.13 ± 0.53 +dL/dt (μm/s) 110.8 ± 14.4 104.4 ± 7.6 –dL/dt (μm/s) –72.0 ± 6.9 –83.5 ± 8.1 TPS (ms) 175.2 ± 4.3 184.3 ± 9.5 TR90 (ms) 323.3 ± 16.3 326.1 ± 21.4 Resting [Ca2+]i (360/380) 0.955 ± 0.014 1.022 ± 0.007* Peak [Ca2+]i (360/380) 1.000 ± 0.017 1.075 ± 0.008* Parameter . Control (n = 46) . Ethanol (n = 46) . ±dL/dt, maximum velocities of myocyte shortening and re-lengthening; TPS, time-to-peak shortening; TR90, time-to-90% re-lengthening. *P < 0.05 versus control. n = number of myocytes. Cell length (CL, μm) 158.0 ± 4.9 148.5 ± 5.0 Peak shortening (% CL) 7.19 ± 0.57 8.13 ± 0.53 +dL/dt (μm/s) 110.8 ± 14.4 104.4 ± 7.6 –dL/dt (μm/s) –72.0 ± 6.9 –83.5 ± 8.1 TPS (ms) 175.2 ± 4.3 184.3 ± 9.5 TR90 (ms) 323.3 ± 16.3 326.1 ± 21.4 Resting [Ca2+]i (360/380) 0.955 ± 0.014 1.022 ± 0.007* Peak [Ca2+]i (360/380) 1.000 ± 0.017 1.075 ± 0.008* Open in new tab Table 3. Baseline mechanical and intracellular characteristics of ventricular myocytes Parameter . Control (n = 46) . Ethanol (n = 46) . ±dL/dt, maximum velocities of myocyte shortening and re-lengthening; TPS, time-to-peak shortening; TR90, time-to-90% re-lengthening. *P < 0.05 versus control. n = number of myocytes. Cell length (CL, μm) 158.0 ± 4.9 148.5 ± 5.0 Peak shortening (% CL) 7.19 ± 0.57 8.13 ± 0.53 +dL/dt (μm/s) 110.8 ± 14.4 104.4 ± 7.6 –dL/dt (μm/s) –72.0 ± 6.9 –83.5 ± 8.1 TPS (ms) 175.2 ± 4.3 184.3 ± 9.5 TR90 (ms) 323.3 ± 16.3 326.1 ± 21.4 Resting [Ca2+]i (360/380) 0.955 ± 0.014 1.022 ± 0.007* Peak [Ca2+]i (360/380) 1.000 ± 0.017 1.075 ± 0.008* Parameter . Control (n = 46) . Ethanol (n = 46) . ±dL/dt, maximum velocities of myocyte shortening and re-lengthening; TPS, time-to-peak shortening; TR90, time-to-90% re-lengthening. *P < 0.05 versus control. n = number of myocytes. Cell length (CL, μm) 158.0 ± 4.9 148.5 ± 5.0 Peak shortening (% CL) 7.19 ± 0.57 8.13 ± 0.53 +dL/dt (μm/s) 110.8 ± 14.4 104.4 ± 7.6 –dL/dt (μm/s) –72.0 ± 6.9 –83.5 ± 8.1 TPS (ms) 175.2 ± 4.3 184.3 ± 9.5 TR90 (ms) 323.3 ± 16.3 326.1 ± 21.4 Resting [Ca2+]i (360/380) 0.955 ± 0.014 1.022 ± 0.007* Peak [Ca2+]i (360/380) 1.000 ± 0.017 1.075 ± 0.008* Open in new tab Fig. 1. Open in new tabDownload slide Post-rest potentiation trials in papillary muscle from control and prenatal ethanol groups. Trials were performed after the stimulus was paused for 10 s (A) and 60 s (B) PTD (peak tension development, which indicates the myocardial force-generating capacity) of each of the first five traces (denoted as trace numbers 1–5) was taken after resuming the electrical stimulus and was normalized to the steady-state PTD value before the cessation of the stimulus. Values are means ± SEM of the numbers (bars) given in parentheses. Fig. 1. Open in new tabDownload slide Post-rest potentiation trials in papillary muscle from control and prenatal ethanol groups. Trials were performed after the stimulus was paused for 10 s (A) and 60 s (B) PTD (peak tension development, which indicates the myocardial force-generating capacity) of each of the first five traces (denoted as trace numbers 1–5) was taken after resuming the electrical stimulus and was normalized to the steady-state PTD value before the cessation of the stimulus. Values are means ± SEM of the numbers (bars) given in parentheses. Fig. 2. Open in new tabDownload slide Peak tension development (PTD) of papillary muscle from control and prenatal ethanol groups at different stimulus frequencies (0.01–5 Hz). PTD was expressed as absolute value (g, panel A), normalized value to respective muscle weight (g/g, panel B) or percentage change from the respective control (0.5 Hz) value (%, panel C). Values are means ± SEM of the numbers (bars) given in parentheses. Control frequency was at 0.5 Hz. Con, control; Rec, recovery. Fig. 2. Open in new tabDownload slide Peak tension development (PTD) of papillary muscle from control and prenatal ethanol groups at different stimulus frequencies (0.01–5 Hz). PTD was expressed as absolute value (g, panel A), normalized value to respective muscle weight (g/g, panel B) or percentage change from the respective control (0.5 Hz) value (%, panel C). Values are means ± SEM of the numbers (bars) given in parentheses. Control frequency was at 0.5 Hz. Con, control; Rec, recovery. Fig. 3. Open in new tabDownload slide Dose-dependent response of extracellular Ca2+ and noradrenaline on myocardial contraction in papillary muscles. Muscles were isolated from control (filled circles) and prenatal ethanol (filled triangles) rat hearts. Peak tension development (PTD) was expressed as absolute value (g, panel A), normalized value to respective muscle weight (g/g, panel B) or percentage change from the respective control (%, panel C). Values are means ± SEM of the numbers (bars) given in parentheses. Control and recovery extracellular Ca2+ concentration was 2.7 mM. Fig. 3. Open in new tabDownload slide Dose-dependent response of extracellular Ca2+ and noradrenaline on myocardial contraction in papillary muscles. Muscles were isolated from control (filled circles) and prenatal ethanol (filled triangles) rat hearts. Peak tension development (PTD) was expressed as absolute value (g, panel A), normalized value to respective muscle weight (g/g, panel B) or percentage change from the respective control (%, panel C). Values are means ± SEM of the numbers (bars) given in parentheses. Control and recovery extracellular Ca2+ concentration was 2.7 mM. Fig. 4. Open in new tabDownload slide Effect of acute ethanol exposure on myocardial contraction in papillary muscles. Muscles were isolated from control (filled circles) and prenatal ethanol (filled triangles) rat hearts. Peak tension development (PTD) was expressed as absolute value (g, panel A), normalized value to respective muscle weight (g/g, panel B) or percentage change from the respective control (%, panel C). Values are means ± SEM of the numbers (bars) given in parentheses. Fig. 4. Open in new tabDownload slide Effect of acute ethanol exposure on myocardial contraction in papillary muscles. Muscles were isolated from control (filled circles) and prenatal ethanol (filled triangles) rat hearts. Peak tension development (PTD) was expressed as absolute value (g, panel A), normalized value to respective muscle weight (g/g, panel B) or percentage change from the respective control (%, panel C). Values are means ± SEM of the numbers (bars) given in parentheses. Fig. 5. Open in new tabDownload slide Effect of acute ethanol exposure (240 and 640 mg/dl) on caffeine (10 mM)-induced increase in intracellular Ca2+ transients in ventricular myocytes. Muscles were isolated from control (open bar) and prenatal ethanol exposure (filled bar) groups. A 10-min interval was given between 1st and 2nd caffeine puff in the control solution to reach complete recovery. Myocytes were then switched to an ethanol-containing solution for 10 min before another caffeine puff was applied. Values are means ± SEM of 12 cells/group, *P < 0.05 versus baseline. SR, sarcoplasmic reticulum. Fig. 5. Open in new tabDownload slide Effect of acute ethanol exposure (240 and 640 mg/dl) on caffeine (10 mM)-induced increase in intracellular Ca2+ transients in ventricular myocytes. Muscles were isolated from control (open bar) and prenatal ethanol exposure (filled bar) groups. A 10-min interval was given between 1st and 2nd caffeine puff in the control solution to reach complete recovery. Myocytes were then switched to an ethanol-containing solution for 10 min before another caffeine puff was applied. Values are means ± SEM of 12 cells/group, *P < 0.05 versus baseline. SR, sarcoplasmic reticulum. Fig. 6. Open in new tabDownload slide Effect of prenatal ethanol exposure on Caspase-3 activation in fresh isolated ventricular myocytes. Myocytes were from control (open bar) and prenatal ethanol exposure (filled bar) rat hearts. The cell lysates were tested for Caspase-3 activation by addition of Caspase-3 colorimetric substrate, DEVD-pNA. The incubation time was 4 h. *P < 0.05 versus control group. Values are means ± SEM for n = 5. O.D., optical density. Fig. 6. Open in new tabDownload slide Effect of prenatal ethanol exposure on Caspase-3 activation in fresh isolated ventricular myocytes. Myocytes were from control (open bar) and prenatal ethanol exposure (filled bar) rat hearts. The cell lysates were tested for Caspase-3 activation by addition of Caspase-3 colorimetric substrate, DEVD-pNA. The incubation time was 4 h. *P < 0.05 versus control group. Values are means ± SEM for n = 5. 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Alcohol and Alcoholism – Oxford University Press

Published: Jan 1, 2002

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