Department of Applied Sciences for Biosystems, Division of Human Physiology and Nutrition, University of Cagliari, Via Porcell 4, I09124 Cagliari and
1 Institute of Human Physiology, Catholic University, Largo F. Vito 1, I00168 Rome, Italy
Received 25 April 2000; in revised form 26 July 2000; accepted 28 July 2000
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ABSTRACT |
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INTRODUCTION |
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In studies on animal models, the effects of chronic ethanol treatment on the heart are similar to those found in man. Indeed, chronic ethanol treatment produces an impaired ventricular function in dogs (Pachinger et al., 1973; Regan et al., 1974
; Thomas et al., 1980
). Moreover, chronic ethanol treatment induces a decrease in cardiac function in isolated rat heart (Weishaar et al., 1977
; Segel et al., 1979
; Chan and Sutter, 1982
; Capasso et al., 1991
, 1992
). Ethanol also produces altered contractile activity associated with a decrease in action potential duration in the left ventricular papillary muscle of rats (Tepper et al., 1986
). However, at variance with humans, rodents do not drink ethanol to the point of intoxication voluntarily. In fact, animals consume intoxicating amounts of ethanol only when forced to (Fadda and Rossetti, 1998
). Thus, in all the above studies chronic ethanol was forcibly administered.
The selectively bred alcohol-preferring animals willingly and freely drink alcohol under free choice with water. Like other selected rat lines, alcohol-preferring Sardinian (sP) rats display a clear preference for ethanol over water and drink 6 g/kg of ethanol or more in a day under the two-bottle, free choice paradigm (Gessa et al., 1991).
The present study was undertaken to assess whether voluntary ethanol consumption for long periods of time also produces an impairment of heart motor performance in sP rats. We investigated the mechanical properties of isolated papillary muscles in terms of lengthtension and forcefrequency relationships. The isolated papillary muscle was chosen for this experiment, because it is an exceptional in vitro preparation for studying the mechanical properties of the rat myocardium without neuronal and hormonal influences. Mechanical properties were determined in isolated papillary muscle of sP rats which consumed ethanol (12% w/v) for 26 weeks in a free choice paradigm with water.
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METHODS |
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Experimental procedure
Animals were killed with a lethal dose of ether anaesthesia and their hearts were excised rapidly and placed initially in modified KrebsHenseleit solution pre-equilibrated with 95% O25% CO2 at room temperature. Hearts were massaged to encourage them to beat and expel residual blood from the ventricles. Left ventricular papillary muscle was carefully dissected and mounted vertically in a water-jacketed bath (10 ml) containing modified KrebsHenseleit solution of the following composition (mM): NaCl (123), NaHCO3 (20), MgSO4 (0.8), KCl (6), CaCl2 (2.52), KH2PO4 (1.16), glucose (11.98). The solution was kept at 32°C and bubbled continuously with a mixture of 95% O25% CO2 maintaining a pH of 7.4. The base of the muscle was fixed firmly to the bottom of the organ bath while its free upper end was tied with a silk thread to a Grass FT03 force-displacement transducer mounted on a moving support allowing minimum length increments of 5 µm.
Stimulation of the muscle was accomplished with platinum mass electrodes (20 x 5 mm) placed along the parallel aspect of the muscle. The muscle was stimulated with a Digit 3T stimulator connected to a multiplexing pulse booster (Basile). Supramaximal square-wave pulses (80150 mA, 4 ms) were used.
A frequency of stimulation of 30 pulses/min was generally employed, except when the response to rate of stimulation was assessed.
Lengthtension responses
The papillary muscle was allowed to equilibrate for a period of 1 h under a preload of 3 mN. After equilibration and before stretching, the length of the muscle was measured using a stereomicroscope with an ocular micrometer. This initial length was called the reference length and indicated as Lr. Recorded tension without stimulation was called resting (or passive) tension. With stimulation, an active contraction was superimposed on the resting tension. This developed tension was termed active tension.
The papillary muscle was gradually stretched in steps of 5% Lr from 100% to 130% Lr, allowing force to stabilize at each new length. Resting and active isometric tensions that developed at each increase in length were recorded.
Frequency response recordings
The papillary muscle was elongated up to 130% Lr and stimulated at the following rates: 0.33, 1, 3, 6, 12, 18, 24, 36, 48, 60, 120, 180 pulses/min for as long as necessary to obtain steady-state amplitude of contraction waves. Outputs from the force-transducer were amplified and displayed continuously on a Grass polygraph. They were collected by a data acquisition board and fed into a personal computer for subsequent analysis.
Hearts and papillary muscles were blotted to remove excess fluid and dried in an oven set at 80°C until they reached a constant weight (dry weight).
Data analysis
At each increment in length of the papillary muscle, the resting tension was measured in the rest period between contractions, from the baseline tension determined at 100% Lr. The developed tensions recorded at each increase in length were calculated from the mean amplitude of the last 10 contractions before the next stretch. Active tensions developed at 130% Lr in response to different electrical stimulation rates were obtained from the mean amplitude of the contractions on the last 10 waves of the steady-state periods at each value of stimulation frequency.
All tension values were normalized to dry weight (mg) of papillary muscle and are presented as means ± SE. Data were analysed using one-way ANOVA. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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DISCUSSION |
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Several studies have shown that rats chronically treated with ethanol have impaired cardiac muscle contractility (Segel et al., 1975; Kino et al., 1981
; Preedy and Peters, 1989
; Capasso et al., 1992
). Measurement of developed isometric tension in papillary muscles dissected from the hearts of rats fed ethanol for periods of 5 weeks to several months, generally demonstrates significant reductions in one or more indices of mechanical performance (Segel et al., 1975
; Kino et al., 1981
; Tepper et al., 1986
). By contrast, Capasso et al. (1992) reported that, in the papillary muscle, no difference in isometric developed tension was found between controls and ethanol-treated rats when either the left or right side was compared. However, these researchers found alterations in cardiac mechanical performance, alterations in myofibrillar Mg2+-ATPase activity and a significant decrease in the total number of myocitic nuclei in the left ventricular myocardium. However, our results are in agreement with those of Tepper et al. (1986), who reported that forced long-term ethanol administration (30 weeks) in unselected Wistar rats produced mechanical alterations (depressed forcevelocity relation) in the left papillary muscle. This altered contractile activity is associated with a decrease in action potential duration. However, in the study by Tepper et al., (1986), rats received a very highly concentrated ethanol solution (40% v/v) as the sole source of fluid, with consumption at the end of the treatment period of a very high amount of ethanol (10.1±1.8 g). In addition, the rats did not have a washout period before being tested. Thus under such experimental conditions, besides the chronic effect, there was also an acute effect of ethanol.
In the present experiments, sP rats drank ethanol solutions voluntarily in a free choice with water and did not consume intoxicating amounts of ethanol (see Fadda and Rossetti, 1998). Nonetheless, the results obtained showed that the quantity of ethanol ingested by sP rats was sufficient to produce alterations in contractile performance in the papillary muscle, and that this manifestation was not reversible after abstinence from alcohol for 1 week. Alterations of contractile performance were also observed in the isolated portal vein motility in sP rats (Licheri et al., 1999), thus indicating that chronic ethanol consumption in a free choice paradigm induces a general depression of motor events in the cardiovascular system.
The mechanism of action of ethanol in the papillary muscular cells of sP rats is at present unknown. Evidence from various laboratories suggests that two major mechanisms appear to be important. One consists of a reduction in the capacity of the sarcoplasmic reticulum to accumulate Ca2+ (Swartz et al., 1974; Retig et al., 1977
; Hara and Kasai, 1977
) together with a decrease in the activity of specific sarcolemmal ion channels (Guarnieri and Lakatta, 1990
). The other consists of an inhibitory effect of ethanol on the contractile proteins (Segel et al., 1975
; Sarma et al., 1976
; Weishaar et al., 1977
; Preedy and Peters, 1989
; Capasso et al., 1992
). Further investigations of these forms of alteration of contractile behaviour in our animal model should not only provide insight into more basic cellular mechanisms responsible for their appearance, but should also lead to possible methods for their prevention or amelioration.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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REFERENCES |
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