Cardiovascular Research, 1975, 9, 649-663.
Effects of chronic graded ethanol consumption on the metabolism, ultrastructure, and mechanical function of the rat heart' LEI GH D . S E G E L , S T E P H E N V . R E N D I G , YVES C H O Q U E T , K U R I E N C H A C K O , E Z R A A . A M S T E R D A M , and D E A N T . MASON
From the Section of Cardiovascular Medicine, Departments of Internal Medicine, Physiology, and Human Anatomy, University of California, School of Medicine Davis and Sacramento, California, U S A
were fed regular chow containing standard vitamins with the ethanol group in each series also receiving a progressively greater alcohol intake for 3 to 6 months: El 5%, E2 lo%, and E3 25% ethanol. Electron microscopy showed swelling of mitochondria, transverse tubules and sarcoplasmic reticulum, dehiscence of intercalated discs and disintegration of myofibrils scattered throughout the ventricular myocardium in El and E2 as early as 7 wk after beginning 5% ethanol; in addition, there were clumping of mitochondria and supercontraction of myofibrils in E,. Concomitant with substructural abnormalities in E,, there were slight but significant depressions of cardiac myofibrillar ATPase activity and mitochondria1 function. Cardiac catecholamines, hydroxyproline, and total bound glycerol were unchanged. Alteration of isometric contraction of isolated, supported left ventricular papillary muscles occurred initially in E2 and was clearly evident in E, by significant reduction of duration of systolic active state (time from onset to peak tension), while total tension generated and peak rate of tension rise were not yet disturbed. Extra vitamin supplementation in additional rats drinking 25% ethanol minimally lessened decline in myofibrillar ATPase activity, but otherwise provided no protection. Thus, chronic daily ingestion of graded quantities of ethanol representing 10 t o 30% of total calories in well-nourished animals exerted toxic effects on microstructure, metabolism and mechanics of the ventricle. These alterations are postulated to be pertinent to early pathogenesis of clinical alcoholic cardiomyopathy. Although the association of chronic alcoholism and heart disease has been recognized clinically for a century (Walshe, 1873), the precise nature of this relationship has not been elucidated and considerable controversy remains concerning the fundamental causative role of ethanol in the This work was supported by NIH Grant MH 19489 (AA00270)and the Vera Button Foundation. Reprint requests to: L.D.S.: Section of Cardiovascular Medicine University of California at Davis, School of Medicine, Davis, California 95616, USA. 1
pathogenesis of myocardial damage. The possibility that alcohol may exert a direct toxic action on heart muscle has been suggested by the increased prevalence of cardiomyopathy in patients with a history of chronic excessive ethanol ingestion (Brigden and Robinson, 1964; Ferrans et-al, 1965; Wendt et al, 1965; Alexander, 1966; Burch and De Pasquale, 1969; Mason el a/, 1971; Regan, 1971; Brigden, 1972; Demakis et al, 1974). Thus, congestive heart
Downloaded from by guest on June 7, 2016
AUTHORS' SYNOPSIS Three sequential sets of ethanolic rats (E) and their matched controls (C)
650 Segel, Rendig, Choquet, Chacko, Amsterdam, and Mason
designed to determine the effects of prolonged, graded consumption of clinically meaningful quantities of ethanol on the integrity of the rat heart by the simultaneous assessment of the biochemistry, ultrastructure and mechanical performance of the myocardium in wellnourished animals, as well as whether augmented vitamin supplementation provides any protection of the heart during chronic ethanol use.
Methods Animals and protocols
Three series of experiments (designated 1, 2, and 3) with progressive intake of alcohol ranging from 5 to 25% oral ethanol for 25 to 45 wk were carried out sequentially for 96 wk on matched male Sprague-Dawley rats obtained from Animal Resources Services, University of California, Davis. The study protocols and alcohol and feeding regimens are outlined in Table 1. Animals were randomly assigned to groups within each series and were housed two per cage until their weights exceeded 400 g and then were housed individually. Lighting was regulated at 10 hr light per day (7.00 am to 5.00 pm) and room temperature at 22°C. All animals were fed Purina Laboratory Rat Chow (Ralston Purina Co, St Louis, Mo) which contains standard requirements of vitamins and, in groups Cz,Ez,C3B, and EaB, this was supplemented with an additional vitamin mixture (No. 1369, Nutritional Biochemicals Corp, Cleveland, Ohio) ground with the chow to a final concentration of 2.2%. Alcohol (95% laboratory ethanol) was supplied in the drinking water of the ethanol groups. Sucrose (1%) was added to the ethanol solutions to disguise the ethanol taste and thus increase fluid intake. The water of groups COAand CBBalso contained 1% sucrose. Food and liquid were allowed nd lib and measurements of consumption were made periodically with random samples of the groups. Biochemical studies Rats were killed by a blow to the head and the heart was rapidly excised. Samples taken from the
left ventricular apex for catecholamine, bound glycerol, and hydroxyproline determinations were immediately frozen in dry ice. A liver sample was also taken and frozen. Frozen samples were stored at -70°C until analyzed. Samples of the liver and apex of the heart were taken for electron microscopy and light microscopy from randomly selected rats. The remainder of the heart was rinsed in the
Downloaded from by guest on June 7, 2016
failure due to ventricular pump dysfunction is frequently observed in alcoholic patients and impaired cardiac performance has been correlated with prolonged ethanol intake in the absence of overt heart disease (Gould et al, 1969; Regan et al, 1969). Necropsy studies of patients with cardiomyopathy, suspected of having been caused by longterm exposure to alcohol, have revealed scattered foci of concurrent hypertrophy and atrophy of muscle fibres with inflammatory cells, intracellular triglyceride accumulation, and ultrastructural and histochemical defects of mitochondria in myocardial cells (Ferrans et al, 1965; Alexander, 1967; Brigden, 1972). Furthermore, the administration of a single dose of alcohol acutely alters myocardial metabolism and transiently depresses cardiac contractility both in experimental animals (Webb and Degerli, 1965; Regan et al, 1966; Mason et al, 1967; Newman and Valicenti, 1971; Horwitz and Atkins, 1974) and clinically in chronic alcoholics and in normal subjects (Wendt et al, 1966; Regan er al, 1969; Gould, 1970; Ahmed et al, 1973). Some investigators, however, have debated whether alcoholic cardiomyopathy is a specific disease entity, since the evidence is as yet inconclusive that prolonged intake of ethanol alone without concomitant nutritional abnormalities results in permanent myocardial mechanical dysfunction. The majority of experimental and human investigations on alcohol and the heart have been concerned with the acute actions of ethanol. However, recent studies have begun to evaluate the cardiac effects of prolonged administration of the agent in experimental animals. Burch et a1 (1971) have demonstrated substructural alterations in the myocardium of mice which consumed 5-20% ethanol for 7-9 wk. Maines and Aldinger (1967) reported depression of cardiac systolic tension in rats drinking 25% ethanol for 4 months. However contractility was not changed in dogs with disturbances of cardiac mitochondrial function following the oral intake of 25% ethanol for 14 weeks (Pachinger et al, 1973). In view of the uncertain relationship between chronic alcohol ingestion and the metabolic and mechanical function of the ventricle, this report presents an integrated series of studies carried out over a two-year period in our laboratories
61Mechanism of alcoholic cardiomyopathy buffer for isolation of either the myofibrils or mitochondria. The atria and great vessels were trimmed away and the ventricles were minced in a fresh aliquot of the buffer.
Catecholamines Epinephrine and norepinephrine were measured according to the method of Sapira et a1 (1971). Recovery of standard epinephrine plus norepinephrine carried through the procedure was 107%. Hydroxyproline Hydroxyproline was analyzed in tissue hydrolysates on a Beckman Model 121 Automatic Amino Acid Analyzer (Spackman et al, 1958) using AA-15 resin in a 0.9 x 50 cm column. The flow rates were 50 ml/hr for buffer (0.20 nmol/l sodium citrate, pH 3.25) and 25 ml/hr for ninhydrin. The analysis was run at 31°C. The conditions of hydrolysis were determined to be optimum for release of hydroxyproline from the heart samples. Recoveryofhydroxyproline standard carried through the procedure was 88%, with 12% appearing in an unidentified peak following 15 min after the hydroxyproline peak.
Myofibrillar A TPase Cardiac myofibrils were isolated at 4°C by modification of the method of Solaro et a1 (1971) using the isolation buffer of 30 mmol/l KCI, 20 mmol/l Protein determination Tris-CI at pH 7.4 with and without Triton X-100. Protein concentration in the mitochondria1 and Myofibrillar ATPase activity was determined from myofibrillar fractions was measured by a modificathe rate of release of Pi in an incubation medium tion of the biuret method (Gornall et a,, 1949) in of 4 mmol/l ATP, 4 mmol/l MgCI2, 4.3 mmol/l which 1.7% sodium deoxycholate was used as a sodium azide, 0.85 mmol/l CaCI,, 81 mmol/l Tris- solubilizing agent. Crystalline bovineserum albumin C1, pH 7.0, and myofibrils (0.1-0.25 mg protein) in was used as the standard. a volume of 1.28 ml. In some tubes 1 mmol/l ethyleneglycol-bis (p-aminoethyl ether)-N,N’ tetra- Papillary muscle mechanics acetic acid replaced the CaClz to test for C a + + Rats were anaesthetized with interperitoneal dependence. After incubation at 37°C in a reciprocal sodium pentobarbital (0.06 mg/g). The heart was shaking water bath for 5 min, the reaction was quickly excised and a left ventricular papillary stopped with perchloric acid and the protein was muscle was removed and suspended in a tissue bath sedimented. The supernate was assayed for PI by with 10 ml of Ringer-Bicarbonate solution conthe method of Fiske and SubbaRow as described taining 0.1% glucose and 21.5 mmol/l NaHCO, by Leloir and Cardini (1957). Activities are (Long, 1961). The bath was maintained at 30°C and expressed as units/mg protein. Mitochondrial was bubbled continuously with 95% 0,: 5% COz ATPase contamination of the myofibrils was less to maintain a pH of 7.4. Modification of a myothan 2% as measured by assaying without azide. graph described in detail previously (Spann et al, Activity in the absence of C a t + was 13% of that 1967) was utilized to study the muscles contracting in the presence of C a + + , in agreement with the isometrically. An electrical stimulus of 5 ms duration was applied at a rate of 6/min by an AEL results of Solaro et a1 (1971).
Downloaded from by guest on June 7, 2016
Mitochondrial function Mitochondria were isolated from the ventricles by the method of Sordahl and Schwartz (1967) and their function determined using the Gilson Model K-IC oxygraph equipped with a Clark oxygen electrode and the reaction components described by Sordahl et a1 (1971). The mitochondrial pellets obtained by centrifugation at 6300 g (Sordahl and Schwartz, 1967) using the Lourdes 9RA rotor were demonstrated by electron microscopy to consist mainly of mitochondrial elements. No variation in mitocondrial function due to differences in protein concentration was observed in the range of protein used (1-2 mg). All assays were completed within 2.5 h after sacrificing the rats. Mitochondria] parameters measured (Estabrook, 1967; Sordahl et al, 1971) included the respiratory control ratio (RCR: ratio of the rate of oxygen consumption in presence of added ADP to that after ADP has been phosphorylated); ADP/O (ratio of nmol of phosphorylated ADP to natom of consumed oxygen); and respiratory quotient (Qoz : oxygen consumption during the phosphorylation of ADP, expressed as natom of oxygen consumed/s.mg-l of mitocondrial protein during state 3 respiration).
Bound glycerol (triglycerides and phospholipids) Tissue samples were homogenized and extracted twice in cold freshly prepared CHC13:CH30H (2: 1). The dried supernates from the extraction were hydrolyzed in alcoholic KOH, then dried and neutralized. The supernate was brought to pH 7.6 with 0.1 mol/l triethanolamine for assay. No free glycerol was extracted from the tissue by this method since fresh CHCI3:CH30H was used. Measurement of the glycerol released from the triglycerides and phospholipids was based on the method of Eggstein and Kreutz (1966). Recovery of triolein standard was 80%.
0 lo*
0 25f 0 257
25 25
25 25 25 25
26 26 26 26
45 45
25 25 0 0
Vitamin supplement
31.3 & 1.4 21.7k 1.7 28.3 f 1.0 20.0 f 1.4 Palc< 0.01
NM
NS NM
NS 37.8 & 1.8 32.650.9 P < 0.02 41.9f3.1 26.3 f 2.0 33.1 f 2.2 25.8 f 1.8 Pal, < 0.01
30.2 f 5.0 28.0 f 5.0
Chow intake (glratlday)
37.5f 1.2 39.0f 3.9
Liquid intake (mllratlday)
1.7 37.4 1.3 36.6
0 19.3
0 12.4
Liquid calories (kcallratl (hY
NS
NS
NM
NM 114.3 115.5 103.2 108.6
NM 112.6 78.1 101.9 72.0 Palc < 0.01
NS
2.07 f 0.05 2.03 f 0.05
NS 530.6 23.1 554.1 f 8.8 0 31.4 0 32.8
677.4 f 19.7 571.4 f 29.9 625.1 f 19.3 579.8 f 27.8 Pa,, < 0.01
*
2.06 f 0.04 2.29 f 0.09 2.10 f0.05 2.18 0.04 Pal, < 0.025
NM NM
(wk)
0
579.1 f 23.4 594.4+ 35.9
NS
0 9.5
total)
NM
&Y)
NS NM
Ventricular weightlbody weight
108.7 113.2
Final body weight ( g )
108.7 100.8
(%
Ethanol calories
Total calories (kcallrat/ hY)
Chow calories (kcallrat/
Study series = 1,2, and 3 ; C = control group ; E =experimental group. 5% Ethanol for 26 weeks, then 7.5% for 4 weeks and 10% for 15 weeks; chow supplemented with vitamins during final 15 weeks. t 5% Ethanol for 1 week, then 10% for 1 week, 15% for 2 weeks, 20% for 3 weeks, and 25% for 19 weeks; chow supplemented with vitamins for all 26 weeks. N M = n o t measured. NS=not significant (P>0.05).
c3B E3B
E3A
C3A
52
5
G
El
0
25 25
C,
Series Animals % Weeks (n) Ethanol of and group study
Alcohol and dietary regimens
TABLE I
Downloaded from by guest on June 7, 2016
E 3"
%
4
"-
ta
653 Mechanism of alcoholic cardiomyopathy model 104A laboratory stimulator. Voltage was set at 15% above the threshold voltage and continuous recording was made with a Hewlett-Packard 7888A recorder. The control muscle data were obtained after 60 rnin equilibration. Recordings were also made after ethanol addition to the bath, after a wash and 30 rnin re-equilibration, after a 30 min period of hypoxia (bubbling with 95% Nz:5% Coal, and after 30 min of recovery (95% O2:5% COz). The muscle length in the bath was then measured with a calibrated low power Edmund Scientific microscope to a precision of 0.12 mm and, after trimming off the clamped ends, the muscle was weighed to 0.1 mg. The following measurements were made from the recordings: peak tension, T(g/mma); peak rate of tension rise, dT/dt (g/s. mm-a); and time to peak tension, TTPT (ms), from the beginning of contraction. After removing a papillary muscle, the atria and great vessels were trimmed away and ventricular wet weight was measured.
Results Animal and organ weights The animals in the three series of experiments consumed ethanol and regular chow diet ad lib. The rats in the 25% ethanol group (E3Aand E3B) received 31-33% of their total calories from
TABLE 2
Cardiac metabolic function. Mitochondria ADPIO
RCR
Group C,, (n=7) (n = 6) (n = 7) ESB(n = 6)
1.74k0.08 1.5250.08 1.63 +O.OS 1.36 k0.07 P,lc
Effects of chronic graded ethanol consumption on the metabolism, ultrastructure, and mechanical function of the rat heart' LEI GH D . S E G E L , S T E P H E N V . R E N D I G , YVES C H O Q U E T , K U R I E N C H A C K O , E Z R A A . A M S T E R D A M , and D E A N T . MASON
From the Section of Cardiovascular Medicine, Departments of Internal Medicine, Physiology, and Human Anatomy, University of California, School of Medicine Davis and Sacramento, California, U S A
were fed regular chow containing standard vitamins with the ethanol group in each series also receiving a progressively greater alcohol intake for 3 to 6 months: El 5%, E2 lo%, and E3 25% ethanol. Electron microscopy showed swelling of mitochondria, transverse tubules and sarcoplasmic reticulum, dehiscence of intercalated discs and disintegration of myofibrils scattered throughout the ventricular myocardium in El and E2 as early as 7 wk after beginning 5% ethanol; in addition, there were clumping of mitochondria and supercontraction of myofibrils in E,. Concomitant with substructural abnormalities in E,, there were slight but significant depressions of cardiac myofibrillar ATPase activity and mitochondria1 function. Cardiac catecholamines, hydroxyproline, and total bound glycerol were unchanged. Alteration of isometric contraction of isolated, supported left ventricular papillary muscles occurred initially in E2 and was clearly evident in E, by significant reduction of duration of systolic active state (time from onset to peak tension), while total tension generated and peak rate of tension rise were not yet disturbed. Extra vitamin supplementation in additional rats drinking 25% ethanol minimally lessened decline in myofibrillar ATPase activity, but otherwise provided no protection. Thus, chronic daily ingestion of graded quantities of ethanol representing 10 t o 30% of total calories in well-nourished animals exerted toxic effects on microstructure, metabolism and mechanics of the ventricle. These alterations are postulated to be pertinent to early pathogenesis of clinical alcoholic cardiomyopathy. Although the association of chronic alcoholism and heart disease has been recognized clinically for a century (Walshe, 1873), the precise nature of this relationship has not been elucidated and considerable controversy remains concerning the fundamental causative role of ethanol in the This work was supported by NIH Grant MH 19489 (AA00270)and the Vera Button Foundation. Reprint requests to: L.D.S.: Section of Cardiovascular Medicine University of California at Davis, School of Medicine, Davis, California 95616, USA. 1
pathogenesis of myocardial damage. The possibility that alcohol may exert a direct toxic action on heart muscle has been suggested by the increased prevalence of cardiomyopathy in patients with a history of chronic excessive ethanol ingestion (Brigden and Robinson, 1964; Ferrans et-al, 1965; Wendt et al, 1965; Alexander, 1966; Burch and De Pasquale, 1969; Mason el a/, 1971; Regan, 1971; Brigden, 1972; Demakis et al, 1974). Thus, congestive heart
Downloaded from by guest on June 7, 2016
AUTHORS' SYNOPSIS Three sequential sets of ethanolic rats (E) and their matched controls (C)
650 Segel, Rendig, Choquet, Chacko, Amsterdam, and Mason
designed to determine the effects of prolonged, graded consumption of clinically meaningful quantities of ethanol on the integrity of the rat heart by the simultaneous assessment of the biochemistry, ultrastructure and mechanical performance of the myocardium in wellnourished animals, as well as whether augmented vitamin supplementation provides any protection of the heart during chronic ethanol use.
Methods Animals and protocols
Three series of experiments (designated 1, 2, and 3) with progressive intake of alcohol ranging from 5 to 25% oral ethanol for 25 to 45 wk were carried out sequentially for 96 wk on matched male Sprague-Dawley rats obtained from Animal Resources Services, University of California, Davis. The study protocols and alcohol and feeding regimens are outlined in Table 1. Animals were randomly assigned to groups within each series and were housed two per cage until their weights exceeded 400 g and then were housed individually. Lighting was regulated at 10 hr light per day (7.00 am to 5.00 pm) and room temperature at 22°C. All animals were fed Purina Laboratory Rat Chow (Ralston Purina Co, St Louis, Mo) which contains standard requirements of vitamins and, in groups Cz,Ez,C3B, and EaB, this was supplemented with an additional vitamin mixture (No. 1369, Nutritional Biochemicals Corp, Cleveland, Ohio) ground with the chow to a final concentration of 2.2%. Alcohol (95% laboratory ethanol) was supplied in the drinking water of the ethanol groups. Sucrose (1%) was added to the ethanol solutions to disguise the ethanol taste and thus increase fluid intake. The water of groups COAand CBBalso contained 1% sucrose. Food and liquid were allowed nd lib and measurements of consumption were made periodically with random samples of the groups. Biochemical studies Rats were killed by a blow to the head and the heart was rapidly excised. Samples taken from the
left ventricular apex for catecholamine, bound glycerol, and hydroxyproline determinations were immediately frozen in dry ice. A liver sample was also taken and frozen. Frozen samples were stored at -70°C until analyzed. Samples of the liver and apex of the heart were taken for electron microscopy and light microscopy from randomly selected rats. The remainder of the heart was rinsed in the
Downloaded from by guest on June 7, 2016
failure due to ventricular pump dysfunction is frequently observed in alcoholic patients and impaired cardiac performance has been correlated with prolonged ethanol intake in the absence of overt heart disease (Gould et al, 1969; Regan et al, 1969). Necropsy studies of patients with cardiomyopathy, suspected of having been caused by longterm exposure to alcohol, have revealed scattered foci of concurrent hypertrophy and atrophy of muscle fibres with inflammatory cells, intracellular triglyceride accumulation, and ultrastructural and histochemical defects of mitochondria in myocardial cells (Ferrans et al, 1965; Alexander, 1967; Brigden, 1972). Furthermore, the administration of a single dose of alcohol acutely alters myocardial metabolism and transiently depresses cardiac contractility both in experimental animals (Webb and Degerli, 1965; Regan et al, 1966; Mason et al, 1967; Newman and Valicenti, 1971; Horwitz and Atkins, 1974) and clinically in chronic alcoholics and in normal subjects (Wendt et al, 1966; Regan er al, 1969; Gould, 1970; Ahmed et al, 1973). Some investigators, however, have debated whether alcoholic cardiomyopathy is a specific disease entity, since the evidence is as yet inconclusive that prolonged intake of ethanol alone without concomitant nutritional abnormalities results in permanent myocardial mechanical dysfunction. The majority of experimental and human investigations on alcohol and the heart have been concerned with the acute actions of ethanol. However, recent studies have begun to evaluate the cardiac effects of prolonged administration of the agent in experimental animals. Burch et a1 (1971) have demonstrated substructural alterations in the myocardium of mice which consumed 5-20% ethanol for 7-9 wk. Maines and Aldinger (1967) reported depression of cardiac systolic tension in rats drinking 25% ethanol for 4 months. However contractility was not changed in dogs with disturbances of cardiac mitochondrial function following the oral intake of 25% ethanol for 14 weeks (Pachinger et al, 1973). In view of the uncertain relationship between chronic alcohol ingestion and the metabolic and mechanical function of the ventricle, this report presents an integrated series of studies carried out over a two-year period in our laboratories
61Mechanism of alcoholic cardiomyopathy buffer for isolation of either the myofibrils or mitochondria. The atria and great vessels were trimmed away and the ventricles were minced in a fresh aliquot of the buffer.
Catecholamines Epinephrine and norepinephrine were measured according to the method of Sapira et a1 (1971). Recovery of standard epinephrine plus norepinephrine carried through the procedure was 107%. Hydroxyproline Hydroxyproline was analyzed in tissue hydrolysates on a Beckman Model 121 Automatic Amino Acid Analyzer (Spackman et al, 1958) using AA-15 resin in a 0.9 x 50 cm column. The flow rates were 50 ml/hr for buffer (0.20 nmol/l sodium citrate, pH 3.25) and 25 ml/hr for ninhydrin. The analysis was run at 31°C. The conditions of hydrolysis were determined to be optimum for release of hydroxyproline from the heart samples. Recoveryofhydroxyproline standard carried through the procedure was 88%, with 12% appearing in an unidentified peak following 15 min after the hydroxyproline peak.
Myofibrillar A TPase Cardiac myofibrils were isolated at 4°C by modification of the method of Solaro et a1 (1971) using the isolation buffer of 30 mmol/l KCI, 20 mmol/l Protein determination Tris-CI at pH 7.4 with and without Triton X-100. Protein concentration in the mitochondria1 and Myofibrillar ATPase activity was determined from myofibrillar fractions was measured by a modificathe rate of release of Pi in an incubation medium tion of the biuret method (Gornall et a,, 1949) in of 4 mmol/l ATP, 4 mmol/l MgCI2, 4.3 mmol/l which 1.7% sodium deoxycholate was used as a sodium azide, 0.85 mmol/l CaCI,, 81 mmol/l Tris- solubilizing agent. Crystalline bovineserum albumin C1, pH 7.0, and myofibrils (0.1-0.25 mg protein) in was used as the standard. a volume of 1.28 ml. In some tubes 1 mmol/l ethyleneglycol-bis (p-aminoethyl ether)-N,N’ tetra- Papillary muscle mechanics acetic acid replaced the CaClz to test for C a + + Rats were anaesthetized with interperitoneal dependence. After incubation at 37°C in a reciprocal sodium pentobarbital (0.06 mg/g). The heart was shaking water bath for 5 min, the reaction was quickly excised and a left ventricular papillary stopped with perchloric acid and the protein was muscle was removed and suspended in a tissue bath sedimented. The supernate was assayed for PI by with 10 ml of Ringer-Bicarbonate solution conthe method of Fiske and SubbaRow as described taining 0.1% glucose and 21.5 mmol/l NaHCO, by Leloir and Cardini (1957). Activities are (Long, 1961). The bath was maintained at 30°C and expressed as units/mg protein. Mitochondrial was bubbled continuously with 95% 0,: 5% COz ATPase contamination of the myofibrils was less to maintain a pH of 7.4. Modification of a myothan 2% as measured by assaying without azide. graph described in detail previously (Spann et al, Activity in the absence of C a t + was 13% of that 1967) was utilized to study the muscles contracting in the presence of C a + + , in agreement with the isometrically. An electrical stimulus of 5 ms duration was applied at a rate of 6/min by an AEL results of Solaro et a1 (1971).
Downloaded from by guest on June 7, 2016
Mitochondrial function Mitochondria were isolated from the ventricles by the method of Sordahl and Schwartz (1967) and their function determined using the Gilson Model K-IC oxygraph equipped with a Clark oxygen electrode and the reaction components described by Sordahl et a1 (1971). The mitochondrial pellets obtained by centrifugation at 6300 g (Sordahl and Schwartz, 1967) using the Lourdes 9RA rotor were demonstrated by electron microscopy to consist mainly of mitochondrial elements. No variation in mitocondrial function due to differences in protein concentration was observed in the range of protein used (1-2 mg). All assays were completed within 2.5 h after sacrificing the rats. Mitochondria] parameters measured (Estabrook, 1967; Sordahl et al, 1971) included the respiratory control ratio (RCR: ratio of the rate of oxygen consumption in presence of added ADP to that after ADP has been phosphorylated); ADP/O (ratio of nmol of phosphorylated ADP to natom of consumed oxygen); and respiratory quotient (Qoz : oxygen consumption during the phosphorylation of ADP, expressed as natom of oxygen consumed/s.mg-l of mitocondrial protein during state 3 respiration).
Bound glycerol (triglycerides and phospholipids) Tissue samples were homogenized and extracted twice in cold freshly prepared CHC13:CH30H (2: 1). The dried supernates from the extraction were hydrolyzed in alcoholic KOH, then dried and neutralized. The supernate was brought to pH 7.6 with 0.1 mol/l triethanolamine for assay. No free glycerol was extracted from the tissue by this method since fresh CHCI3:CH30H was used. Measurement of the glycerol released from the triglycerides and phospholipids was based on the method of Eggstein and Kreutz (1966). Recovery of triolein standard was 80%.
0 lo*
0 25f 0 257
25 25
25 25 25 25
26 26 26 26
45 45
25 25 0 0
Vitamin supplement
31.3 & 1.4 21.7k 1.7 28.3 f 1.0 20.0 f 1.4 Palc< 0.01
NM
NS NM
NS 37.8 & 1.8 32.650.9 P < 0.02 41.9f3.1 26.3 f 2.0 33.1 f 2.2 25.8 f 1.8 Pal, < 0.01
30.2 f 5.0 28.0 f 5.0
Chow intake (glratlday)
37.5f 1.2 39.0f 3.9
Liquid intake (mllratlday)
1.7 37.4 1.3 36.6
0 19.3
0 12.4
Liquid calories (kcallratl (hY
NS
NS
NM
NM 114.3 115.5 103.2 108.6
NM 112.6 78.1 101.9 72.0 Palc < 0.01
NS
2.07 f 0.05 2.03 f 0.05
NS 530.6 23.1 554.1 f 8.8 0 31.4 0 32.8
677.4 f 19.7 571.4 f 29.9 625.1 f 19.3 579.8 f 27.8 Pa,, < 0.01
*
2.06 f 0.04 2.29 f 0.09 2.10 f0.05 2.18 0.04 Pal, < 0.025
NM NM
(wk)
0
579.1 f 23.4 594.4+ 35.9
NS
0 9.5
total)
NM
&Y)
NS NM
Ventricular weightlbody weight
108.7 113.2
Final body weight ( g )
108.7 100.8
(%
Ethanol calories
Total calories (kcallrat/ hY)
Chow calories (kcallrat/
Study series = 1,2, and 3 ; C = control group ; E =experimental group. 5% Ethanol for 26 weeks, then 7.5% for 4 weeks and 10% for 15 weeks; chow supplemented with vitamins during final 15 weeks. t 5% Ethanol for 1 week, then 10% for 1 week, 15% for 2 weeks, 20% for 3 weeks, and 25% for 19 weeks; chow supplemented with vitamins for all 26 weeks. N M = n o t measured. NS=not significant (P>0.05).
c3B E3B
E3A
C3A
52
5
G
El
0
25 25
C,
Series Animals % Weeks (n) Ethanol of and group study
Alcohol and dietary regimens
TABLE I
Downloaded from by guest on June 7, 2016
E 3"
%
4
"-
ta
653 Mechanism of alcoholic cardiomyopathy model 104A laboratory stimulator. Voltage was set at 15% above the threshold voltage and continuous recording was made with a Hewlett-Packard 7888A recorder. The control muscle data were obtained after 60 rnin equilibration. Recordings were also made after ethanol addition to the bath, after a wash and 30 rnin re-equilibration, after a 30 min period of hypoxia (bubbling with 95% Nz:5% Coal, and after 30 min of recovery (95% O2:5% COz). The muscle length in the bath was then measured with a calibrated low power Edmund Scientific microscope to a precision of 0.12 mm and, after trimming off the clamped ends, the muscle was weighed to 0.1 mg. The following measurements were made from the recordings: peak tension, T(g/mma); peak rate of tension rise, dT/dt (g/s. mm-a); and time to peak tension, TTPT (ms), from the beginning of contraction. After removing a papillary muscle, the atria and great vessels were trimmed away and ventricular wet weight was measured.
Results Animal and organ weights The animals in the three series of experiments consumed ethanol and regular chow diet ad lib. The rats in the 25% ethanol group (E3Aand E3B) received 31-33% of their total calories from
TABLE 2
Cardiac metabolic function. Mitochondria ADPIO
RCR
Group C,, (n=7) (n = 6) (n = 7) ESB(n = 6)
1.74k0.08 1.5250.08 1.63 +O.OS 1.36 k0.07 P,lc
