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Alcoholic cardiomyopathy II. The inhibition of cardiac microsomal protein synthesis by acetaldehyde
Journal
II.
of Molecular
The
and Cellular
Cardiology
(1974)
6, 207-213
Alcoholic Cardiomyopathy of Cardiac Mkrosomal Acetaldehyde*
Inhibition
Protein
Synthesis
by
SIDNEY S. SCHREIBER, MURRAY ORATZ, MARCUS A. ROTHSCHILD, FRANCINE REFF AND CAROLE EVANS Department of Nuclear Medicine, New York Veterans Hospital and the Department of Medicine New Tork University School of Medicine, New York, U.S.A. (Received
1 June 1973,
and accepted
10 August
1973)
S. S. SCHREIBER, M. ORATZ, M. A. ROTHSCHILD, F. REFF AND C. EVANS. Alcoholic Cardiomyopathy, II. The Inhibition of Cardiac Microsomal Protein Synthesis by Acetaldehyde,3ournaI of Molecular and Cellular Cardiology (1974) 6,207-2 13. Previous studies in this laboratory had shown that while ethanol at levels of 200 to 300 mg/lOO ml had no effect on cardiac protein synthesis, acetaldehyde (3.5 mg/lOO ml or 0.8 rrm) markedly inhibited cardiac protein synthesis in the intact heart in u&o. In order to localiie further the action of acetaldehyde and to separate the protein synthetic effects from contractile function, studies on cell free systems with cardiac muscle microsomes were carried out at concentrations of acetaldehyde seen in humans after moderate ethanol ingestion. There was a significant reduction of microsomal protein synthesis even at these levels of acetaldehyde. Thus, with an acetaldehyde concentration of 0.53 mg/ 100 ml (0.12 nnr) the protein synthesis was reduced to 52 + 5.3% of the control microsomes. With acetaldehyde concentrations of 0.13 to 0.26 mg/lOO ml (0.03 to 0.06 nm), the microsomal protein synthesis was 65 f 8.6% of the controls. The differences from the controls were statistically significant. These data show that at concentrations seen in humans following ethanol ingestion, acetaldehyde interferes with normal cardiac protein synthesis independent of contractile action and thus may play a role in the ultimate development of ethanolic cardiomyopathy.
KEY WORDS : Alcoholic
cardiomyopathy;
Acetaldehyde;
Protein
synthesis.
I. Introduction The occurrence of cardiac diseasein alcoholics is well known, but the causesare still unclear. Accumulation of triglycerides and decreasedextraction of fatty acids [7] have been suggestedascausesof the cardiomyopathy, but it hasalsobeen postulated that a metabolite, acetaldehyde, may play a role in the development of alcoholic cardiomyopathy [4, 5, 91. In recent studies with the isolated perfused guinea pig heart, perfusion with 0.8 mM-acetaldehyde (3.5 mg%) evoked a marked inhibition * Aided in part by a grant from the National Heart and Lung Institute HLO9562, from the Licensed Beverage Industries, and from a grant from the Louise and Bernard Palitz Research Fund.
208
S. S. SCHREIBER
ETAL.
of cardiac protein synthesis despite a concomitant inotropic and chronotropic effect [9]. In order to localize further the site of action of acetaldehyde on protein synthesis, studies were carried out on cell free systems with cardiac muscle microsomes to see if there was direct inhibition of the cardiac protein synthetic mechanism apart from the effects of acetaldehyde on contractile function. Furthermore, since earlier studies used concentrations of acetaldehyde higher than those found in humans after ethanol ingestion [S, 131, it was of interest to determine whether the lower levels of acetaldehyde could also effect alterations in cardiac protein synthesis. The present report presents data obtained from studies with cardiac muscle microsomes in cell free systems exposed to such low levels.
2. Methods
Microsome protein synthesis: The ability of microsomes obtained from normal unperfused hearts to incorporate labelled amino acids into protein was testedin cell free systemswith and without acetaldehyde. Guinea pigs, weighing 300 to 350 g approximately 3 to 4 weeks after weaning were used in the studies. The animals were fed ad lib on standard laboratory chow and were not fasted prior to the experiment. On the day of the study, they were anesthetized with Nembutal [g-11], the chest cage rapidly opened, the heart washed free of blood with ice cold oxygenated Krebs-Henseleit solution and removed. The auricles were excised, and the ventricles homogenized and microsomesisolated as previously described [I,?]. The methods for measuring microsomal protein synthesiswere as previously used in this laboratory [11, 121 with the modification that the cell sap used for incubation media obtained from the post microsomal supematant was passed through a Sephadex G-25 column and then fortified with essential and nonessential L-amino acids each in concentration of 10-4 M. (No [rsC]leucine was added). Incorporation of L-[W]leucine (specific activity 255 mCi/mmol) into microsomal protein was used as an index of protein synthesis. Since it has been demonstrated that radioactivity may be trapped by precipitated pellets in proportion to the activity added [I, 11, 121, it was essentialthat this non-specific binding be eliminated as an artifact. For this reason, different quantities of radioactivity were added on different days to vary the incorporation by control microsomes 30 to 40 fold. On any single day, the controls and acetaldehyde incubated received identical quantities of [W]leucine. Each control and experimental incubation tube contained a final volume of 0.8 ml which included cell sap and amino acids, ATP generating system and enzymes [I.?], and identical quantities of the same batch of microsomesin each study. The concentrations of ingredients in the 0.8 ml volume in the incubation tubes were as follows: sucrose0.15 M; KC1 0.025 M; MgCl2 0.01 M; Tris buffer 0.05 M; ATP 1 mM; GTP 0.5 mu, PK 12.5 p&ml; PEP 10 mu; mercaptoethanol
ALCOHOLIC
CARDIOMYOPATHY
209
0.6 mM. Microsomal RNA was approximately 45 pg/ml of final volume for all studies. All tubes were kept at 1°C until incubation was initiated. Acetaldehyde (redistilled, Eastman Organic Chemicals, stored in sealed ampules at -60°C) was dissolved in homogenizing buffer in sufficient concentration so that 0.1 ml added to the incubation tubes would give a final concentration of 0.12, 0.06, or 0.03 mM (0.53, 0.26 or 0.13 mg%). In the controls, 0.1 ml buffer was added without the acetaldehyde. Since evaporation of acetaldehyde from solution is very. rapid [4] each tube was layered with 0.5 ml of mineral oil to prevent loss from the incubation solution for the next hour [9]. Control tubes were similarly treated. The incubation tubes and the appropriate blanks (incubates with all ingredients but microsomes missing) were placed in a shaker bath and gently agitated for 1 h at 37°C. It has been previously demonstrated that incorporation of lGamino acids into cardiac muscle microsomal protein is linear for 1 h [II]. At the end of this period, the incubation was stopped by the addition of 2 ml of ice cold trichloro-acetic acid (TCA) containing 1.2 x 10-s M non-radioactive L-leucine as carrier. After precipitation in TCA, the tubes were centrifuged, the protein pellets washed repeatedly with TCA and stable leucine to remove free [%]leucine, and the washed pellet was then dissolved in 1 M-NaOH. Following solution of protein in the NaOH, a known quantity was pipetted on a l-in square of Whatman 3MM filter paper, the paper square dried and then treated with cold 10% TCA, washed with 10% TCA, heated for 15 min at 95°C in 10% TCA to hydrolyze tRNAamino acid-r4C, washed and then treated with ethanol, ethanol+ther and ether as described previously [II, 121. Radioactivity in the resultant solution was determined with a liquid scintillation counter by standard methods. (Samples in butyl PBD-toluene with added standards of 14C-amino acids to correct for quenching [IO, 21, 121) . RNA in the microsomal samples was determined in microsomal aliquots by the method of Fleck and Begg [3]. Perfusion studies were carried out as recently described with higher levels of acetaldehyde [9], with the modification that the acetaldehyde level was 0.2 mM (0.88 mg%) and the duration of the experiment was 5 h. The perfusate was an L-amino acid fortified Krebs-Henseleit solution oxygenated with 95% 0~5% COz with the pH at 7.45 at 37°C and containing uniformly labelled L-[iG]lysine (specific activity 0.025 @i/pmol, obtained from New England Nuclear Co., with greater than 98% purity as L-lysine by chromatography). The fluid load to the perfused heart was 45 to 50 ml min-1 g-1 dry heart weight and the aortic pressure was maintained at 40 to 50 mmHg. Evaporation of acetaldehyde from the perfusate reservoir was prevented by layering mineral oil over the solution after it had been maximally oxygenated. The maintenance of oxygen tension and pH by this method without 10s~ of acetaldehyde has been previously documented [9]. Oxidation of acetaldehyde to acetic acid in the perfusate was probably negligible since measurements of acetaldehyde by enzymic methods [9] showed no alteration in acetaldehyde concentration in the perfusate after 3 to 5 h and the pH was
210
S. S. SCHREIBER
ETAL.
stable throughout. Furthermore, quantities of acetic acid sufficient to alter pH were not found in the subcellular incubates. After completion of perfusion, protein synthesis was determined from the fraction : L-[i%]lysine/g L-[%]lysine/~mol
protein N in left ventricle L-lysine in intracellular
The determination of the intracellular [i%]lysine pool was as described in earlier studies [9, IO].
ventricle
pool
specific activity in the free lysine
3. Results Microsome
studies
The results of the studiesare shown in Table 1. On different days, the quantities of [‘“Cl leucine used in the experiments was varied to assurethat the incorporation of radioactive leucine into the microsomal protein was not concentration dependent. In ten studieswith the acetaldehyde concentration of 0.12 mM cardiac microsomes obtained from unperfused normal hearts showed a marked inhibition of protein synthesis (52% of the control, P < 0.001). When the level of acetaldehyde was reduced to 0.03-0.06 mM, protein synthesiswas still inhibited to nearly 65% of that in controls. Since the meansfor the two lower groups was the same,the values were pooled and the mean showed significant decreasefrom the control (P < 0.01).
Perfwion
studies
Left ventricular contractility was maintained without failure similar to studies previously reported [9, 121 and acetaldehyde perfusion at 0.2 mM levels gave a similar chronotropic and inotropic effect. Furthermore, in 12 perfusion experiments, protein synthesis of mixed left ventricular protein was 37.5 f 2.1 pmol lysine incorporated/g protein N in the controls after 5 h of perfusion with constant perfusate lysine specific activity while the acetaldehyde perfused hearts showedonly 28.3 f 2.4 p.mol incorporated during the sameperiod. The difference was significant (P < 0.05) and similar to the findings reported with higher levels of 0.8 mMacetaldehyde [9]. 4. Discussion
Previous studies in our laboratory have shown that acute exposure to ethanol at levels of 200 mg/lOO ml was sufficient to depressmarkedly hepatic albumin synthesis[8] while the samelevel neither altered measuredparameters of cardiac function
ALCOHOLIC
TABLE Exp
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Mean 11. 12. 13. 14. 15. 16. 17. 18. Mean
1. Effect of acetaldehyde Controls synthesis (ct min-l/mg RNA)
No.
12 200 2600 29 700 27 600 4460 41800 13 750 1221 1507 1500 Nos.
on cardiac microsome
protein synthesis
Acetaldehyde Concentration synthesis (ct min-l/mg bM) RNA) 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12
3700 1400 16 900 16400 1540 5380 8250 902 1178 755
1 to 10 1221 13 750 1507 1500 1221 13 750 1507 1500
Nos.
211
CARDIOMYOPATHY
11 to 18
0.06 0.06 0.06 0.06 0.03 0.03 0.03 0.03
544 9340 1537 585 715 11 780 1310 592
o/0 of control
30 54 57 59 35 13 60 74 78 50 52 (P < 45 68 101 39 56 86 87 39 65 (P <
jy 5.3 0.001)
& 8.6 0.01)
In any one experiment the same group of microsomes was used for control and experimentaks. Different quantities of radioactivity were used on different days (see Methods). Synthesis was deiined as the activity of L-[Wlleucine incorporated into microsomal protein: P values indicate significance from the controls. Controls for Experiments Nos. 7 to 10 were the same for Nos. 11 to 14 and 15 to 18.
or protein synthesis in the perfused heart [9]. However, 0.8 rnM-acetaldehyde effected a marked decrease in protein synthesis while it produced a chronotropic and inotropic effect [9]. Recent data from our laboratory have also shown that when livers from fed rabbit donors were perfused with 200 mg/lOO ml ethanol, the acetaldehyde concentration in the perfusate averaged 0.3 mM (unpublished data), Furthermore, blood concentrations of acetaldehyde in humans after alcohol ingestion have been found in the range of 0.03 to 0.04 mu [S] to levels of 0.16 to 0.3 mM [12]. The present report presents data to show that even at these lower levels, acetaldehyde inhibits cardiac protein synthesis. Although studies with acetaldehyde had suggested that the primary effect of this metabolite of ethanol was inotropic and chronotropic stimulation of contraction possibly leading to cardiomyopathy [5], later studies in our laboratory suggested
212
S. S. SCHREIBER
ETAL.
that decreased protein synthesis by acetaldehyde may play a role in the development of this myopathy [9]. Th e results of the microsome studies presented here emphasize that the inhibitition of protein synthesis by acetaldehyde is directly on the protein synthetic machinery and apparently independent of its effects on contraction. In the perfused intact heart, inhibition of protein synthesis by acetaldehyde was not manifest until 3-5 h of perfusion [9], while microsomal inhibition in cell free systems was seen with lower levels after 1 h of incubation. One may speculate that this difference in apparent time of acetaldehyde effect may be due to the lag in appearance of acetaldehyde at the site of protein synthesis within the cell due to diffusion delays or oxidation of the acetaldehyde [Z, 141 before it reaches the synthetic sites, and only after the NAD is used up, is the aldehyde able to reach the critical area. In contrast, in the cell free system, acetaldehyde is at the site of protein synthesis almost immediately. It may be suggested that the inhibitory action of acetaldehyde is due to interference of acetaldehyde with the ATP generating system. Inhibition of protein synthesis occurred in the intact heart in uitro as well as in subcellular systems. The isolated perfused baby guinea pig heart is exquisitely sensitive to decreased ATP levels, e.g. in anoxia or decreased coronary flow, contractility is almost immediately diminished and complete failure in asystole occurs by 10 or 15 min (unpublished data). In contrast, the present studies and those reported previously show a continued stimulation of contractility with acetaldehyde above that of controls with perfusion for 3 or 5 h. It would seem therefore that any diminution in ATP generation by acetaldehyde would have to be just enough to allow enhanced contraction but to inhibit protein synthesis or that only a compartmentalized ATP related to protein synthesis is affected. At this time, ATP data with acetaldehyde are not available to support either contention. Although the site of acetaldehyde action on the microsomes is still not clarified, the data presented here indicate that cardiac protein synthesis is sensitive to the levels of acetaldehyde seen in humans after moderate alcohol ingestion [S, 131 or manufactured by the mammalian liver in vitro. In view of the findings that cardiac protein turnover may be very rapid [lo], it is suggested that continued exposure even to these low levels of acetaldehyde may play a role in the development of cardiomyopathy in chronic alcoholism due to interference with normal cardiac protein synthesis. REFERENCES 1.
CAMPBELL,
albumin 262-266 2.
DIETRICH, capacity.
P. N. and
tiRNOT,
B. A. The
by the isolated microsome (1961). R. A. Tissue
and subcellular
Biochemical Pharmmolo~
of [l%]leucine into serum from rat liver. Biochemical Journal 82,
incorporation
fraction distribution
15, 1911 (1966).
of mammalian
aldehyde
oxidizing
ALCOHOLIC
3. 4. 5. 6. 7.
8.
9.
10. 11. 12. 13. 14.
CARDIOMYOPATHY
213
FLECK, A. & BEGG, D. The estimation of ribonucleic acid using ultraviolet absorption measurements. Biochimica et Bi@hysica Acta 108, 333-339 (1965). GAILIS, L. & VERDY, M. The effect of ethanol and acetaldehyde on the metabolism and vascular resistance of the perfused heart. Canadian Journal of Biochemistry 49, 227-233 (1971). JAMES, T. N. & BEAR, E. S. Effects of ethanol and acetaldehyde on the heart. American Heart Journal 74, 243-255 (1967). MAJCHROWICZ, E. & MENDELSON, J. H. Blood concentrations of acetaldehyde and ethanol in chronic alcoholics. Science 168, 1100-l 102 (1970). REGAN, T. J., KOROXENIDIS, G., MOSCHOS, C. B., OLDEWURTEL, H. A., LEHAN, P. H. HELLEMS, H. K. The acute metabolic and hemodynamic responses of the left ventricle to ethanol. 3oumE of Clinical Investigation 45, 270-280 (1966). ROTHSCHILD, M. A., ORATZ, M., MONGELLI, J. & SCHREIBER, S. S. Alcohol-induced depression of albumin synthesis : reversal by tryptophan. Journal of Clinical Investigation 50,1812-1818 (1971). SCHREIBER, S. S., BRIDEN, K., ORATZ, M. & ROTHSCHILD, M. A. Ethanol, acetaldehyde and myocardial protein synthesis. 3ournd of Clinical Investigation 51,2820-2826 (1972). SCHREIBER, S. S., ORATZ, M., EVANS, C., REFF, F., KLEIN, I. & ROTHSCHILD, M, A. Cardiac protein degradation in acute overload in vitro: reutilization of amino acids. American 3ounzal of Physiology 224, 338-345 ( 1973). SCHREIBER, S. S., ORATZ, M., EVANS, C., SILVER, E. & ROTHSCHILD, M. A. Effect of acute overload on cardiac muscle mRNA. American Journal of Physiology 215, 12501259 (1968). SCHREIBER, S. S., ORATZ, M. & ROTHSCHILD, M. A. Effect of acute overload on protein synthesis in cardiac muscle microsomes. American Journal of Physiology 213, 1552-1555 (1967). &OR, E. A calorimetric determination of acetaldehyde in blood. Journal of Biological Chemistry 148, 585-59 1 ( 1943). VON WARTBURG, J. P. The metabolism of alcohol in normals and alcoholics: Enzymes. 7% Biology of Alcoholism. (Kissin, B. and Begleiter, H., Eds). Plenum Press, New York. 63-102 (1971).
II.
of Molecular
The
and Cellular
Cardiology
(1974)
6, 207-213
Alcoholic Cardiomyopathy of Cardiac Mkrosomal Acetaldehyde*
Inhibition
Protein
Synthesis
by
SIDNEY S. SCHREIBER, MURRAY ORATZ, MARCUS A. ROTHSCHILD, FRANCINE REFF AND CAROLE EVANS Department of Nuclear Medicine, New York Veterans Hospital and the Department of Medicine New Tork University School of Medicine, New York, U.S.A. (Received
1 June 1973,
and accepted
10 August
1973)
S. S. SCHREIBER, M. ORATZ, M. A. ROTHSCHILD, F. REFF AND C. EVANS. Alcoholic Cardiomyopathy, II. The Inhibition of Cardiac Microsomal Protein Synthesis by Acetaldehyde,3ournaI of Molecular and Cellular Cardiology (1974) 6,207-2 13. Previous studies in this laboratory had shown that while ethanol at levels of 200 to 300 mg/lOO ml had no effect on cardiac protein synthesis, acetaldehyde (3.5 mg/lOO ml or 0.8 rrm) markedly inhibited cardiac protein synthesis in the intact heart in u&o. In order to localiie further the action of acetaldehyde and to separate the protein synthetic effects from contractile function, studies on cell free systems with cardiac muscle microsomes were carried out at concentrations of acetaldehyde seen in humans after moderate ethanol ingestion. There was a significant reduction of microsomal protein synthesis even at these levels of acetaldehyde. Thus, with an acetaldehyde concentration of 0.53 mg/ 100 ml (0.12 nnr) the protein synthesis was reduced to 52 + 5.3% of the control microsomes. With acetaldehyde concentrations of 0.13 to 0.26 mg/lOO ml (0.03 to 0.06 nm), the microsomal protein synthesis was 65 f 8.6% of the controls. The differences from the controls were statistically significant. These data show that at concentrations seen in humans following ethanol ingestion, acetaldehyde interferes with normal cardiac protein synthesis independent of contractile action and thus may play a role in the ultimate development of ethanolic cardiomyopathy.
KEY WORDS : Alcoholic
cardiomyopathy;
Acetaldehyde;
Protein
synthesis.
I. Introduction The occurrence of cardiac diseasein alcoholics is well known, but the causesare still unclear. Accumulation of triglycerides and decreasedextraction of fatty acids [7] have been suggestedascausesof the cardiomyopathy, but it hasalsobeen postulated that a metabolite, acetaldehyde, may play a role in the development of alcoholic cardiomyopathy [4, 5, 91. In recent studies with the isolated perfused guinea pig heart, perfusion with 0.8 mM-acetaldehyde (3.5 mg%) evoked a marked inhibition * Aided in part by a grant from the National Heart and Lung Institute HLO9562, from the Licensed Beverage Industries, and from a grant from the Louise and Bernard Palitz Research Fund.
208
S. S. SCHREIBER
ETAL.
of cardiac protein synthesis despite a concomitant inotropic and chronotropic effect [9]. In order to localize further the site of action of acetaldehyde on protein synthesis, studies were carried out on cell free systems with cardiac muscle microsomes to see if there was direct inhibition of the cardiac protein synthetic mechanism apart from the effects of acetaldehyde on contractile function. Furthermore, since earlier studies used concentrations of acetaldehyde higher than those found in humans after ethanol ingestion [S, 131, it was of interest to determine whether the lower levels of acetaldehyde could also effect alterations in cardiac protein synthesis. The present report presents data obtained from studies with cardiac muscle microsomes in cell free systems exposed to such low levels.
2. Methods
Microsome protein synthesis: The ability of microsomes obtained from normal unperfused hearts to incorporate labelled amino acids into protein was testedin cell free systemswith and without acetaldehyde. Guinea pigs, weighing 300 to 350 g approximately 3 to 4 weeks after weaning were used in the studies. The animals were fed ad lib on standard laboratory chow and were not fasted prior to the experiment. On the day of the study, they were anesthetized with Nembutal [g-11], the chest cage rapidly opened, the heart washed free of blood with ice cold oxygenated Krebs-Henseleit solution and removed. The auricles were excised, and the ventricles homogenized and microsomesisolated as previously described [I,?]. The methods for measuring microsomal protein synthesiswere as previously used in this laboratory [11, 121 with the modification that the cell sap used for incubation media obtained from the post microsomal supematant was passed through a Sephadex G-25 column and then fortified with essential and nonessential L-amino acids each in concentration of 10-4 M. (No [rsC]leucine was added). Incorporation of L-[W]leucine (specific activity 255 mCi/mmol) into microsomal protein was used as an index of protein synthesis. Since it has been demonstrated that radioactivity may be trapped by precipitated pellets in proportion to the activity added [I, 11, 121, it was essentialthat this non-specific binding be eliminated as an artifact. For this reason, different quantities of radioactivity were added on different days to vary the incorporation by control microsomes 30 to 40 fold. On any single day, the controls and acetaldehyde incubated received identical quantities of [W]leucine. Each control and experimental incubation tube contained a final volume of 0.8 ml which included cell sap and amino acids, ATP generating system and enzymes [I.?], and identical quantities of the same batch of microsomesin each study. The concentrations of ingredients in the 0.8 ml volume in the incubation tubes were as follows: sucrose0.15 M; KC1 0.025 M; MgCl2 0.01 M; Tris buffer 0.05 M; ATP 1 mM; GTP 0.5 mu, PK 12.5 p&ml; PEP 10 mu; mercaptoethanol
ALCOHOLIC
CARDIOMYOPATHY
209
0.6 mM. Microsomal RNA was approximately 45 pg/ml of final volume for all studies. All tubes were kept at 1°C until incubation was initiated. Acetaldehyde (redistilled, Eastman Organic Chemicals, stored in sealed ampules at -60°C) was dissolved in homogenizing buffer in sufficient concentration so that 0.1 ml added to the incubation tubes would give a final concentration of 0.12, 0.06, or 0.03 mM (0.53, 0.26 or 0.13 mg%). In the controls, 0.1 ml buffer was added without the acetaldehyde. Since evaporation of acetaldehyde from solution is very. rapid [4] each tube was layered with 0.5 ml of mineral oil to prevent loss from the incubation solution for the next hour [9]. Control tubes were similarly treated. The incubation tubes and the appropriate blanks (incubates with all ingredients but microsomes missing) were placed in a shaker bath and gently agitated for 1 h at 37°C. It has been previously demonstrated that incorporation of lGamino acids into cardiac muscle microsomal protein is linear for 1 h [II]. At the end of this period, the incubation was stopped by the addition of 2 ml of ice cold trichloro-acetic acid (TCA) containing 1.2 x 10-s M non-radioactive L-leucine as carrier. After precipitation in TCA, the tubes were centrifuged, the protein pellets washed repeatedly with TCA and stable leucine to remove free [%]leucine, and the washed pellet was then dissolved in 1 M-NaOH. Following solution of protein in the NaOH, a known quantity was pipetted on a l-in square of Whatman 3MM filter paper, the paper square dried and then treated with cold 10% TCA, washed with 10% TCA, heated for 15 min at 95°C in 10% TCA to hydrolyze tRNAamino acid-r4C, washed and then treated with ethanol, ethanol+ther and ether as described previously [II, 121. Radioactivity in the resultant solution was determined with a liquid scintillation counter by standard methods. (Samples in butyl PBD-toluene with added standards of 14C-amino acids to correct for quenching [IO, 21, 121) . RNA in the microsomal samples was determined in microsomal aliquots by the method of Fleck and Begg [3]. Perfusion studies were carried out as recently described with higher levels of acetaldehyde [9], with the modification that the acetaldehyde level was 0.2 mM (0.88 mg%) and the duration of the experiment was 5 h. The perfusate was an L-amino acid fortified Krebs-Henseleit solution oxygenated with 95% 0~5% COz with the pH at 7.45 at 37°C and containing uniformly labelled L-[iG]lysine (specific activity 0.025 @i/pmol, obtained from New England Nuclear Co., with greater than 98% purity as L-lysine by chromatography). The fluid load to the perfused heart was 45 to 50 ml min-1 g-1 dry heart weight and the aortic pressure was maintained at 40 to 50 mmHg. Evaporation of acetaldehyde from the perfusate reservoir was prevented by layering mineral oil over the solution after it had been maximally oxygenated. The maintenance of oxygen tension and pH by this method without 10s~ of acetaldehyde has been previously documented [9]. Oxidation of acetaldehyde to acetic acid in the perfusate was probably negligible since measurements of acetaldehyde by enzymic methods [9] showed no alteration in acetaldehyde concentration in the perfusate after 3 to 5 h and the pH was
210
S. S. SCHREIBER
ETAL.
stable throughout. Furthermore, quantities of acetic acid sufficient to alter pH were not found in the subcellular incubates. After completion of perfusion, protein synthesis was determined from the fraction : L-[i%]lysine/g L-[%]lysine/~mol
protein N in left ventricle L-lysine in intracellular
The determination of the intracellular [i%]lysine pool was as described in earlier studies [9, IO].
ventricle
pool
specific activity in the free lysine
3. Results Microsome
studies
The results of the studiesare shown in Table 1. On different days, the quantities of [‘“Cl leucine used in the experiments was varied to assurethat the incorporation of radioactive leucine into the microsomal protein was not concentration dependent. In ten studieswith the acetaldehyde concentration of 0.12 mM cardiac microsomes obtained from unperfused normal hearts showed a marked inhibition of protein synthesis (52% of the control, P < 0.001). When the level of acetaldehyde was reduced to 0.03-0.06 mM, protein synthesiswas still inhibited to nearly 65% of that in controls. Since the meansfor the two lower groups was the same,the values were pooled and the mean showed significant decreasefrom the control (P < 0.01).
Perfwion
studies
Left ventricular contractility was maintained without failure similar to studies previously reported [9, 121 and acetaldehyde perfusion at 0.2 mM levels gave a similar chronotropic and inotropic effect. Furthermore, in 12 perfusion experiments, protein synthesis of mixed left ventricular protein was 37.5 f 2.1 pmol lysine incorporated/g protein N in the controls after 5 h of perfusion with constant perfusate lysine specific activity while the acetaldehyde perfused hearts showedonly 28.3 f 2.4 p.mol incorporated during the sameperiod. The difference was significant (P < 0.05) and similar to the findings reported with higher levels of 0.8 mMacetaldehyde [9]. 4. Discussion
Previous studies in our laboratory have shown that acute exposure to ethanol at levels of 200 mg/lOO ml was sufficient to depressmarkedly hepatic albumin synthesis[8] while the samelevel neither altered measuredparameters of cardiac function
ALCOHOLIC
TABLE Exp
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Mean 11. 12. 13. 14. 15. 16. 17. 18. Mean
1. Effect of acetaldehyde Controls synthesis (ct min-l/mg RNA)
No.
12 200 2600 29 700 27 600 4460 41800 13 750 1221 1507 1500 Nos.
on cardiac microsome
protein synthesis
Acetaldehyde Concentration synthesis (ct min-l/mg bM) RNA) 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12
3700 1400 16 900 16400 1540 5380 8250 902 1178 755
1 to 10 1221 13 750 1507 1500 1221 13 750 1507 1500
Nos.
211
CARDIOMYOPATHY
11 to 18
0.06 0.06 0.06 0.06 0.03 0.03 0.03 0.03
544 9340 1537 585 715 11 780 1310 592
o/0 of control
30 54 57 59 35 13 60 74 78 50 52 (P < 45 68 101 39 56 86 87 39 65 (P <
jy 5.3 0.001)
& 8.6 0.01)
In any one experiment the same group of microsomes was used for control and experimentaks. Different quantities of radioactivity were used on different days (see Methods). Synthesis was deiined as the activity of L-[Wlleucine incorporated into microsomal protein: P values indicate significance from the controls. Controls for Experiments Nos. 7 to 10 were the same for Nos. 11 to 14 and 15 to 18.
or protein synthesis in the perfused heart [9]. However, 0.8 rnM-acetaldehyde effected a marked decrease in protein synthesis while it produced a chronotropic and inotropic effect [9]. Recent data from our laboratory have also shown that when livers from fed rabbit donors were perfused with 200 mg/lOO ml ethanol, the acetaldehyde concentration in the perfusate averaged 0.3 mM (unpublished data), Furthermore, blood concentrations of acetaldehyde in humans after alcohol ingestion have been found in the range of 0.03 to 0.04 mu [S] to levels of 0.16 to 0.3 mM [12]. The present report presents data to show that even at these lower levels, acetaldehyde inhibits cardiac protein synthesis. Although studies with acetaldehyde had suggested that the primary effect of this metabolite of ethanol was inotropic and chronotropic stimulation of contraction possibly leading to cardiomyopathy [5], later studies in our laboratory suggested
212
S. S. SCHREIBER
ETAL.
that decreased protein synthesis by acetaldehyde may play a role in the development of this myopathy [9]. Th e results of the microsome studies presented here emphasize that the inhibitition of protein synthesis by acetaldehyde is directly on the protein synthetic machinery and apparently independent of its effects on contraction. In the perfused intact heart, inhibition of protein synthesis by acetaldehyde was not manifest until 3-5 h of perfusion [9], while microsomal inhibition in cell free systems was seen with lower levels after 1 h of incubation. One may speculate that this difference in apparent time of acetaldehyde effect may be due to the lag in appearance of acetaldehyde at the site of protein synthesis within the cell due to diffusion delays or oxidation of the acetaldehyde [Z, 141 before it reaches the synthetic sites, and only after the NAD is used up, is the aldehyde able to reach the critical area. In contrast, in the cell free system, acetaldehyde is at the site of protein synthesis almost immediately. It may be suggested that the inhibitory action of acetaldehyde is due to interference of acetaldehyde with the ATP generating system. Inhibition of protein synthesis occurred in the intact heart in uitro as well as in subcellular systems. The isolated perfused baby guinea pig heart is exquisitely sensitive to decreased ATP levels, e.g. in anoxia or decreased coronary flow, contractility is almost immediately diminished and complete failure in asystole occurs by 10 or 15 min (unpublished data). In contrast, the present studies and those reported previously show a continued stimulation of contractility with acetaldehyde above that of controls with perfusion for 3 or 5 h. It would seem therefore that any diminution in ATP generation by acetaldehyde would have to be just enough to allow enhanced contraction but to inhibit protein synthesis or that only a compartmentalized ATP related to protein synthesis is affected. At this time, ATP data with acetaldehyde are not available to support either contention. Although the site of acetaldehyde action on the microsomes is still not clarified, the data presented here indicate that cardiac protein synthesis is sensitive to the levels of acetaldehyde seen in humans after moderate alcohol ingestion [S, 131 or manufactured by the mammalian liver in vitro. In view of the findings that cardiac protein turnover may be very rapid [lo], it is suggested that continued exposure even to these low levels of acetaldehyde may play a role in the development of cardiomyopathy in chronic alcoholism due to interference with normal cardiac protein synthesis. REFERENCES 1.
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