Department of Clinical Chemistry, Helsinki University Central Hospital, Haartmaninkatu 4, SF-00290 Helsinki,
1 Department of Mental Health and Alcohol Research, (C.J.P.E.) National Public Health Institute, PL 719, SF-00101 Helsinki, Finland and
2 Department of Clinical Chemistry, Odense University Hospital, Odense, Denmark
Received 23 August 1999; in revised form 14 February 2000; accepted 17 February 2000
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ABSTRACT |
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INTRODUCTION |
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Acetaldehyde, the first metabolite of ethanol, has been shown to have significant cardiovascular effects (James and Bear, 1967) and has been shown to interfere with protein synthesis in heart muscle (Siddiq et al., 1993
). Therefore, in the present study models of elevating the ethanol-derived blood-acetaldehyde levels by the aldehyde dehydrogenase inhibitor, calcium carbimide, were used and compared with effects of ethanol alone. Ventricular ANP and BNP gene expression, blood-ANP concentration as well as heart and body weights were studied in ethanol- and/or calcium carbimide-treated rats.
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MATERIALS AND METHODS |
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In the experiment designed to clarify the results of our preliminary observation, 64 2-month-old male Wistar rats (Laboratory Animal Centre, University of Helsinki, Finland) were assigned randomly to eight experimental groups. Animals were housed in a temperature-controlled room with a 12 h light:12 h dark cycle and provided with chow and water ad libitum. The four experimental groups were as follows: controls (half of the group: saline 8.3 ml/kg i.p. once a day; half of the group no treatment); ethanol (1 g/kg body weight, i.p. as a 12% w/v solution in saline once a day); calcium carbimide (half of the group: 100 mg/kg of diet; half of the group: calcium carbimide 100 mg/kg of diet + saline, i.p.) and ethanol + calcium carbimide (ethanol 1 g/kg i.p. once a day + calcium carbimide 100 mg/kg of diet). The calcium carbimide diet started in the morning on day 1, ethanol on day 2. This way, in the 2-day experiment, rats received calcium carbimide for 2 days and ethanol for 1 day, and in the 8-day experiment rats received calcium carbimide for 8 days and ethanol for 7 days. Blood samples for ethanol and acetaldehyde measurement were taken from the tail vein 2 h after alcohol injection 1 day before decapitation and for N-terminal pro-ANP (NT-ANP) at the time of decapitation. After decapitation, hearts were removed within 60 s, weighed and the free walls of the left ventricles were carefully dissected and immediately frozen in liquid nitrogen for RNA isolation. All tissue samples were stored at 80°C until mRNA analysis. The study protocols were approved by the National Public Health Institution Animal Care and Use Committee.
Blood-ethanol and blood-acetaldehyde measurements
Tail blood samples were haemolysed, and their ethanol and acetaldehyde contents measured with head-space gas chromatography on the day that the samples were collected (Eriksson et al., 1977).
Radioimmunoassay (RIA) for plasma NT-ANP
Blood for NT-ANP assay was taken in tubes containing aprotinin (500 IU/ml) and EDTA (1 mg/ml) and immediately placed on ice. After centrifugation at 1000 g for 10 min at 4°C, aliquots of plasma were stored at 80°C and later subjected to RIA by a commercial kit (Biotop, Oulu, Finland). All the samples were run within the same assay.
Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using internal standards
RT-PCR was performed as described in our earlier study (Jänkälä et al., 1997). Synthetic control RNA was produced by in vitro transcription, was 120 bases long and included sequences complementary for the ANP and BNP primers used (Feldman et al., 1991
). The cDNA was amplified in a DNA Thermal Cycler (Perkin Elmer) using 2.5 U Dynazyme DNA polymerase (Finnzymes, Finland). A trace amount of
[32P]-labelled 3' primer was added to provide about 1.5 x 106 cpm per reaction to label the DNA. The first cycle started with a 4 min denaturation at 96°C. In the following cycles, each step lasted for 1 min: the denaturation at 96°C, primer annealing at 54°C (ANP) or 58°C (BNP), and the synthesis step at 72°C. Oligonucleotide PCR primers complementary to the rat genes encoding ANP (Seidman et al., 1984
) and BNP (Kojima et al., 1989
) are the following: 5'-TCG AGC AGA TCG CAA AAG ATC-3' (ANP sense), 5'-CAC ACT AAA CCA CTC ATC TAC-3' (ANP antisense), 5'-CAG AAG ATA GAC CGG ATC g-3' (BNP sense), 5'-CAG GAT CAC TTG AGA GGT T-3' (BNP antisense). Since internal controls also contained the complementary sequence, the PCR primers amplified both sample and control cDNAs. Comparing the levels of radioactivity, the mRNA level of interest could then be calculated from the known amount of control RNA by using values from the exponential cycles (Feldman et al., 1991
).
Northern blot hybridization
Total RNA was isolated from ventricles using a modification of the acid guanidium thiocyanate/phenol/chloroform extraction (RNAzol B, Tel-Test Inc., Friedswood, TX, USA). Fifteen µg of total RNA were loaded onto a 1.2% (w/v) agarose gel containing formaldehyde and, after electrophoresis, transferred onto Zeta-Probe GT membranes (BioRad, Hercules, CA, USA) by capillary transfer. The 1.0 kb bands of preproANP mRNAs were detected by hybridization with appropriate randomly primed [32P]-dCTP-labelled cDNA probe (Seidman et al., 1984
). The 1.6 kb glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were analysed identically to estimate the possible differences in the amounts of total RNA loaded.
Drugs
Calcium carbimide (Dipsan®) was purchased from Cyanamide Canada Inc., Montreal, Canada.
Statistical analysis
All the data are presented as means ± SEM. The differences between study groups were analysed by the two-tailed Mann-Whitney U-test. P values < 0.05 were considered to be significant.
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RESULTS |
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ANP mRNA levels
As shown in Fig. 2(a), ethanol treatment (1 g/kg) daily increased left ventricular ANP mRNA levels by 5% and calcium carbimide treatment (100 mg/kg diet) by 22%, compared with the control group at 2 days (not significant). Combined ethanol + calcium carbimide treatment increased ANP mRNA levels by 60%, compared with the control group (0.96 ± 0.09 x 107 vs 0.60 ± 0.07 x 107; P < 0.05). In the 8-day experiment, the ethanol and the calcium carbimide-treated groups had 18% and 13% higher ANP mRNA levels respectively, compared with the control group (not significant). Combined ethanol + calcium carbimide treatment, however, elevated ANP mRNA levels by 41%, compared with the control group (0.96 ± 0.10 x 107 vs 0.68 ± 0.03 x 107; P < 0.05) (Fig. 2b
).
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NT-ANP levels in plasma
The ANP concentrations were measured in the 8-day experiment. No statistically significant differences in plasma NT-ANP between experimental groups were detected. Levels were as follows (pmol/l); controls: 0.73 ± 0.13 ethanol-treated: 0.60 ± 0.05, calcium carbimide-treated: 0.61 ± 0.07 and ethanol + calcium carbimide-treated: 0.86 ± 0.15.
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DISCUSSION |
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Chronic alcoholism is associated with dysfunction of the heart in as many as one-third of patients (Urbano-Marquez et al., 1989). The duration of alcoholism reported by most authors is usually at least 10 years before cardiac symptoms appear (Regan, 1971
). The transition from alcohol-induced injury, which is reversible, to permanent organ damage is not well understood. Clinical evidence indicates that the effects of alcohol on the myocardium are reversible if recognized and managed early and if alcohol intake is completely eliminated. Undoubtedly, there is considerable individual variation in susceptibility to alcohol and alcoholic heart disease, which is probably the result of several factors acting in concert in a susceptible person (Regan, 1971
).
In earlier studies, acetaldehyde has been proposed to participate in the development of alcoholic heart disease by impairing myocardial protein synthesis (Siddiq et al., 1993). This finding is significant in the light of the observation that inhibition of myocardial protein synthesis leads to the development of myocardial failure in rabbit hearts with increased afterload (Zühlke et al., 1965
). It is possible that ANP could mediate the negative effect of acetaldehyde on protein synthesis, and it has been shown that ANP induces apoptosis in neonatal rat cardiac myocytes (Wu et al., 1997
). It is of interest that acetaldehyde has positive chronotropic and inotropic effects on the heart (Preedy et al., 1994
). Cardiac stimulation occurs at levels which normally develop in man after ingestion of ethanol and the stimulating effect of acetaldehyde can be blocked with propranolol, which suggests that it is due to the release of noradrenaline (James and Bear, 1967
), which is known to be a stimulus for ANP synthesis (Ruskoaho, 1992
). Acutely, ethanol intake increases susceptibility for arrhythmias, possibly due to sympathetic overdrive (Mäki et al., 1998
). Withdrawal of chronic alcohol abuse induces a period of hypersensitivity to catecholamines (Mäki et al., 1990
). However, there have been numerous reports on various effects of acetaldehyde on cellular and molecular levels, independent of catecholamines.
In the 2- and 8-day experiments of the present study, combined calcium carbimide + ethanol treatment had a statistically significant inhibitory effect on total body weight increase and calcium carbimide treatment alone had an inhibitory effect on body weight increase at 2 days. At 8 days, total body weights as well as heart weights of ethanol + calcium carbimide-treated rats were markedly lower, compared to values in the control group, but heart to body weight ratios, usually measured to detect clinical heart failure, were unchanged. An earlier study reported a 12% decrease in heart weights in rats, paralleling the reduction in body weight after 8 months of ethanol ingestion (Capasso et al., 1991) and another group showed that 12 weeks of ethanol ingestion reduced both heart weight and heart weight:body weight ratio in rats (Brown et al., 1996
). In rats, combined ethanol + calcium carbimide treatment also decreases food consumption, and calcium carbimide has been shown to be anorectic per se (Eriksson, 1985
). In isolated heart preparations, addition of acetaldehyde to the perfusate medium reduced rates of protein synthesis. An acute ethanol dose reduced protein synthesis by approximately 20%, as did treatment with both ethanol and 4-methylpyrazole. When treated with both ethanol and calcium carbimide, more marked reductions in translation rates occurred, implicating acetaldehyde as a powerful protein synthesis inhibitor (Siddiq et al., 1993
) and therefore a potential inducer of heart failure.
In summary, this study shows that 2 days to 3 weeks combined treatment with ethanol and calcium carbimide elevated blood-acetaldehyde concentrations and increased rat heart left ventricular ANP gene expression as typically seen in various overload and hypertrophy models of the heart. This suggests a potential role for acetaldehyde in the development of alcoholic heart disease. However, only studies performed on tissue and cellular levels will establish whether acetaldehyde is the actual triggering molecule of ANP induction and elucidate the molecular mechanisms involved. Moreover, further animal and human studies are needed to confirm whether the chronic exposure to much lower blood-acetaldehyde concentrations during prolonged alcohol abuse could be responsible for similar actions, and for alcoholic heart disease.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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REFERENCES |
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Capasso, J. M., Li, P., Guideri G. and Anversa, P. (1991) Left ventricular dysfunction induced by chronic alcohol ingestion in rats. American Journal of Physiology 261, H212H219.
Eriksson, C. J. P. (1985) Endogenous acetaldehyde in rats. Effects of exogenous ethanol, pyrazole, cyanamide and disulfiram. Biochemical Pharmacology 34, 39793982.[ISI][Medline]
Eriksson, C. J. P., Sippel, H. W. and Forsander, O. A. (1977) The determination of acetaldehyde in biological samples by head-space gas chromatography. Analytical Biochemistry 80, 116124.[ISI][Medline]
Eriksson, C. J. P., Koivisto, T., Sriwatanawongsa, V., Martelius, T., Mäkisalo, H. and Höckerstedt, K. (1997) Manipulation of alcohol drinking by liver transplantation. Alcoholism: Clinical and Experimental Research 21, 763765.[ISI][Medline]
Feldman, A. M., Ray, P. E., Silan, C. M., Mercer, J. A., Minobe W. and Bristow, M. R. (1991) Selective gene expression in failing human heart. Quantification of steady-state levels of messenger RNA in endomyocardial biopsies using the polymerase chain reaction. Circulation 83, 18661872.[Abstract]
James, T. N. and Bear, E. S. (1967) Effects of ethanol and acetaldehyde on the heart. American Heart Journal 74, 243255.[ISI][Medline]
Jänkälä, H., Harjola, V-P., Petersen, N. E. and Härkönen, M. (1997) Myosin heavy chain mRNA transform to faster isoforms in immobilized skeletal muscle: a quantitative PCR study. Journal of Applied Physiology 82, 977982.
Kojima, M., Minamino, N., Kangawa, K. and Matsuo, H. (1989) Cloning and sequence analysis of rat cDNA encoding a precursor for rat brain natriuretic peptide. Biochemical and Biophysical Research Communications 159, 14201426.[ISI][Medline]
Levin, E. R. (1997) Natriuretic peptides as antigrowth factors. Natriuretic peptides in health and disease. Contemporary Endocrinology 5, 223237.
Mäki, T., Heikkonen, E., Härkönen, T., Kontula, K., Härkönen, M. and Ylikahri, R. (1990) Reduction of lymphocytic ß-adrenoceptor level in chronic alcoholics and rapid reversal after ethanol withdrawal. European Journal of Clinical Investigation 20, 313316.[ISI][Medline]
Mäki, T., Toivonen, L., Koskinen, P., Näveri, H., Härkönen, M. and Leinonen, H. (1998) Effect of ethanol drinking, hangover, and exercise on adrenergic activity and heart rate variability in patients with a history of alcohol-induced atrial fibrillation. American Journal of Cardiology 82, 317322.[ISI][Medline]
Preedy, V. R., Siddiq, T., Why, H. J. F. and Richardson, P. J. (1994) Ethanol toxicity and cardiac protein synthesis in vivo. American Heart Journal 127, 14321439.[ISI][Medline]
Regan, T. J. (1971) Ethyl alcohol and the heart. Circulation 44, 957963.[ISI][Medline]
Ruskoaho, H. (1992) Atrial natriuretic peptide: synthesis, release, and metabolism. Pharmacological Review 44, 479602.
Seidman, C. E., Duby, A. D., Choi, E., Graham, R. M., Haber, E., Homcy, C., Smith, J. A. and Seidman, J. G. (1984) The structure of rat preproatrial natriuretic factor as defined by a complementary DNA clone. Science 225, 324326.[ISI][Medline]
Siddiq, T., Salisbury, J. R., Richardson, P. J. and Preedy, V. R. (1993) Synthesis of ventricular mitochondrial proteins in vivo: effect of acute ethanol toxicity. Alcoholism: Clinical and Experimental Research 17, 894899.[ISI][Medline]
Urbano-Marquez, A., Estruch, R., Navarro-Lopez, F., Grau, J. M., Mont, L. and Rubin, E. (1989) The effects of alcoholism on skeletal and cardiac muscle. New England Journal of Medicine 320, 409415.[Abstract]
Wigle, D. A., Watson, J. D., Pang, S. C., Sarda, I. R., Roy, R. N. and Flynn, T. G. (1995) Gene expression of A- and B-type natriuretic peptides in response to acute ethanol ingestion. Alcoholism: Clinical and Experimental Research 19, 13171320.[ISI][Medline]
Wu, C.-F., Bishopric, N. H. and Pratt, R. E. (1997) Atrial natriuretic peptide induces apoptosis in neonatal rat cardiac myocytes. Journal of Biological Chemistry 272, 1486014866.
Yoshimura, M., Yasue, H., Okumura, K., Ogawa, H., Jougasaki, M., Mukoyama, M., Nakao, K. and Imura, H. (1993) Different secretion patterns of atrial natriuretic peptide and brain natriuretic peptide in patients with congestive heart failure. Circulation 87, 464469.[Abstract]
Zühlke, V., du Mesnil de Rochemont, W., Gudbjarnason, S. and Bing, R. J. (1965) Inhibition of protein synthesis in cardiac hypertrophy and its relation to myocardial failure. Circulation Research 18, 558572.[ISI][Medline]