Department of Pharmacology, Physiology and Therapeutics and 1 North Dakota Fetal Alcohol Syndrome Center, University of North Dakota School of Medicine and Health Science, Grand Forks, ND 58203, USA
* Author to whom correspondence should be addressed at: Center for Cardiovascular Research and Alternative Medicine, Division of Pharmaceutical Sciences, University of Wyoming, Laramie, WY 82071, USA. Fax: +307 766 2953; E-mail: jren{at}uwyo.edu
(Received 24 March 2004; first review notified 23 April 2004; in revised form 12 May 2004; accepted 25 June 2004)
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ABSTRACT |
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INTRODUCTION |
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Recent evidence suggested that depletion of vitamins as a consequence of alcohol use may play a role in alcohol-induced organ and tissue damage (Ba et al., 1996, 1999). Deficiencies in group B vitamins and folic acid following alcohol intake are among the key causative factors associated with alcoholic organ and tissue damage (Ba et al., 1999
), consistent with the observation of idiopathic dilated cardiomyopathy in patients with vitamin deficiency (Whyte et al., 1982
; Tobias et al., 1989
). Group B vitamins are important water-soluble vitamins often obtained through diet. They are essential for DNA synthesis and repair. In addition, vitamins B6 and B12 are required for biological formation of homocysteine. The aim of this study was to investigate the influence of group B vitamin supplementation on short-term acetaldehyde exposure-induced cardiac mechanical dysfunction in isolated rat ventricular myocytes. Direct measurement of cardiomyocyte mechanics on a beat-to-beat basis is essential to the evaluation of cardiac excitationcontraction coupling under pathophysiological conditions such as alcoholism (Ren and Brown, 2000
; Aberle and Ren, 2003
).
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MATERIALS AND METHODS |
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Cell shortening/relengthening
Mechanical properties of ventricular myocytes were assessed using an IonOptix MyoCam® system (IonOptix Incorporation, Milton, MA) as described (Aberle et al., 2003). In brief, cells were placed in a chamber mounted on the stage of an inverted microscope (Olympus IX-70) and superfused (
2 ml/min at 25°C) with a buffer containing (in mM): 131 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, at pH 7.4. The cells were field stimulated to contract at a frequency of 0.5 Hz. Changes in cell length during shortening and relengthening were captured and converted to digital signal. Cell shortening and relengthening were assessed using the following indices: peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocities of shortening and relengthening (±dL/dt).
Protein carbonyl assay
To assess protein damage, the carbonyl content of protein extracted from cardiac myocytes was determined as described (Hintz et al., 2003; Ren et al., 2003
). Briefly, myocyte number was counted and proteins were extracted. Nucleic acids were eliminated by treating the samples with 1% streptomycin sulfate for 15 min, followed by a 10 min centrifugation (11 000 g). Protein was precipitated by adding an equal volume of 20% TCA to protein (0.5 mg) and centrifuged for 1 min. The TCA solution was removed and the cells were resuspended in 10 mM 2,4-dinitrophenylhydrazine (2,4-DNPH) solution. Myocytes were incubated at room temperature for 20 min and were centrifuged for 3 min following addition of 500 µl of 20% TCA. The supernatant was discarded and the pellet washed in ethanol:ethyl acetate and allowed to incubate at room temperature for 10 min. The cells were centrifuged again for 3 min and the ethanol:ethyl acetate steps repeated twice more. The precipitate was resuspended in 6 M guanidine solution and centrifuged for 3 min. The maximum absorbance (360390 nm) of the supernatant was read against appropriate blanks (water, 2 M HCl) and the carbonyl content was calculated using the molar absorption coefficient of 22 000 M1cm1.
Caspase-3 activation assay
Caspase-3 plays a critical role in apoptotic signaling; induction of Bax may lead to the activation of caspase-3 (Telford et al., 1994). Ventricular myocytes were plated on 100 mm petri dishes. Caspase-3 activity was determined using the colorimetric kit purchased from R&D System (Minneapolis, MN). Myocytes were harvested and washed once with phosphate-buffered saline. After the myocytes were lysed, reaction buffer was added to the myocytes followed by the additional 5 µl of caspase-3 colorimetric substrate (DEVD-pNA) and incubated in a 96-well plate for 4 h at 37°C in a CO2 incubator. The plate was then read with a microplate reader at 405 nm (Ren et al., 2002
).
Experimental protocols
Cardiac myocytes were incubated with acetaldehyde (10 µM) in the presence or absence of vitamin B1 (thiamin, 10 µM), vitamin B6 (pyridoxine, 10 µM) or vitamin B12 (cyanocobalmin, 1 mM) in 20-ml sealed vials that possess silicone septa (VWR) for 46 h before mechanical and biochemical properties were evaluated.
Data analysis
Data were mean ± SEM. Differences between means between groups were assessed using analysis of variance (ANOVA). When an overall significance was determined, a Dunnetts post hoc analysis was incorporated. P-value <0.05 was considered significant.
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RESULTS |
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DISCUSSION |
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Vitamin B1 and vitamin B6 levels are reduced in 33% and 25% of the patients, respectively, with chronic alcohol exposure. Many of these vitamin-deficient patients displayed cardiovascular dysfunction including idiopathic dilated cardiomyopathy (Whyte et al., 1982; Tobias et al., 1989
). The most common forms of severe complications of vitamin B1 deficiency are beriberi and WernickeKorsakoff syndrome (Singleton and Martin, 2001
). Similar to ethanol, acetaldehyde itself has been shown to reduce thiamin levels in blood within 12 h following intravenous injection. Thiamin levels decreased by acetaldehyde injection returned to normal levels 72 h after injection (Takabe and Itokawa, 1983
). Therefore, it is plausible to speculate that an acute ethanol ingestion-induced decrease in thiamin may be due to its metabolism into acetaldehyde and subsequently, catabolism of acetaldehyde. The mechanism of action behind ethanol or acetaldehyde-induced deficiency in group B vitamin may be related to the reduced activity of thiamin pyrophosphokinase and the thiamin pyrophosphate synthetic enzyme (Rindi et al., 1986
; Singleton and Martin, 2001
). Thiamin administration reverses ethanol-induced cytoplasmic damage by protecting against ethanol-induced change in membrane fluidity and stability (Ba et al., 1996
). This received support from our current study that vitamin B1 abolished acetaldehyde-induced protein damage and apoptotic cell death. It is plausible that interruption of membrane fluidity may serve as one of the mechanisms behind the acetaldehyde-induced cardiac mechanical and biochemical defects. Acetaldehyde has been demonstrated to inhibit the activity of a number of membrane ion transporting proteins, which contribute to alteration of the membrane fluidity (Tillotson et al., 1981
). Our results observed that vitamin B6 and vitamin B12 only partially attenuated protein carbonyl formation and exhibited no effect on caspase-3 activation following acetaldehyde exposure, consistent with the minor effect of these two B group vitamins on acetaldehyde-induced cardiac mechanical dysfunctions. Although it is beyond the scope of the present study, it can be postulated that vitamin B1-elicited cardiac protection is related to antagonism against oxidative stress in acetaldehyde-mediated cell damage (Aberle and Ren, 2003
).
In summary, results from our present study confirmed that short-term acetaldehyde exposure directly leads to cardiac mechanical dysfunction, protein damage and apoptosis in ventricular myocytes. More importantly, our findings suggest that acetaldehyde-induced cardiac mechanical dysfunction, protein damage and apoptotic cell death in ventricular myocytes may be prevented by vitamin B1 supplementation, but not by vitamin B6 or vitamin B12. These data indicate a relative specificity of vitamin B1 or its deficiency over other B group vitamins in acetaldehyde-induced cardiac defects and possibly onset of alcoholic cardiomyopathy. Further study is warranted to depict the role of vitamin B group deficiency, especially vitamin B1, on the propensity of alcohol-induced cardiac disorders, and the therapeutic potential of vitamin B1 (thiamin) in alcoholic complications in the cardiovascular system.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Aberle, N. S. II, Privratsky, J. R., Burd, L. and Ren, J. (2003) Combined acetaldehyde and nicotine exposure depresses cardiac contraction in ventricular myocytes: prevention by folic acid. Neurotoxicology and Teratology 25, 731736.[CrossRef][ISI][Medline]
Ba, A., Seri, B. V. and Han, S. H. (1996) Thiamine administration during chronic alcohol intake in pregnant and lactating rats: effects on the offspring neurobehavioural development. Alcohol and Alcoholism 31, 2740.[Abstract]
Ba, A., Seri, B. V., Aka, K. J., Glin, L. and Tako, A. (1999) Comparative effects of developmental thiamine deficiencies and ethanol exposure on the morphometry of the CA3 pyramidal cells. Neurotoxicology and Teratology 21, 579586.[CrossRef][ISI][Medline]
Hintz, K. K., Relling, D. P., Saari, J. T., Borgerding, A. J., Duan, J., Ren, B. H., Kato, K., Epstein, P. N. and Ren, J. (2003). Cardiac overexpression of alcohol dehydrogenase exacerbates cardiac contractile dysfunction, lipid peroxidation, and protein damage after chronic ethanol ingestion. Alcoholism: Clinical and Experimental Research 27, 10901098.[ISI][Medline]
Laposata, E. A. and Lange, L. G. (1986) Presence of nonoxidative ethanol metabolism in human organs commonly damaged by ethanol abuse. Science 231, 497499.[ISI][Medline]
Preedy, V. R. and Richardson, P. J. (1994) Ethanol induced cardiovascular disease. British Medical Bulletin 50, 152163.[Abstract]
Preedy, V. R., Patel, V. B., Reilly, M. E., Richardson, P. J., Falkous, G. and Mantle, D. (1999) Oxidants, antioxidants and alcohol: implications for skeletal and cardiac muscle. Frontiers in Bioscience 4, e58e66.[Medline]
Ren, J. and Brown, R. A. (2000) Influence of chronic alcohol ingestion on acetaldehyde-induced depression of rat cardiac contractile function. Alcohol and Alcoholism 35, 554560.
Ren, J., Davidoff, A. J. and Brown, R. A. (1997) Acetaldehyde depresses shortening and intracellular Ca2+ transients in adult rat ventricular myocytes. Cellular and Molecular Biology 43, 825834.[ISI]
Ren, J., Wold, L. E., Natavio, M., Ren, B. H., Hannigan, J. H. and Brown, R. A. (2002) Influence of prenatal alcohol exposure on myocardial contractile function in adult rat hearts: role of intracellular calcium and apoptosis. Alcohol and Alcoholism 37, 3037.
Ren, J., Roughead, Z. K., Wold, L. E., Norby, F. L., Rakoczy, S., Mabey, R. L. and Brown-Borg, H. M. (2003) Increases in insulin-like growth factor-1 level and peroxidative damage after gestational ethanol exposure in rats. Pharmacological Research 47, 341347.[CrossRef][ISI][Medline]
Rindi, G., Imarisio, L. and Patrini, C. (1986) Effects of acute and chronic ethanol administration on regional thiamin pyrophosphokinase activity of the rat brain. Biochemical Pharmacology 35, 39033908.[CrossRef][ISI][Medline]
Singleton, C. K. and Martin, P. R. (2001) Molecular mechanisms of thiamine utilization. Current Molecular Medicine 1, 197207.[Medline]
Spies, C. D., Sander, M., Stangl, K., Fernandez-Sola, J., Preedy, V. R., Rubin, E., Andreasson, S., Hanna, E. Z. and Kox, W. J. (2001) Effects of alcohol on the heart. Current Opinion in Critical Care 7, 337343.[CrossRef][Medline]
Takabe, M. and Itokawa, Y. (1983) Thiamin depletion after ethanol and acetaldehyde administration to rabbits. Journal of Nutritional Science and Vitaminology 29, 509514.[ISI][Medline]
Telford, W. G., King, L. E. and Fraker, P. J. (1994) Rapid quantitation of apoptosis in pure and heterogeneous cell populations using flow cytometry. Journal of Immunological Methods 172, 116.[CrossRef][ISI][Medline]
Thomas, A. P., Rozanski, D. J., Renard, D. C. and Rubin, E. (1994) Effects of ethanol on the contractile function of the heart: a review. Alcoholism: Clinical and Experimental Research 18, 121131.[ISI][Medline]
Tillotson, L. G., Carter, E. A., Inui, K. I. and Isselbacher, K. J. (1981) Inhibition of Na+-stimulated glucose transport function and perturbation of intestinal microvillus membrane vesicles by ethanol and acetaldehyde. Archives of Biochemistry and Biophysics 207, 360370.[ISI][Medline]
Tobias, S. L., van der Westhuyzen, J., Davis, R. E., Icke, G. C. and Atkinson, P. M. (1989) Alcohol intakes and deficiencies in thiamine and vitamin B6 in black patients with cardiac failure. South African Medical Journal 76, 299302.[ISI][Medline]
Whyte, K. F., Dunnigan, M. G. and McIntosh, W. B. (1982) Excessive beer consumption and beri-beri. Scottish Medical Journal 27, 288291.[ISI][Medline]