Effects of methionine on endogenous antioxidants in the heart

Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R2H 2A6

	ABSTRACT

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The deficiency of methionine, an essential amino acid, is associated with cardiovascular lesions. Because different types of cardiac pathologies are caused by a decrease in antioxidants, we examined the effects of methionine on myocardial antioxidant enzymes in hemodynamically assessed rats that were treated with methionine (10 mg/ml) in drinking water for 12, 24, and 48 h. Glutathione peroxidase (GSHPx) activity was significantly increased to 150.5 ± 12.2 and 191.7 ± 13.7% of the control value at 12 and 24 h, respectively, followed by a decline to 120 ± 24.6% at 48 h. The mRNA levels of GSHPx at these time points were 151.2 ± 12.0, 218.7 ± 35.3, and 173.5 ± 25.2%, respectively. Superoxide dismutase (SOD) activity was 144.3 ± 3.7, 114.3 ± 10.1, and 143.1 ± 11.2% at 12, 24, and 48 h, respectively. Catalase (Cat) activity was 272.4 ± 5.4, 237.8 ± 16.6, and 224.1 ± 17.3% of the control value. The expression of Cat and SOD mRNA was unchanged at 12, 24, and 48 h. The lipid peroxidation was decreased by 24.4 ± 11.2, 54.9 ± 0.1, and 6.4 ± 2.1% at 12, 24, and 48 h, respectively. Methionine had no effect on the ventricular or aortic pressures, heart rate, and myocardial glutathione levels at any of the time points. The study shows that methionine has a significant effect on the myocardial antioxidant enzyme activities, and only changes in GSHPx enzyme activity correlated with the mRNA changes. These antioxidant changes may have a role in the beneficial effects of methionine in pathological rather than physiological conditions.

lipid peroxidation; gene expression

	INTRODUCTION

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METHIONINE, AN ESSENTIAL amino acid, is used as a supplement in health foods. Its deficiency has been associated with a variety of cardiac and vascular changes (6, 17, 19). Spontaneously hypertensive rats fed with a low methionine diet showed increased incidence of cerebral lesions, and treatment with methionine in the drinking water reversed these changes (17). Methionine was also protective against atherosclerotic lesions caused by dietary fats in rats (6). In ex vivo studies, perfusion of hearts with methionine significantly increased the developed force in a dose-dependent manner (24).

It has been reported that reactive oxygen species (ROS) contribute to cardiac dysfunction and myocardial cell damage under a variety of conditions such as ischemia-reperfusion (3), catecholamine-induced arrhythmias (29), and adriamycin-induced cardiomyopathy (30, 35). A host of defense mechanisms, including antioxidant enzymes, have evolved that have been shown to offer protection in these conditions (15, 31). Antioxidant enzymes such as glutathione peroxidase (GSHPx), superoxide dismutase (SOD), and catalase (Cat) play an important role in the detoxification of ROS generated under normal metabolic conditions (15, 32). Improved or sustained cardiac function under a variety of physiological or pathophysiological conditions, such as exercise (14), hypertrophy (11), and adriamycin-induced cardiomyopathy (30, 34, 35), has been suggested to be due to an increase in the myocardial antioxidant reserve reflected by improved antioxidant enzymes and reduced oxidative stress (15). However, it is not known whether the beneficial effects of methionine (6, 17, 19) are associated with changes in the activities or mRNA levels for antioxidant enzymes such as GSHPx, SOD, and Cat.

In this study, we examined the effects of methionine supplementation in the drinking water on the myocardial GSHPx, SOD, and Cat enzyme activities and their mRNA expression at 12, 24, and 48 h after treatment. Corresponding lipid peroxidation and total glutathione levels were also determined. Whether methionine treatment influenced in vivo hemodynamic function was examined. The results show that methionine supplementation to rats increases all three enzyme activities in a characteristic fashion, and only the GSHPx mRNA abundance correlated with its enzyme activity change. Methionine treatment did not have any effect on the left ventricular function, aortic pressures, or heart rate in these normal rats.

	MATERIALS AND METHODS

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Animals and Methionine Treatment

Male Sprague-Dawley rats, body weight 215 ± 10 g, were randomly divided into control and treatment groups. The animals were provided food and water ad libitum. For methionine treatment, animals were provided with water containing L-methionine (10 mg/ml). Animals in the control and treatment groups were hemodynamically assessed at 12, 24, and 48 h, and their hearts were collected for the study of GSHPx, SOD, and Cat enzyme activities, mRNA abundance, glutathione levels, and lipid peroxidation.

Hemodynamic Studies

Animals were anesthetized with pentobarbital sodium (50 mg/kg ip). A miniature pressure transducer (Millar Micro-Tip) was inserted in the left ventricle via the right carotid artery (11). Left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), aortic systolic pressure (ASP), aortic diastolic pressure (ADP), and heart rate were recorded by an on-line computer data acquisition and analysis system.

Biochemical Studies

GSHPx assay. GSHPx activity was expressed as nanomoles of NADPH oxidized to NADH per minute per milligram protein, with a molar extinction coefficient for NADPH of 6.22 × 10⁶ (23). Cytosolic GSHPx was assayed in a 3-ml cuvette containing 2.4 ml of 75 mM phosphate buffer (pH 7.0). The following solutions were then added: 50 µl of 60 mM GSH, 100 µl of glutathione reductase (30 U/ml), 50 µl of 120 mM NaN₃, 100 µl of 15 mM Na₂EDTA, 100 µl of 3.0 mM NADPH, and 100 µl of cytosolic fraction obtained after centrifugation of the heart homogenate at 20,000 g for 25 min. The reaction was initiated by the addition of 100 µl of 7.5 mM H₂O₂, and the conversion of NADPH to NADP was assayed by measuring the absorbance at 340 nm at 1-min intervals for 5 min.

SOD assay. Supernatant (20,000 g for 20 min) was assayed for SOD activity by following the inhibition of pyrogallol autooxidation (21). Pyrogallol (24 mM) was prepared in 10 mM HCl and was stored at 4°C. Cat (30 µM stock solution) was made in an alkaline buffer (pH 9.0). Aliquots of supernatant (150 µg protein) were added to Tris · HCl buffer containing 25 µl pyrogallol and 10 µl Cat stock solutions. The total reaction mixture was made to 3 ml using the same Tris · HCl buffer. Autooxidation of pyrogallol was monitored by measuring absorbance at 420 nm at 1-min intervals for 5 min.

Cat assay. Extraneous fat and atrial tissue were removed, and the ventricles were homogenized in 50 mM potassium phosphate buffer (pH 7.4) using a weight-to-volume ratio of 1:10. The homogenate was centrifuged at 40,000 g for 30 min. Supernatant of 50 µl was added to a cuvette containing 2.95 ml of 19 mM H₂O₂ solution prepared in potassium phosphate buffer (7). The disappearance of H₂O₂ was monitored at 240 nm wavelength at 1-min intervals for 5 min. Specific activity of the enzyme was expressed as micromoles per milligram protein.

Glutathione assay. Myocardial tissue was homogenized in 5% sulfosalicylic acid. The tissue homogenate was centrifuged for 10 min at 10,000 g. Concentration of total glutathione (GSSG + GSH) was measured by the glutathione reductase-5,5'-dithio-bis(2-nitrobenzoic acid) recycling assay. The rate of 5-thio-2-nitrobenzoic acid formation was measured at 412 nm and is proportional to the sum of GSH and GSSG present (1).

Thiobarbituric acid reactive substances assay. Measurement of lipid peroxidation by determining thiobarbituric acid reactive substances (TBARS) was performed by using a modified thiobarbituric acid method as described previously (33).

RNA Isolation and Northern Blot Analysis

Total RNA was isolated from rat heart samples using the guanidium isothiocyanate-cesium chloride method (5). The RNA (50 µg) was separated electrophoretically on 1% agarose-2.2 M formaldehyde gels and was transferred to nitrocellulose membranes. The membranes were prehybridized for 16 h at 42°C in a solution containing 50% formamide, 20 mM NaH₂PO₄ (pH 7), 4× saline sodium citrate (SSC), 2 mM EDTA, 5× Denhardt's solution (1× = 0.02% BSA, Ficoll, and polyvinylpyrrolidone), 0.01% SDS, and 100 µg/µl sonicated salmon sperm DNA. Hybridization was performed at 42°C in the same solution. The membranes were washed initially at room temperature with 2× SSC-0.01% SDS and finally washed for 30 min at 65°C in 0.1× SSC-0.1% SDS (28). Gel-purified cDNA inserts of human GSHPx (8), human Cat (27), and human manganese-SOD (Mn-SOD; ATCC, Bethesda, MA; see Ref. 39) were radiolabeled with a labeling kit (GIBCO-BRL) to a specific activity of 10⁸ dpm/µg DNA and were used as probes. The same filters were rehybridized with an insert encoding mouse 28S ribosomal RNA as a control for RNA loading.

Protein Assay and Statistical Analysis

Protein concentration was determined by the method of Lowry and associates (18). The results are expressed as means ± SE. Statistical comparison of data was assessed using ANOVA and Bonferroni's test to identify differences between treatment groups and the control group. P < 0.05 was considered significant.

	RESULTS

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Hemodynamic Function

Animals were examined for left ventricular function (LVSP, LVEDP), aortic pressures (ASP, ADP), and heart rates at 12, 24, and 48 h of methionine treatment, and these data are shown in Table 1. Methionine did not alter any of the above-mentioned hemodynamic parameters compared with those in the control group.

View this table: [in this window] [in a new window]   Table 1.   Hemodynamic parameters in rats supplemented with L-methionine

Enzyme Activities and mRNA Abundance

Treatment of rats with methionine (10 mg/ml) through drinking water for 12 h resulted in an increase of GSHPx enzyme activity to 150.5 ± 12.2% of the control (P < 0.05). At 24 h, this value rose further to 191.7 ± 13.7% (P < 0.05) followed by a 120.5 ± 24.6% (P > 0.05) decline at 48 h (Fig. 1). The GSHPx mRNA expression levels increased in a correlative manner to that of the enzyme activity. At 12, 24, and 48 h of methionine treatment, the mRNA expression was increased to 151.2 ± 12.0, 218.7 ± 35.3, and 173.5 ± 25.2%, respectively, of the control, and in all cases the increments were statistically significant (P < 0.05; Fig. 2, A and B).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effects of methionine on myocardial glutathione peroxidase (GSHPx), superoxide dismutase (SOD), and catalase (Cat) activities. Data are means ± SE of 4-5 hearts at each time point. * Significantly different from control (Cont) values (P < 0.05). Control values for enzyme activities were as follows: GSHPx, 52.2 ± 2.5 nM/mg protein; SOD, 34.5 ± 2.2 U/mg protein; and Cat, 29.6 ± 3.6 µM/mg protein.

View larger version (44K): [in this window] [in a new window]   Fig. 2.   A: representative autoradiogram showing time course of effects of methionine on GSHPx, manganese-SOD (Mn-SOD), and Cat mRNA expression at 12, 24, and 48 h of treatment. RNA from 4 control and 5 methionine-treated hearts was isolated, and each was subjected to Northern blot analysis. The membranes were reprobed with mouse 28S ribosomal cDNA for loading control. All five bands for Mn-SOD mRNA are from a single gene and result from alternative polyadenylation. B: densitometric analysis is plotted as percentage of control value (±SE). Each time point represents mean of 4-5 hearts. Densitometric data of Mn-SOD mRNA included all five bands. * Significantly different from controls (P < 0.05).

Data on SOD enzyme activity and mRNA expression are shown (Figs. 1 and 2, A and B). SOD enzyme activity increased to 144.3 ± 3.7% of the control at 12 h of methionine treatment (P < 0.05). At 24 h, the enzyme activity was 114.3 ± 10.1% (P > 0.05), and by 48 h the activity was increased again to 143.15 ± 11.2% (P < 0.05). In contrast, the SOD mRNA expression was 95.6 ± 12.9, 103.5 ± 12.9, and 97.9 ± 19.9% of the control at 12, 24, and 48 h of methionine treatment, respectively (P > 0.05). It should be noted that Mn-SOD mRNA consists of five bands because of alternative polyadenylation. However, all of the transcripts are derived from a single functional gene (12). For densitometeric analysis, all five bands were read.

The Cat enzyme activity was 272.4 ± 5.4% of the control at 12 h of methionine treatment (Fig. 1). The methionine-induced enzyme activity remained elevated at 24 (237.8 ± 16.6%) and 48 (224.1 ± 17.3%) h of the treatment, and in all groups the increases were statistically significant (P < 0.05). Cat mRNA expression at 12, 24, and 48 h was 122.3 ± 17.7, 98.7 ± 17.7, and 135.6 ± 11.7%, and the changes were not significantly different from control values (Fig. 2, A and B).

Oxidative Stress

TBARS, a measure of lipid peroxidation, were decreased by 24.4 ± 11.2, 54.9 ± 0.1, and 6.4 ± 2.1%, respectively, at 12, 24, and 48 h of methionine treatment (Fig. 3). There was no significant change in the total glutathione levels at any time point (Fig. 4).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effects of methionine on myocardial lipid peroxidation examined as thiobarbituric acid reactive substances (TBARS) at 12, 24, and 48 h of treatment. Each time point represents the mean ± SE of 4-5 hearts. * Significantly different from control (P < 0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effects of methionine on myocardial total glutathione at 12, 24, and 48 h of treatment. Each time point represents the mean ± SE of 4-5 hearts.

	DISCUSSION

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By using this subchronic model in which the normal rats were supplemented with methionine in drinking water, the study demonstrates for the first time that this essential amino acid has a significant effect on the myocardial antioxidant enzyme activities. In the heart, GSHPx is important in the reduction of not only H₂O₂ but also other organic peroxides. Furthermore, the cardiac muscle is relatively rich in this enzyme (9), and it is able to remove H₂O₂ in the micromolar range as opposed to Cat, which is more efficient in the millimolar concentration of H₂O₂ (15). Methionine influenced both GSHPx enzyme activity and its mRNA abundance. The magnitude of mRNA regulation was higher than that of the enzyme activity, which suggests that the GSHPx expression may be predominantly regulated at the mRNA level. This could be achieved by increasing the half-life of the mRNA (25) and/or increasing the gene transcription (20, 22). If the methionine regulation of GSHPx expression is at the transcription level, it will be of importance to map the relevant response element because of the potential to regulate a major antioxidant enzyme with an essential amino acid. Because myocardial glutathione levels were not influenced by methionine treatment, the change in enzyme activity may not have been influenced by the substrate availability.

The Cat activity is relatively low in the heart compared with other tissue such as liver (9). The two antioxidant enzymes, GSHPx and Cat, are capable of completely reducing H₂O₂ and other organic peroxides (15). Both of these antioxidant enzyme activities remained elevated at 12 and 24 h, and Cat was also elevated at 48 h of methionine treatment. This induction in the antioxidant capacity may explain reduced lipid peroxidation seen after methionine treatment. Lipid peroxidation is postulated to be the most potent mechanism of cell injuries caused by ROS. Therefore, methionine may be useful in combating such cell damage. The decrease in lipid peroxidation was most marked at 24 h of methionine treatment (Fig. 3), also coinciding with the peak GSHPx and Cat enzyme activities. Conversely, the smallest effect on lipid peroxidation was at 48 h of methionine (6.4% lower than the control), coinciding with the lowest GSHPx activity. In this regard, at low concentrations of H₂O₂, its predominant scavenger in the heart is GSHPx (15). Thus the increase in the Cat activity alone may be insufficient to counter any low concentrations of H₂O₂-based lipid peroxidation, which may explain the marginal (6.4%) decrease in lipid peroxidation at 48 h of methionine exposure. Although there was a good correlation between increased GSHPx and reduced lipid peroxidation, the cause and effect in this relationship remains to be established.

In contrast to GSHPx enzyme activity, both Cat and SOD enzyme regulation by methionine appears to be posttranscriptional, i.e., translational or posttranslational, because mRNA expression of both enzymes was not altered but the enzyme activities were increased. Posttranslational regulation of gene expression occurs frequently in nature (10, 13, 40). Methionine is a methylating agent for proteins (4, 36), and such methylation may be involved in the regulation of Cat and SOD enzyme activities. Because methionine is an amino acid containing a sulfhydryl group, under oxidative stress conditions, it may also offer some direct protection.

Of the three enzyme activities studied, SOD showed the smallest percent change. In cardiac myocytes, cystolic copper and zinc-containing SOD represents 59% and Mn-SOD ~41% of the total SOD activity (16). Under oxidative stress conditions, it is Mn-SOD that changes the most (16). Furthermore, cytosolic SOD expression is also reported to be relatively stable in other tissues (37, 38). Thus, in the present study, we focussed on Mn-SOD expression, which in methionine-treated animals showed relatively stable, steady-state levels at all time points.

Methionine treatment did not affect the left ventricular functions (LVSP, LVEDP), aortic pressures (ASP, ADP), and the heart rates (Table 1). However, under stress conditions such as ischemia, increased cardiac workload subsequent to infarction, or adriamycin-induced cardiomyopathy, the methionine-induced increase in antioxidants may manifest as improved hemodynamic function. However, such a possibility remains to be tested.

Methionine clearly improved "endogenous antioxidant reserve," and the latter has been suggested to maintain and/or improve myocardial structure and function under a variety of conditions. Although the mechanisms for the methionine-induced increase in antioxidant enzyme activities and mRNA abundance are not clear, it is suggested that methionine may provide protection against oxidative stress-induced myocardial cell damage and heart failure.

	ACKNOWLEDGEMENTS

This study was supported by a group grant from the Medical Research Council of Canada. P. K. Singal is a Career Investigator of the Medical Research Council. N. Khaper and T. Li were supported by student fellowships from the Heart and Stroke Foundation of Canada and by the Faculty of Medicine, University of Manitoba.

	FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. K. Singal, Institute of Cardiovascular Sciences, St. Boniface General Hospital, Research Centre, 351 Tache Ave., Winnipeg, Canada, R2H 2A6 (E-mail: psingal{at}sbrc.umanitoba.ca).

Received 27 April 1999; accepted in final form 8 July 1999.

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