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Am J Physiol Heart Circ Physiol 287: H2122-H2131, 2004. First published July 22, 2004; doi:10.1152/ajpheart.00378.2004
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Decreased cardiac mitochondrial NADP+-isocitrate dehydrogenase activity and expression: a marker of oxidative stress in hypertrophy development

Mohamed Benderdour,1 Guy Charron,2 Blandine Comte,1 Riwa Ayoub,1 Diane Beaudry,3 Sylvain Foisy,1 Denis deBlois,3 and Christine Des Rosiers1

Departments of 1Nutrition, 2Medicine, and 3Pharmacology, University of Montreal, Montreal, Quebec, Canada H1T 1C8

Submitted 26 April 2004 ; accepted in final form 15 July 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mitochondrial dysfunction subsequent to increased oxidative stress and alterations in energy metabolism is considered to play a role in the development of cardiac hypertrophy and its progression to failure, although the sequence of events remains to be elucidated. This study aimed at characterizing the impact of hypertrophy development on the activity and expression of mitochondrial NADP+-isocitrate dehydrogenase (mNADP+-ICDH), a metabolic enzyme that controls redox and energy status. We expanded on our previous finding of its inactivation through posttranslational modification by the lipid peroxidation product 4-hydroxynonenal (HNE) in 7-wk-old spontaneously hypertensive rat (SHR) hearts before hypertrophy development (Benderdour et al. J Biol Chem 278: 45154–45159, 2003). In this study, we used 7-, 15-, and 30-wk-old SHR and Sprague-Dawley (SD) rats with abdominal aortic coarctation. Compared with age-matched control Wistar-Kyoto (WKY) rats, SHR hearts showed a significant 25% decrease of mNADP+-ICDH activity, which preceded in time 1) the decline in its protein and mRNA expression levels (between 10% and 35%) and 2) the increase in hypertrophy markers. The chronic and persistent loss of mNADP+-ICDH activity in SHR was associated with enhanced tissue accumulation of HNE-mNADP+-ICDH and total HNE-protein adducts at all ages and contrasted with the profile of changes in the activity of other mitochondrial enzymes involved in antioxidant or energy metabolism. Two-way ANOVA of the data also revealed a significant effect of age on most parameters measured in SHR and WKY hearts. The mNADP+-ICDH activity, protein, and mRNA expression were reduced between 25% and 35% in coarctated SD rats and were normalized by treatment of SHR or coarctated SD rats with renin-angiotensin system inhibitors, which prevented or attenuated hypertrophy. Altogether, our data show that cardiac mNADP+-ICDH activity and expression are differentially and sequentially affected in hypertrophy development and, to a lesser extent, with aging. Decreased cardiac mNADP+-ICDH activity, which is attributed at least in part to HNE adduct formation, appears to be a relevant early and persistent marker of mitochondrial oxidative stress-related alterations in hypertrophy development. Potentially, this could also contribute to the aetiology of cardiomyopathy.

energy metabolism; 4-hydroxynonenal; lipoperoxidation; renin-angiotensin system; aging

CARDIAC HYPERTROPHY, an early milestone in the clinical course of heart failure, is an independent risk factor for future cardiac events associated with mortality and morbidity. Increased oxidative stress and/or alterations of fuel metabolism are among factors that contribute to the development of cardiac hypertrophy and its progression to failure, regardless of etiology (3, 16, 29, 37, 58, 63). Both factors could independently impair the capacity of the mitochondria to fulfil their crucial role in energy production necessary for contraction (42) and thereby contribute to the activation of signaling pathways governing cell death by apoptosis and/or necrosis (1, 9). However, additional work is needed to clarify how the various changes integrate themselves in relation with the various adaptive and maladaptive stages of the disease (from hypertrophy compensated and decompensated to failure). There is also a need to identify markers of the various stages of disease development to define more targeted therapies and to increase our ability to predict whether a given intervention will lead to beneficial or detrimental outcomes.

In a recent study, we obtained data in spontaneously hypertensive rats (SHR) that are of potential relevance to our better understanding of the sequence of oxidative stress-related pathophysiological events linked to cardiac disease development. The SHR is a well-established model of genetic hypertension, which develops cardiac hypertrophy between 9 and 12 wk of age (60) and shows functional symptoms of decompensated hypertrophy after 18 to 24 mo (47). We found that mitochondrial NADP+-isocitrate dehydrogenase (mNADP+-ICDH), a metabolic enzyme controlling energy and redox status, is inactivated through 4-hydroxynonenal (HNE) adduct formation before hypertrophy development (5). HNE, the major {alpha},{beta}-unsaturated aldehyde formed from free radical-induced peroxidation of {omega}-6 polyunsaturated fatty acids (24), reacts readily with cysteine, lysine, and histidine residues of proteins. While aldehydes have long been considered merely as markers of tissue damage, their role as "second toxic messengers," as initially hypothesized by Esterbauer et al. (25), is becoming increasingly accepted. Indeed, it is now believed that aldehydes, and HNE among them, are responsible for many cytopathological effects observed during oxidative stress in vivo (13, 21, 41, 52, 55, 67, 74). In this regard, the results of our recent study emphasized the involvement of posttranslational modifications of mitochondrial metabolic enzymes by HNE in the early oxidative stress-related events linked to cardiac hypertrophy development (5).

Additional work is, however, necessary to assess the significance of cardiac mNADP+-ICDH inactivation caused by HNE binding within the context of disease development. Could mNADP+-ICDH inactivation be a marker and/or player in oxidative stress-related events during cardiac hypertrophy development? mNADP+-ICDH, which catalyzes reversible interconversion between isocitrate and {alpha}-ketoglutarate, shows its highest activity and expression in the heart, where it is confined to cardiomyocytes (31, 36, 43, 72). Another NADP+-ICDH isoform is located in the cytosol, but in the heart it represents <5% (54). The high level of homology of mNADP+-ICDH polypeptide and cDNA sequences between species (~95% between humans, pigs, cattle, mice, and rats) (36, 45, 72) suggests that it may play a critical role in cellular metabolism. However, much remains to be learned about the role and regulation of mNADP+-ICDH, especially in the heart. There is a current resurgence of interest in this enzyme following the study by Jo et al. (37) in NIH3T3 cells, which supported its antioxidant role through the generation of NADPH (36). However, an alternative role for this enzyme is also proposed, where it operates in the reverse direction, i.e., generating isocitrate and NADP+, thereby participating in a substrate cycle regulating citric acid cycle (CAC) activity (59). The latter role is specifically relevant in the heart (14), where a high mitochondrial NADPH-to-NADP+ ratio (>50 vs. <1 in NIH3T3) could restrict CAC flux (crucial for contraction).

The objective of this study, which expands on our previous one (5), was to assess the impact of hypertrophy development on myocardial mNADP+-ICDH activity, protein, and mRNA expression as well as tissue accumulation of HNE-mNADP+-ICDH and total HNE-protein adducts in 7-, 15-, and 30-wk-old SHR. Age-matched Wistar-Kyoto (WKY) rats served as controls. In addition, we used another model of cardiac hypertrophy elicited in Sprague-Dawley (SD) rats by pressure overload induced by abdominal aortic coarctation. Finally, in both animal models, we evaluated the effect of a drug treatment that prevents or attenuates hypertrophy through inhibition of the renin-angiotensin system (RAS).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Animals and Treatments

Animal experimentation was approved by the local ethics committee in compliance with guidelines of the Canadian Council on Animal Care.

Genetic model of cardiac disease. Male SHR and age-matched control WKY rats purchased from Charles River (St. Constant, Quebec, Canada) were killed at 7, 15, and 30 wk of age. Body weights at death were, respectively (n = 6), 210 ± 10, 320 ± 26, and 680 ± 47 g for SHR and 215 ± 15, 330 ± 30, and 710 ± 65 g for WKY rats. Before the day of the experiments, the rats were housed for ≥7 days in a 12:12-h light-dark cycle facility with unlimited access to water and standard chow. The hearts were isolated under pentobarbital sodium anesthesia (65 mg/kg ip, MTC Pharmaceuticals; Cambridge, Ontario, Canada), cannulated rapidly, flushed with a cold saline solution, freeze clamped, and stored in liquid nitrogen until further analyses. Freeze-clamped heart tissue samples were also obtained from a subgroup of 15-wk-old SHR that had been treated for 4 wk with enalapril [an angiotensin-converting enzyme (ACE) inhibitor, 30 mg·kg–1·day–1, a generous gift from Merck-Frosst; Montreal, Quebec, Canada] in drinking water. This same subgroup of SHR was part of a previously published study (20). The body weights of untreated and treated SHR at death were 314 ± 4 and 306 ± 5 g, respectively (n = 6).

Surgery model of cardiac disease. Heart tissue samples were obtained from 16-wk-old SD rats 14 days after sham operation or after aortic coarctation with or without treatment with the RAS inhibitor valsartan, an angiotensin II type 1 (AT1) receptor antagonist. Data from this same subgroup of SD rats were obtained within the context of another study (4). Briefly, after rats were anesthetized with pentobarbital sodium, a 0.6-mm band was placed around the abdominal aorta. The controls had sham surgery without band insertion. A subgroup of SD rats underwent aortic coarctation and oral treatment with valsartan (10 mg·kg–1·day–1, a generous gift from Novartis; Montreal, Quebec, Canada). All rats were killed 14 days after surgery. Body weights at death were 379 ± 10, 379 ± 6, and 373 ± 4 g (n = 4) for rats that underwent sham surgery or coarctation with or without treatment, respectively.

Measurements of Cardiomyocyte Size

The NIH Image 1.61 program (http://rsb.info.nih.gov/nih-image/) served to measure the cross-sectional area of the ventricles as described previously (20).

Enzyme Assays

Total activities of myocardial enzymes were assessed in 100 mg of powdered tissues that were homogenized on ice in 1 ml buffer containing 180 mM KCl, 5 mM MOPS, and 2 mM EDTA (pH 7) and centrifuged for 10 min at 800 g at 4°C. Supernatants were used for enzyme assays after 10-min centrifugation at 6,000 g at 4°C. Commercial kits were employed for mNADP+-ICDH (Sigma Diagnostics; Oakville, Ontario, Canada) and manganese superoxide dismutase (MnSOD; Dojindo Molecular Technologies; Gaithersburg, MD) assays. MnSOD was measured by the addition of 0.1 mM sodium cyanide to the sample before the assay for 10 min to inhibit Cu/ZnSOD. Glutathione (GSH) peroxidase and reductase were assayed according to the standard Sigma protocol (EC 1.11.1.9 [EC] , MB-765). Aconitase and citrate synthase activities were measured as described previously by Comte et al. (14). Protein levels were quantified with a kit (Bio-Rad; Mississauga, Ontario, Canada), and BSA (Sigma) was the standard. Enzyme activities were expressed in units per milligram of protein, where 1 unit was defined as the amount of enzyme catalyzing the conversion of 1 µmol substrate/min at 37°C.

Western Blot Analysis

Twenty micrograms of tissue proteins, extracted in cold extraction buffer containing 1 mM phenylmethysulfonyl fluoride, 10 µM each of aprotinin, leupeptin, and pepstatin, and 1 mM orthovanadate, were subjected to discontinuous 4–12% SDS-PAGE under reducing conditions. Protein transfer, immunodetection, and semiquantitative measurements were performed as described previously (5). Rabbit-specific anti-mouse mNADP+-ICDH was a generous gift from Dr. T. L. Huh (Kyungpook National University; Taegu, South Korea), and goat anti-rabbit IgG-horseradish peroxidase conjugate was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).

Semiquantitative RT-PCR

mRNA levels of mNADP+-ICDH, atrial natriuretic factor (ANF), and GAPDH were analyzed semiquantitatively with a programmable thermal controller (MJ Research; Waltham, MA) as previously described (5). The following sense- and antisense-specific primers were used: mouse mNADP+-ICDH sense 5'-GGCTGTGAGCTCGCTCTGCAGAGC-3' and antisense 5'-TCTTGGTGCTCAAGTAGAGCGGC-3'; rat ANF sense 5'-ATCTGATGGATTTCAAGAACC-3' and antisense 5'-CTCCAATCCTGTCAATCCTACC-3'; and rat GAPDH sense 5'-AGTGGACATTGTTGCCATCAACGACC-3' and antisense 5'-GTCATGAGCCCTTCCACGATGCCAA-3'. The number of cycles (20–25) was optimized for each gene to fall within the linear range of PCR amplification (data not shown). After PCR amplification, the samples were subject to electrophoresis on a 1.8% agarose gel, transferred to nylon membranes, and quantified as described previously (5).

Tissue Levels of HNE-mNADP+-ICDH Adducts

Five hundred micrograms of tissue protein extract from 7-, 15-, 30-wk-old SHR and WKY hearts were subjected to immunoprecipitation (5). An aliquot of the immunoprecipitated proteins (20 µl ) was heated at 95°C for 3 min before Western blot analysis employing rabbit anti-HNE antibody (1:1,000 dilution, Calbiochem; Mississauga, Ontario, Canada) as the primary antibody.

Tissue Levels of Total HNE/Protein Adducts

Tissue protein extracts were concentrated fivefold by house (ELISA), as described by Toyokuni et al. (64) and modified by Benderdour et al. (5).

Statistical Analyses

Data are expressed as means ± SE. The statistical significance of differences between mean values at P < 0.05 was assessed by the unpaired t-test or one-way ANOVA, followed by the Bonferroni multiple-comparison posttest. Two-way ANOVA was used to test for significant differences in the effect of 1) hypertrophy development and progression in SHR, which we referred to as the disease factor throughout the text; 2) age; and 3) the interaction of disease x age. The Bonferroni multiple-comparison posttest was performed to assess significant differences in the effect of disease at 7, 15, and 30 wk.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Studies of SHR

Myocardial ANF mRNA levels were significantly elevated in 15- and 30-wk-old SHR compared with age-matched WKY rats (Fig. 1A). To confirm the development of hypertrophy in SHR, cardiomyocyte sizes were measured in a number of heart tissue samples from SHR and WKY rats (n = 3 of each strain). SHR hearts showed an increase in cardiomyocyte size (Fig. 1B), and, according to two-way ANOVA, this effect of disease was significant and independent of age.



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  		Fig. 1. Markers of hypertrophy development and progression (A and B) in the hearts of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats at various ages. Hearts isolated from 7-, 15-, and 30-wk-old SHR and WKY rats were processed as described in METHODS for the following analyses. A: atrial natriuretic factor (ANF) mRNA levels. Data are from representative semiquantitative RT-PCR analysis of total myocardial RNA using ANF-specific primers. Corresponding data on GAPDH-specific primers are shown in Fig. 2. mRNA levels were quantitated in arbitrary units by the densitometric analysis of autoradiograph bands. ANF mRNA levels were normalized to those of GAPDH mRNA and are expressed relative to those of WKY control rats. Data are means ± SE of 6 rat hearts. B: cardiomyocyte area. Data were obtained by quantitative microplanimetry in sections of ventricles. Data are means ± SE of 3 rat hearts. Statistics are as follows: one-way ANOVA followed by the Bonferroni multiple-comparison posttest (A) (SHR vs. WKY: *P < 0.05 and #P < 0.001) or two-way ANOVA for the effect of disease, age, or disease x age (B). NS, not significant.

 
mNADP+-ICDH Activity and Expression Are Sequentially Affected During Hypertrophy Development

Figure 2 shows mNADP+-ICDH activity (A), protein levels (B), and mRNA expression (C) in the hearts of 7-, 15-, and 30-wk-old SHR compared with age-matched WKY control rats. At all ages, myocardial mNADP+-ICDH activity in SHR was 25% lower than in WKY rats (Fig. 2A). In contrast, at 7 wk of age, myocardial mNADP+-ICDH protein and mRNA levels in SHR were comparable to those in WKY rats, but at 15 and 30 wk, they were significantly reduced in SHR hearts. Two-way ANOVA revealed an effect of disease that was independent of age for mNADP+-ICDH activity and protein expression but not for mNADP+-ICDH mRNA levels. Two-way ANOVA also disclosed an effect of age on all parameters.



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  		Fig. 2. Cardiac mitochondrial NADP+-isocitrate dehydrogenase (mNADP+-ICDH) activity (A), protein (B), and mRNA levels (C) in SHR and WKY rats at various ages. Total proteins and RNA were extracted from the hearts of 7-, 15-, and 30-wk-old SHR and WKY rats and processed as described in METHODS for the following analyses: mNADP+-ICDH activity (A), protein (B), and mRNA level (C). Western blot analysis was performed on samples of total myocardial proteins and of pig heart mNADP+-ICDH standard (Std; ~46 kDa). Protein levels were quantitated in arbitrary units by the densitometric analysis of autoradiograph bands. RT-PCR analysis and quantitation were conducted as described in Fig. 1. All values are expressed as a percentage of the mean value for 7-wk-old WKY rats. Data are means ± SE of 6 rat hearts. {phi}Data from Ref. 5. Statistics are as follows: two-way ANOVA followed by the Bonferroni multiple-comparison posttest (SHR vs. WKY: *P < 0.05, **P < 0.01, and #P < 0.001).

 
Loss of mNADP+-ICDH Activity in SHR Hearts Is Associated With Enhanced Tissue Accumulation of HNE-mNADP+-ICDH and HNE-Protein Adducts

To follow on our recent finding of mNADP+-ICDH inactivation through HNE adduct formation in 7-wk-old SHR (5), we assessed myocardial HNE-mNADP+-ICDH levels in SHR and control WKY rats of all ages. As shown in Fig. 3A, the relative level of HNE-mNADP+-ICDH adducts was increased significantly at all ages. The highest increase was observed at 7 wk of age (2.1-fold) compared with 1.28- and 1.4-fold at 15 and 30 wk, respectively. Tissue levels of total HNE-protein adducts were also significantly elevated in SHR at all ages compared with control WKY rats (Fig. 3B). However, the magnitude of the increase varied with age in the following order: 7 wk (2.7-fold) > 30 wk (2.5-fold) > 15 wk (1.5-fold). Two-way ANOVA of the data revealed a significant effect of age, with the impact of the disease being significant and dependent on age.



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  		Fig. 3. Cardiac levels of 4-hydroxynonenal (HNE)-mNADP+-ICDH (A) and HNE-protein (B) adducts in SHR and WKY rats of various ages. Total proteins from 7-, 15-, and 30-wk-old SHR and WKY rat heart extracts were immunoprecipitated and subjected to Western blot analysis (A) or concentrated fivefold, coated on microtiter plates, and subjected to ELISA (B), as described in METHODS. Data are means ± SE of 3 (A) or 6 (B) rat hearts. {phi}Data from Ref. 5. Statistics are as follows: one-way (A) and two-way ANOVA (B) followed by the Bonferroni multiple-comparison posttest (SHR vs. WKY: *P < 0.05, **P < 0.01, and #P < 0.001).

 
Changes in mNADP+-ICDH Activity During Hypertrophy Development Contrast With That of Other Antioxidant and Metabolic Enzymes

Because hypertrophy development is associated with alterations in mitochondrial energy and oxidative stress status, we evaluated whether changes in mNADP+-ICDH activity were paralleled by similar modifications in the activities of other mitochondrial enzymes involved in antioxidant and energy metabolism. Figure 4 depicts the variations in activities of the antioxidant enzyme MnSOD (A) and the CAC enzymes aconitase (B), which is highly susceptible to free radical inactivation (50), and citrate synthase (C). Two-way ANOVA showed a significant effect of disease on MnSOD and aconitase activity, which was independent of age for MnSOD. Compared with control WKY rat hearts, aconitase activity was reduced significantly in 7- and 30- but not 15-wk-old SHR hearts (Fig. 4B), whereas that of MnSOD was increased in 15- and decreased in 30-wk-old SHR (Fig. 4A). Two other antioxidant enzymes, GSH peroxidase and reductase, presented a disease and age-dependent activity profile that was identical to that of MnSOD (data not reported). In contrast to the oxidative stress-related enzymes, tissue citrate synthase did not differ significantly between SHR and WKY rats, nor did its activity vary with age (Fig. 4C). Altogether, the data shown in Figs. 2 and 4 indicate that the profile of changes in mNADP+-ICDH activity in SHR with disease progression differs from that of measured antioxidant and energy metabolic enzymes.



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  		Fig. 4. Activity of mitochondrial enzymes involved in antioxidant defences (A and B) and in mitochondrial energy metabolism (B and C) in the hearts of SHR and WKY rats at various ages. Hearts isolated from 7-, 15-, and 30-wk-old SHR and WKY rats were processed as described in METHODS for activity measurements of MnSOD (A), aconitase (B), and citrate synthase (C). Data are means ± SE of 6 rat hearts. Statistics are as follows: two-way ANOVA followed by a Bonferroni multiple-comparison posttest (SHR vs. WKY: *P < 0.05 and #P < 0.001).

 
Loss of mNADP+-ICDH Activity and Expression in SHR Hearts Is Attenuated by Treatment With a RAS Inhibitor

We evaluated whether mNADP+-ICDH activity and expression in SHR hearts could be modulated by a drug that attenuates cardiac hypertrophy (20). For this purpose, we analyzed heart tissues from 15-wk-old SHR that had been exposed for 4 wk to 30 mg·kg–1·day–1 enalapril, an ACE inhibitor (20). In our earlier study, it was shown that such treatment decreased systolic blood pressure and induced regression of cardiac hypertrophy as reflected by a reduction of cardiac mass and DNA (20). As illustrated in Fig. 5A, myocardial mNADP+-ICDH activity in enalapril-treated SHR was 1.3-fold higher than in age-matched, untreated SHR. The increase of mNADP+-ICDH activity was accompanied by a parallel 1.5-fold elevation of mNADP+-ICDH protein and mRNA levels (Figs. 5, B and C). Comparison of data from Figs. 2 and 5 suggests that after 4-wk enalapril treatment, 15-wk-old SHR showed values for cardiac mNADP+-ICDH activity and expression that were close to those of age-matched WKY control rats.



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  		Fig. 5. Effect of enalapril (E) treatment on cardiac mNADP+-ICDH activity (A), protein (B) and mRNA level (C) in 15-wk-old SHR. Total proteins and mRNA were extracted from the hearts of untreated and enalapril-treated 15-wk-old SHR and processed as described in Figs. 1 and 2 for the determination of mNADP+-ICDH activity, mNADP+-ICDH protein expression, and mNADP+-ICDH and GAPDH mRNA expression. Data are means ± SE of 4 rat hearts. Statistics are as follows: the effect of enalapril treatment was determined using the unpaired t-test (*P < 0.05 and **P < 0.01).

 
Studies of SD Rats Subjected to Aortic Coarctation

We considered that it was essential to exclude the possibility that changes in mNADP+-ICDH activity and expression observed in SHR were a distinct feature of this genetic model of hypertension. We therefore analyzed heart tissues from 16-wk-old SD rats 14 days after sham operation or after abdominal aortic coarctation with or without treatment with a RAS inhibitor, specifically, the AT1 receptor antagonist valsartan (4). In the same subgroup of SD rats with coarctation, left ventricular hypertrophy was documented by an increase of 14% in the left ventricular weight-to-body weight ratio (2.04 ± 0.03 x 10–3, P < 0.05 vs. sham). In rats treated with valsartan, however, the left ventricular mass index (2.09 ± 0.08 x 10–3) presented no significant change compared with sham controls. As an additional marker of hypertrophy, we documented the cardiac level of ANF mRNA and found that it was increased 1.5-fold after 14 days of aortic coarctation (Fig. 6A) compared with sham controls. Valsartan prevented the elevation of ANF mRNA in agreement with the drug's capacity to prevent cardiac hypertrophy.



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  		Fig. 6. Effect of coarctation (C) and coarctation with valsartan treatment (Cv) on cardiac ANF mRNA levels (A) and mNADP+-ICDH activity (B), protein (C), and mRNA level (D) in Sprague-Dawley (SD) rats. Total proteins and mRNA were extracted from the hearts of SD rats subjected to either sham operation (S), coarctation, or coarctation with valsartan treatment. Heart samples were processed as described in Figs. 1 and 2 for the determination of mNADP+-ICDH activity, mNADP+-ICDH protein level, and mNADP+-ICDH, ANF and GAPDH mRNA level. Data are means ± SE of 4 rat hearts. Statistics are as follows: one-way ANOVA followed by a Bonferroni multiple-comparison posttest (all vs. sham: **P < 0.01 and #P < 0.001).

 
mNADP+-ICDH Activity and Expression Are Decreased in Coarctated SD Rats

mNADP+-ICDH activity (Fig. 6B), protein (Fig. 6C), and mRNA expression (Fig. 6D) were all reduced, between 25% and 35%, in coarctated compared with sham-operated SD rats. The decrease in mNADP+-ICDH activity and expression after abdominal aortic coarctation was not observed when the rats were treated with valsartan. Together, the results obtained in this surgical model of cardiac hypertrophy concur with those in SHR.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we evaluated the impact of cardiac hypertrophy on the activity and expression of mNADP+-ICDH, an enzyme regulating mitochondrial energy and redox status. The first set of experiments was conducted in SHR, a well-established genetic model of cardiomyopathy associated with hypertension, hypertriglyceridemia, and insulin resistance, which develops hypertrophy between 9 and 12 wk of age. The results from this study demonstrate the following alterations in SHR compared with control WKY rats. First, a significant reduction of mNADP+-ICDH activity in SHR hearts was apparent as early as 7 wk, before hypertrophy develops (Figs. 1 and 2A), and persisted with hypertrophy development and progression. Second, the decline in mNADP+-ICDH activity preceded that of its expression at the protein and mRNA levels, which was apparent at 15 wk (Fig. 2, B and C). Hence, collectively, these data demonstrate that in SHR, mNADP+-ICDH activity and expression are affected sequentially in the heart during hypertrophy development and progression.

The reduction of mNADP+-ICDH activity in SHR hearts at all ages was associated with enhanced accumulation of HNE-mNADP+-ICDH and total HNE-protein adducts. These data are in agreement with mNADP+-ICDH inactivation through HNE binding, possibly at a cysteine residue near the substrate's binding site (5). The enhanced accumulation of HNE-mNADP+-ICDH and total HNE/protein adducts indicated that a high level of oxidative stress already prevails at 7 wk of age in SHR hearts, specifically in the mitochondria, and persists with the progression of hypertrophy. The highest percent increase of myocardial HNE-mNADP+-ICDH adducts observed in SHR at 7 wk than at 15 or 30 wk can be explained by considerations of changes in the activity of the free radical target aconitase and of antioxidant enzymes (see below for details). Our results of an increased accumulation of HNE-protein adducts in SHR hearts concur with other literature data indicating that lipid peroxidation products, especially HNE, contribute to the pathophysiological events associated with cardiac disease development (7, 23, 49, 55, 66, 74). Specifically, our data emphasize the importance of including the effects of HNE at the posttranslational level on mitochondrial metabolic enzymes determining energy and NAD(P)H redox status in the network of events triggered by HNE in the context of cardiac hypertrophy development. The mitochondria are viewed both as a target and the principal source of free radicals because of electron leakage from the respiratory chain to oxygen-forming superoxide anions (35). However, HNE formation from free radical-induced lipid peroxidation could also occur at extramitochondrial sites and diffuse within the cells to propagate damage to the mitochondria. In SHR, enhanced superoxide anion production, reported as early as 4 wk, has been attributed to dysfunctional nitric oxide synthase activity and/or angiotensin II-stimulated NAD(P)H oxidase activity (15, 17, 44, 56, 73). However, the contribution of the various site(s) of HNE formation remains to be clarified.

In our study, we considered it essential to address the confounding effect of hypertrophy development and age in SHR. Indeed, both conditions are characterized by mitochondrial dysfunction subsequent to increased oxidative stress and energy deficits (18, 42). For this purpose, the data were analyzed by two-way ANOVA, which revealed an effect of disease on mNADP+-ICDH activity and protein, although not on mRNA expression levels, which was independent of age. However, our analysis also detected a significant effect of age on all parameters presented in Figs. 2 and 3. An age-dependent accumulation of HNE-protein adducts in the mitochondria (Fig. 3B) has been reported recently by other investigators (46, 62). Collectively, these findings concur with the notion of progressive oxidative stress enhancement with age. The fact that two-way ANOVA revealed an interaction between disease and age for this parameter supports the notion that these two conditions share some oxidative stress-related components.

The chronic loss of mNADP+-ICDH activity with hypertrophy development and progression in SHR contrasted with the observed changes in other mitochondrial enzymes involved in energy (aconitase, citrate synthase) or antioxidant (MnSOD) metabolism (Fig. 4). Collectively, the observed changes in enzyme activities, whether expressed per milligram of protein or relative to that of citrate synthase, which was unchanged with hypertrophy development or aging (Fig. 4 and Ref. 40), suggest the following oxidative stress-related events in SHR hearts: 1) oxidative stress already increased at 7 wk, preceding hypertrophy; 2) antioxidant defences are upregulated at 15 wk, in line with a compensated hypertrophy; and 3) the activities of the CAC enzyme aconitase and of antioxidant enzymes are decreased at 30 wk, suggesting the beginning of a decompensated hypertrophy. Upregulation of antioxidant enzymes in 15-wk-old SHR hearts could explain the lower increases in tissue levels of HNE-mNADP+-ICDH and HNE-protein adducts. However, because the tissue levels of these adducts remain higher in SHR than in WKY rat hearts, upregulation of the antioxidant enzymes MnSOD, GSH peroxidase, and GSH reductase provided only partial protection against the increased oxidative stress. Other detoxification enzymes could be differentially affected by disease. In fact, hearts from SHR were shown to have a lower capacity to metabolize exogenous HNE than those of WKY rats (28). Furthermore, in a dog model of pacing-induced heart failure, a progressive and sequential decline in activity and expression at the protein and mRNA levels was reported for aldose reductase, an enzyme proposed to be involved in HNE detoxification (61).

While the decreased of mNADP+-ICDH activity in 7-wk-old SHR can be attributed to posttranslational modification of mNADP+-ICDH by HNE binding, other factors should be considered in 15-and 30-wk-old SHR because there was also a reduction of mNADP+-ICDH mRNA and protein expression. We do not have an explanation for these findings, although they appear to be a consequence of enhanced oxidative stress. Numerous factors governing mRNA transcription and turnover as well as protein translation and turnover could be altered by conditions prevailing in the diseased and/or aging heart and should be considered in future studies. Jo et al. (36) have proposed that the transcription factor NF-{kappa}B, whose nuclear binding activity is inhibited by HNE (51), could regulate mNADP+-ICDH expression. However, to the best of our knowledge, the involvement of this regulatory mechanism remains to be demonstrated. To seek other factors that could regulate mNADP+-ICDH expression, we conducted a search in the Ensemble database (www.ensembl.org) to obtain sequence information on the 250-bp proximal 5'-flanking region of the human, mouse, and rat gene (Fig. 7). Our analysis revealed high GC content but no sequence homology for the NF-{kappa}B binding site or the consensus sequence for TATA or CCAAT binding sites. However, this does not exclude the existence of distal NF-{kappa}B regulating site(s).



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  		Fig. 7. Promoter sequence analysis of the human, mouse, and rat mNADP+-ICDH gene. Two hundred and fifty base pairs of the proximal 5' region of the mNADP+-ICDH gene were analyzed; {bullet} represents a putative Sp1 binding site, and exon sequences are depicted as a shaded box. Human (ENSG00000182054) and mouse (ENSMUSG00000030541) mNADP+-ICDH gene and promoter sequences were obtained through a search in the Ensembl database (www.ensembl.org). The rat promoter homolog for mNADP+-ICDH gene has not been cloned yet, but a Novel Ensembl prediction localized it at the ENSRNOG00000013949 locus. This locus presents 100% primary sequence homology at exon sequences with rat ICDH2 cDNA (45) and similar intron/exon organization with both human and mouse mNADP+-ICDH. All promoter sequences were analyzed with Alibaba 2.1 software (http://www.gene-regulation.com/pub/programs/alibaba2/index.html) and the Transfac 4.0 transcription factor motif database.

 
Interestingly, our analysis showed several putative Sp1 sites in the homolog mNADP+-ICDH promoter region of all species. This promoter structure is reminiscent of a housekeeping gene promoter (Fig. 7) (2, 8, 26, 57). A Sp1 cluster near the transcription start site could compensate for the lack of TATA box consensus for the initiation of transcription, another feature of a housekeeping gene promoter. The abundance and conservation of Sp1 binding sites suggest that this transcription factor might play a critical role in regulating mNADP+-ICDH gene expression. Sp1, which is a ubiquitous transcription factor that belongs to the Sp family, regulates the expression of several types of genes, including those coding for metabolic enzymes (6, 53). Sp1 protein is characterized by the presence of three zinc finger motifs of 30 amino acids of the Cys2His2 type, which are essential for DNA binding. Interestingly, oxidation of the cysteine and histidine residues of Sp1 zinc finger protein by oxygen- and nitrogen-derived free radicals has been shown to decrease Sp1 binding to DNA (38, 69). Thus it is tempting to speculate that the reduced mNADP+-ICDH mRNA level in the hearts of 15- and 30-wk-old SHR could result from lower activation of its promoter due to attack by oxidative stress-derived molecules, including HNE, to cysteine/histidine amino acid residues of Sp1 (27, 38, 69). However, the existence of this regulatory mechanism, especially the capacity of HNE to alter mNADP+-ICDH expression via its binding to Sp1, remains to be demonstrated.

Other sets of experiments provided additional evidence for a link between hypertrophy development and mNADP+-ICDH activity and expression. First, our finding of decreased mNADP+-ICDH activity and expression after 2 wk of coarctation in SD rats indicates that these modifications are not a specific characteristic of SHR hearts (Figs. 6, B–D). Second, we found that attenuation or prevention of hypertrophy development by treatment with RAS inhibitors of SHR (enalapril; Fig. 5) or of coarctated SD rats (valsartan; Fig. 6), respectively, normalized or prevented the decrease in mNADP+-ICDH activity and expression. It is well established that RAS activation by an agonist of angiotensin II receptors results in cardiac hypertrophy, fibrosis, and congestive heart failure via AT1 receptor activation (71). The latter mediates effects such as vasoconstriction, cellular proliferation, and matrix deposition (11, 70), most likely via a mechanism that involves enhanced free radical production due to stimulated NADPH oxidase activity (75). Although in this study we used tissues of rats that received RAS inhibitors that act by different mechanisms, namely, inhibition of ACE (enalapril) and antagonism of AT1 receptor (valsartan), respectively, we observed similar effects of both drugs on mNADP+-ICDH activity and expression. Der Sarkissian et al. (20) showed that both drugs also 1) reduced systolic blood pressure, left ventricular hypertrophy, and cardiomyocyte cross-sectional area and 2) abolished noncardiomyocyte hyperplasia in the SHR left ventricle by apoptosis induction. It remains to be seen, however, whether the capacity of the RAS inhibitor to improve myocardial antioxidant defences and contractile activity (10, 40) could be attributed, in part, to the observed changes in mNADP+-ICDH activity, which, in turn, could be subsequent to reduced free radical production by NADPH oxidase or other mechanisms (56).

We recognize that several factors should be kept in mind in the interpretation of data of our study. Indeed, for our measurements, we used whole heart tissue homogenates. While cardiomyocytes represent >85% of the heart cell mass, the heart contains also noncontractile cells (i.e., fibroblasts and endothelial cells) whose relative number and/or size can be affected with hypertrophy development, progression, and regression. For example, compared with age-matched WKY rats, 15-wk-old SHR hearts contain more fibroblasts and larger cardiomyocytes, and these cellular changes are abolished by prior treatment with enalapril (20). Clearly, further work is necessary to address the issue of heterogeneity in cell size and composition in our study. For mNADP+-ICDH, activity changes are likely to be restricted to cardiomyocytes (31), but for the other measured enzymes, the contribution of other cell types to total activity remains to be clarified. Nevertheless, the impact of oxidative stress on the measured activities is expected to be similar irrespective of the cell origin or size.

With regard to the potential significance of our finding of an early and persistent loss of mitochondrial mNADP+-ICDH activity in SHR hearts, collectively our results support its relevance as an early and persistent marker of oxidative stress-related mitochondrial alterations in hypertrophy development. However, clarification of the (patho)physiological significance of decreased cardiac mNADP+-ICDH would require an entirely different approach, most likely using transgenic, cardiomyocyte-specific knockout animals. Potentially, mNADP+-ICDH inactivation by HNE could contribute to the impaired mitochondrial energy and GSH status of hypertrophied and failing hearts (16, 37, 61). However, because cardiac mNADP+-ICDH is abundant inside cardiomyocyte mitochondria, it could represent an easy target for HNE binding to spare other enzymes such as the {alpha}-ketoglutarate dehydrogenase, whose activities are even more crucial for energy production. Such a mechanism has been also proposed for GSH transferase in the liver (65) and albumin in blood (30). We previously alluded to the possible participation of mNADP+-ICDH to the coordinated mitochondrial response to oxidative stress initially hypothesized by Szweda et al. (33, 34) for the CAC enzyme {alpha}-ketoglutarate dehydrogenase. The latter enzyme appears to be less susceptible to inactivation by HNE than mNADP+-ICDH (50% inhibition at 40 vs. 20 µM, respectively; Refs. 33 and 5). Therefore, in the short term, changes in mNADP+-ICDH activity may be considered partly adaptive, but in the long term, they would be maladaptive leading to mitochondrial energy deficits. Interestingly, when perfused ex vivo in the working mode, 15-wk-old SHR hearts maintain functional parameters that are similar to control Wistar or WKY rats (68), although they show an impaired capacity to respond and withstand an acute adrenergic stimulation (39), suggesting that hypertrophy development in these rats include some maladaptive components.

In conclusion, the major findings of this study can be summarized as follows. mNADP+-ICDH activity and expression are reduced during cardiac hypertrophy development and can be normalized by treatment with drugs that inhibit the RAS. The decline in mNADP+-ICDH activity, which occurred before hypertrophy development in SHR, preceded that of its protein and mRNA expression in time, indicating that activity and expression are differentially modulated during hypertrophy development and progression. The early and persisting decline in mNADP+-ICDH activity is associated with the formation of HNE-mNADP+-ICDH adducts and contrasts with the profile of activity changes of other mitochondrial metabolic enzymes involved in energy production and antioxidant defences. Hence, myocardial mNADP+-ICDH inactivation through HNE adduct formation appears to be a relevant early and persistent marker of mitochondrial oxidative stress-related alterations linked to hypertrophy development. Whether this contributes also to the etiology of the cardiomyopathy remains to be evaluated. Such studies appears to be warranted, given that HNE binding to mNADP+-ICDH, if predominantly occurring at cysteine residues (5), could potentially be reversed by aldehyde-sequestering drugs (12, 19, 22, 32). In fact, the reversal of HNE binding to proteins could be part of the mechanism by which N-acetylcysteine prevents cardiac hypertrophy in rats infused with angiotensin II (48). Finally, additional work appears also warranted to expand on the interesting peripheral finding of this study, namely, that hypertrophy and aging results in similar directional changes in mNADP+-ICDH activity and expression.


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This study was supported by Canadian Institutes of Health Research Grant 10816 (to C. Des Rosiers). D. deBlois is a scholar of Fonds de la Recherche en Santé du Québec.


   ACKNOWLEDGMENTS
 
The authors thank Dr. T. L. Huh and Dr. J. Wu for the generous gifts of mNADP+-ICDH antibody and cDNA, respectively, and David Duguay for providing tissues from enalapril-treated and untreated SHR. Thanks are also due to Ovid Da Silva of the Research Support Office, Centre Hospitalier de l’Université de Montréal Research Centre, for editorial assistance.

Part of this work was presented as an abstract at the 9th Oxygen Society Meeting (2002) and at the European section of the Society for Free Radical Research Meeting (2003).

Present address of M. Benderdour: Centre de Recherche, Hôpital Sacré-coeur, 5400 Boul. Gouin ouest, Montréal, Quebec, Canada H4J 1C5.

Present address of S. Foisy: Diploïd.net. 7013 rue Jogues, Montréal, Quebec, Canada H4E 2W9.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Des Rosiers, Institut de cardiologie de Montréal, Centre de Recherche (S-5350), 5000 rue Bélanger est, Montréal, Québec, Canada H1T 1C8 (E-mail: christine.des.rosiers{at}umontreal.ca)

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. Section 1734 solely to indicate this fact.


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