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European Journal of Heart Failure 2008 10(3):226-232; doi:10.1016/j.ejheart.2008.01.001
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© 2008 European Society of Cardiology

Enhanced myocardial vitamin C accumulation in left ventricular hypertrophy in rats is not attenuated with transition to heart failure

Susanne Rohrbacha,*, Antonio Martinb, Bernd Niemanna and Antonio Cherubinic

a Institute of Pathophysiology, Martin-Luther–University Halle–Wittenberg Germany
b Jean Mayer USDA Nutrition Research Center on Aging, Tufts University Boston, USA
c Institute of Gerontology and Geriatrics, University of Perugia Medical School Italy

* Corresponding author. Institute of Pathophysiology, Martin-Luther–University Halle–Wittenberg, Ernst-Grube-Str. 40, FG06 E02, 06097 Halle/Saale, Germany. Tel.: +49 345 557 1454; fax: +49 345 557 1404. E-mail address: susanne.rohrbach{at}medizin.uni-halle.de (S. Rohrbach).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Indirect observations are compatible with cardiac vitamin C deficiency as one contributory factor to oxidative stress in heart failure, but data on ventricular vitamin C content are lacking. Here, we used the well established model of aortic-banded rats at the stage of compensated hypertrophy (6 weeks after banding) and at the transition to cardiac failure (22 weeks after banding) to analyze vitamin C, vitamin E, protein carbonyls and malondialdehyde tissue content together with the respective plasma concentrations. Furthermore, we investigated the expression of the vitamin C transporters SVCT1 and SVCT2 in the left ventricle (LV).

Aortic-banded rats, independently from their age, had higher malondialdehyde and protein carbonyl levels in plasma and LV tissue compared to sham-operated animals indicating increased oxidative stress. Plasma vitamin C remained unaffected from cardiac overload, while LV vitamin C was elevated in both stages of hypertrophy together with an increased expression of the vitamin C transporter SVCT2 suggesting increased vitamin C uptake. The levels of antioxidants and lipid peroxides were similar 6 and 22 weeks after aortic banding. Therefore, the accumulation of vitamin C in compensated hypertrophy and in decompensated failure excludes cardiac vitamin C deficiency as a primary factor to oxidative stress in this model.

Key Words: Vitamin C • Vitamin C transporter • Heart failure • Oxidative stress • Malondialdehyde

Received August 16, 2007; Revised November 26, 2007; Accepted January 7, 2008


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Sustained pressure overload of the heart results in ventricular hypertrophy, previously regarded as an adaptation, and is inexorably followed by transition to cardiac failure. This transition from compensated hypertrophy to failure is characterized by enhanced oxidative stress in the myocardium [1,2] and by activation of apoptosis in cardiomyocytes [3]. In several experimental heart failure models, cardiomyocyte apoptosis and the progression of cardiac dysfunction are attenuated by treatments with vitamin C and/or other antioxidant vitamins [1,4].

In patients, enhanced oxidative stress in failing myocardium has been proposed on the basis of significant correlations between parameters of ventricular dysfunction and indices of oxidative stress in plasma or pericardial fluid [5,6]. Reduced circulating vitamin C levels in heart failure are occasionally reported, and plasma ascorbate levels are inversely related to all-cause mortality and cardiovascular mortality [7], which is probably only in part explained by a reduction in the risk of myocardial infarction. Chronic intake of vitamin C together with other vitamins and micronutrients improved ventricular function in a prospective, controlled trial in elderly patients with heart failure [8].

While these observations appear supportive to the hypothesis of myocardial vitamin C deficiency as contributory factor in heart failure progression, any data on ventricular vitamin C content in experimental or clinical heart failure and their modulation by chronic intake are not available. Therefore, we analyzed LV vitamin C content in compensated LV hypertrophy and subsequent transition to failure in a rat model of pressure overload following aortic banding, which had been used previously for the analysis of this transition to failure [9].


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Preparation of animals and in vivo left ventricular measurements
Ascending aortic banding was performed in male Wistar weanling rats (body weight 50-70 g, 3-4 weeks), using a titanium clip (Weck, Inc., 0.6 mm internal diameter), as previously described [9]. Age-matched animals underwent the same procedure without placement of the clip to serve as sham-operated controls. All rats were subsequently fed regular chow containing 55 IU/kg vitamin E but no vitamin C, and received water ad libitum. Aortic-banded animals and age-matched sham-operated controls were sacrificed after 6 weeks (14 aortic-banded, 12 sham-operated) or after 22 weeks (13 aortic-banded, 12 sham-operated) following surgery. At this time in vivo LV pressure measurements were performed in all animals. Under intraperitoneal pentobarbital anesthesia, the LV apex was punctured from the abdomen with an 18-gauge needle connected to a Statham pressure transducer (Gould Instruments). LV systolic pressure (LVSP) and LV end-diastolic pressure (LVEDP) were recorded for at least 10 s. All treatments of animals throughout this study received local institutional animal care and use committee approval and the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

2.2. Malondialdehyde (MDA) in tissue and plasma
MDA content in the blood and the left ventricular tissue was estimated by means of the reaction with thiobarbituric acid using HPLC-fluorometric analysis. The heart tissue was homogenized in potassium phosphate buffer 100 mM, pH 7.4 containing 0.4% butylated hydroxytoluene (BHT). Malondialdehyde bis (dimethyl acetal) (TMP) was used as standard (Sigma-Aldrich). Detection of MDA was performed using an HPLC system coupled with a Waters 470 fluorescence detector (excitation 530 nm; emission 552 nm). The mobile phase consisted of 40% methanol, 60% 50 mM potassium dihydrogen phosphate, pH 7.0, with a flow rate of 0.9 ml/min. The retention time for the peak of interest under the chromatographic conditions used was 6.7 min. MDA concentration is expressed as nmol/L in plasma or pmol/mg protein in tissue.

2.3. Measurement of carbonylated proteins in tissue and plasma
For determination of protein carbonyl content in rat LV tissue and plasma, we used the Protein Carbonyl Assay Kit (Cayman Chemical), which utilizes the reaction between 2,4-dinitrophenylhydrazine (DNPH) and protein carbonyls, forming a Schiff' base. The resulting reaction product was measured spectrophotometrically at 385 nm.

2.4. Vitamin E in tissue and plasma
The concentration of vitamin E ({alpha}- and {gamma}-tocopherol) in plasma and in LV tissue was measured by reversed-phase HPLC [10]. Briefly, 100 {varepsilon}l plasma sample and 100 {varepsilon}l homogenized tissue were mixed with 100 {varepsilon}l ethanol. Tocopherols were extracted into 500 {varepsilon}l hexane containing 0.002% butylated hydroxytoluene. Tocol (a gift from Hoffmann-La Roche) was added to the mixture as an internal standard. Samples were centrifuged at 800 rpm for 5 min at 4 °C. The supernatant was collected and desiccated under a stream of nitrogen gas, and reconstituted in 100 {varepsilon}l of methanol. {alpha}- and {gamma}-Tocopherol were separated by using a 3 {varepsilon}m C18 reverse phase column. The eluted peaks of {alpha}- and {gamma}-tocopherol and the internal standard were detected at an applied potential of + 0.6V by a LC 4B amperometric electrochemical detector (Bioanalytical Systems, West Lafayette, IN). Tocopherols eluted as well separated peaks with a retention time between 2 and 6 min. Peaks were integrated with a ChemStation (Hewlett-Packard). The concentration of tocopherols is expressed in {varepsilon}mol/L in plasma and as pmol/mg protein in tissue.

2.5. Vitamin C in tissue and plasma
Tissue was homogenized in chelex-treated PBS containing 1 mmol/L diethylene-triaminepentaacetic acid. Both tissue and plasma samples were mixed with an equal volume of cold 5% metaphosphoric acid and centrifuged to remove precipitated proteins. An aliquot of the supernatant was chromatographed on a LC8 column using 40 mmol/l sodium acetate and 1.5 mmol/l dodecyltriethylammonium phosphate as the mobile phase. Vitamin C was detected at an applied potential of + 0.6V by a LC4B amperometric electrochemical detector (Bioanalytical Systems). Vitamin C eluted as a single peak with a retention time of 5.5 min [11].

2.6. Real-time RT-PCR
Reverse transcription (RT) of RNA samples was carried out for 30 min at 42 °C. Real-time PCR and data analysis were performed using the Mx3000P Multiplex Quantitative PCR System (Stratagene) as described previously from our laboratory [12]. Fluorescence data were collected at the end of the annealing stage of amplification. We performed Real-time PCR of the sodium-dependent vitamin C transporters SVCT-1 (forward 5'-CTC TCA CAG TCA CCC CCA CT-3', reverse 5'-AAA CAG GCA ACA GGA AGG TG-3') and SVCT-2 (forward 5'-CTT TGG GCT TGT TCT TCC AA -3', reverse 5'-GTT CCT GGG ATG GTG TTG TC-3') in samples derived from rat left ventricles. For the purpose of quantification, a standard curve was generated with six different amounts of target cDNA cloned into plasmid. Each assay was performed in duplicate and validation of PCR-runs was assessed by evaluation of the melting curve and by slope and error obtained with the standard curve. Data were analyzed with Stratagene MX3000P v1.20 Build 160 software. All data of mRNA are given as relative units of 18S rRNA concentrations.

2.7. Western blotting
LV tissue was rapidly homogenized in a buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% Na-deoxycholate, protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail (Sigma). Proteins were quantified using the BCA Protein Assay (Pierce). 50 µg of protein in 6x Laemmli SDS sample buffer were boiled for 5 min and after centrifugation loaded on a SDS-PAGE gel. After electrophoresis, proteins were transfered to a nitrocellulose membrane at 100 V for 60 min. The filters were blocked with 0.01% Tween, 2% nonfat dry milk and then incubated with antibodies directed against SVCT1 and SVCT2 (both Abcam) and GAPDH (Abcam) was used to demonstrate equal loading of the gels. After incubation with peroxidase-conjugated secondary antibody, blots were subjected to the enhanced chemiluminescent detection method (Amersham) and exposed to a film.

2.8. Plasma BNP, TIMP-1 and MMP-2
Plasma BNP, TIMP-1 and MMP-2 were measured by Enzyme-Linked-Immunosorbent-Assays (Rat BNP ELISA-kit, Assay Pro; Rat TIMP-1 ELISA-kit, RayBiotech; MMP-2 Biotrak Assay; Amersham Pharmacia Biotech).

2.9. Statistical analysis
All values are expressed as mean ± SEM. Statistical analysis of the differences observed between the aortic-banded groups (6 and 22 weeks after banding) and the sham-operated control groups were done by ANOVA comparison, using the Fisher test for post-hoc analysis. Statistical significance was accepted at the level of p < 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. LV hypertrophy and hemodynamics
LV weights were significantly increased 6 (+ 57%) and 22 weeks (+ 50%) after ascending aortic banding compared with sham-operated controls (Table 1). The LV weight to body weight ratio was also significantly increased by 80% in the 6 weeks group, and by 59% in the 22 weeks group of banded rats compared with sham-operated rats. 6 weeks after aortic banding surgery, LV developed pressure per gram of LV (LVdevP/g) and end-diastolic LV pressure (Table 2) were significantly elevated in the animals with aortic stenosis compared to age-matched sham-operated controls. 22 weeks after aortic banding, LVdevP/g was slightly lower than in age-matched sham-operated controls, and LV end-diastolic pressure was drastically increased compared to the age-matched controls (Table 2), consistent with prior data in this model [9]. Echocardiographic studies in this model have shown that compensatory hypertrophy with normal LV cavity dimensions and contractile indices is present 6 weeks after banding, whereas animals develop heart failure by 22 weeks after banding, which is characterized by onset of LV cavity enlargement, mild depression of ejection indices and pressure development per gram of LV mass [13]. Clinical markers of heart failure, including the presence of tachypnea, ascites, percardial and pleural effusions as well as increased lung weight (Table 1) were observed only 22 weeks after banding. The ANP and BNP mRNA expression and BNP plasma concentration were increased 6 weeks after banding with a further amplification after 22 weeks, indicating a progression of heart failure. Serum parameters such as matrix metalloproteases (MMP) or tissue inhibitors of MMPs (TIMP), which correlate with LV diastolic filling or the development of chronic heart failure [14], were significantly increased in animals 22 weeks after aortic banding (Table 4). While the TIMP-1 increase is not yet detectable 6 weeks after surgery, MMP-2 is activated at both stages of heart disease (Table 4).


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  		Table 1 LV and body weight

 


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  		Table 2 In vivo LV hemodynamics

 
3.2. Malondialdehyde and protein carbonyls in tissue and plasma
Animals with aortic stenosis showed higher levels of MDA and protein carbonyls in the plasma both at 6 weeks and 22 weeks compared to age-matched sham-operated controls (Table 3). MDA and protein carbonyls are increased at 6 weeks, but we observed no further increase 22 weeks after aortic banding in the LV tissue (Fig. 1). When MDA concentration was normalized to total plasma protein concentration, similar results were obtained. Plasma protein carbonyls were increased in older sham animals, suggesting an age-dependent increase in this marker of oxidative stress, which was not observed for plasma MDA levels (Table 3).


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  		Table 3 Plasma levels of antioxidants, malondialdehyde and protein carbonyls

 


Figure 01
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  		Fig. 1 Tocopherol, vitamin C and MDA content in the left ventricle. LV {alpha}-tocopherol (upper left), {gamma}-tocopherol (upper right), vitamin C (lower left), malondialdehyde tissue (lower middle) and protein carbonyl content (lower right). Data are mean±SEM, *: p<0.05, **: p<0.01, ***: p<0.001.

 
3.3. Vitamin E and Vitamin C in tissue and plasma
The concentrations of {alpha}- and {gamma}-tocopherol both in plasma and in tissue were similar among the four groups (Fig. 1 and Table 3). Both {alpha}- and {gamma}-tocopherol concentration in plasma were highly correlated with the respective tissue levels in the LV.

6 weeks after aortic banding surgery, animals had significantly higher levels of vitamin C in LV tissue than age-matched sham-operated controls (Fig. 1). There was a trend for an age-related decrease in LV vitamin C concentration within the sham-operated control animals, which did not reach the level of significance. However, such a trend for age-related declines was not detectable in the rats with aortic banding: animals operated 22 weeks previously, now suffering from heart failure, actually showed higher levels of vitamin C than the 6 weeks banded animals at the stage of compensated LV hypertrophy (not significant). No differences were observed in plasma vitamin C concentrations among any of the 4 groups (Table 3).

3.4. Expression of sodium-dependent vitamin C transporters
Real-time PCR analyses revealed no difference in the expression of SVCT1 among the groups (not shown). The recently described dominant negative, shorter splice-isoform of SVCT2 [15] was not detected in any of the samples by RT-PCR (not shown). The expression of SVCT2 mRNA, however, is increased 6 weeks after aortic banding at the stage of compensated LV hypertrophy compared to age-matched sham-operated controls (3.2 ± 0.3 vs. 1.8 ± 0.2, p < 0.05) as well as after 22 weeks following surgery in failing LV (2.9 ± 0.4 vs. 1.6 ± 0.3, p < 0.05). Similarly, Western blot analyses revealed an increased expression of SVCT2 protein 6 and 22 weeks after aortic banding while SVCT1 is not altered (Fig. 2).


Figure 02
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  		Fig. 2 SVCT1 and SVCT2 protein expression in the left ventricle. Left and middle panel: Densitometry of protein data normalized to GAPDH. Homogenates of LV tissue from 6 or 22 weeks old sham-operated and aortic-banded rats probed with SVCT1 or SVCT2 antibody. Data are mean±SEM, *: p<0.05, **: p<0.01. Right panel: Representative examples of Western blots. S=sham-operated rats; L=aortic-banded rats.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
This study was performed in a rat model of cardiac overload with a well-characterized transition to failure, confirmed by anatomic, hemodynamic (Tables 1 and 2) serum or transcriptional parameters (Table 4), showing a clear difference between the stage of compensated LV hypertrophy and heart failure. Plasma and LV MDA and protein carbonyl levels (Table 3 and Fig. 1), as two independent signs of oxidative stress, appear to be elevated with cardiac overload. However, MDA does penetrate cell membranes and can leave the tissue in which it is generated [16]. Therefore, MDA tissue content might underestimate the degree of local oxidative stress. Tocopherols, both in tissue and in plasma, are not affected in this model (Tables 3 and 4), neither by cardiac overload nor by the transition to failure. The rats were on a standard diet, which contained vitamin E (55 IU of vitamin E/kg diet) and had an uptake of roughly 1.7 IU/die. This amount is lower than that used previously to prevent heart failure in pressure overloaded guinea pigs (6 IU/die) [1].


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  		Table 4 Markers of heart failure

 
In contrast to unaffected tocopherol levels, two principal and somewhat unexpected findings of this study relate to vitamin C: 1.) The content in LV tissue is significantly higher in aortic-banded rats with compensated LV hypertrophy than in age-matched controls. There is no decline in LV vitamin C content with the transition to decompensated cardiac failure. 2.) The overload-associated increase in ventricular vitamin C is not mirrored by enhanced plasma levels. Thus, any depletion in cardiac vitamin C content cannot have contributed to the transition from compensated overload hypertrophy to cardiac failure in this model. However, this must not exclude the possibility that even the increased tissue vitamin C levels are insufficient to exclude enhanced oxidative stress. Vitamin C/E and a variety of other molecules account for the total anti-oxidative activity of the heart. The balance of this anti-oxidative defence with the excess production of ROS determines the oxidative damage. Indeed, the analyses of the expression of some anti-oxidative enzymes in our animals indicate that mitochondrial superoxide dismutase is upregulated at both stages of heart disease, while the expression of the cytosolic thioredoxin reductase (TrxR1) is increased only in failing hearts and mitochondrial TrxR2, Cu/ZnSOD or GPX are not altered (not shown). Besides cardiac mitochondria other sources of free radicals such as xanthine oxidase, uncoupled nitric oxide synthase and NADPH oxidase are involved in the pathogenesis of heart failure. Increased oxidative stress, NADPH oxidase activity and interstitial fibrosis coexist in heart failure. Accordingly, mice lacking p47phox have reduced cardiomyocyte hypertrophy, apoptosis and interstitial fibrosis leading to improved LV function after myocardial infarction [17]. In the present study, we did not attempt to define the source of radicals. However, we can not exclude that among different sources of free radicals NADPH oxidase may also have contributed to the increased oxidative stress in the diseased heart. As described previously by others, Nox-2 and p47phox are upregulated in compensated hypertrophy and at the transition to heart failure (not shown).

Vitamin C is considered as the principal water soluble antioxidant in plasma and, after uptake into cells, is able to exert many protective anti-apoptotic and anti-inflammatory actions. Important aspects of the protective ascorbate effects are the intramitochondrial accumulation, which inhibits oxidative damage of mitochondrial DNA [18], and the antagonism against peroxynitrite-induced oxidation reactions, which occurs partially by inhibition of the interaction of NO and superoxide, and partially by repair catalysis of peroxynitrite-induced damage [19,20]. Vitamin C uptake in all mammalian cells is possible via facilitated diffusion through glucose transporters [21] and has also been documented in acutely isolated adult rat ventricular cardiomyocytes [22]. Interestingly, however, comparison of vitamin C levels in plasma and ventricular tissue (Table 3 and Fig. 1) from our rats reveals a several-fold higher vitamin C concentration in myocardium, which is not easily compatible with facilitated transport alone. Six weeks after operation, LV vitamin C concentration in sham-operated rats (0.6 nmol/mg protein, equivalent to approximately 120 {varepsilon}mol/kg myocardium) is substantially higher than the respective plasma value of 31.3 {varepsilon}mol/L, and an even larger gradient between ventricular tissue and plasma exists in animals with aortic banding (Table 3 and Fig. 1).

Presently, several mechanisms potentially affecting this accumulation above plasma levels should be considered. Recently, two mammalian sodium-dependent vitamin C transporters (SVCT1 and SVCT2) have been identified, mediating concentrative, stereoselective high-affinity uptake of L-ascorbate [23]. SVCT2 has a 10-fold higher affinity for ascorbate [23], and SVCT2 mRNA is expressed in many tissues including the human heart [24]. In the present study, we were able to demonstrate a significant increase in SVCT2 mRNA and protein in LV of rats 6 and 22 weeks after aortic banding (Fig. 2) while no difference in the expression of SVCT1 and the shorter splice-isoform of SVCT2 among the groups was observed. SVCT1 appears to be involved in whole-body homeostasis of vitamin C, while SVCT2 protects metabolically active and specialized cells against oxidative stress [25]. Accordingly, C2C12 skeletal myotubes have recently been shown to accumulate vitamin C, and the SVCT2 transporter for ascorbic acid was shown to be induced by the presence of oxidant compounds in vitro [26]. Although no functional data are available for this transporter in cardiomyocytes so far, adult cardiomyocytes have been shown to accumulate vitamin C resulting in enhanced resistance against oxidative as well as hypoxic stress in vitro [22]. Therefore, the increased expression of SVCT2 and accumulation of vitamin C as observed in our study may represent a protective mechanism against increased oxidative stress in heart failure. Accordingly, in experimental heart failure models, cardiomyocyte apoptosis and the progression of cardiac dysfunction are attenuated by treatments with vitamin C and/or other antioxidant vitamins [1,4], suggesting that antioxidant therapy may be beneficial in heart failure. Furthermore, a study by Bell et al. [27] provides evidence that vitamin C treatment of aortic-banded guinea pigs reduced oxidative stress and improved endothelial function. However, these data are difficult to compare to our study, since guinea pigs, but not rats, are unable to synthesize vitamin C.

Data concerning vitamin E and vitamin C tissue content are scarce. The cardiac vitamin C levels in our sham-operated rats are in the magnitude range reported in rats [28], while cardiac tissue levels in mammalian models of overload and/or failure are not available. Decreased levels of plasma and tissue levels of vitamin C [28] but increased vitamin E [29] concentrations have been described in a rat model of ageing. However, in a dog model of congestive heart failure, vitamin E concentrations were reported to be significantly lower, while vitamin C concentrations were higher in dogs with heart failure than in controls [30]. In contrast to human tissues, rat cells can synthesize vitamin C from glucose, but this occurs only in the liver and has not been shown in the heart. Ascorbate can also be used as a source of energy by entering the glycolytic pathway via the pentose-phosphate pathway. In terminally failing myocardium, glycolytic flux and glucose oxidation are enhanced secondary to a suppression of fatty acid oxidation. It remains speculative whether this enhanced glycolytic flux also includes enhanced ascorbate uptake and metabolism. While this study identifies a hitherto unknown augmentation of vitamin C uptake in hypertrophied and failing myocardium, it has some limitations. Although we observed an increased SVCT2 mRNA and protein expression in hypertrophied and failing myocardium (Fig. 2) which may have caused the increased accumulation of vitamin C, the functional relevance of these changes requires further elucidation. The known species-specifities of ascorbate metabolism argue against an uncritical extrapolation of these data to cardiac overload and failure in humans. Nevertheless, the unexpected augmentation in the accumulation of the putatively protective ascorbate in overloaded and failing myocardium appears important and deserves further consideration and evaluation.


   Acknowledgements
 
This study was supported by a Konrad-Adenauer Fellowship given to SR and the DFG (RO 2328/2-1). Initial experiments were performed in the laboratory of Prof. Dr. Beverly Lorell at Harvard Medical School, and we are grateful for her support.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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