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Clinical Chemistry 53: 773-780, 2007. First published February 15, 2007; 10.1373/clinchem.2006.077859
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(Clinical Chemistry. 2007;53:773-780.)
© 2007 American Association for Clinical Chemistry, Inc.

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Hyperhomocysteinemia and Myocardial Expression of Brain Natriuretic Peptide in Rats

Markus Herrmann1, Omid Taban-Shoma1, Ulrich Hübner1, Anette Pexa3, Heiko Kilter2, Natalia Umanskaya1, Rainer Hans Straub4, Michael Böhm2 and Wolfgang Herrmann1,a

Departments of1 Clinical Chemistry and Laboratory Medicine and 2 Department of Internal Medicine III, University Hospital of Saarland, Homburg/Saar, Germany.
3 Institute of Physiology, Medical Faculty Carl Gustav Carus, Technical University Dresden, Dresden, Germany.
4 Department of Internal Medicine I, University Hospital of Regensburg, Regensburg, Germany.

aAddress correspondence to this author at: Wolfgang Herrmann, Abteilung für Klinische Chemie und Laboratoriumsmedizin / Zentrallabor, Universitätsklinikum des Saarland, D-66421 Homburg/Saar, Germany. Fax +496841 1630703, E-mail kchwher{at}uniklinik-saarland.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Hyperhomocysteinemia (HHcy) has been linked to impaired left ventricular function and clinical class in patients with chronic heart failure. We hypothesized that HHcy stimulates myocardial brain natriuretic peptide (BNP) expression and induces adverse left ventricular remodeling.

Methods: We randomized 50 rats into 5 groups. Groups Co1 and Co2 (controls) received a typical diet. Groups Meth, Hcy1, and Hcy2 were fed the same diet supplemented with 2.4% methionine, 1% homocystine, and 2% homocystine, respectively. After 12 weeks, we measured total plasma homocysteine (tHcy) and BNP in plasma and tissue, and we performed histomorphometric analyses.

Results: All animals had comparable baseline body weight [mean (SD) 234 (26) g] and total circulating Hcy [4.7 (1.7) µmol/L]. After 12 weeks of treatment, total circulating Hcy increased in Meth, Hcy1, and Hcy2 [27.3 (8.8), 40.6 (7.0), and 54.0 (46.0) µmol/L, respectively] and remained unchanged in Co1 and Co2. Serum BNP significantly increased in 1 of 10 animals in Meth, 3 of 10 animals in Hcy1, and 3 of 10 animals in Hcy2. Median (25th–75th percentile) BNP tissue concentrations in Hcy1 and Hcy2 were 55% higher than in the corresponding controls [Co1 vs Hcy1, 225 (186–263) vs 338 (262–410) pg/mg protein, P = 0.05; Co2 vs Hcy2, 179 (107–261) vs 308 (192–429) pg/mg protein, P = 0.12]. In the Meth group, BNP expression was comparable to that of controls [200 (159–235) vs 225 (186–263) pg/mg protein, P = 0.32]. The percentage of perivascular and interstitial collagen and mast cell infiltration were comparable in all groups, indicating no adverse cardiac remodeling.

Conclusion: Three months of intermediate HHcy stimulated increased cardiac BNP expression that was not accompanied by adverse cardiac remodeling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hyperhomocysteinemia (HHcy) and chronic heart failure (CHF) are frequent problems of elderly individuals and often coincide (1). Recent epidemiologic studies from Europe and the US consistently found that the frequency of HHcy was >25% in persons ≥55 years old (2) and increased continuously with age. CHF affects 5 million patients in the US, with ~550 000 newly diagnosed cases each year (3). Hospital discharges for CHF increased from 399 000 in 1979 to 1 093 000 in 2003, and the 2003 overall death rate for CHF was 19.7% (3). A similar situation exists in Europe(4). Consequently, prevention of CHF by identifying and modifying risk factors is a major issue.

Previous analyses found hypertension, smoking, diabetes mellitus, obesity, and advancing age to be the most important risk factors for CHF (5). Recent clinical and experimental studies suggest that HHcy is also a risk factor for CHF (1)(6)(7). The prevalence of HHcy in CHF patients is 50%–60%, depending on the cutoff applied (8)(9). Results from the prospective Framingham Heart Study demonstrated an almost doubled incidence of CHF in individuals with a total circulating Hcy (tHcy) above the sex-specific median (1). In previous studies from our group, analyzing >1000 individuals, we found a consistent relation of tHcy with the severity of CHF (6)(9). The closest relation could be observed between tHcy and N-terminal pro–brain natriuretic peptide (NT-proBNP). A recent study from Korea confirmed this finding (10). In animal experiments from the group of Joseph and Kennedy (11)(12), chronic intermediate HHcy induced cardiac hypertrophy, pathological remodeling (11)(13)(14), and diastolic as well as systolic dysfunction (11)(12). These findings provide strong evidence for a causal role of HHcy in the pathogenesis of left ventricular dysfunction.

Brain natriuretic peptide (BNP) and NT-proBNP are laboratory markers of CHF with a high negative predictive value (15)(16)(17). They are increased in systolic and diastolic dysfunction (18)(19) and have been found to be useful for differential diagnosis of dyspnea in CHF and pulmonary diseases (20). Especially in early disease stages, NT-proBNP seems to be more sensitive than echocardiography (21)(22). Moreover, measurements of BNP and NT-proBNP are free from subjective influences and have good reproducibility (23). In this study, we investigated myocardial BNP expression and cardiac remodeling in healthy adult rats after 3 months of moderate or intermediate HHcy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
animals and study design
After 7 days of acclimatization, 50 10- to 12-week-old female Wistar rats (180 to 292 g; Charles River GmbH) were randomized into 5 groups of 10 animals each: 2 control groups (Co1 and Co2) and 3 groups with HHcy. Animals were maintained on a 12 h:12 h light-dark cycle in our institutional stable with free access to food and water. HHcy was induced by 3 different dietary regimens. The first HHcy group (Meth) received a diet that was enriched with 24 g methionine (Sigma) per kg food (corresponds to 2.4% methionine). The 2 other groups were fed with homocystine-enriched diets, containing 10 g (Hcy1) or 20 g (Hcy2) homocystine (Sigma) per kg food (corresponding to 1% and 2% Hcy). Except for the added methionine and homocystine, all diets were of identical composition. Diets were purchased from Altromin. Because of limited space in the stable, we performed experiments in 2 consecutive runs. The first run comprised the groups Co1, Meth, and Hcy1; in the second run, we analyzed Co2 and Hcy2. The study was approved by the Institutional Animal Care and Use Committee of the Saarpfalz-Kreis.

We monitored body weight, food consumption, and fluid intake weekly. We performed capillary blood sampling from the retrobulbar plexus before and at the end of the 12-week feeding period by use of heparin-coated capillaries. Immediately after blood sampling, plasma was separated by centrifugation (10 min at 1500g) and stored at –80 °C until analysis. For blood sampling, animals were anesthetized using Ketavet 0.1% (Pharmacia GmbH) and Rompun 2% (Bayer Healthcare). After the final blood sampling, hearts and lungs were isolated, explanted, and rinsed thoroughly to remove adherent blood. The left ventricles were isolated, and the left ventricular weight and lung weight were measured using a Sartorius Genius scale (Sartorius AG). Left ventricles were divided into 4 aliquots. One aliquot was fixed with 4% formalin and used for histomorphometric analyses. The remaining 3 aliquots were shock-frozen with liquid nitrogen and stored at –80 °C until measurement. One of these aliquots was used for the quantification of tissue BNP concentration.

blood characteristics
We used heparin plasma samples to measure tHcy, BNP, folate, vitamin B12, and creatinine. tHcy was analyzed with an enzymatic fluorescence polarization immunoassay on an Axsym analyzer (Abbott). Intra- and interassay CVs of this method were 4.5% and 5.1%, respectively. We quantified BNP by use of the rat BNP-45 ELISA reagent set (Gentaur), using a polyclonal antibody specific for BNP-45. BNP-45 represents the circulating (active) form of BNP in rats and is equivalent to BNP-32 in humans. Intra- and interassay CVs of the BNP ELISA were 5.3% and 8.5% at concentrations of 100 ng/L. For this assay, the recovery rate from plasma is 89%, and the reference range of healthy untreated rats is below the detection limit of 10 ng/L. Folate and vitamin B12 were detected with chemiluminescence immunoassays on an ACS Centaur analyzer (Bayer Healthcare). Intra- and interassay CVs were 4.0% and 4.4% at a concentration of 208 ng/L for vitamin B12, and 5.3% and 5.5% at a concentration of 5.3 µg/L for folate. We measured creatinine by use of a colorimetric assay using the alkaline picrate method on a Hitachi 917 analyzer (Roche Diagnostics).

bnp tissue concentration
One deep frozen tissue aliquot per ventricle was thawed and homogenized with a mortar. The homogenized tissue was dissolved in phosphate buffered saline (PAA Laboratories; 0.2 g/L KCl, 0.2 g/L KH2PO4, 8.0 g/L NaCl, 1.15 g/L Na2HPO4), and we measured the total protein concentration using a Bradford assay (Bio-Rad). We quantified BNP with the same method that was previously described for the quantification in plasma. The recovery rate from tissue is 85% with this assay. The stability of tissue BNP-45 at –20 °C is 3 months. Because tissue aliquots had different volumes and masses, BNP values were expressed relative to the protein content of each aliquot.

histomorphometry
We performed morphometric analyses as previously described by Southern et al. (24). After dehydration with increasing alcohol concentrations, aliquots were embedded in paraffin, and 5-µm sections from 2 locations 100 µm apart were prepared. After removal of the paraffin, sections were stained with picrosirius red (Sigma), which is specific for fibrillar collagen (25).

To avoid subjective influences on morphometric results, sections were blinded before analysis. We prepared digital images from 15 microscopic fields per section and analyzed 2 sections per ventricle, 1 per location. The interstitial collagen fraction was calculated as the percentage of red-colored interstitial regions relative to the total area. For the quantification of perivascular collagen, we analyzed 5 vessels per section with a diameter of 50–200 µm and expressed perivascular collagen relative to the vessel luminal area. Serial measurements of the same section revealed a variation of 1% for the analysis of perivascular and interstitial collagen.

Previous work by Joseph et al. showed a protective role of mast cells in Hcy-induced cardiac remodeling (26). Therefore, we also analyzed myocardial mast cell infiltration using toluidine blue staining (27). Sections from both locations were rehydrated and incubated for 30 min in 0.001% toluidine blue solution (Sigma). In 2 sections per ventricle, 1 per location, mast cells (blue) were counted and expressed relative to the analyzed area.

bnp expression in ex vivo superfused rat myocardium
To confirm the BNP results in vivo, we performed superfusion experiments with freshly prepared rat myocardium from three 10- to 12-week-old Wistar rats (mean weight 260 g; Charles River) as described previously by Jeron et al. (28). Briefly, after explantation of the heart and isolation of the left ventricle, we prepared tissue slices (0.35 mm thick) from the left ventricle (48 slices per heart) and transferred them immediately to minisuperfusion chambers (volume 80 µL). We performed superfusion for 13 h at a temperature of 37 °C and a flow rate of 66 µL/min (1 slice per chamber, 48 chambers in parallel) with culture medium containing RPMI 1640 (Sigma), 25 mmol/L HEPES (Roth), 5% fetal calf serum (PAA Laboratories), 30 µmol/L mercaptoethanol (Sigma), 0.57 mmol/L ascorbic acid (Merck), 1.3 mmol/L calcium (Merck), 100 000 IU/L penicillin, and 100 mg/L streptomycin (Invitrogen Gibco).

During the first 2 h of superfusion, we superfused tissue slices with culture medium without further stimulation (wash-in period). From 2–13 h, Hcy-conditioned medium was applied continuously at concentrations of 0, 10, 50, and 200 µmol/L to modulate BNP secretion. Each Hcy concentration was applied to 8 tissue slices per rat (8 slices in 3 rats = 24 total).

We collected superfusate aliquots for 15 min at 2, 3, 5, 7, 9, 11, and 13 h and immediately froze them at –80 °C until measurements. We quantified BNP with the same rat BNP-45 ELISA reagent set (Gentaur) as used for serum and tissue samples. After the end of the superfusion period, tissue slices were homogenized and protein concentration was measured using the BCA-Protein assay (Pierce). BNP secretion was expressed as picograms per microgram protein.

statistics
Based on results of a Kolmogorov–Smirnov test, all variables, except of tissue BNP, showed gaussian distribution. Results of the descriptive statistics are expressed as mean (SD). Group comparisons of gaussian-distributed variables were performed using 1-way ANOVA with least significant difference post hoc test. Tissue BNP, which was not gaussian distributed, was compared using a Kruskal–Wallis or a Mann–Whitney test. All calculations were performed using the SPSS 11.0 software package (SPSS Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
animal model
At the beginning of the study, animals had comparable baseline characteristics. Mean (SD) body weight was 234 (26) g and tHcy was 4.7 (1.7) µmol/L. After 12 weeks of treatment, Meth, Hcy1, and Hcy2 animals exhibited significant HHcy (Table 1 ). The Meth group developed moderate HHcy, and the Hcy1 and Hcy2 groups had intermediate HHcy, with the highest Hcy concentrations in the Hcy2 group. We excluded 3 animals from the Co2 group because they exhibited spontaneously increased Hcy concentrations >10 µmol/L. We excluded 2 animals from the Hcy2 group because their Hcy concentration at the end of the treatment was <10 µmol/L.


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  		Table 1. Characterization of experimental groups and controls at the end of the study.

Plasma creatinine was within reference intervals in all animals [50.4 (9.7) µmol/L]. Moreover, Meth, Hcy1, and Hcy2 groups did not show higher plasma creatinine than controls (Table 1Up ). All animals exhibited high plasma concentrations of folate and vitamin B12 that did not differ between groups (Table 1Up ). Body weight was significantly lower in Meth and Hcy2 compared with controls, owing to reduced food intake in these groups. Heart rate and blood pressure did not differ between groups.

bnp expression
At the end of the study, plasma BNP was below the detection limit of 10 ng/L in all controls. One Meth animal, 3 Hcy1 animals, and 3 Hcy2 animals showed an increase in circulating BNP (Fig. 1 ). Because many of the animals in the HHcy groups exhibited undetectable BNP concentrations at the end of the study, the calculation of mean values was not meaningful. Therefore, we categorized animals as BNP positive and negative. The number of BNP-positive animals increased with higher circulating Hcy concentrations (Fig. 1 ). Introducing categorized BNP values in a cross table, Pearson {chi}2 test revealed a significant relation between circulating Hcy and plasma BNP ({chi}2 = 7.89, P = 0.048). Pooling all controls and all HHcy animals in 2 groups, we confirmed a higher number of BNP-positive animals in the HHcy group ({chi}2 = 5.03, P = 0.025).


Figure 1
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  		Figure 1. Distribution of BNP plasma concentrations after 12 weeks of feeding.

{blacktriangleup} plasma BNP <10 ng/L, {circ} increased plasma BNP. Co1, control group 1st run; Co2, control group 2nd run; Meth, methionine group; Hcy1, homocystine 1% group; Hcy2, homocystine 2% group.

Because most of the animals had undetectable BNP plasma concentrations, we quantified tissue concentrations of BNP in the myocardium (Fig. 2 ). For animals in the Hcy1 and Hcy2 groups, BNP tissue concentrations were ~55% higher than in controls. In the Hcy1 group, this difference was significant (P = 0.05). Group comparison between Co2 and Hcy2 revealed only a trend (P = 0.12) because of larger interindividual variation of BNP tissue concentrations in the Hcy2 group.


Figure 2
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  		Figure 2. Box plot of median (25th–75th percentile) tissue BNP expression in relation to total protein concentration after 12 weeks of feeding.

Co1, control group 1st run; Co2, control group 2nd run; Meth, methionine group; Hcy1, homocystine 1% group; Hcy2, homocystine 2% group. *P ≤0.05 vs Co1, $P = 0.12 vs Co2.

To confirm the results described above and to demonstrate causality between circulating Hcy and BNP expression, we performed superfusion experiments stimulating freshly prepared rat myocardium slices with increasing concentrations of Hcy. The mean BNP secretion during the 11 h of Hcy superfusion increased significantly with higher Hcy concentrations in the superfusion medium (Fig. 3 ).


Figure 3
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  		Figure 3. Mean BNP secretion [95% confidence interval (CI)] of freshly prepared rat myocardium slices during 13 h of superfusion with Hcy-enriched RPMI 1640 medium.

The experiment was performed with 3 animals and 8 slices in each animal. For each Hcy concentration, 24 tissue slices (8 per animal) were analyzed. The P value above the horizontal line represents the result of the 1-way ANOVA including all experimental groups. **P <0.01 vs 0 µmol/L Hcy (least significant difference post hoc test).

left ventricular remodeling
The relative weight of the left ventricle in Meth and Hcy1 groups was comparable to the corresponding controls. Only Hcy2 showed a significant increase, by 15% (Fig. 4A ). Similarly, lung weight, as a measure of interstitial fluid, did not differ from controls in the Meth and Hcy1 groups (Fig. 4B ). Only the Hcy2 group exhibited a significantly higher lung weight, indicating congestion.


Figure 4
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  		Figure 4. Relative heart (A) and lung (B) weights after 12 weeks of feeding.

Co1, control group 1st run; Co2, control group 2nd run; Meth, methionine group; Hcy1, homocystine 1% group; Hcy2, homocystine 2% group. **P < 0.01 vs Co1; $$P < 0.01 vs Co2.

Because ventricular and lung weights are relatively raw measures, we performed morphometric analyses of interstitial and perivascular collagen. Interstitial collagen was 2.8%–5.1% and was not different between groups (Fig. 5A ). Perivascular collagen was comparable in Co1, Co2, Meth, and Hcy1 groups, at 2.0%– 2.1% (Fig. 5B ). The Hcy2 group exhibited a perivascular collagen of 2.7%, ~35% higher than the corresponding control Co2 (Fig. 5B ). Because of large interindividual variation, however, this difference was not statistically significant (P = 0.265).


Figure 5
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  		Figure 5. Histomorphometric analysis of indicators of left ventricular remodeling after 12 weeks of feeding.

A, Percentage of interstitial collagen. B, percentage of perivascular collagen. Co1, control group 1st run; Co2, control group 2nd run; Meth, methionine group; Hcy1, homocystine 1% group; Hcy2, homocystine 2% group.

In this study, mean mast cell infiltration was 11–15 cells/mm2 and was comparable in all groups [see Fig. 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue4].


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main finding of the present study was a significant induction of cardiac BNP expression by chronically increased tHcy concentrations. Higher BNP concentrations were not accompanied by an adverse remodeling of the left ventricle, the main source of BNP. The causality between HHcy and increased BNP expression was demonstrated by ex vivo superfused left ventricular tissue slices, which were stimulated with various Hcy concentrations. HHcy was induced by 3 different models. According to current concepts, in humans HHcy can be classified as moderate (12–30 µmol/L), intermediate (30–100 µmol/L), or severe (>100 µmol/L) (29)(30). Based on this classification, moderate HHcy was induced in the Meth group by the addition of 2.4% methionine to the typical diet. A comparable model of moderate HHcy has been described by Woo et al. (31). The mean tHcy in Meth was comparable with the findings of Woo et al. (27.3 vs 25.3 µmol/L). Intermediate HHcy was created with a homocystine-enriched diet (1%), as published by Joseph et al. (12)(14). tHcy was similar to tHcy values reported by Joseph et al. [40.6 vs 32.7 µmol/L (12) and vs 47.1 µmol/L (14)]. To further increase tHcy, we augmented the homocystine concentration (2%) in the diet of the 3rd group. This diet caused a mean tHcy of 54.0 µmol/L; however, animals of the Hcy2 group exhibited large interindividual variations in circulating Hcy, and 3 animals had to be excluded because they did not show increased Hcy concentrations. Compared with the corresponding controls (Co2), weight gain was significantly lower, indicating a reduced tolerance to this diet and suggesting that a homocystine concentration of 2% is probably the maximum that can be administered.

This study demonstrates a significant stimulation of BNP expression, a sensitive marker of myocardial dysfunction and increased wall stress (21)(22), with a high negative predictive value (15)(16)(17) by intermediate HHcy (Hcy1 and Hcy2), but not by moderate HHcy (Meth). This BNP-inducing effect was demonstrated in plasma as well as in myocardial tissue and confirms the results of 2 previous studies, in which we demonstrated a significant positive relation between Hcy and NT-proBNP in CHF patients (6)(9). The changes in BNP were not accompanied by adverse cardiac remodeling, as indicated by lack of change in interstitial and perivascular collagen in all groups. Moreover, the number of mast cells did not differ between controls and HHcy groups. The discrepancy between BNP expression and cardiac remodeling is not surprising, since BNP probably represents a more sensitive indicator to detect cardiac changes. Increases of BNP and NT-proBNP can frequently be found in diastolic dysfunction (18)(19)(32) in which the ejection fraction is normal, as well as in mild systolic dysfunction (21)(22). In addition, modern concepts consider the heart to be a multifunctional and interactive organ that is part of a complex network (33)(34). Several studies have shown that BNP has antifibrotic and cytoprotective properties (35)(36). Obviously, these counterregulatory actions of BNP are present before clinical symptoms and before adverse cardiac remodeling.

Contrary to histomorphometry, BNP assessment is also free from subjective modification. The sensitivity of currently available rat BNP assays is limited, however. The ELISA used in the current study could not quantify plasma BNP in control animals (all control animals had undetectable plasma BNP concentrations). Consequently, a reference interval could not be defined. Because of the limited sensitivity of the rat BNP assay, we could not see potential changes of plasma BNP below the detection limit. Therefore, the measurement of tissue BNP has to be considered more reliable, since all animals exhibited concentrations above the detection limit and in the linear range of the assay. Hcy1 and Hcy2 groups showed a 55% increase of BNP tissue expression, which supports the result in plasma. Unchanged BNP tissue concentrations were observed in the Meth group. The causality between Hcy and BNP expression was demonstrated by the ex vivo superfusion of freshly prepared rat myocardial slices with Hcy-enriched medium. Hcy concentrations of 50 and 200 µmol/L caused a significant increase of BNP secretion by 60% and 140%, respectively.

What is the explanation for the absence of BNP induction in the Meth group? An obvious reason could be the lower tHcy in these animals. However, even if tHcy was significantly lower than in the Hcy1 and Hcy2 animals, it was clearly above control concentrations. At baseline, all controls had tHcy <10 µmol/L. Therefore, a mean tHcy of 27.3 µmol/L corresponds to at least triple normal values. A more probable explanation is that methionine and homocystine caused HHcy by different biochemical mechanisms with different consequences. Homocystine is the disulfide of 2 Hcy molecules. Addition of homocystine to the diet probably increases tHcy directly and leads to a product excess in methionine metabolism, inhibiting transmethylation and thereby the utilization of methionine. In contrast, a high intake of methionine induces a substrate excess in methionine metabolism that is probably accompanied by increased transmethylation activity. Because hypomethylation is a major mechanism of adverse Hcy effects (37)(38), this biochemical difference between the diets could explain the absent BNP induction in the Meth group. However, this explanation is still not proven.

Contrary to previous work by the group of Joseph et al. (11)(12)(13)(14)(26), we could not find any morphologic evidence for adverse cardiac remodeling. This result was surprising, because we used exactly the same dietary model and the same methods to detect cardiac remodeling. The increase of perivascular and interstitial collagen observed by Joseph et al. was 50%–100% in normotensive rats (12) and 100%–500% in hypertensive rats (14). Both studies included fewer than 10 animals per group. Our investigation comprised 28 HHcy animals and 17 controls. These relatively large numbers lend our study a strong impact. At present, we cannot find any explanation for the discrepancy between the results by Joseph et al. and ours. Because Joseph et al. confirmed their findings in several studies (11)(12)(13)(14)(26), there is no reason to doubt them. However, our experiments were performed in 2 independent runs, and all variables analyzed consistently suggested no relevant cardiac remodeling after 12 weeks of HHcy. These conflicting findings must be clarified by further studies.


   Acknowledgments
 
Supported by the Kompetenznetzwerk Herzinsuffizienz by the BMBF (TP11) to Michael Böhm and Heiko Kilter.


   Footnotes
 
1 Nonstandard abbreviations: HHcy, hyperhomocysteinemia; CHF, chronic heart failure; Hcy, homocysteine; tHcy, total circulating homocysteine; NT-proBNP, N-terminal pro-BNP; BNP, brain natriuretic peptide; Co1, Co2, control group 1 and 2; Meth, methionine group; Hcy1, homocystine 1% group; Hcy2, homocystine 2% group.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Vasan RS, Beiser A, D’Agostino RB, Levy D, Selhub J, Jacques PF, et al. Plasma homocysteine and risk for congestive heart failure in adults without prior myocardial infarction. JAMA 2003;289:1251-1257.[Abstract/Free Full Text]
  2. Herrmann W, Quast S, Ullrich M, Schultze H, Bodis M, Geisel J. Hyperhomocysteinemia in high-aged subjects: relation of B-vitamins, folic acid, renal function and the methylenetetrahydrofolate reductase mutation. Atherosclerosis 1999;144:91-101.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  3. Thom T, Haase N, Rosamond W, Howard VJ, Rumsfeld J, Manolio T, et al. Heart disease and stroke statistics–2006 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 2006;113:e85-e151.[Free Full Text]
  4. Cleland JG, Khand A, Clark A. The heart failure epidemic: exactly how big is it?. Eur Heart J 2001;22:623-626.[Free Full Text]
  5. Kenchaiah S, Narula J, Vasan RS. Risk factors for heart failure. Med Clin North Am 2004;88:1145-1172.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  6. Herrmann M, Kindermann I, Muller S, Georg T, Kindermann M, Bohm M, et al. Relationship of plasma homocysteine with the severity of chronic heart failure. Clin Chem 2005;51:1512-1515.[Free Full Text]
  7. Kennedy RH, Melchert RB, Joseph J. Cardiovascular effects of hyperhomocysteinemia in conscious unrestrained rats. Am J Hypertens 2006;19:94-97.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  8. Ventura P, Panini R, Verlato C, Scarpetta G, Salvioli G. Hyperhomocysteinemia and related factors in 600 hospitalized elderly subjects. Metabolism 2001;50:1466-1471.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  9. Herrmann M, Mueller S, Kindermann I, Guenther L, Koenig J, Boehm M, et al. Plasma B-vitamins and their relation to the severity of chronic heart failure. Am J Clin Nutr 2007;in print..
  10. Cho SE, Sook HK, Shin GJ, Chung WS. The methylenetetrahydrofolate reductase C677T gene mutation is associated with hyperhomocysteinemia, cardiovascular disease and plasma B-type natriuretic peptide concentrations in Korea. Clin Chem Lab Med 2006;44:1070-1075.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  11. Devi S, Kennedy RH, Joseph L, Shekhawat NS, Melchert RB, Joseph J. Effect of long-term hyperhomocysteinemia on myocardial structure and function in hypertensive rats. Cardiovasc Pathol 2006;15:75-82.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  12. Joseph J, Joseph L, Shekhawat NS, Devi S, Wang J, Melchert RB, et al. Hyperhomocysteinemia leads to pathological ventricular hypertrophy in normotensive rats. Am J Physiol Heart Circ Physiol 2003;285:H679-H686.[Abstract/Free Full Text]
  13. Joseph J, Washington A, Joseph L, Kennedy RH. Hyperhomocysteinaemia-induced atrial remodeling in hypertensive rats. Clin Exp Pharmacol Physiol 2004;31:331-337.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  14. Joseph J, Washington A, Joseph L, Koehler L, Fink LM, Hauer-Jensen M, et al. Hyperhomocysteinemia leads to adverse cardiac remodeling in hypertensive rats. Am J Physiol Heart Circ Physiol 2002;283:H2567-H2574.[Abstract/Free Full Text]
  15. Hobbs FD, Davis RC, Roalfe AK, Hare R, Davies MK. Reliability of N-terminal proBNP assay in diagnosis of left ventricular systolic dysfunction within representative and high risk populations. Heart 2004;90:866-870.[Abstract/Free Full Text]
  16. Nielsen LS, Svanegaard J, Klitgaard NA, Egeblad H. N-terminal pro-brain natriuretic peptide for discriminating between cardiac and non-cardiac dyspnoea. Eur J Heart Fail 2004;6:63-70.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  17. McDonagh TA, Holmer S, Raymond I, Luchner A, Hildebrant P, Dargie HJ. NT-proBNP and the diagnosis of heart failure: a pooled analysis of three European epidemiological studies. Eur J Heart Fail 2004;6:269-273.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  18. Yamaguchi H, Yoshida J, Yamamoto K, Sakata Y, Mano T, Akehi N, et al. Elevation of plasma brain natriuretic peptide is a hallmark of diastolic heart failure independent of ventricular hypertrophy. J Am Coll Cardiol 2004;43:55-60.[Abstract/Free Full Text]
  19. Lubien E, DeMaria A, Krishnaswamy P, Clopton P, Koon J, Kazanegra R, et al. Utility of B-natriuretic peptide in detecting diastolic dysfunction: comparison with Doppler velocity recordings. Circulation 2002;105:595-601.[Abstract/Free Full Text]
  20. Mueller C, Scholer A, Laule-Kilian K, Martina B, Schindler C, Buser P, et al. Use of B-type natriuretic peptide in the evaluation and management of acute dyspnea. N Engl J Med 2004;350:647-654.[Abstract/Free Full Text]
  21. Mueller T, Gegenhuber A, Dieplinger B, Poelz W, Haltmayer M. Capability of B-type natriuretic peptide (BNP) and amino-terminal proBNP as indicators of cardiac structural disease in asymptomatic patients with systemic arterial hypertension. Clin Chem 2005;51:2245-2251.[Abstract/Free Full Text]
  22. Hammerer-Lercher A, Ludwig W, Falkensammer G, Muller S, Neubauer E, Puschendorf B, et al. Natriuretic peptides as markers of mild forms of left ventricular dysfunction: effects of assays on diagnostic performance of markers. Clin Chem 2004;50:1174-1183.[Abstract/Free Full Text]
  23. Collinson PO, Barnes SC, Gaze DC, Galasko G, Lahiri A, Senior R. Analytical performance of the N terminal pro B type natriuretic peptide (NT-proBNP) assay on the Elecsys 1010 and 2010 analysers. Eur J Heart Fail 2004;6:365-368.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  24. Southern FN, Cruz N, Fink LM, Cooney CA, Barone GW, Eidt JF, et al. Hyperhomocysteinemia increases intimal hyperplasia in a rat carotid endarterectomy model. J Vasc Surg 1998;28:909-918.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  25. Junqueira LC, Bignolas G, Brentani RR. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J 1979;11:447-455.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  26. Joseph J, Kennedy RH, Devi S, Wang J, Joseph L, Hauer-Jensen M. Protective role of mast cells in homocysteine-induced cardiac remodeling. Am J Physiol Heart Circ Physiol 2005;288:H2541-H2545.[Abstract/Free Full Text]
  27. Roberts IS, Brenchley PE. Mast cells: the forgotten cells of renal fibrosis. J Clin Pathol 2000;53:858-862.[Abstract/Free Full Text]
  28. Jeron A, Kaiser T, Straub RH, Weil J, Riegger GA, Muders F. Myocardial IL-6 regulation by neurohormones: an in vitro superfusion study. Brain Behav Immun 2003;17:245-250.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  29. Durand P, Prost M, Loreau N, Lussier-Cacan S, Blache D. Impaired homocysteine metabolism and atherothrombotic disease. Lab Invest 2001;81:645-672.[Web of Science][Medline] [Order article via Infotrieve]
  30. Stanger O, Herrmann W, Pietrzik K, Fowler B, Geisel J, Dierkes J, et al. DACH-LIGA Homocystein (German, Austrian and Swiss Homocysteine Society): Consensus paper on the rational clinical use of homocysteine, folic acid and B vitamins in cardiovascular and thrombotic diseases: guidelines and recommendations. Clin Chem Lab Med 2003;41:1392-1403.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  31. Woo CW, Siow YL. O K. Homocysteine activates cAMP-response element binding protein in HepG2 through cAMP/PKA signaling pathway. Arterioscler Thromb Vasc Biol 2006;26:1043-1050.[Abstract/Free Full Text]
  32. Celinski R, Cholewinski W, Stefaniak B, Tarkowska A. Relationship between plasma BNP levels and left ventricular diastolic function as measured by radionuclide ventriculography in patients with coronary artery disease. Nucl Med Rev Cent East Eur 2004;7:123-128.[Medline] [Order article via Infotrieve]
  33. Clerico A, Recchia FA, Passino C, Emdin M. Cardiac endocrine function is an essential component of the homeostatic regulation network: physiological and clinical implications. Am J Physiol Heart Circ Physiol 2006;290:H17-H29.[Abstract/Free Full Text]
  34. Schulze CP, Gielen S, Adams V, Linke A, Möbius-Winkler S, Erbs S, et al. Muscular levels of proinflammatory cytokines correlate with a reduced expression of insulin like growth factor-I in chronic heart failure. Bas Res Cardiol 2006;98:267-274.
  35. Rosenkranz AC, Woods RL, Dusting GJ, Ritchie RH. Antihypertrophic actions of the natriuretic peptides in adult rat cardiomyocytes: importance of cyclic GMP. Cardiovasc Res 2003;57:515-522.[Abstract/Free Full Text]
  36. D’Souza SP, Davis M, Baxter GF. Autocrine and paracrine actions of natriuretic peptides in the heart. Pharmacol Ther 2004;101:113-129.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  37. Zaina S, Lindholm MW, Lund G. Nutrition and aberrant DNA methylation patterns in atherosclerosis: more than just hyperhomocysteinemia?. J Nutr 2005;135:5-8.[Abstract/Free Full Text]
  38. James SJ, Melnyk S, Pogribna M, Pogribny IP, Caudill MA. Elevation in S-adenosylhomocysteine and DNA hypomethylation: potential epigenetic mechanism for homocysteine-related pathology. J Nutr 2002;132(Suppl 8):2361S-2366S.[Abstract/Free Full Text]



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M. Shimano, Y. Inden, Y. Tsuji, H. Kamiya, T. Uchikawa, R. Shibata, and T. Murohara
Circulating homocysteine levels in patients with radiofrequency catheter ablation for atrial fibrillation
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