Published online before print March 24, 2008, doi: 10.1161/CIRCULATIONAHA.107.725481
(Circulation. 2008;117:1810-1819.)
© 2008 American Heart Association, Inc.
Heart Failure |
High-Dose Folic Acid Pretreatment Blunts Cardiac Dysfunction During Ischemia Coupled to Maintenance of High-Energy Phosphates and Reduces Postreperfusion Injury
From the Johns Hopkins Medical Institutions, Divisions of Cardiology (A.L.M., H.C.C., A.H., M.Z., D.B., K.L.G., N.P., D.A.K.) and Pathology (R.S.E.), Baltimore, Md; University of Antwerp, Departments of Cardiology (A.L.M., M.J.C., D.J.B., P.C., C.J.V.), Cell Biology and Histology (L.V.N., J.-P.T.), and Biomedical Statistics (F.L.W.), Antwerp, Belgium; University of Rome, Department of Chemical Science, Rome, Italy (B.T., G.L.); and New York Medical College, Department of Physiology, Valhalla (P.M.K., M.W.S.).
Correspondence to David A. Kass, MD, Johns Hopkins Medical Institutions, Division of Cardiology, Ross Research Bldg, Room 835, 720 Rutland Ave, Baltimore, MD 21205. E-mail dkass{at}jhmi.edu
Received July 5, 2007; accepted January 11, 2008.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
Background— The B vitamin folic acid (FA) is important to mitochondrial protein and nucleic acid synthesis, is an antioxidant, and enhances nitric oxide synthase activity. Here, we tested whether FA reduces myocardial ischemic dysfunction and postreperfusion injury.
Methods and Results— Wistar rats were pretreated with either FA (10 mg/d) or placebo for 1 week and then underwent in vivo transient left coronary artery occlusion for 30 minutes with or without 90 minutes of reperfusion (total n=131; subgroups used for various analyses). FA (4.5x10–6 mol/L IC) pretreatment and global ischemia/reperfusion (30 minutes/30 minutes) also were performed in vitro (n=28). After 30 minutes of ischemia, global function declined more in controls than in FA-pretreated rats (
dP/dtmax, –878±586 versus –1956±351 mm Hg/s placebo; P=0.03), and regional thickening was better preserved (37.3±5.3% versus 5.1±0.6% placebo; P=0.004). Anterior wall perfusion fell similarly (–78.4±9.3% versus –71.2±13.8% placebo at 30 minutes), yet myocardial high-energy phosphates ATP and ADP reduced by ischemia in controls were better preserved by FA pretreatment (ATP: control, 2740±58 nmol/g; ischemia, 947±55 nmol/g; ischemia plus FA, 1332±101 nmol/g; P=0.02). Basal oxypurines (xanthine, hypoxanthine, and urate) rose with FA pretreatment but increased less during ischemia than in controls. Ischemic superoxide generation declined (3124±280 cpm/mg FA versus 5898±474 cpm/mg placebo; P=0.001). After reperfusion, FA-treated hearts had smaller infarcts (3.8±1.2% versus 60.3±4.1% placebo area at risk; P<0.002) and less contraction band necrosis, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling positivity, superoxide, and nitric oxide synthase uncoupling. Infarct size declined similarly with 1 mg/d FA.
Conclusions— FA pretreatment blunts myocardial dysfunction during ischemia and ameliorates postreperfusion injury. This is coupled to preservation of high-energy phosphates, reducing subsequent reactive oxygen species generation, eNOS-uncoupling, and postreperfusion cell death.
Key Words: contractility • folic acid • ischemia • infarction • nitric oxide synthase • reperfusion • superoxide
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
During acute coronary occlusion, myocardial perfusion and oxygen delivery become insufficient to support active muscle contraction. In the ischemic zone, myocardial high-energy phosphate (HEP) content falls, inorganic phosphate rises, and the tissue is rendered acidotic,1 resulting in regional dyskinesis and global dysfunction. Coronary reperfusion after ischemia limits tissue damage but confers toxicity from the activation of reactive oxygen species (ROS), calcium-dependent proteases such as calpain, myofilament contracture, microvascular dysfunction, and inflammatory cytokines.2 ATP depletion during ischemia also may contribute to reperfusion injury.3 In addition, ROS generated during both ischemia4 and reperfusion5 is thought to play a central role, and various antioxidant strategies have been tested to offset this damage.
Editorial p 1772
Clinical Perspective p 1819
Ischemia/reperfusion (IR) damage can be diminished by subjecting hearts to brief ischemia before more prolonged exposure, a phenomenon called preconditioning. This involves activation of protein kinase C,6 mitochondrial KATP channels, and nitric oxide synthesis (NOS),7 with NOS playing a central role. Infarct size after IR is greater in endothelial NOS (eNOS) –deficient mice8 and reduced in mice overexpressing eNOS.9 This role of NOS has led to efforts to enhance its function, including administration of its obligate cofactor, tetrahydrobiopterin (BH4),10 to help maintain NOS in a functionally coupled (NO synthesis, little ROS generation) state.11,12
A far less costly alternative may be folic acid (FA), a B vitamin that stabilizes BH4 by augmenting its binding affinity to eNOS13 and enhances BH4 regeneration from oxidized and inactive BH2. FA or its active metabolite is thought to enhance endothelial function by this mechanism.14,15 FA also is essential for normal mitochondrial protein and purine synthesis16 and can enhance total HEP levels in chronically hypertrophied right ventricles.17 In the present study, we tested the hypothesis that high-dose FA pretreatment ameliorates IR injury and explored the mechanisms for such an effect. The data show postreperfusion benefits but more strikingly reveal a marked and surprising effect of FA pretreatment on reducing regional and chamber dysfunction and ROS generation during the period of ischemia itself. This benefit appears to be linked to alterations in purine catabolism and preservation of HEP levels during ischemia.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication 85-23) and approved by the ethics committees of the University of Antwerp and the Johns Hopkins Medical Institutions.
In Vivo Ischemia Model
Adult Wistar rats received FA (10 mg/d unless otherwise stated) or placebo by oral gavage for 7 days before the IR experiment. A total of 131 rats were used, reflecting the multiple assays that could not be performed in each one. Animals were anesthetized (pentobarbital 60 mg/kg), intubated via tracheotomy, and ventilated (Harvard Apparatus, Holliston, Mass). The ECG was monitored, and temperature was maintained at 37.5°C. The left anterior descending coronary artery (LAD) was exposed through the fourth to fifth intercostal space with a suture placed around it, and transient coronary artery ligation performed for 30 minutes with (n=85) or without (n=46) 90 minutes of reperfusion. In 1 subgroup of reperfused animals (n=9), FA was provided 10 minutes after the onset of LAD occlusion (ie, 20 minutes before the onset of reperfusion), delivered as an intravenous bolus.
In Vivo Hemodynamics
Left ventricular (LV) function was assessed in vivo by pressure-volume loops (n=14) both during ischemia and after reperfusion. A 1.4F pressure-volume catheter (SciSense, London, Ontario, Canada) was advanced through the apex, positioned along the longitudinal axis, and attached to a stimulator/analyzer (IOX 1.8.9.19, Emka Technologies, Paris, France). Volume data were calibrated with the hypertonic saline method, assuming a gain of 1. Two animals had catheter dislocation during ischemia; their volume data were not used. Open-chest myocardial anterior wall motion also was measured with a Sequoia Acuson C256 equipped with a 15-MHz linear transducer (Sequoia C256 Echocardiography System, Acuson Corp, Mountain View, Calif) at the parasternal view of the LV chamber as described.
Assessment of Redox and Energy Metabolism
Snap-frozen samples (n=24) from the anterior wall (with or without FA pretreatment, with or without 30 minutes of ischemia, no reperfusion) were deproteinized and subjected to high-performance liquid chromatography analysis of water-soluble low-molecular-weight compounds reflecting tissue oxidoreductive and energy status. HEPs, oxypurines (hypoxanthine, xanthine, and uric acid), nucleosides (inosine and adenosine), malondialdehyde, and reduced and oxidized glutathione were measured by ion-pairing high-performance liquid chromatography as described.18
Myocardial Flow Measurements
Regional myocardial blood flow was assessed (n=12) by nuclear-activated microspheres (15 µmol/L diameter, BioPal, Worcester, Mass) injected into the left atrium (0.3 mL of 2.5x106 spheres per 1 mL) at baseline and after 5 and 30 minutes of ischemia.19 Total counts per minute were normalized to weight, and results from the ischemic zone were normalized to the remote region to provide relative blood flow before and during ischemia.
In Vitro IR Model
Adult Wistar rats (n=28) were anesthetized (pentobarbital 60 mg/kg), and hearts were rapidly excised and mounted onto a retrograde perfusion system (Emka Technologies) with warmed, oxygenated, buffered Krebs-Henseleit solution at a constant perfusion pressure (75 mm Hg). Hearts were paced at 300 bpm and maintained unloaded. Coronary flow was measured by an inline ultrasonic flow probe (Transonic Systems, Ithaca, NY). After 30 minutes of equilibration, bradykinin or sodium nitroprusside was infused by bolus injection (50 µL, 10–8 to 10–5.5 mol/L IC), and coronary flow reserve was assessed at constant perfusion pressure. After baseline was reestablished, hearts received FA (4.5x10–6 mol/L IC) or vehicle for 30 minutes and then were subjected to 40 minutes of zero-flow ischemia followed by 40 minutes of reperfusion. Coronary effluent was collected, concentrated (Sartorius-Sipan, Lier, Belgium), and analyzed for lactate dehydrogenase (Vitros 950AT, OCD, Beerse, Belgium). Coronary vasodilator reserve studies were then repeated.
Infarct Size Analysis and Histology
Infarct size was assessed (n=31) with area at risk (AAR) determined by Evans blue–negative staining and triphenyl tetrazolium chloride staining to detect myocardial necrosis. Regions were planimetered, digitized, and quantified. AAR was not assessed for the in vitro studies because global ischemia was created. Contraction band necrosis was examined in vivo (n=22) by fixation in Carnoy solution and Masson’s trichrome staining. Serial adjacent fields were examined throughout the LV to calculate the percent myocardium with contraction bands present. A similar analysis was performed for terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) positivity (Chemicon Intern, Temecula, Calif), also expressed as percent LV area.
ROS Determination
Superoxide was assessed by lucigenin (5 µmol/L) -enhanced chemiluminescence (Beckman LS6000IC, Beckman Coulter, Fullerton, Calif; n=23)20 and fluorescent microtopography (dichlorodihydrofluorescein diacetate, n=16; dihydroethidium, n=24).18 The direct antioxidant effects of FA were analyzed with an in vitro xanthine/xanthine oxidase system21 and compared with Tempol.
eNOS Monomer/Dimer Formation and Enzymatic Activity
SDS-resistant eNOS dimers and monomers were assayed on IR tissue (n=16) with low-temperature SDS-PAGE as previously described.18 NOS enzymatic activity was assessed by arginine-to-citrulline conversion assay from extracts obtained from frozen myocardium (n=15).18
Data Analysis
Data are presented as mean±SEM, and values of P<0.05 were considered statistically significant. Coronary flow reserve and in vivo hemodynamic data were analyzed by repeated-measures ANOVA. Other comparisons used either 1-way ANOVA or Kruskal-Wallis tests to compare multiple independent groups, with a Bonferroni correction for multiple comparisons. Analysis used SPS6 version 11.0 (SPSS Inc, Chicago, Ill). The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
FA pretreatment Improves Cardiac Function With IR
Figure 1A displays example and summary LV pressure-volume data in hearts with or without FA (10 mg/d) pretreatment. Data were measured at initial baseline in open-chest rats, during 30 minutes of coronary occlusion, and after 90 minutes of reperfusion. Control hearts displayed markedly reduced function with a rightward downshift of pressure-volume loops at 30 minutes of ischemia that persisted with reperfusion (Figure 1A, top). With FA pretreatment, cardiac function was better preserved during ischemia and reperfusion (Figure 1A, bottom). Peak systolic pressure changed little despite LAD occlusion in FA-pretreated rats but fell nearly 25% in controls. Similar disparities were observed in dP/dtmax. Cardiac output and stroke work also declined less in FA-pretreated animals, particularly in late ischemia and reperfusion.
|
The relative preservation of global function during ischemia was somewhat surprising and suggested less regional dysfunction despite coronary occlusion in FA-pretreated hearts. This was tested by echocardiography measured before and during LAD occlusion (Figure 2). Example M-mode tracings (top left) show marked reductions in anterior wall thickening during ischemia in controls but preserved thickening in FA-treated animals (5±0.6% versus 37±5.3%; P=0.004). Ejection fraction was much higher despite ischemia (72.8±1.2% versus 27.4±2.2% placebo; P<0.001 at 30 minutes), consistent with the pressure-volume loop data.
|
FA and Myocardial Flow
Because FA pretreatment improved both regional and global function during LAD occlusion, we tested whether it enhanced myocardial blood flow to reduce the ischemic insult per se. However, after 5 minutes of LAD ligation, the ratio of ischemic to remote zone myocardial perfusion obtained by microsphere analysis declined similarly in placebo- and FA-pretreated groups (–73.7±6.0% and –77.7±5.1%, respectively). Flow remained low in both groups at 30 minutes (–78.4±9.3% versus –71.2±13.8% placebo).
FA Preserves Myocardial Levels of HEPs
Because improved perfusion could not explain the FA treatment effect, we next tested whether FA altered HEP metabolism at baseline and/or during ischemia. As shown in Figure 3, FA pretreatment did not alter HEP at baseline but did elevate levels of inositol monophosphate (IMP) and its catabolites (oxypurines: xanthine, hypoxanthine, uric acid). During ischemia, myocardial ATP and ADP declined >66% in controls, consistent with reported data.1 However, both were better maintained in FA-pretreated hearts (P<0.001 for drug interaction effect). Oxypurines rose markedly during ischemia in controls, consistent with reduced HEP and enhanced AMP catabolism, but changed little or declined in FA-treated hearts. Redox state indexed by malondialdehyde (a marker of lipid peroxidation) and ratio of reduced to oxidized glutathione were little changed by FA pretreatment with or without myocardial ischemia.
|
FA Pretreatment Reduces Myocardial Infarct Size
A potential consequence of improving both function and HEP metabolism during ischemia is a reduction in infarct size. Infarct size was 60.3±4.1% of the AAR in placebo-treated animals versus 3.8±1.2% with FA pretreatment (P<0.002; Figure 4A). A similar reduction was observed with 40% or 10% of the FA dose (1 or 4 mg/d), although this dose is still fairly high compared with that typically used in humans. Contraction band necrosis was found in 26.7±2.6% of the LV in controls versus 4.6±1.2% in FA-treated hearts (P=0.001; Figure 4B). Similarly, TUNEL-positive myocytes were prevalent (63.0±5.8% of LV fields) in controls but rare (4.3±1.3%) with FA treatment (P=0.001; Figure 4C). Lethal ventricular arrhythmia was fairly common during ischemia in controls but not FA-treated rats (36.7% versus 8.3%) and was reduced in frequency during reperfusion (6.1% versus 0%; P<0.01 for both).
|
Because postreperfusion infarct size in vivo is related partially to coupling of function with coronary perfusion, we also tested the impact of FA pretreatment in isolated hearts. FA reduced infarct size markedly (7.7±2.8% versus 41.1±4.9%; P<0.0001; Figure 4D) with less lactate dehydrogenase in the coronary effluent (Figure 4E), consistent with reduced necrosis.
Folate Pretreatment Versus Acute Folate Administration
In a separate group of 9 animals, FA was administered acutely starting after 10 minutes of coronary occlusion (10 mg IV) when functional responses first appeared to diverge (see Figures 1 and 2
) and continued for the remaining 20-minute ischemic period. Infarct size relative to AAR also was reduced (n=5; 3.0±2.2%; P<0.001 versus placebo) with AAR itself similar to placebo (52.4±5.5%). Histology (n=4) found reduced TUNEL staining (4.3±1.3% LV) and contraction band necrosis (5.1±0.7% LV; both P<0.001 versus placebo). Thus, the effect of FA on infarct reduction did not appear to be a classic preconditioning effect because it could be generated by FA administration after ischemia had begun.
Folate Pretreatment Reduces ROS Generation
Myocardial superoxide (lucigenin chemiluminescence) declined 
50% in FA-pretreated animals during ischemia and after 90 minutes of reperfusion (Figure 5A). When extracts were preincubated with 100 µmol/L BH4, O2– generation declined 90.9±0.7% in vehicle-controls but less so in FA-pretreated hearts (52.1±11.3%; P<0.03; Figure 5B). This suggested that an antioxidant pathway targeted by BH4 (eg, NOS coupling) either was lacking in FA-pretreated hearts or was already ameliorated by FA therapy. Dihydroethidium- and dichlorodihydrofluorescein diacetate–stained myocardial slices also showed marked ROS generation in the placebo group that was reduced with FA pretreatment (Figure 5C through 5E). To test for direct antioxidant effects of FA, we performed an in vitro assay using a xanthine/xanthine oxidase O2– generating system (Figure 5F). FA antioxidant effects were substantial in this assay and were similar to the superoxide dismutase mimetic Tempol.
|
Folate Pretreatment Improves eNOS Dimerization and Activity and Endothelial Function
Because FA and its metabolites have been linked to the BH4-mediated improvement in NOS coupling and decline in ROS generation,14,21 we examined NOS coupling in IR hearts. Immunoblots showed a decline in the ratio of NOS dimer to monomer that was preserved at nearly normal in FA-pretreated hearts (Figure 6A through 6C). Total eNOS was similar among the conditions (Figure 6B). NOS activity (arginine-to-citrulline conversion) was borderline improved by FA pretreatment (P=0.08; data not shown).
|
Coronary endothelial function also improved with FA pretreatment. Bradykinin induced a maximal 108.3±9.2% rise in coronary flow at baseline but 67.1±8.1% after IR (P<0.001; Figure 6D). This decline was absent in FA-pretreated hearts (122.0±11.3%). Basal flow before (placebo, 10.2±0.7 mL/min; FA, 11.4±13.8 mL/min; P=0.5) and after (placebo, 9.8±0.7 mL/min; FA, 8.9±1.4 mL/min; P=0.5) ischemia was similar between groups. Coronary flow rose similarly before and after ischemia with sodium nitroprusside (Figure 6E), supporting the endothelium dependence of the prior effect.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
This study demonstrates that pretreatment with high doses of oral FA markedly reduces ischemic dysfunction during coronary occlusion and enhances function and diminishes infarct size after reperfusion. These were associated with better-preserved HEPs during ischemia despite a flow reduction, sustaining regional function and reducing oxidative stress, with preserved eNOS coupling after reperfusion. Preserved function during ischemia is unusual and quite different from the influence of antioxidants and the classic preconditioning agents that typically benefit the heart only after reperfusion. For that matter, the benefit of FA in reducing infarct size even when delivered after ischemia had begun implies a different mechanism.
During normoxia, ATP synthesis is highly regulated and levels are maintained. With ischemia, ATP supplies from anaerobic glycolysis and HEP reserves such as phosphocreatine are insufficient to meet demand, and the net ATP level falls. Theoretically, interventions that could maintain ATP even in ischemic myocardium should delay the advent of lethal injury because low tissue ATP levels (<5 µmol/g dry weight) are associated with depressed HEP resynthesis, failure of cell volume regulation, and irreversible myocardial injury. FA is known to regulate mitochondrial function and plays an important role as a methylation cofactor for the synthesis of thymidylate, purines, and methionine. With regard to purine synthesis, the metabolite N10-formyl tetrahydrofolate contributes 2 carbons (C2 and C8) to the ring structure, with the primary end point being formation of IMP. IMP is then further metabolized to generate AMP or GMP or is catabolized to oxypurines.
The present results support a role of FA in HEP generation. The increased basal level of oxypurines suggests that high doses of FA drove their synthesis (via IMP) by mass action, but because HEP was adequate, IMP catabolites were enhanced with AMP levels unchanged. During ischemia, however, ATP and ADP levels fell, leading to increased AMP and AMP catabolism (IMP, oxypurinols) in controls. In FA-treated hearts, however, purine synthesis would be primed to help rederive HEP (FA-treated hearts had virtually no decline in ADP during ischemia, and the ATP level decline was about half). Actual ATP generation was probably higher in the FA-treated hearts because the anterior wall was still actively contracted and therefore was using more ATP despite LAD occlusion compared with controls. The concept of FA-enhanced HEP is supported by a recent study by Lamberts et al,22 who found that chronic high-dose FA restored total HEPs (ATP plus ADP plus AMP) accompanied by improved diastolic function in hypertrophied rat hearts. Our finding that FA administered intravenously 10 minutes into the ischemic period still diminished infarct size also could be explained by such a mass action effect because it may occur quickly. This remains to be confirmed but has intriguing therapeutic implications.
High-dose FA may have potent antioxidant effects both directly and via weak but competitive inhibition of xanthine oxidase,13 which also could benefit the heart during ischemic and postischemic periods. Although our in vitro data showed similar antioxidant effects for FA and Tempol, in vivo, scavenging effects of FA are thought to be modest even at high doses.14 Importantly, reduced oxidant stress per se has not previously been shown to enhance HEP; rather, a decline in HEP is linked to mitochondrial damage and ROS generation.23 Klawitter et al4 administered 1,2-dihydroxybenzene-3,5-disulfonate (Tiron), a superoxide scavenger, or N-acetyl-L-cysteine before ischemia and found no change in energetic recovery compared with untreated hearts. Tempol reduces postreperfusion infarct size in vivo in rat and rabbit by 50% and 33%, respectively,24 but only modestly improves LV pressure recovery after reperfusion.25 Similar results are reported with other free radical scavengers.26 Thus, it seems unlikely that the sole or primary mechanism for FA benefit was its antioxidant capacity.
Another potential mechanism is the recoupling of NOS. NOS uncoupling is thought to contribute to the pathophysiology of diseases such as hypertension, atherosclerosis, and cardiac hypertrophy.18,27–29 Uncoupling involves a decline in the normal electron transfer from the reductase to oxidase domains, leading to reduced NO synthesis and greater ROS generation by NOS.30 BH4 plays a key role in maintaining normal eNOS coupling, and FA (or 5-methyltetrahydrofolate) can increase BH4 levels by facilitating enzymatic reduction of its oxidized forms31 and are associated with improved endothelial function.14 The present study supports this mechanism on the basis of the diminished eNOS uncoupling after reperfusion and an in vitro analysis showing that the addition of BH4 reduced ROS generation far more in untreated than in FA-treated myocardium. Improved NOS coupling also could contribute to improved HEP generation because endogenous NO generation reduces oxygen consumption and improves energetic efficiency32 partly by enhancing HEP synthesis itself.
This is the first study to test the effects of FA on in vivo ischemia and IR injury. Several recent studies have found that FA and/or its active metabolite 5-methyltetrahydrofolate improve endothelial function,21,33 although the dose required has been somewhat controversial. Tawakol et al34 used high-dose FA (30 mg PO) in patients with coronary artery disease and found that it increased both adenosine-stimulated myocardial blood flow and flow reserve in segments with impaired dilator reserve but also acutely lowered arterial pressure. The antinecrotic effect of FA observed in the present study is consistent with earlier in vitro data in which high-dose folate (>1 mmol/L) suppressed apoptosis from oxidant injury in U937 cells.35 The beneficial effect of FA on in vivo ischemia and reperfusion-induced arrhythmias is similar to the reduction of in vitro reperfusion-induced arrhythmias by FA described by Manning et al.36 One prior study10 examined the effects of BH4 on post-IR infarct size and found more modest changes than we observed with folate. This can be due to dose differences but also to specific influences that folate has that are unrelated to BH4.
FA has been studied in clinical trials, particularly to test its potential to lower cardiovascular risk in patients with myocardial vascular disease. For example, Oster37 demonstrated that long-term FA treatment (10 years) at a dose far higher than typically used (40 to 80 mg/d) reduced the incidence of myocardial infarction, angina pectoris, and the requirement for nitroglycerin in patients with coronary artery disease. This observation was not confirmed by a placebo-controlled trial, nor was the mechanism explored. Other studies focused on the ability of FA to lower homocysteine and found a 3-µmol/L decrease in serum homocysteine (achievable with 0.8 mg/d). Despite these positive results, meta-analyses of multiple FA trials for use in cardiovascular prevention have been unimpressive,38,39 and it remains unclear whether dose, study duration, target population, or other factors explain this. The doses of FA used in the present study are far above those used in prior cardiovascular interventional studies (5 to 25 mg · 70 kg–1 · d–1) or prevention trials (500 µg to 1 mg). Even taking into account that rodents have a higher metabolic turnover, the present dose would likely correlate with between 0.25 and 2.5 g · 70 kg–1 · d–1 in a human. Although long-term treatment at this level may have adverse off-target effects, short-term dosing in a peri-infarct period should be well tolerated. Admittedly, this remains to be determined.
A few limitations to our study should be noted. Although FA pretreatment improved HEP balance and function during a 30-minute coronary occlusion, it might become less effective if the ischemic period would have been more prolonged. It is also unknown how long after an occlusion we might intervene with FA and still see a benefit. Finally, our data support but do not prove that FA directly helps rederive HEP by mass action effects on purine synthesis. This requires future studies using 14C-labeled folate in which the carbons can be tracked and the direct involvement can be tested.
|
Conclusions |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
We have found marked and novel evidence for improved ischemic and post-IR myocardial function and reduced infarction by pretreatment with high-dose FA. The relation to preservation of myocardial HEP during the ischemic period is intriguing and suggests that FA can alter the manner by which HEPs are synthesized despite a decline in myocardial perfusion. This may relate to changes in purine synthesis, enhanced NOS coupling modulated by interactions between folate and BH4, and/or effects of FA on improving mitochondrial function during ischemia. Given that FA is inexpensive, safe even at high doses, and readily available, it is possible that such treatment in high-risk patients could provide a novel means to limit ischemic damage.
|
Acknowledgments |
|---|
We thank Professor C. Van Campenhout for help with the lactate dehydrogenase measurements on the coronary effluent; F. Terloo, R. Spillemaeckers, and D. Vindevogel for technical assistance; and J. Van Daele and D. De Rijck for graphic assistance.
Sources of Funding
This work was supported by the research fund of the University of Antwerp, the Belgian-American Education Foundation (Collen) grant, and an American Heart Association Postdoctoral Fellowship Grant (Dr Moens); the Fund for Scientific Research FWO–Flanders, No. 2204/4951 (Drs Vrints and Timmermans); MIN NIH National Heart, Lung, and Blood grants HL31069, HL43023, and HL66331 (Dr Wolin); an American Heart Association Scientist Development Grant (Dr Champion); and NIH National Heart, Lung, and Blood grants RO1-AG18324, HL-47511, and PO1-HL59408 (Dr Kass).
Disclosures
None.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
1. Jennings RB, Steenbergen C Jr. Nucleotide metabolism and cellular damage in myocardial ischemia. Annu Rev Physiol. 1985; 47: 727–749.[CrossRef][Medline] [Order article via Infotrieve]
2. Gross ER, Gross GJ. Ligand triggers of classical preconditioning and postconditioning. Cardiovasc Res. 2006; 70: 212–221.
3. Gunduz D, Kasseckert SA, Hartel FV, Aslam M, Abdallah Y, Schafer M, Piper HM, Noll T, Schafer C. Accumulation of extracellular ATP protects against acute reperfusion injury in rat heart endothelial cells. Cardiovasc Res. 2006; 71: 764–773.
4. Klawitter PF, Murray HN, Clanton TL, Angelos MG. Reactive oxygen species generated during myocardial ischemia enable energetic recovery during reperfusion. Am J Physiol Heart Circ Physiol. 2002; 283: H1656–H1661.
5. Zweier JL, Talukder MA. The role of oxidants and free radicals in reperfusion injury. Cardiovasc Res. 2006; 70: 181–190.
6. Yamamura K, Steenbergen C, Murphy E. Protein kinase C and preconditioning: role of the sarcoplasmic reticulum. Am J Physiol Heart Circ Physiol. 2005; 289: H2484–H2490.
7. Jones SP, Bolli R. The ubiquitous role of nitric oxide in cardioprotection. J Mol Cell Cardiol. 2006; 40: 16–23.[CrossRef][Medline] [Order article via Infotrieve]
8. Jones SP, Girod WG, Palazzo AJ, Granger DN, Grisham MB, Jourd’Heuil D, Huang PL, Lefer DJ. Myocardial ischemia-reperfusion injury is exacerbated in absence of endothelial cell nitric oxide synthase. Am J Physiol. 1999; 276: H1567–H1573.[Medline] [Order article via Infotrieve]
9. Jones SP, Greer JJ, Kakkar AK, Ware PD, Turnage RH, Hicks M, van Haperen R, de Crom R, Kawashima S, Yokoyama M, Lefer DJ. Endothelial nitric oxide synthase overexpression attenuates myocardial reperfusion injury. Am J Physiol Heart Circ Physiol. 2004; 286: H276–H282.
10. Wajima T, Shimizu S, Hiroi T, Ishii M, Kiuchi Y. Reduction of myocardial infarct size by tetrahydrobiopterin: possible involvement of mitochondrial KATP channels activation through nitric oxide production. J Cardiovasc Pharmacol. 2006; 47: 243–249.[CrossRef][Medline] [Order article via Infotrieve]
11. Hevel JM, Marletta MA. Macrophage nitric oxide synthase: relationship between enzyme-bound tetrahydrobiopterin and synthase activity. Biochemistry. 1992; 31: 7160–7165.[CrossRef][Medline] [Order article via Infotrieve]
12. Vasquez-Vivar J, Kalyanaraman B, Martasek P, Hogg N, Masters BS, Karoui H, Tordo P, Pritchard KA Jr. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci U S A. 1998; 95: 9220–9225.
13. Stroes ES, van Faassen EE, Yo M, Martasek P, Boer P, Govers R, Rabelink TJ. Folic acid reverts dysfunction of endothelial nitric oxide synthase. Circ Res. 2000; 86: 1129–1134.
14. Shirodaria C, Antoniades C, Lee J, Jackson CE, Robson MD, Francis JM, Moat SJ, Ratnatunga C, Pillai R, Refsum H, Neubauer S, Channon KM. Global improvement of vascular function and redox state with low-dose folic acid: implications for folate therapy in patients with coronary artery disease. Circulation. 2007; 115: 2262–2270.
15. Moat SJ, Clarke ZL, Madhavan AK, Lewis MJ, Lang D. Folic acid reverses endothelial dysfunction induced by inhibition of tetrahydrobiopterin biosynthesis. Eur J Pharmacol. 2006; 530: 250–258.[CrossRef][Medline] [Order article via Infotrieve]
16. Depeint F, Bruce WR, Shangari N, Mehta R, O’Brien PJ. Mitochondrial function and toxicity: role of B vitamins on the one-carbon transfer pathways. Chem Biol Interact. 2006; 163: 113–132.[CrossRef][Medline] [Order article via Infotrieve]
17. Lamberts RR, Caldenhoven E, Lansink M, Witte G, Vaessen RJ, St Cyr JA, Stienen GJ. Preservation of diastolic function in monocrotaline-induced right ventricular hypertrophy in rats. Am J Physiol Heart Circ Physiol. 2007; 293: H1869–H1876.
18. Takimoto E, Champion HC, Li M, Ren S, Rodriguez ER, Tavazzi B, Lazzarino G, Paolocci N, Gabrielson KL, Wang Y, Kass DA. Oxidant stress from nitric oxide synthase-3 uncoupling stimulates cardiac pathologic remodeling from chronic pressure load. J Clin Invest. 2005; 115: 1221–1231.[CrossRef][Medline] [Order article via Infotrieve]
19. Reinhardt CP, Dalhberg S, Tries MA, Marcel R, Leppo JA. Stable labeled microspheres to measure perfusion: validation of a neutron activation assay technique. Am J Physiol Heart Circ Physiol. 2001; 280: H108–H116.
20. Gupte SA, Kaminski PM, Floyd B, Agarwal R, Ali N, Ahmad M, Edwards J, Wolin MS. Cytosolic NADPH may regulate differences in basal Nox oxidase-derived superoxide generation in bovine coronary and pulmonary arteries. Am J Physiol Heart Circ Physiol. 2005; 288: H13–H21.
21. Antoniades C, Shirodaria C, Warrick N, Cai S, de Bono J, Lee J, Leeson P, Neubauer S, Ratnatunga C, Pillai R, Refsum H, Channon KM. 5-Methyltetrahydrofolate rapidly improves endothelial function and decreases superoxide production in human vessels: effects on vascular tetrahydrobiopterin availability and endothelial nitric oxide synthase coupling. Circulation. 2006; 114: 1193–1201.
22. Lamberts RR, Caldenhoven E, Lansink M, Witte G, Vaessen RJ, St Cyr JA, Stienen GJ. Preservation of diastolic function in monocrotaline-induced right ventricular hypertrophy in rats. Am J Physiol Heart Circ Physiol. 2007; 293: H1869–H1876.
23. Hyslop PA, Hinshaw DB, Halsey WA Jr, Schraufstatter IU, Sauerheber RD, Spragg RG, Jackson JH, Cochrane CG. Mechanisms of oxidant-mediated cell injury: the glycolytic and mitochondrial pathways of ADP phosphorylation are major intracellular targets inactivated by hydrogen peroxide. J Biol Chem. 1988; 263: 1665–1675.
24. McDonald MC, Zacharowski K, Bowes J, Cuzzocrea S, Thiemermann C. Tempol reduces infarct size in rodent models of regional myocardial ischemia and reperfusion. Free Radic Biol Med. 1999; 27: 493–503.[CrossRef][Medline] [Order article via Infotrieve]
25. Kutala VK, Khan M, Mandal R, Potaraju V, Colantuono G, Kumbala D, Kuppusamy P. Prevention of postischemic myocardial reperfusion injury by the combined treatment of NCX-4016 and Tempol. J Cardiovasc Pharmacol. 2006; 48: 79–87.[CrossRef][Medline] [Order article via Infotrieve]
26. Penumathsa SV, Thirunavukkarasu M, Koneru S, Juhasz B, Zhan L, Pant R, Menon VP, Otani H, Maulik N. Statin and resveratrol in combination induces cardioprotection against myocardial infarction in hypercholesterolemic rat. J Mol Cell Cardiol. 2007; 42: 508–516.[CrossRef][Medline] [Order article via Infotrieve]
27. Alp NJ, McAteer MA, Khoo J, Choudhury RP, Channon KM. Increased endothelial tetrahydrobiopterin synthesis by targeted transgenic GTP-cyclohydrolase I overexpression reduces endothelial dysfunction and atherosclerosis in ApoE-knockout mice. Arterioscler Thromb Vasc Biol. 2004; 24: 445–450.
28. Bendall JK, Alp NJ, Warrick N, Cai S, Adlam D, Rockett K, Yokoyama M, Kawashima S, Channon KM. Stoichiometric relationships between endothelial tetrahydrobiopterin, endothelial NO synthase (eNOS) activity, and eNOS coupling in vivo: insights from transgenic mice with endothelial-targeted GTP cyclohydrolase 1 and eNOS overexpression. Circ Res. 2005; 97: 864–871.
29. Adlam D, Bendall JK, De Bono JP, Alp NJ, Khoo J, Nicoli T, Yokoyama M, Kawashima S, Channon KM. Relationships between nitric oxide-mediated endothelial function, eNOS coupling and blood pressure revealed by eNOS-GTP cyclohydrolase 1 double transgenic mice. Exp Physiol. 2007; 92: 119–126.
30. Forstermann U, Munzel T. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation. 2006; 113: 1708–1714.
31. Hyndman ME, Verma S, Rosenfeld RJ, Anderson TJ, Parsons HG. Interaction of 5-methyltetrahydrofolate and tetrahydrobiopterin on endothelial function. Am J Physiol Heart Circ Physiol. 2002; 282: H2167–H2172.
32. Loke KE, Laycock SK, Mital S, Wolin MS, Bernstein R, Oz M, Addonizio L, Kaley G, Hintze TH. Nitric oxide modulates mitochondrial respiration in failing human heart. Circulation. 1999; 100: 1291–1297.
33. Moat SJ, Madhavan A, Taylor SY, Payne N, Allen RH, Stabler SP, Goodfellow J, McDowell IF, Lewis MJ, Lang D. High- but not low-dose folic acid improves endothelial function in coronary artery disease. Eur J Clin Invest. 2006; 36: 850–859.[CrossRef][Medline] [Order article via Infotrieve]
34. Tawakol A, Migrino RQ, Aziz KS, Waitkowska J, Holmvang G, Alpert NM, Muller JE, Fischman AJ, Gewirtz H. High-dose folic acid acutely improves coronary vasodilator function in patients with coronary artery disease. J Am Coll Cardiol. 2005; 45: 1580–1584.
35. Huang RF, Yaong HC, Chen SC, Lu YF. In vitro folate supplementation alleviates oxidative stress, mitochondria-associated death signalling and apoptosis induced by 7-ketocholesterol. Br J Nutr. 2004; 92: 887–894.[CrossRef][Medline] [Order article via Infotrieve]
36. Manning A, Bernier M, Crome R, Little S, Hearse D. Reperfusion-induced arrhythmias: a study of the role of xanthine oxidase-derived free radicals in the rat heart. J Mol Cell Cardiol. 1988; 20: 35–45.[Medline] [Order article via Infotrieve]
37. Oster K. Atherosclerosis treated with folic acid. Fed Proc. 1981; 40: 865. Abstract.
38. Wang X, Qin X, Demirtas H, Li J, Mao G, Huo Y, Sun N, Liu L, Xu X. Efficacy of folic acid supplementation in stroke prevention: a meta-analysis. Lancet. 2007; 369: 1876–1882.[CrossRef][Medline] [Order article via Infotrieve]
39. Wald DS, Wald NJ, Morris JK, Law M. Folic acid, homocysteine, and cardiovascular disease: judging causality in the face of inconclusive trial evidence. BMJ. 2006; 333: 1114–1117.
 |
|
 |
|
  C. S. Thompson-Torgerson, H. C. Champion, L. Santhanam, Z. L. Harris, and A. A. Shoukas Cyclohexanone contamination from extracorporeal circuits impairs cardiovascular function Am J Physiol Heart Circ Physiol, June 1, 2009; 296(6): H1926 - H1932. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Antoniades, A. S. Antonopoulos, D. Tousoulis, K. Marinou, and C. Stefanadis Homocysteine and coronary atherosclerosis: from folate fortification to the recent clinical trials Eur. Heart J., January 1, 2009; 30(1): 6 - 15. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. R. Greyson Letter by Greyson Regarding Article, "High-Dose Folic Acid Pretreatment Blunts Cardiac Dysfunction During Ischemia Coupled to Maintenance of High-Energy Phosphates and Reduces Postreperfusion Injury" Circulation, November 18, 2008; 118(21): e703 - e703. [Full Text] [PDF]
|
|
|
|
|
|
A. L. Moens, H. C. Champion, A. Haile, M. Zviman, D. Bedja, K. L. Gabrielson, N. Paolocci, D. A. Kass, R. S. Elsaesser, M. J. Claeys, et al. Response to Letter Regarding Article, "High-Dose Folic Acid Pretreatment Blunts Cardiac Dysfunction During Ischemia Coupled to Maintenance of High-Energy Phosphates and Reduces Postreperfusion Injury" Circulation, November 18, 2008; 118(21): e704 - e704. [Full Text] [PDF]
|
|
|
|
|
|
R. Tian and J. S. Ingwall How Does Folic Acid Cure Heart Attacks? Circulation, April 8, 2008; 117(14): 1772 - 1774. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||