Uncoupling of nitric oxide synthase (NOS) has been implicated in left ventricular (LV) remodeling and dysfunction after myocardial infarction (MI). We hypothesized that inducible NOS (iNOS) plays a crucial role in LV remodeling after MI, depending on its coupling status. MI was created in wild-type, iNOS-knockout (iNOS−/−), endothelial NOS-knockout (eNOS−/−), and neuronal NOS-knockout (nNOS−/−) mice. iNOS and nNOS expressions were increased after MI associated with an increase in nitrotyrosine formation. The area of myocardial fibrosis and LV end-diastolic volume and ejection fraction were more deteriorated in eNOS−/− mice compared with other genotypes of mice 4 wk after MI. The expression of GTP cyclohydrolase was reduced, and tetrahydrobiopterin (BH4) was depleted in the heart after MI. Oral administration of sepiapterin after MI increased dihydrobiopterin (BH2), BH4, and BH4-to-BH2 ratio in the infarcted but not sham-operated heart. The increase in BH4-to-BH2 ratio was associated with inhibition of nitrotyrosine formation and an increase in nitrite plus nitrate. However, this inhibition of NOS uncoupling was blunted in iNOS−/− mice. Sepiapterin increased capillary density and prevented LV remodeling and dysfunction after MI in wild-type, eNOS−/−, and nNOS−/− but not iNOS−/− mice. Nω-nitro-l-arginine methyl ester abrogated sepiapterin-induced increase in nitrite plus nitrate and angiogenesis and blocked the beneficial effects of sepiapterin on LV remodeling and function. These results suggest that sepiapterin enhances angiogenesis and functional recovery after MI by activating the salvage pathway for BH4 synthesis and increasing bioavailable nitric oxide predominantly derived from iNOS.
- nitric oxide synthase
- left ventricular
cardiac remodeling remains an important primary therapeutic target in patients with myocardial infarction (MI). Strategies to prevent or halt adverse left ventricular remodeling after MI include pharmacotherapy, cell-based therapy, percutaneous interventions, and surgical procedures. Despite the use of evidence-based strategies to post-MI, heart failure supervenes and is attended by an unacceptably high mortality rate. Despite the multitude of agents available for the treatment of heart failure such as the inhibitors of renin-angiotensin-aldosterone system, β-blockers, G protein receptor kinase inhibition, administration of growth factors, and cytokines, it remains a highly prevalent clinical syndrome with substantial morbidity and mortality, necessitating alternative pharmacological tools of targeted management. One such area of interest is the ability to modulate the bioavailability of nitric oxide (NO).
Accumulating evidence indicates that NO plays a central role in cardiac cell death/survival and myocardial repair/remodeling (39). Biosynthesis of NO is dependent on enzymatic activity of NO synthase (NOS). Three major NOS isoforms have been identified by molecular cloning: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). NOS is a homodimeric oxidoreductase containing iron protoporphyrin IX (heme), flavin adenine dinucleotide, flavin mononucleotide, and tetrahydrobiopterin (BH4), which is a cofactor essential for the catalytic activity of these NOS isoforms (36, 48). The coupling between the NO substrate, l-arginine, and the heme site requires BH4 to bind in the dimer interface of NOS. BH4 is synthesized de novo by the action of GTP cyclohydrolase-1 (GTPCH-1) or by the salvage pathway that converts sepiapterin to BH4 via the action of sepiapterin reductase and dihydrofolate reductase (52). BH4 depletion, which results from its oxidation and/or reduced synthesis, causes functional uncoupling of NOS. Uncoupled NOS generates more superoxide and less NO, shifting the nitroso-redox balance and leading to adverse consequences on the cardiovascular system (20).
The essential role of eNOS in LV remodeling and dysfunction after MI has been recognized for many years. eNOS-derived NO appears to provide a beneficial effect on the heart after MI, because eNOS-deficient mice developed more severe LV remodeling and dysfunction after MI than wild-type mice do (46), and vice versa, endothelial overexpression of eNOS has been shown to attenuate LV dysfunction in mice after MI (26). On the contrary, eNOS uncoupling has been implicated in the pathophysiology of heart failure after experimental MI (3, 42, 55). Thus, reversal of eNOS uncoupling has become a therapeutic target for heart failure after MI. Indeed, Masano et al. (34) demonstrated that administration of BH4 inhibited uncoupling of eNOS and ameliorated LV remodeling in rats with MI.
In contrast with the established role of uncoupling of eNOS in adverse LV remodeling, the role of iNOS and nNOS in LV remodeling and function after MI has been poorly understood. The prevailing hypothesis is that overexpression of iNOS contributes to detrimental cardiac remodeling in the failing heart (12, 16, 19, 21). However, this concept appeared to be at odds with a large body of evidence indicating that iNOS-derived NO plays a beneficial role in cardioprotection against ischemia-reperfusion injury by ischemic preconditioning (5, 6) or pharmacological preconditioning with resveratrol (18, 23). In the late preconditioning phenomenon, upregulation of iNOS was associated with simultaneous activation of the antioxidant defense system (22), which may presumably inhibit uncoupling of iNOS by preventing oxidation and depletion of BH4. Thus, whether iNOS exerts a protective or injurious effect seems to depend on the coupling status of iNOS. Therefore, it is suggested that inhibition of iNOS uncoupling switches iNOS from a detrimental to beneficial phenotype. Indeed, our recent study demonstrated that exogenous BH4 unmasks tolerance of the diabetic heart to ischemia-reperfusion injury by inhibiting uncoupling of iNOS (37). On the other hand, the role of nNOS in the regulation of cardiovascular function has recently emerged. Xu et al. (57) found nNOS in the sarcoplasmic reticulum of several mammalian hearts, including humans. Although it has been established that nNOS-derived NO regulates myocardial inotropy and relaxation (1, 2, 28, 47), the mechanisms underlying these actions and their potential relevance to myocardial disease state are still debated. Dawson et al. (11) have demonstrated that nNOS plays a crucial role in preventing adverse LV remodeling and maintaining myocardial β-adrenergic reserve after MI. Thus it is also interesting to investigate the role of nNOS uncoupling in LV remodeling and dysfunction after MI.
Supplementation with BH4 after MI may represent an effective approach to ameliorate LV remodeling and dysfunction not only by increasing eNOS-derived NO but also iNOS- or nNOS-derived NO. Sepiapterin may be a more preferable pharmacological tool than BH4 to investigate the role of uncoupling of NOS in the regulation of cardiovascular function, because it is a stable precursor of BH4 and much more permeable across the cell membrane than BH4 (45). Indeed, Tiefenbacher et al. (53) first demonstrated that sepiapterin was effective in ameliorating postischemic injury in the rat heart. Moreover, oral administration of sepiapterin in rats has been shown to increase BH4 levels by threefold in the transplanted heart (41), suggesting that the salvage pathway for BH4 synthesis is activated in the heart. In addition, unlike BH4, sepiapterin by itself does not act as an antioxidant, which may confer a cardioprotective effect independent of inhibition of NOS uncoupling. Therefore, we used sepiapterin to investigate the role of uncoupling of each NOS isoform in LV remodeling and dysfunction after MI. The results of the present study suggest that uncoupling of iNOS predominates over that of eNOS or nNOS after MI, and sepiapterin increases myocardial BH4 content and inhibits uncoupling of NOS. The resultant increase in NO especially derived from iNOS may enhance angiogenesis and improve LV remodeling and function after MI.
MATERIAL AND METHODS
Male iNOS knockout (iNOS−/−) mice, eNOS knockout (eNOS−/−) mice, nNOS knockout (nNOS−/−) mice, and their wild-type (WT) littermates (based on a C57BL/6 background) mice (8–10 wk of age) were obtained from Jackson Laboratory (Bar Harbor, ME). All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the National Institutes of Health, National Research Council, and published by the National Academy Press, revised 1996. The study was approved by the Animal Care Committee of Kansai Medical University (Moriguchi, Japan).
The trachea of mice was cannulated with a polyethylene tube connected to a respirator (Shinano, Tokyo, Japan) with a tidal volume set at 1.2 ml and a rate set at 100/min. The mice were then anesthetized with 1.5–2.0% isoflurane under controlled ventilation with a respirator for the remainder of the surgical procedure. A left thoracotomy was performed between the fourth and fifth ribs, and the pericardial sac was removed. The left anterior descending coronary artery was ligated with 6-0 prolene sutures to produce MI. The mice receiving sham surgery underwent the same procedure except the suture was passed under the coronary artery and then removed. The chest was then closed in three layers (ribs, muscle, and skin), and the animal was allowed to recover. Body temperature was maintained at 37°C throughout the surgical procedure. Buprenorphin (0.1 mg/kg ip) was administered after surgery to alleviate pain.
The mice surviving the operation were randomized to receive oral administration of sepiapterin (10 mg·kg−1·day−1) or the vehicle (drinking water) for 28 days. The animals did not have free access to water until they drank up of the water containing required dose of sepiapterin. Some WT mice were randomly assigned to receive sepiapterin together with NG-nitro-l-arginine methyl ester (l-NAME; Sigma Chemical, St. Louis, MO,), a nonselective inhibitor of all NOS isoforms, at a dose of 100 mg·kg−1·day−1.
Blood pressure measurement and echocardiography.
Before and 28 days after MI, blood pressure was measured by the tail-cuff system (BP-98A; Softron, Tokyo, Japan). The mice were then lightly anesthetized with 1.0% isoflurane inhalation, and transthoracic echocardiography was performed using a SONOS-7500 echocardiography system (Philips Medical Systems, Andover, MA) equipped with a 15-MHz transducer. Measurements were made by an observer who was blinded to the experimental groups. LV end-diastolic volume (LVEDV), LV end-systolic volume, and LV ejection fraction (LVEF) were calculated using three parasternal short-axis views and the parasternal long-axis views as described previously (46). Body weight (BW) was then measured, and the mice were euthanized by intraperitoneal injection with overdose pentobarbital sodium. The heart was quickly removed, weighed, and served for histological and biochemical analysis.
Immunoblot analysis for NOS and GTPCH-1.
Frozen myocardial tissue samples were ground with a mortar and pestle and subsequently placed into a tissue grinder with lysis buffer containing 30 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail Complete (Roche Diagnostics, Mannheim, Germany). The protein concentration was determined using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). The lysate samples were separated by 7.5% SDS-PAGE, and the separated proteins were transferred to a polyvinylidene-difluoride membrane with a transfer buffer containing 25 mM Tris, 192 mM glycine, and 10% methanol. The membranes were blocked with 5% skimmed milk and incubated in a primary antibody against nNOS, iNOS, or eNOS (BD Biosiences, San Jose, CA) at a dilution of 1:1,000, or GTPCH-1 (Invitrogen, Carlsbad, CA) at a dilution of 1:500 and then were subsequently incubated with a peroxidase-conjugated secondary antibodies at a dilution of 1: 5,000 and developed using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's instructions. Equivalent sample loading was confirmed by reprobing the membranes with an anti-β-actin antibody (Sigma), and consistency in the data analysis was ensured by normalization of each immunoblot signal to the corresponding β-actin signal. The immunolabeling intensity was quantified by densitometric analysis using the Win Roof image analyzing software system (Mitani, Fukui, Japan).
Measurement of myocardial biopterin concentrations.
BH4 and dihydrobiopterin (BH2; the oxidized and inactive form of BH4) were measured in cardiac homogenates by high performance liquid chromatography analysis developed by Tani and Ohno (50) after iodine oxidation in acidic or alkaline conditions as described previously (34).
Immunohistochemistry for NOS and nitrotyrosine.
Myocardial tissue slices measuring 2 mm in thickness were obtained from the mid-LV level, and frozen sections were made for immunofluorescence microscopy. Briefly, frozen samples were cut into 6 μm sections and mounted on glass slides, incubated in acetone and hydrogen peroxide, rinsed with PBS, and blocked with 10% normal rabbit serum. The sections were incubated for 1 h at room temperature with a mouse monoclonal antibody against eNOS, iNOS, nNOS (BD Biosciences), or nitrotyrosine (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:100 and washed with PBS. They were then incubated for 2 h at room temperature with FITC-conjugated rabbit anti-mouse immunoglobulin at a dilution of 1:100. The slides were viewed with a confocal laser microscope (Fluo View; Olympus, Tokyo, Japan).
Measurement of nitrite and nitrate generation.
Nitrite and nitrate (NOx), stable oxidation metabolites of NO, have been measured as indexes for the bioavailability of NO (25). The myocardial level of NOx was measured by an HPLC method. Heart tissue, sampled from the frozen and subsequently powdered left ventricle, was homogenized in 500 μl of extraction buffer containing 50 mM Tris (pH 7.4), 1 mM DTT, and 1 mM EDTA. The samples were centrifuged at 10,000 g at 4°C for 10 min. A 300 μl aliquot of supernatant was removed, and NOx was measured using an HPLC system (Shimadzu Kyoto, Japan) according to the method described previously by Green et al. (17).
ELISA assay for nitrotyrosine formation.
Nitrotyrosine formation in the heart was also measured by the ELISA method. Heart tissue, sampled from the frozen and subsequently powdered left ventricle, was homogenized in 200 μl of radioimmunoprecipitation assay buffer containing 50 mM Tris (pH 7.4), 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS. The samples were centrifuged at 10,000 g at 4°C for 10 min. A 50 μl aliquot of supernatant was removed and 3-nitrotyrosine was quantified using a nitrotyrosine ELISA kit (Cell Biolabs, San Diego, CA) according to the manufacturer's instructions.
Measurement of capillary density.
The frozen sections were stained for endothelial marker CD-31 using a primary antibody (BD Biosciences) and a FITC-conjugated secondary antibody, and viewed with a confocal laser microscope at a magnification ×600. For the quantitative measurement, the number of CD-31-positive cells was counted on the infarct border area from the endocardium through epicardium of the midportion of the LV free wall. Eight nonoverlapping random fields from four sections of each heart were examined. Counts of capillary density per squared millimeter were obtained after superimposing a calibrated morphometric grid on each digital image using Win Roof.
Measurement of the area of fibrosis.
The area of fibrosis was determined by a Masson trichrome staining method. The heart was cut in 2 mm thickness at the mid-LV level, fixed with 10% formalin, embedded in paraffin, and sectioned at a 6 μm thickness. The section was stained with Masson trichrome, and the gross morphology of the heart was viewed under a low power field (×0.5). The area of fibrosis was quantified by morphometric analysis as described previously (13).
Statistical analyses were conducted with a commercially available software package (StatView 5.0; SAS Institute, Cary, NC). Differences between the groups were assessed by one-way ANOVA followed by Tukey post hoc test. Two-way repeated-measures ANOVA was applied to compare serial measurements of variables. All numerical data are expressed as the means ± SE. The differences were considered to be significant at a P value < 0.05.
Both systolic and diastolic blood pressures were significantly higher in the eNOS−/− mice than the WT mice at baseline and 28 days after sham surgery (Table 1). In contrast, heart rate was significantly smaller in the eNOS−/− mice than the WT mice at baseline and after sham surgery. These hemodynamic differences are consistent with the previous study using the same genotypes of mice (24). Although heart rate and blood pressures did not significantly change after MI in the WT and iNOS−/− mice, blood pressures significantly decreased after MI in the eNOS−/− mice compared with baseline and the time-matched WT and eNOS−/− mice with sham surgery. Blood pressures also significantly decreased after MI in the nNOS−/− mice compared with baseline. Sepiapterin had no significant effects on blood pressures and heart rate in any genotypes of mice with sham surgery and MI except for the eNOS−/− mice, in which systolic blood pressure was restored by sepiapterin.
Body weight and heart weight.
In the sham-operated groups, heart weight (HW) was significantly higher in the eNOS−/− mice than the WT mice with sham surgery (Table 2). HW significantly increased in any genotypes of mice after MI without a significant intergroup difference. HW-to-BW ratio was significantly higher in any genotypes of mice with MI compared with sham surgery except for the iNOS−/− mice. HW-to-BW ratio was not significantly different between the groups 28 days after MI. Sepiapterin significantly decreased HW in any genotypes of mice with MI except for the iNOS−/− mice, although it did not significantly affect HW-to-BW ratio in any genotypes of mice with MI.
Immunohistochemical analysis demonstrated that iNOS expression markedly increased in the infarct border zone 28 days after MI in the WT, eNOS−/−, and nNOS−/− mice (Fig. 1A). iNOS expression was not observed in the remote myocardium (posterior wall) in the infarcted heart. iNOS appeared to be localized in the cell membrane of cardiomyocytes and endothelial cells. In contrast, eNOS was expressed in the sham-operated heart except for the eNOS−/− mice and localized predominantly in endothelial cells (Fig. 1C). eNOS expression did not change after MI in any genotypes of mouse hearts. nNOS was faintly expressed in the sham-operated heart and predominantly localized in endothelial cells (Fig. 1E). nNOS expression increased in the infarct border zone in any genotypes of mice except for the nNOS−/− mice.
Immunoblot analysis showed that iNOS expression was significantly increased 28 days after MI in any genotypes of mice compared with the sham-operated WT mice except for the iNOS−/− mice (Fig. 1B). There was no significant change in eNOS expression in the heart in any genotypes of mice after MI (Fig. 1D). nNOS expression was significantly increased in the heart after MI in the WT, iNOS−/−, and eNOS−/− mice without a significant intergroup difference (Fig. 1F). Sepiapterin did not significantly affect the increase in iNOS and nNOS expressions in the heart after MI in any genotypes of mice.
GTPCH-1 expression and myocardial biopterin content.
The expression of GTPCH-1, which is the rate limiting step for BH4 synthesis in the de novo pathway, was significantly reduced in the infarcted heart in any genotypes of mice (Fig. 2A), suggesting that the de novo pathway for BH4 synthesis is inactivated in the infarcted heart.
We measured myocardial BH2 and BH4 content and calculated BH4-to-BH2 ratio, which is a primary determinant of the coupling status of NOS (29). Myocardial BH2 content significantly increased 28 days after MI in any genotypes of mice, whereas BH4 content decreased (Fig. 2, B and C). This reciprocal change in myocardial BH2 and BH4 level was associated with a marked decrease in the BH4-to-BH2 ratio (Fig. 2D). Sepiapterin had an only a marginal effect on BH2 and BH4 content and BH4-to-BH2 ratio in the sham-operated heart in any genotypes of mice. However, sepiapterin significantly increased BH2 and more prominently BH4 content in the heart after MI, giving rise to a significant increase in the BH4-to-BH2 ratio. These results suggest that the salvage pathway for BH4 synthesis is activated in the infarcted heart.
Nitrotyrosine was formed from the reaction of protein tyrosine with peroxynitrite and has been measured as a marker of NOS uncoupling (39). Immunohistochemical analysis demonstrated that expression of nitrotyrosine was markedly increased in the infarct border zone and appeared to be localized in the cell membrane of cardiomyocytes and endothelial cells (Fig. 3A). There was no appreciable difference in the localization and intensity of nitrotyrosine expression in the heart after MI between the mice except for the iNOS−/− mice, which showed reduced expression of nitrotyrosine especially in cardiomyocytes. Sepiapterin inhibited the expression of nitrotyrosine in the infarct border zone in all genotypes of mice. Nitrotyrosine expression was not observed in the remote myocardium in the infarcted heart.
Quantitative analysis demonstrated that nitrotyrosine formation was significantly increased in the heart after MI in all genotypes of mice, but the magnitude of increase was significantly attenuated in the iNOS−/− mice (Fig. 3B). Sepiapterin inhibited the increase in nitrotyrosine formation in the heart without a significant intergroup difference.
There was no significant change in NOx generation in the infarcted myocardium in all genotypes of mice compared with the sham-operated mice (Fig. 4). Sepiapterin significantly increased NOx generation in the infarcted myocardium in all genotypes of mice, but the magnitude of increase was significantly attenuated in the iNOS−/− mice compared with other genotypes of mice.
Capillary density in the sham-operated heart was similar between any genotypes of mice (Fig. 5, A and B). Capillary density significantly decreased in the infarct border zone after MI in any genotypes of mice without a significant intergroup difference. Although sepiapterin did not affect capillary density in the sham-operated heart in any genotypes of mice, it significantly increased capillary density in the infarct border zone after MI in any genotypes of mice except for the iNOS−/− mice, in which the increase in capillary density by sepiapterin was significantly attenuated compared with the WT mice. Capillary density was similar between the remote myocardium in the infarcted heart and the comparable region in the sham-operated heart, and sepiapterin did not increase capillary density in the remote myocardium in these mice (not shown).
There was no significant difference in the area of fibrosis after MI between any genotypes of mice (Fig. 6). Sepiapterin significantly reduced the area of fibrosis in the WT, eNOS−/−, and nNOS−/− but not iNOS−/− mice. We could not find any differences in interstitial fibrosis between the remote myocardium and the comparable region of the sham-operated heart (not shown).
LVEDV significantly increased after MI in any genotypes of mice, especially in the eNOS−/− mice, which showed a more significant increase in LVEDV compared with the WT mice (Fig. 7A). Sepiapterin had no effect on LVEDV in the sham-operated heart in any genotypes of mice. However, sepiapterin significantly reduced LVEDV after MI in any genotypes of mice except for iNOS−/− mice. LVEF significantly decreased after MI in any genotypes of mice (Fig. 7B). Particularly, the eNOS−/− mice had lower LVEF after MI compared with the WT mice. Sepiapterin significantly improved LVEF after MI in any genotypes of mice except for iNOS−/− mice. LVEF remained significantly lower in the eNOS−/− mice treated with sepiapterin after MI compared with the WT mice.
Effect of l-NAME.
To confirm that the observed cardiac effect of sepiapterin was mediated by NOS, l-NAME was coadministered with sepiapterin to the WT mice. l-NAME had no significant effect on nitrotyrosine formation, NOx generation, and capillary density in the sham-operated heart (Fig. 8, A–C). However, l-NAME abolished the increase in NOx generation and capillary density and the decrease in the area of fibrosis induced by sepiapterin treatment (Fig. 8D). l-NAME did not significantly affect LVEDV and LVEF in the sham-operated mice (Fig. 8, E and F). However, l-NAME abolished the sepiapterin-induced decrease in LVEDV and the increase in LVEF after MI.
We investigated the role of NOS uncoupling in LV remodeling and dysfunction after MI in mice and the relative contribution of three major NOS isoforms to this pathogenesis. The salient findings of the present study are that 1) iNOS expression was markedly increased in cardiac cells including endothelial cells and cardiomyocytes after MI associated with an increase in nitrotyrosine formation, which was blunted in iNOS−/− mice; 2) the expression of GTPCH-1 was reduced in the infarcted heart; 3) oral administration of sepiapterin increased BH4 content and BH4-to-BH2 ratio in the infarcted but not sham-operated heart; 4) the increase in the BH4-to-BH2 ratio was associated with inhibition of NOS uncoupling as demonstrated by decreased nitrotyrosine formation and increased bioavailability of NO; 5) the inhibitory effect of sepiapterin on NOS uncoupling was attenuated in the iNOS−/− mice compared with other genotypes of mice; 6) sepiapterin increased capillary density, reduced the area of fibrosis, and improved LV remodeling and function after MI except for iNOS−/− mice; and 7) l-NAME inhibited nitrotyrosine formation and sepiapterin-induced increase in NOx generation and angiogenesis and cancelled the beneficial effect of sepiapterin on LV remodeling and function after MI. These results are consistent with the hypothesis that uncoupling of iNOS predominates over that of eNOS or nNOS in the heart after MI, and inhibition of uncoupling of NOS and an increase in NO, especially derived from iNOS, promotes angiogenesis and protects the heart from adverse LV remodeling after MI. This study also points to the efficacy of sepiapterin in increasing BH4 via the salvage pathway and inhibiting NOS uncoupling in the heart after MI.
We found that NO levels remained similar in the infarcted heart. We presume that upregulated iNOS maintained NO generation in the infarcted heart despite the occurrence of uncoupling. Unlike eNOS or nNOS, which are constitutively expressed in the heart under physiological conditions, iNOS expression is induced only under pathological conditions that impose oxidative stress on the heart (38, 39). However, care must be taken to evaluate the activity of NOSs based on the expression, because activity of eNOS and nNOS is regulated not only by expression but also by phosphorylation. Because the present study did not examine the phosphorylation of eNOS and nNOS, uncoupling of these constitutive NOSs may be over- or underestimated than we expected from the expression study.
The role of enhanced expression of iNOS in the infarcted heart remains controversial. Feng et al. (14) demonstrated that increased iNOS expression contributes to myocardial dysfunction after MI in mice. The detrimental effect of iNOS on the infarcted heart has also been suggested by the experiments using the iNOS−/− mice, which showed reduced apoptotic cell death and LV remodeling after MI (33, 44). On the contrary, it has been demonstrated that adverse LV remodeling and congestive heart failure were not attenuated in the iNOS−/− mice (27). The latter findings are similar to those obtained by our study demonstrating that the area of fibrosis and the extent of LV remodeling were comparable between the WT and iNOS−/− mice. Although the mechanism for such discordant observations using iNOS−/− mice remains to be clarified, the present study demonstrating that sepiapterin reduced infarct size and improved LV remodeling and function after MI in iNOS−/− mice suggests that the difference in the coupling status of iNOS seems to determine whether iNOS plays a beneficial or detrimental role in the infarcted heart.
eNOS plays a pivotal role in maintaining cardiovascular homeostasis under physiological conditions, and the deficiency of eNOS is known to cause a variety of cardiovascular disorders. The present study confirmed higher blood pressure in eNOS−/− mice compared with other genotypes of mice at baseline. The present study also demonstrated that adverse LV remodeling and LV dysfunction was exaggerated in the eNOS−/− mice compared with other genotypes of mice as has been demonstrated by Scherrer-Crosbie et al. (46). In contrast with the effect on iNOS−/− mice, sepiapterin significantly improved LV remodeling and LV dysfunction after MI in the eNOS−/− mice. However, the beneficial effect of sepiapterin on LV function was blunted in these mice compared with the WT mice. Such a limiting beneficial effect of sepiapterin was observed despite similar inhibition of oxidative/nitrosative stress and increase in the bioavailability of NO and angiogenesis. These findings suggest that recoupling of iNOS and/or nNOS did not fully compensate the detrimental effect of the absence of eNOS on LV remodeling and dysfunction after MI, and eNOS still exerted a beneficial effect on the infarcted heart even with uncoupling.
When compared with eNOS and iNOS, the role of nNOS in the pathophysiology of LV remodeling and dysfunction after MI is even more obscure. However, the recent experimental studies point to an important role of nNOS in the regulation of basal and β-adrenergic cardiac function (8, 9, 31). The present study demonstrated that nNOS expression was significantly increased after MI. This finding is consistent with previous studies demonstrating that MI induces upregulation of nNOS (4, 10, 11). Although nNOS is known to be localized in the sarcoplasmic reticulum (57) and plays an important role in fine-tuning the myocardial response to stress and β-adrenergic stimulation (35, 47), the present study suggests that nNOS is predominantly localized in the cell membrane of endothelial cells. It is possible that our immunohistochemical technique may not identify nNOS localized in the sarcoplasmic reticulum in cardiomyocytes. Because nNOS gene deletion was associated with more severe LV remodeling, functional deterioration, and ventricular arrhythmias in the mouse model of MI, nNOS overexpression in the infarcted heart has been suggested to be an adaptive mechanism to prevent adverse LV remodeling and functional deterioration by maintaining intracellular Ca2+ homeostasis and increasing β-adrenergic reserve (7, 11). Although the present study did not reveal accelerated LV remodeling after MI in the nNOS−/− mice compared with the WT mice, it may be necessary to employ more sophisticated techniques to detect a subtle difference in cardiac function after MI between the nNOS−/− and WT mice. On the other hand, we found that sepiapterin significantly attenuated nitrotyrosine formation and increased NOx generation and capillary density after MI in the nNOS−/− mice associated with the improvement of LV remodeling and function to the degree comparable with the WT mice, indicating that inhibition of iNOS and/or eNOS uncoupling may compensate the absence of nNOS after MI. The exact localization and the role of nNOS uncoupling in the infarcted heart remain to be investigated.
The present study demonstrated that BH4 content decreased in the heart after MI associated with a reciprocal increase in BH2, giving rise to a marked decrease in the BH4-to-BH2 ratio. Such a change in BH4 and BH2 after MI was similarly observed in any genotypes of mice. Interestingly, sepiapterin administration increased BH2 and more prominently BH4 only in the infarcted but not sham-operated heart. Consequently, the BH4-to-BH2 ratio significantly increased in the infarcted heart in any genotypes of mice receiving sepiapterin. Such an increase in BH2 and BH4 in the infarcted heart by administration of sepiapterin suggests that the salvage pathway for BH4 synthesis through sepiapterin reductase and dihydrofolate reductase is activated in the infarcted heart. Intracellular BH4 levels are regulated by the activity of the de novo biosynthetic pathway and the salvage pathway. In the de novo biosynthetic pathway, GTPCH-1 catalyzes GTP to dihydroneopterin triphosphate. BH4 is generated by further steps catalyzed by 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase (52). GTPCH-1 appears to be the rate-limiting enzyme in BH4 biosynthesis in the de novo pathway. However, we provided the evidence for the first time that the expression of GTPCH-1 is attenuated in the infarcted heart as has been demonstrated in endothelial cells exposed to hyperglycemia (54, 56). Thus BH4 synthesis via the de novo pathway is inhibited, and alternative means to supply BH4 may be required to increase the BH4-to-BH2 ratio and inhibit uncoupling of NOS in the infarcted heart. However, exogenous BH4 is labile in physiological solution, and in vivo half-life of BH4 is 3.3–5.1 h in the plasma of healthy adult humans (15). Therefore, oral administration of sepiapterin may represent a promising approach to increase BH4-to-BH2 ratio and inhibit NOS uncoupling in the heart after MI.
The mechanism by which NO increases angiogenesis in the infarcted heart remains elusive. The present study demonstrated that not only eNOS but also iNOS and nNOS were localized in endothelial cells in the infarcted heart, suggesting that NO derived from endothelial cells may play a crucial role in promoting angiogenesis. It has been demonstrated that NO potentiates angiogenesis in the infarcted myocardium through VEGF/Flt-1 signaling (40, 51), and this mechanism may be mediated by S-nitrosylation of hypoxia inducible factor-1α, which is an oxygen-sensitive transcriptional factor essential for expression of VEGF (32). In addition, NO may increase mobilization of endothelial progenitor cells from the bone marrow and their recruitment to the heart after MI (30), suggesting a critical role for restored NO production in endothelial progenitor cells mobilization and neovascularization after MI. On the other hand, the beneficial effect of NO in inhibiting LV remodeling may at least in part be mediated by the NO-dependent guanylyl cyclase/cGMP system. It has been demonstrated that genetically increased synthesis of cGMP inhibits pressure load-induced pathological remodeling (49). Moreover, the phosphodiesterase-5 inhibitor sildenafil prevented cardiac hypertrophy and ameliorated heart failure through enhanced generation of cGMP (43). Signaling mechanisms involved in NO-mediated angiogenesis in the infarcted heart remain to be investigated.
In conclusion, oral administration of sepiapterin increases BH4 content and BH4-to-BH2 ratio in the infarcted heart and inhibits uncoupling of NOS, leading to the increase in NO especially derived from iNOS. This increase in the bioavailability of NO enhances angiogenesis and improves LV remodeling and function in the heart after MI. Sepiapterin, therefore, may represent a potential therapeutic tool for prevention of post-MI LV remodeling and dysfunction.
This study was supported in part by a Research Grant (No. 20590847) from the Ministry of Education, Science, and Culture of Japan.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: T. S., K. Y., M. F., T. O., and T. I. performed experiments; H. O. was involved in conception and design of research.
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