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(Circulation. 2003;107:1598.)
© 2003 American Heart Association, Inc.

Clinical Investigation and Reports

Effect of the Asp²⁹⁸ Variant of Endothelial Nitric Oxide Synthase on Survival for Patients With Congestive Heart Failure

Dennis M. McNamara, MD; Richard Holubkov, PhD; Lisa Postava, MBA; Ravi Ramani, MD; Karen Janosko, MSN; Michael Mathier, MD; Guy A. MacGowan, MD; Srinivas Murali, MD; Arthur M. Feldman, MD, PhD; Barry London, MD, PhD

From the Cardiovascular Institute, University of Pittsburgh Medical Center (D.M.M., L.P., R.R., K.J., M.M., G.A.M., S.M., B.L.), Pittsburgh, Pa; the Department of Family and Preventive Medicine, School of Medicine, University of Utah (R.H.), Salt Lake City, Utah; and the Department of Medicine, Thomas Jefferson Medical Center (A.M.F.), Philadelphia, Pa.

Correspondence to Dennis M. McNamara, MD, Heart Failure Section, Cardiovascular Institute, University of Pittsburgh Medical Center, 558 Scaife Hall, 200 Lothrop St, Pittsburgh, PA 15213. E-mail mcnamaradm{at}msx.upmc.edu

	Abstract

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Background— Significant variation exists within the endothelial nitric oxide synthase (NOS3) gene that may influence cardiovascular risk. The Asp²⁹⁸ variant of NOS3 has a shorter half-life in endothelial cells. Given the importance of nitric oxide in the heart failure syndrome, we evaluated the effect of this variant on event-free survival in a population with systolic dysfunction.

Methods and Results— Four hundred sixty-nine patients (72% male, 49% ischemic; mean age, 56±12 years) with systolic dysfunction (left ventricular ejection fraction 0.45) were enrolled in a study of Genetic Risk Assessment of Cardiac Events (GRACE). The polymorphism in exon 7 of NOS3, a G-T transition at position 894 that results in a Glu to Asp amino acid substitution for codon 298, was genotyped and subjects were followed prospectively to the end point of death or heart transplantation. Event-free survival was compared on the basis of the presence (group 1, n=266) or absence (group 2, n=203) of the Asp²⁹⁸ variant. Event-free survival was significantly poorer in patients with the Asp²⁹⁸ variant (percent event-free survival group 1 at 1/2/3 years=78/65/54; group 2=82/72/64, P=0.03). In subset analysis, the adverse impact of the Asp²⁹⁸ variant was primarily in patients with nonischemic cardiomyopathy (group 1=82/73/63; group 2=87/79/71, P=0.03) and was not apparent among patients with ischemic heart disease (group 1=75/59/47; group 2=74/62/54, P=0.71).

Conclusions— For patients with heart failure caused by systolic function, the Asp²⁹⁸ variant of NOS3 is associated with poorer event-free survival, particularly in patients with nonischemic cardiomyopathy.

Key Words: heart failure • cardiomyopathy • nitric oxide synthase • genetics • survival

	Introduction

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Nitric oxide (NO) is a ubiquitous molecule that plays an important role in the modulation of cardiovascular physiology. NO is synthesized from L-arginine by a family of three enzymes, the nitric oxide synthases (NOS).¹ Endothelial nitric oxide synthase (NOS3) and the neuronal isoform (NOS1) are constitutive enzymes that produce low levels of basal NO production in a calcium-dependent fashion. In contrast, inducible nitric oxide synthase (NOS2, primarily found in macrophages) produces high levels of NO production when given appropriate physiological stimuli in a calcium-independent fashion. All three isoforms are present in the heart, NOS1 in sympathetic nerve endings, NOS3 in vascular endothelium and the endocardium, and NOS2 in activated macrophages and myocytes.

NO plays an important role in the pathophysiology of several cardiac disease states, including heart failure.^2,3 The heart failure syndrome is characterized by an increase in peripheral vascular resistance, which both limits cardiac output and increases myocardial workload. NO release results in relaxation of smooth muscle cells through activation of guanylyl cyclase and modulates both peripheral vascular tone and coronary blood flow. Clinical investigations in subjects with heart failure demonstrate a decrease in the L-arginine-NO metabolic pathway⁴ and abnormal vascular response to NOS inhibitors,⁵ suggesting that impairment of NOS activity may underlie endothelial dysfunction seen in this disorder and contribute to disease progression.

NO is also an important regulator of myocardial contractility.⁶ TNF administration to myocytes induces expression of NOS2,⁷ which mediates its cardiodepressant effects. NO diminishes the effect of ß-agonist stimulation on contractility,^8,9 potentially limiting the impact of sympathetic activation on disease progression. Given the effects on adrenergic response and the peripheral vasculature, an increase in NOS activity and NO production can be postulated to have a cardioprotective effect in heart failure. Pharmacological or genetic reductions in NOS activity would be predicted to have an adverse impact on heart failure progression.

NOS3 is encoded by a 26-exon gene located on chromosome 7.¹⁰ A common polymorphism exists in nucleotide 894 (G-T) that results in the conversion of glutamate to aspartate for codon 298. The Asp²⁹⁸ variant has been shown to have a shorter half-life in endothelial cell culture as the result of increased enzymatic cleavage.¹¹ In addition, clinical studies have demonstrated that vascular responsiveness is altered in subjects with this variant, as Asp²⁹⁸ patients have an increased vasoconstrictive response to phenylephrine¹² consistent with decreased NOS activity. The Asp²⁹⁸ variant has been in implicated as a risk factor for both hypertension^13,14 and coronary disease,^15,16 although conflicting reports exist.^17–19 Previous studies of this variant in cardiovascular disease have been predominantly case-control–designed, and few studies have evaluated the effect of this polymorphism on disease progression.

To evaluate the importance of genetic heterogeneity of NOS3 in heart failure progression, we investigated the impact of the Asp²⁹⁸ variant on event-free survival in a population of patients with systolic dysfunction. We postulated that NO plays a cardioprotective role in limiting heart failure progression and therefore predicted that the Asp²⁹⁸ variant of NOS3 associated with diminished activity would adversely affect heart failure outcomes.

	Methods

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Study Population
This study was approved by the Institutional Review Board of the University of Pittsburgh Medical Center. A series of 469 patients with heart failure caused by systolic dysfunction referred to the Cardiomyopathy Clinic at the University of Pittsburgh Medical Center were enrolled in a study of Genetic Risk Assessment of Cardiac Events (GRACE) between April 1996 and January 2001. Informed consent was obtained and peripheral blood was drawn for DNA isolation and genotyping. At the time of study entry, demographic information, New York Heart Association class, previous cardiovascular evaluation and medical therapy were recorded. Patients were prospectively followed up to an end point of either death or heart transplantation. Medical personnel participating in treatment and follow-up were blinded to the genotype status of individual subjects.

In all patients, the most recent clinical assessment demonstrated left ventricular systolic dysfunction, defined as left ventricular ejection fraction (LVEF) 0.45 (n=439) or qualitative assessment documenting moderate to severe left ventricular dysfunction (n=30). Left ventricular systolic function was estimated by radionuclide scan in 174 patients (37.1%), left ventricular angiography in 25 (5.3%), and by echocardiography in 270 (57.6%). All patients enrolled had previously undergone diagnostic evaluation for coronary artery disease that consisted of coronary angiography in the vast majority (>90%) and noninvasive assessment in the remainder. Patients with angiographic evidence of coronary disease (defined as >50% stenosis of a major epicardial coronary artery) or a noninvasive assessment positive for ischemia or previous myocardial infarction were classified as ischemic. No differences in the extent of testing were apparent between groups.

Genotyping of the NOS III Polymorphism
Genomic DNA was extracted from peripheral blood with the use of a Puregene Kit (Gentra Systems Inc). Primers 5' AAG GCA GGA GAC AGT GGA TGG A-3' and 5' CCC AGT CAA TCC CTT TGG TGC TCA-3' were used to amplify a 248-bp DNA fragment from exon 7 including the G/T polymorphism in codon 298. Polymerase chain reactions were run for 35 cycles: 94°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute. The product (20 µL) was digested with 3 U Ban II, which cuts the G (Glu²⁹⁸ but not the T allele (Asp²⁹⁸, at 37° for >4 hours, then subjected to gel electrophoresis for genotyping. The NOS3 G allele gives two fragments of 163 and 85 base pairs, and the NOS3 T allele yields a single 248-base pair fragment.

Statistical Analysis
Continuous variables are presented as mean±SD. Subjects were grouped on the basis of presence or absence of the Asp²⁹⁸ variant (Asp²⁹⁸ homozygotes and heterozygotes were combined and compared with Glu²⁹⁸ homozygotes). Distributions of continuous variables were compared between two groups by use of the Mann-Whitney test; distributions of categoric variables were compared by genotype status using Pearson’s ² (unordered variables) or Mantel-Haentzel ² (ordered categoric variables). For outcome analysis, Kaplan-Meier freedom from event curves were computed for subgroups, and these curves were compared between subgroups by using the log rank test. Cox regression analysis was used to quantify the relative risk of an event over time by genotype class; reported probability values for relative risk were calculated by means of the Wald test.

To evaluate whether the Asp²⁹⁸ variant effect on heart failure progression was influenced by the cause of heart failure, outcome analysis was repeated separately in ischemic and nonischemic subsets. This subset analysis sought to investigate whether the impact of NOS3 variation was specific to ischemic cardiomyopathy or produced a general modulation of the heart failure syndrome, which would be evident in nonischemics and ischemics.

	Results

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Baseline Demographics
The mean age of the cohort was 56±12 years (range, 20 to 84); 72% percent were male, 49% were ischemic, and 90% were white. Ninety-five percent of patients were treated with either an ACE inhibitor or an angiotensin receptor blocker, and 42% were taking ß-blockers. Demographics and baseline medical therapy are listed by genotype and overall in Table 1. Of note, patients with the Asp variant were slightly older (57.0±12.3 versus 53.9±11.6, P=0.004).

View this table: [in this window] [in a new window]   TABLE 1. Demographics and Medical Therapy by Genotype

The prevalence of the Asp²⁹⁸ variant was significantly higher in patients with an ischemic cardiomyopathy when compared with those who were nonischemic (ischemic: 14% Asp²⁹⁸ homozygotes, 50% heterozygotes, 36% Glu²⁹⁸homozygotes; nonischemic: 12% Asp²⁹⁸ homozygotes, 38% heterozygotes, 50% Glu²⁹⁸ homozygotes; P=0.008 for Mantel-Haentzel ²). In addition, and consistent with previous reports,²⁰ significant racial differences were seen in the frequency of the Asp²⁹⁸ variant, with a much lower prevalence noted in nonwhite patients (nonwhites: 2% Asp²⁹⁸homozygotes, 31% heterozygotes, 67% Glu²⁹⁸ homozygotes; whites: 14% Asp²⁹⁸ homozygotes, 45% heterozygotes, 41% Glu²⁹⁸ homozygotes; P<0.001 for Mantel-Haentzel ²). Testing for Hardy-Weinberg equilibrium demonstrated no statistical evidence of disequilibrium overall (prevalence of Asp²⁹⁸ allele 35%, ², 0.47, P=0.50) nor by ischemic strata (ischemic: 39%, 0.58, P=0.45; nonischemic: 31%, 2.55, P=0.11), or by race (white: 37%, 0.24, P=0.62; nonwhite: 18%, 0.19, P=0.67).

Clinical Characteristics
Comparisons of LVEF, baseline heart rate and blood pressure demonstrated no significant differences by genotype class (Table 2). In patients who underwent functional assessment by metabolic stress testing (n=198), the presence of Asp²⁹⁸ was associated with a poor functional capacity (O₂ max equal to 15.9±5.5 versus 17.7±4.8 mL/kg per minute, P=0.006). In a similar fashion, there was a trend toward higher NYHA class in patients with the Asp²⁹⁸ variant that failed to reach significance (P=0.07).

View this table: [in this window] [in a new window]   TABLE 2. Clinical Characteristics by Genotype

Outcomes: Event-Free Survival
The median follow-up time for all patients was 22 months; for patients alive and transplant-free at last follow-up, median follow-up was 33 months (range, 3 to 62 months). During the course of follow-up there were 192 events, including 134 deaths and 58 transplants (Table 3). For the entire cohort, the presence of the Asp²⁹⁸ variant was associated with poor transplant-free survival (1/2/3 year percent transplant-free survival group 1=78/65/54; group 2=82/72/64; P=0.027, Figure, A). Corresponding Cox regression analysis for patients with the Asp²⁹⁸ variant revealed an increased relative risk compared with Glu homozygotes (RR, 1.39; 95% CI, 1.03 to 1.86; P=0.029). Adjusting for race in the overall model, the risk remains significant for the Asp²⁹⁸ variant (race-adjusted RR=1.36; 95% CI, 1.01 to 1.83, P=0.042).

View this table: [in this window] [in a new window]   TABLE 3. Study Events by Genotype

View larger version (22K): [in this window] [in a new window]   Event-free survival by NOS3 codon 298 polymorphism. Solid line indicates subjects with Asp298 variant; dashed line, Glu298 homozygotes. A, All subjects (n=469): Asp298 variant (n=266), Glu298 homozygotes (n=203). Event-free survival was significantly poorer in subjects with the Asp298 variant, P=0.03. B, Subjects with nonischemic cardiomyopathy (n=241): Asp298 variant (n=120), Glu298 homozygotes (n=121). Event-free survival remains significantly poorer in subjects with the Asp298 variant, P=0.03. C, Subjects with ischemic cardiomyopathy (n=228): Asp298 variant (n=146), Glu298 homozygotes (n=82). Event-free survival was similar in both groups, P=0.71.

When analysis was limited to subjects with an LVEF 0.35 (n=393), the Asp²⁹⁸ variant still increased the relative risk of events compared with Glu homozygotes (RR=1.44; 95% CI, 1.05 to 1.96; P=0.023). The increase in relative risk with this variant appeared stronger when analysis was limited to subjects with an LVEF 0.30 (n=344, RR=1.50; CI, 1.08 to 2.09; P=0.017). Adjusting for O₂, NYHA class and LVEF in the subset of patients with O₂ max (n=185 with LVEF) does not diminish the relative risk of the Asp²⁹⁸ variant (RR=1.64; CI, 0.98 to 2.73; P=0.057); however, the significance is reduced as a result of loss in study number.

Overall event-free survival was substantially poorer for patients with ischemic cardiomyopathy than for nonischemics (1/2/3 year percent transplant-free survival nonischemics=85/76/67; ischemics=74/60/49; P<0.001). Subset analysis revealed the impact of the Asp²⁹⁸ variant on survival was primarily evident in patients with nonischemic cardiomyopathy (n=241). For nonischemics, the event-free survival remained significantly poorer with the Asp²⁹⁸ variant (group 1) when compared with Glu homozygotes (1/2/3 year percent transplant-free survival group 1=82/73/63; group 2=87/79/71; P=0.028, Figure, B). In contrast, the adverse effect of Asp²⁹⁸on event-free survival was not apparent for patients with ischemic cardiomyopathy (n=228, 1/2/3 year event-free survival group 1=75/59/47; group 2=74/62/54; P=0.71, Figure, C).

Cox regression analysis in the nonischemic subgroup of patients revealed a relative risk with the Asp variant of 1.65 (95% CI, 1.05 to 2.60, P=0.03). Whereas patients with the Asp variant were somewhat older than other nonischemics (53±14 versus 50±12 years, P=0.15), adjustment for age and race had no effect on this increased relative risk (adjusted RR=1.70, 95% CI=1.06 to 2.74, P=0.027).

	Discussion

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In this study of patients with systolic dysfunction, the presence of the NOS3 Asp²⁹⁸ variant was associated with poorer event-free survival, particularly in those with nonischemic cardiomyopathy. In addition, this variant appeared to alter the severity of the heart failure phenotype, as subjects with this variant was had a significantly lower O₂ max and a trend toward higher NYHA class. The findings of the current study suggests genetic variation in NOS3 alters the rate of heart failure progression and emphasizes the importance of NO as a modulator of this clinical syndrome.

Plasma NO metabolites are increased in patients with heart failure in proportion to BNP levels,²¹ suggesting a close relation to the functional severity of disease. Myocardial expression of NOS is increased in subjects with heart failure, although the relative contribution of NOS3 and NOS2 remains uncertain.² Initial studies of gene expression from endomyocardial biopsy specimens demonstrated upregulation in vivo of inducible NOS2 in the myocardium of patients with idiopathic dilated cardiomyopathy, peripartum disease, and postmyocarditis but not in those with ischemic causes of heart failure.²² In contrast, Stein and associates²³ reported upregulation of NOS3 expression in patients with heart failure with both ischemic and nonischemic causes but reported no alterations of NOS2. These investigations demonstrate that an increase in NO production is an important element of heart failure syndrome, and the impact of the Asp²⁹⁸ variant in the current study supports a significant regulatory role for NOS3.

In a murine infarct model, NOS3-deficient mice (NOS3^-/-) have increased LV dilation, hypertrophy, and mortality rates when compared with littermate controls,²⁴ suggesting the activity of NOS3 diminishes LV remodeling in response to injury. In a similar model, treatment with ACE inhibitors in wild-type mice diminished infarct size but had no impact in NOS3^-/- mice.²⁵ In a murine model in which exercise enhances NOS3 expression, heterozygous NOS3^-/+ mice had normal levels under basal conditions but failed to increase activity during exercise.²⁶ This exercise model demonstrates that one absent or defective NOS3 allele can affect NO production under stress. This current clinical study and the murine models both support the hypothesis that increased NOS3 activity helps to limit heart failure development and that deficiencies of NOS activation may hasten progression.

The prevalence of the NOS3 Asp²⁹⁸ was significantly higher in ischemics than nonischemics, consistent with previous reports suggesting this variant is a risk factor for coronary disease.^15,16 Within the ischemic subset, the NOS3 Asp²⁹⁸ variant did not appear to influence event-free survival. However, overall outcomes were markedly worse for patients with ischemic cardiomyopathy than for those with nonischemic causes, with an event rate exceeding 50% by 3 years. In this high-risk subset with ischemic disease, the risk of death as the result of reinfarction or ischemia may diminish the impact of NOS3 heterogeneity on heart failure progression and subsequent outcomes. Indeed, the increased prevalence of the Asp²⁹⁸ variant suggests a potential pathogenic role of NOS3 variation in subjects with ischemic cardiomyopathy that warrants further study, despite the apparent absence of an outcomes effect in the current analysis.

Importantly, the influence of the Asp²⁹⁸ variant on outcomes in the current study was evident in the nonischemic population, suggesting that this NOS3 variant modulates heart failure progression and not simply development of coronary pathology. This may represent a primary endothelial effect; as in nonischemic cardiomyopathy, preserved endothelial function has been associated with improved clinical outcomes,²⁷ and exacerbation of endothelial dysfunction may underlie the adverse impact of the Asp²⁹⁸ variant in this subset. Alternatively, given the modulation of adrenergic signaling by NO,^8,9 the impact of the Asp²⁹⁸ variant in nonischemic outcomes may reflect a primary influence on cardiomyocyte function.

This study demonstrates that for patients with heart failure, genetic variation of NOS3 contributes to the observed heterogeneity of clinical outcomes. In particular, this investigation suggests a potential role of the NOS3 Asp²⁹⁸ variant as a predictor of outcomes for subjects with nonischemic cardiomyopathy that requires additional study. Although these findings support the hypothesis that NOS3 plays a critical role in the modulation the heart failure phenotype, NOS2 is also involved in the pathogenesis of this complex syndrome. Future studies addressing functional variation at both genetic loci may help to clarify their interactions and relative contributions in mediating heart failure progression.

	Acknowledgments

 
This study was supported in part by grants from the National Heart, Lung, and Blood Institute: contracts HL-62300 (Dr London) and HL-69912 (Dr McNamara). The authors gratefully acknowledge the assistance of Marge Altvater in the preparation of the manuscript.

Received October 1, 2002; revision received January 10, 2003; accepted January 14, 2003.

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This Article Abstract Full Text (PDF) All Versions of this Article: 107/12/1598    most recent 01.CIR.0000060540.93836.AAv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McNamara, D. M. Articles by London, B. Search for Related Content PubMed PubMed Citation Articles by McNamara, D. M. Articles by London, B. Related Collections Clinical genetics Congestive