(Hypertension. 2000;36:885.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
From the Pennington Biomedical Research Center (T. Rankinen, C.B.), Human Genomics Laboratory, Baton Rouge, La; Division of Biostatistics (T. Rice, D.C.R.), Washington University Medical School, St Louis, Mo; Physical Activity Sciences Laboratory (L.P., Y.C.C., J.G.) and Laboratory of Molecular Endocrinology (J.G.), Laval University, Ste-Foy, Québec, Canada; School of Kinesiology and Leisure Studies (A.S.L.), University of Minnesota (Minneapolis); Department of Kinesiology (J.S.S.), Indiana University, Bloomington, Ind; Department of Health and Kinesiology (J.H.W.), Texas A&M University, College Station, Tex; and Departments of Genetics and Psychiatry (D.C.R.), Washington University Medical School, St Louis, Mo.
Correspondence to Tuomo Rankinen, PhD, Pennington Biomedical Research Center, Human Genomics Laboratory, 6400 Perkins Rd, Baton Rouge, LA 70808-4124. E-mail rankint{at}pbrc.edu
| Abstract |
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Key Words: exercise genetics blood pressure endothelial-derived factor nitric oxide
| Introduction |
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The BP-lowering effect of endurance training in hypertensive subjects is well documented.7 8 Although the precise mechanisms are unknown, changes in vascular structure and function, and thereby in peripheral resistance, could be involved.9 10 These changes are at least in part mediated by endothelial NO production. A single bout of exercise has been shown to enhance NOS activity and NO production,11 and this increase seems to contribute to vasodilation during steady-state exercise.12 13 In addition to acute responses, endurance training has been reported to increase NOS3 gene expression in coronary resistance arteries and to enhance basal NO production.14 15 16 This effect seems to be essential for the beneficial effects of endurance training on BP in both normotensive and hypertensive subjects,17 18 as well as in patients with chronic heart failure.19
Both the physiological role of NOS3 in the regulation of endothelial function and BP and the potential of physical exercise to enhance NO synthesis in vascular endothelium make NOS3 an attractive candidate gene for exercise hemodynamic phenotypes. Thus, the purpose of the present study was to investigate the associations between the NOS3 Glu298Asp polymorphism and endurance traininginduced changes in resting and submaximal exercise BP in normotensive, sedentary white subjects of the HERITAGE Family Study.
| Methods |
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Exercise Training Program
The details of the 20-week endurance training program have been
published elsewhere.20 21 Briefly, during the first 2
weeks, training was carried out at a heart rate (HR) that corresponds
to 55% of the baseline
O2max
for 30 minutes per session. Duration and intensity of the training
sessions were gradually increased to 50 minutes and 75% of the HR
associated with baseline
O2max, which were then
sustained for the last 6 weeks. The average training frequency was 3
times per week, and all training was performed on cycle ergometers
under supervision in the laboratory. HR was monitored during all
training sessions with a computerized cycle ergometer system (Universal
FitNet System), which adjusted the ergometer resistance to maintain
target HR.
Hemodynamic Phenotypes
All BP phenotypes were measured using Colin STBP-780
automated units, and recordings were confirmed by technicians
wearing headphones. Resting BP was measured on 2 separate days before
11 AM in the postabsorptive state. Subjects were asked not
to use any caffeine-containing or tobacco products for 2 hours
before measurements were made. Measurements were taken in a quiet room
at a neutral ambient temperature (24° to 26°C) with the lights
dimmed. Subjects rested for 5 minutes before the initial measurement in
a reclining chair with legs slightly elevated and back support reclined
at
45° from the ground. After the rest period,
4 BP readings
were taken at 2-minute intervals between measurements. The first
recording was automatically discarded, and 3 valid measurements
were kept. SBP and DBP were defined as the mean of all valid readings
taken on both days (ie, a maximum of 6).
Submaximal exercise BP was measured during 2 cycle ergometer tests after 8 to 12 minutes at a constant power output (50 W) in relative steady state, both before and after a 20-week endurance training program. BP was recorded twice during each test, and the mean of 4 readings was used for analyses. Steady-state HR was recorded with ECG. Rate-pressure product (RPP), an index of myocardial workload, was calculated by multiplying SBP by HR. Cardiac output (Q) was determined twice at 50 W with the Collier CO2 rebreathing technique,22 as described by Wilmore et al.23 A mean of the 2 measurements was used for the analyses. SV was calculated by dividing Q by HR.
In summary, the following hemodynamic phenotypes were available for the analyses: SBP, DBP, and RPP at rest and during steady-state submaximal exercise at 50 W (SBP50, DBP50 and RPP50, respectively) and Q and SV during submaximal exercise (SV50 and Q50).
Other Phenotypes
Stature was measured to the nearest 0.1 cm with the subject
standing erect on a flat surface; the heels, buttocks, and back pressed
against the stadiometer; and the head positioned in the Frankfort
horizontal plane. Body mass was recorded to the nearest 100 g
with a balance scale with subjects clothed in only a lightweight
bathing suit. Body mass index (BMI) was calculated by dividing body
mass (kg) by stature squared (m2). Body surface
area (BSA) was obtained from the following equation: BSA=weight
(kg)0.5378xheight
(cm)0.3964x 0.024265.24
Genotype Determinations
Genomic DNA was prepared from permanent lymphoblastoid cells
according to the proteinase K and phenol/chloroform technique. DNA was
dialyzed 4 times against 10 mmol/L Tris1 mmol/L EDTA (pH
8.0) buffer for 6 hours at 4°C and ethanol precipitated.
The Glu298Asp polymorphism of the NOS3 gene was typed with PCR, followed by digestion with BanII, as previously described.25 The PCR was performed in standard buffer (QIAGEN Inc), and each 20-µL PCR contained 100 ng genomic DNA, 0.2 µmol/L concentration of each primer, 200 µmol/L concentration of each dNTPs, and 0.5 U Taq polymerase (QIAGEN Inc). The reactions were incubated at 94°C for 3 minutes, 60°C for 1 minute, and 72°C for 1 minute, followed by 35 cycles of 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 45 seconds, and finally 1 cycle of 72°C for 10 minutes (model 9600 thermal cycler; Perkin-Elmer Cetus). The PCR product was digested with 5 U BanII (New England Biolabs) at 37°C for 4 hours. The resulting fragments were separated on 2.5% agarose gel and visualized under UV light after ethidium bromide staining.
Statistical Analyses
A
2 test was used to confirm that
the observed genotype frequencies were in a Hardy-Weinberg
equilibrium. Normality of the distributions was checked with the
Shapiro-Wilk statistic of the UNIVARIATE procedure of the SAS
statistical software package (SAS Institute Inc). The associations
between the NOS3 Glu298Asp polymorphism and
hemodynamic phenotypes were tested with ANCOVA
with the GLM procedure. Baseline BP phenotypes were adjusted
for age, gender, and BMI. and BP training response phenotypes,
obtained as the difference between pretraining and posttraining values,
were adjusted for age, gender, baseline BMI, and baseline value of the
BP phenotype. In addition, we tested the effects of
training-induced changes in BMI (
BMI) on the associations between
the NOS3 genotype and BP training responses. However, because
BMI showed either no (exercise BP) or only weak (resting BP)
correlations with BP responses and because baseline BMI was usually a
stronger predictor of BP training responses, the final GLM models
included only baseline BMI. Gender-specific associations between the
genotype and hemodynamic phenotypes
were tested by adding a genderxgenotype interaction term into
the GLM model. However, no significant interactions were observed, and
therefore the analyses were performed with the entire cohort
and with gender as a covariate. Values are given as mean and SEM.
| Results |
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None of the hemodynamic phenotypes measured in the sedentary state were associated with the NOS3 Glu298Asp polymorphism (Tables 2 and 3). However, DBP50 training response showed a highly significant (P=0.0005) association with the NOS3 genotype. The homozygotes for the Glu298 allele showed a 3.1 mm Hg greater reduction in DBP50 than the Asp298 homozygotes, whereas the heterozygotes showed an intermediate response (Table 3). The association was similar in men and women (data not shown) and was independent of age, baseline BMI, and initial DBP50 level. The NOS3 genotype explained 2.3% of the variance in DBP50 training response. The Glu298 homozygotes also showed a greater reduction in SBP50 than did the Asp/Asp genotype. However, the association (P=0.004) became nonsignificant (P=0.090) after adjustment for the baseline SBP50 values, which tended to be higher in the subjects carrying the Glu/Glu genotype. Both the Glu298 homozygotes and the heterozygotes had a greater training-induced reduction in RPP50 than the Asp298 homozygotes. Training responses in submaximal exercise SV and Q were not associated with the NOS3 genotype.
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| Discussion |
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Both linkage and association studies have shown that DNA sequence
variation at the NOS3 locus contributes significantly to the plasma
levels of NO metabolites.33 34 However, there are no data
available on the associations between the Glu298Asp polymorphism
and plasma NO metabolite levels. Moreover, the biological significance
of the Asp-to-Glu substitution in codon 298 of the NOS3 gene locus is
still unclear. The codon is located within the amino-terminal
oxygenase domain of the endothelial NOS,
which includes the binding sites for heme, tetrahydrobiopterin, and
L-arginine. The codon 298 falls between the critical
residues of the heme domain (
100 to 200) and the binding sites for
L-arginine and tetrahydrobiopterin (
350 to
450).35 Whether the Glu298Asp variant has any effect on
the binding properties or other functions of the oxygenase
domain or whether it is in linkage disequilibrium with another
functional mutation remains to be explored in future studies.
Our data suggest that the Glu298Asp polymorphism has a role in the long-term adaptation of hemodynamic phenotypes to endurance training rather than in the short-term response to a single bout of exercise. Previous studies have shown that regular endurance training increases endothelial NO production both in humans16 18 and in animals14 15 and that the enhanced NO production capacity mediates several beneficial effects of regular aerobic exercise.17 Shear stress and cholinergic nerve activity are 2 potential mechanisms that may mediate the greater NO production after endurance training.36 Increased blood flow during exercise generates greater laminar shear stress on vascular endothelium and thereby activates the expression of several genes, including NOS3.37 Acetylcholine is a neurotransmitter that induces vasodilation indirectly through an NO-dependent pathway, and endurance training has been reported to enhance cholinergic vasodilation.36 Further studies are needed to clarify whether the DNA sequence variation in the NOS3 locus affects these pathways and, if so, via which mechanisms.
At this point, we can only speculate on the possible clinical implications of the present findings. However, in consideration of the greater reduction in submaximal exercise DBP and myocardial workload (estimated as RPP50), the Glu allele of the NOS3 Glu298Asp polymorphism could be a marker of normotensive sedentary white individuals, who are most likely to benefit from endurance training in terms of reduction in the hemodynamic load during moderate-intensity physical activity. If this observation, derived from a cohort of normotensive, sedentary subjects, is applicable to resting BP in subjects with elevated BP or hypertension, the NOS3 marker could be useful for the screening of patients who are likely to derive the greatest benefits from regular physical activity. Naturally, the hypothesis itself and whether an endurance training program with different intensity, frequency, and duration conditions could induce greater training responses in the homozygotes for the Asp allele remain to be tested in future studies.
Another aspect of the NOS3 polymorphism that warrants further studies is the possible interactions with other genetic and environmental factors. NOS has several cofactors, such as tetrahydrobiopterin and calmodulin, that are necessary for the optimal function of the enzyme. The genes that encode these proteins are also potential candidates for themselves and because of their interactions with NOS3. Another interesting possibility involves the level of oxidative stress to which the individual is exposed. Superoxide anions inactivate NO and thereby block its physiological effects.38 On the other hand, dietary antioxidants, such as vitamin C, have been shown to restore endothelial NO activity in hypertensive subjects.39 Moreover, oxidized LDL may directly impair endothelial NOS activation.40 Finally, the suggestion that hypertensives may exhibit a selective defect in endothelial NO synthesis emphasizes the need to explore the interactions with the cholinergic and ß-adrenergic pathways of NOS3 stimulation.41
In summary, these data from the HERITAGE Family Study cohort suggest that in previously sedentary normotensive adult whites, the DNA sequence variation in the NOS3 locus is associated with the responsiveness of submaximal exercise DBP and RPP to regular endurance training.
| Acknowledgments |
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Received March 15, 2000; first decision April 20, 2000; accepted May 24, 2000.
| References |
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Asp] is a major risk factor for coronary
artery disease in the UK. Circulation. 1999;100:15151520.This article has been cited by other articles:
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P. W. Franks, J. Luan, I. Barroso, S. Brage, J. L. G. Sanchez, U. Ekelund, M. S. Rios, A. J. Schafer, S. O'Rahilly, and N. J. Wareham Variation in the eNOS Gene Modifies the Association Between Total Energy Expenditure and Glucose Intolerance Diabetes, September 1, 2005; 54(9): 2795 - 2801. [Abstract] [Full Text] [PDF] |
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T. Kimura, T. Yokoyama, Y. Matsumura, N. Yoshiike, C. Date, M. Muramatsu, and H. Tanaka NOS3 Genotype-Dependent Correlation Between Blood Pressure and Physical Activity Hypertension, February 1, 2003; 41(2): 355 - 360. [Abstract] [Full Text] [PDF] |
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