Produção Nacional
Revisado por pares
Endothelial nitric oxide gene haplotype reduces the effect of a single bout of exercise on the vascular reactivity in healthy subjects
2012; Elsevier BV; Volume: 161; Issue: 1 Linguagem: Inglês
10.1016/j.trsl.2012.05.004
ISSN1931-5244
AutoresBruno M. Silva, Fabricia J. Neves, Natália G. Rocha, Allan R. K. Sales, Renata Frauches Medeiros, Thales C. Barbosa, Felipe S. Pereira, Fabiane Toste Cardoso, Antônio Cláudio Lucas da Nóbrega,
Tópico(s)Cardiovascular Effects of Exercise
ResumoPolymorphisms in the endothelial nitric oxide synthase (eNOS) gene reduce shear stress–induced nitric oxide production. Thus, we investigated the individual and combined impact of 3 variants in the eNOS gene (−786T>C, intron 4b4a, and 894G>T) on vascular reactivity before and after exercise. Sedentary, healthy subjects were studied (105 women/26 men, age 32 ± 1 years [mean ± standard error of the mean]). Genotypes were determined by polymerase chain reaction restriction fragment length polymorphism, and haplotypes were determined by a Bayesian-based algorithm. Vascular reactivity was evaluated by the percentage of change in forearm vascular conductance provoked by 5 minutes of circulatory occlusion before (baseline) and 10, 60, and 120 minutes after a maximal cardiopulmonary exercise test. Vascular reactivity increased 10 minutes after exercise in the entire sample (baseline: 218 ± 11% vs 10 minutes: 284 ± 15%, P < 0.001), remained increased at 60 minutes (239 ± 12%, P = 0.02 vs baseline), and returned to baseline at 120 minutes (210 ± 10%, P = 0.83 vs baseline). Genotype analysis showed that subjects with the 894G>T polymorphism had lower vascular reactivity than wild counterparts (group effect, P = 0.05). Furthermore, subjects with haplotype 2 (H2), containing the −786T>C and 894G>T polymorphisms, had lower vascular reactivity than wild counterparts (haplotype 1 [H1]) (group effect, P = 0.05), whereas subjects with haplotype 4 (H4), containing only the 894G>T polymorphism, had vascular reactivity similar to that of wild counterparts (H1) (group effect, P = 0.35). Altogether, these results indicate that the 894G>T polymorphism reduced exercise-mediated increase in vascular reactivity, particularly when it occurred concomitantly with the −786T>C polymorphism. Polymorphisms in the endothelial nitric oxide synthase (eNOS) gene reduce shear stress–induced nitric oxide production. Thus, we investigated the individual and combined impact of 3 variants in the eNOS gene (−786T>C, intron 4b4a, and 894G>T) on vascular reactivity before and after exercise. Sedentary, healthy subjects were studied (105 women/26 men, age 32 ± 1 years [mean ± standard error of the mean]). Genotypes were determined by polymerase chain reaction restriction fragment length polymorphism, and haplotypes were determined by a Bayesian-based algorithm. Vascular reactivity was evaluated by the percentage of change in forearm vascular conductance provoked by 5 minutes of circulatory occlusion before (baseline) and 10, 60, and 120 minutes after a maximal cardiopulmonary exercise test. Vascular reactivity increased 10 minutes after exercise in the entire sample (baseline: 218 ± 11% vs 10 minutes: 284 ± 15%, P < 0.001), remained increased at 60 minutes (239 ± 12%, P = 0.02 vs baseline), and returned to baseline at 120 minutes (210 ± 10%, P = 0.83 vs baseline). Genotype analysis showed that subjects with the 894G>T polymorphism had lower vascular reactivity than wild counterparts (group effect, P = 0.05). Furthermore, subjects with haplotype 2 (H2), containing the −786T>C and 894G>T polymorphisms, had lower vascular reactivity than wild counterparts (haplotype 1 [H1]) (group effect, P = 0.05), whereas subjects with haplotype 4 (H4), containing only the 894G>T polymorphism, had vascular reactivity similar to that of wild counterparts (H1) (group effect, P = 0.35). Altogether, these results indicate that the 894G>T polymorphism reduced exercise-mediated increase in vascular reactivity, particularly when it occurred concomitantly with the −786T>C polymorphism. Dynamic exercise performed with large muscle groups requires complex integrative cardiovascular responses that leads to systemic increase in shear stress.1Thijssen D.H. Dawson E.A. Black M.A. Hopman M.T. Cable N.T. Green D.J. Brachial artery blood flow responses to different modalities of lower limb exercise.Med Sci Sports Exerc. 2009; 41: 1072-1079Crossref PubMed Scopus (132) Google Scholar This exercise-mediated increase in shear stress stimulates nitric oxide (NO) production in the whole circulatory system,2Allen J.D. Miller E.M. Schwark E. Robbins J.L. Duscha B.D. Annex B.H. Plasma nitrite response and arterial reactivity differentiate vascular health and performance.Nitric Oxide. 2009; 20: 231-237Crossref PubMed Scopus (58) Google Scholar, 3Allen J.D. Cobb F.R. Gow A.J. Regional and whole-body markers of nitric oxide production following hyperemic stimuli.Free Radic Biol Med. 2005; 38: 1164-1169Crossref PubMed Scopus (37) Google Scholar, 4Haram P.M. Adams V. Kemi O.J. et al.Time-course of endothelial adaptation following acute and regular exercise.Eur J Cardiovasc Prev Rehabil. 2006; 13: 585-591Crossref PubMed Scopus (98) Google Scholar which takes several minutes or hours to return to pre-exercise baseline values.2Allen J.D. Miller E.M. Schwark E. Robbins J.L. Duscha B.D. Annex B.H. Plasma nitrite response and arterial reactivity differentiate vascular health and performance.Nitric Oxide. 2009; 20: 231-237Crossref PubMed Scopus (58) Google Scholar, 3Allen J.D. Cobb F.R. Gow A.J. Regional and whole-body markers of nitric oxide production following hyperemic stimuli.Free Radic Biol Med. 2005; 38: 1164-1169Crossref PubMed Scopus (37) Google Scholar, 4Haram P.M. Adams V. Kemi O.J. et al.Time-course of endothelial adaptation following acute and regular exercise.Eur J Cardiovasc Prev Rehabil. 2006; 13: 585-591Crossref PubMed Scopus (98) Google Scholar, 5Bousquet-Santos K. Soares P.P. Nórega A.C. Subacute effects of a maximal exercise bout on endothelium-mediated vasodilation in healthy subjects.Braz J Med Biol Res. 2005; 38: 621-627Crossref PubMed Scopus (21) Google Scholar Thus, after a single bout of exercise the vascular reactivity is augmented, which is largely dependent on NO2Allen J.D. Miller E.M. Schwark E. Robbins J.L. Duscha B.D. Annex B.H. Plasma nitrite response and arterial reactivity differentiate vascular health and performance.Nitric Oxide. 2009; 20: 231-237Crossref PubMed Scopus (58) Google Scholar, 3Allen J.D. Cobb F.R. Gow A.J. Regional and whole-body markers of nitric oxide production following hyperemic stimuli.Free Radic Biol Med. 2005; 38: 1164-1169Crossref PubMed Scopus (37) Google Scholar, 4Haram P.M. Adams V. Kemi O.J. et al.Time-course of endothelial adaptation following acute and regular exercise.Eur J Cardiovasc Prev Rehabil. 2006; 13: 585-591Crossref PubMed Scopus (98) Google Scholar, 5Bousquet-Santos K. Soares P.P. Nórega A.C. Subacute effects of a maximal exercise bout on endothelium-mediated vasodilation in healthy subjects.Braz J Med Biol Res. 2005; 38: 621-627Crossref PubMed Scopus (21) Google Scholar and has been associated with favorable after-effects of exercise on the cardiovascular system,6Nóbrega A.C. The subacute effects of exercise: concept, characteristics, and clinical implications.Exerc Sport Sci Rev. 2005; 33: 84-87Crossref PubMed Scopus (54) Google Scholar such as inhibited blood pressure response during sympathoexcitatory maneuvers.6Nóbrega A.C. The subacute effects of exercise: concept, characteristics, and clinical implications.Exerc Sport Sci Rev. 2005; 33: 84-87Crossref PubMed Scopus (54) Google Scholar, 7Hamer M. Taylor A. Steptoe A. The effect of acute aerobic exercise on stress related blood pressure responses: a systematic review and meta-analysis.Biol Psychol. 2006; 71: 183-190Crossref PubMed Scopus (136) Google Scholar, 8Lindqvist M. Melcher A. Hjemdahl P. Hemodynamic and sympathoadrenal responses to mental stress during nitric oxide synthesis inhibition.Am J Physiol Heart Circ Physiol. 2004; 287: H2309-H2315Crossref PubMed Scopus (16) Google ScholarSilva B, et al.At a Glance CommentaryBackgroundRecently it was shown that subjects carrying the 894G>T polymorphism in the eNOS gene had blunted vascular reactivity to ischemia after exercise in comparison with wild counterparts. Nevertheless, the impact of other eNOS gene polymorphisms, isolated or combined, on the vascular reactivity after exercise is still unknown.Translational SignificanceThe present study showed that only the 894G>T polymorphism reduces the exercise-mediated increase in vascular reactivity, particularly when it occurs concomitantly with the −786T>C polymorphism. Therefore, these findings contribute to translate the impact of eNOS genetic variations on the after-effect of exercise on vascular function. Recently it was shown that subjects carrying the 894G>T polymorphism in the eNOS gene had blunted vascular reactivity to ischemia after exercise in comparison with wild counterparts. Nevertheless, the impact of other eNOS gene polymorphisms, isolated or combined, on the vascular reactivity after exercise is still unknown. The present study showed that only the 894G>T polymorphism reduces the exercise-mediated increase in vascular reactivity, particularly when it occurs concomitantly with the −786T>C polymorphism. Therefore, these findings contribute to translate the impact of eNOS genetic variations on the after-effect of exercise on vascular function. The enzyme that catalyzes NO production in response to shear stress over the endothelium is the endothelial nitric oxide synthase (eNOS).9Balligand J.L. Feron O. Dessy C. eNOS activation by physical forces: from short-term regulation of contraction to chronic remodeling of cardiovascular tissues.Physiol Rev. 2009; 89: 481-534Crossref PubMed Scopus (347) Google Scholar The gene that codes this enzyme is located at chromosome 7 (location 7q36) and contains 21 kb. Since the characterization of the eNOS gene in the mid-1990s,10Marsden P.A. Heng H.H. Scherer S.W. et al.Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene.J Biol Chem. 1993; 268: 17478-17488Abstract Full Text PDF PubMed Google Scholar many allelic variations were identified. Nevertheless, only some of these have been consistently associated with functional impairments11Dias R.G. Alves M.J. Pereira A.C. et al.Glu298Asp eNOS gene polymorphism causes attenuation in nonexercising muscle vasodilatation.Physiol Genomics. 2009; 37: 99-107Crossref PubMed Scopus (26) Google Scholar, 12Neves F.J. Silva B.M. Rocha N.G. Sales A.R. Ribeiro G.S. Nóbrega A.C. Effect of the 894G>T polymorphism of the endothelial nitric oxide synthase on vascular reactivity following maximal dynamic exercise.J Hypertens. 2010; 28: 764-770Crossref PubMed Scopus (8) Google Scholar, 13Rocha N.G. Neves F.J. Silva B.M. Sales A.R. Nóbrega A.C. The 894G>T endothelial nitric oxide synthase genetic polymorphism affects hemodynamic responses to mental stress performed before and after exercise.Eur J Appl Physiol. 2012; 112: 877-886Crossref PubMed Scopus (5) Google Scholar, 14Silva B.M. Neves F.J. Negrão M.V. et al.eNOS polymorphisms and adaptation of parasympathetic modulation to exercise training.Med Sci Sports Exerc. 2011; 43: 1611-1618Crossref PubMed Scopus (10) Google Scholar and clinical end points.15Casas J.P. Bautista L.E. Humphries S.E. Hingorani A.D. Endothelial nitric oxide synthase genotype and ischemic heart disease: meta-analysis of 26 studies involving 23028 subjects.Circulation. 2004; 109: 1359-1365Crossref PubMed Scopus (278) Google Scholar Among these variations are a single nucleotide polymorphism (SNP) in the promoter region (−786T>C, rs2070744), a variable number of tandem repeats polymorphism in the intron 4 (4b4a), and an SNP in the exon 7 (894G>T, rs1799983). The −786T>C polymorphism has been shown to reduce promoter activity of the eNOS gene, which reduces efficiency of eNOS transcription.16Nakayama M. Yasue H. Yoshimura M. et al.T-786-->C mutation in the 5’-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm.Circulation. 1999; 99: 2864-2870Crossref PubMed Scopus (689) Google Scholar The 4b4a polymorphism impairs the eNOS mRNA splicing process, which can also reduce efficiency of eNOS transcription.17Nakamura Y. Koyama K. Matsushima M. VNTR (variable number of tandem repeat) sequences as transcriptional, translational, or functional regulators.J Hum Genet. 1998; 43: 149-152Crossref PubMed Scopus (101) Google Scholar Finally, the 894G>T polymorphism alters the structure of the eNOS enzyme and has been associated with altered eNOS localization at endothelial caveolae,18Joshi M.S. Mineo C. Shaul P.W. Bauer J.A. Biochemical consequences of the NOS3 Glu298Asp variation in human endothelium: altered caveolar localization and impaired response to shear.FASEB J. 2007; 21: 2655-2663Crossref PubMed Scopus (105) Google Scholar leading to reduced response to shear stress and impaired coordination of the enzyme regulatory cycle.18Joshi M.S. Mineo C. Shaul P.W. Bauer J.A. Biochemical consequences of the NOS3 Glu298Asp variation in human endothelium: altered caveolar localization and impaired response to shear.FASEB J. 2007; 21: 2655-2663Crossref PubMed Scopus (105) Google Scholar Therefore, it is conceivable that these polymorphisms in the eNOS gene could blunt the enhancement of vascular reactivity that is usually observed after a single bout of exercise. Our group recently showed that healthy subjects, who carried the 894G>T polymorphism, had blunted vascular reactivity to ischemia12Neves F.J. Silva B.M. Rocha N.G. Sales A.R. Ribeiro G.S. Nóbrega A.C. Effect of the 894G>T polymorphism of the endothelial nitric oxide synthase on vascular reactivity following maximal dynamic exercise.J Hypertens. 2010; 28: 764-770Crossref PubMed Scopus (8) Google Scholar and mental stress13Rocha N.G. Neves F.J. Silva B.M. Sales A.R. Nóbrega A.C. The 894G>T endothelial nitric oxide synthase genetic polymorphism affects hemodynamic responses to mental stress performed before and after exercise.Eur J Appl Physiol. 2012; 112: 877-886Crossref PubMed Scopus (5) Google Scholar after a single bout of exercise in comparison with wild counterparts (ie, subjects without the polymorphism). Nevertheless, the impact of other eNOS gene polymorphisms on the vascular reactivity after exercise is still unknown. Most important, the impact of the interaction among eNOS gene polymorphisms on the vascular reactivity after exercise is not known, which is a relevant issue, because the influence of genetic variations on physiologic traits can be more informative when SNPs are analyzed concomitantly as haplotypes (combinations of genetic markers within a chromosome cluster location).19Metzger I.F. Sertorio J.T. Tanus-Santos J.E. Modulation of nitric oxide formation by endothelial nitric oxide synthase gene haplotypes.Free Radic Biol Med. 2007; 43: 987-992Crossref PubMed Scopus (95) Google Scholar, 20Nejatizadeh A. Kumar R. Stobdan T. et al.Endothelial nitric oxide synthase gene haplotypes and circulating nitric oxide levels significantly associate with risk of essential hypertension.Free Radic Biol Med. 2008; 44: 1912-1918Crossref PubMed Scopus (42) Google Scholar On the basis of this background, the aim of the present study was to investigate the effect of 3 polymorphisms in the eNOS gene (−786T>C, intron 4b4a, and 894G>T), analyzed individually as genotypes and concomitantly as haplotypes, on the vascular reactivity to an ischemic stimulus performed before and after a single bout of exercise. Subjects were recruited through advertisements at the university and in local newspapers. Approximately 1000 people volunteered to participate, but only 105 women and 26 men met the inclusion criteria and completed the study. Most of these subjects participated in previous studies from our group.12Neves F.J. Silva B.M. Rocha N.G. Sales A.R. Ribeiro G.S. Nóbrega A.C. Effect of the 894G>T polymorphism of the endothelial nitric oxide synthase on vascular reactivity following maximal dynamic exercise.J Hypertens. 2010; 28: 764-770Crossref PubMed Scopus (8) Google Scholar, 13Rocha N.G. Neves F.J. Silva B.M. Sales A.R. Nóbrega A.C. The 894G>T endothelial nitric oxide synthase genetic polymorphism affects hemodynamic responses to mental stress performed before and after exercise.Eur J Appl Physiol. 2012; 112: 877-886Crossref PubMed Scopus (5) Google Scholar The eligibility requirements were verified through clinical history assessment, physical examination, blood pressure measurement on 2 different days, biochemical blood analyses, resting electrocardiogram, and maximal cardiopulmonary exercise testing. Subjects had to fulfill the following criteria to be included in the study: age 18 to 49 years, women with regular menstrual cycles, absence of any diagnosed disease and no recent infections, body mass index (BMI) between 18.5 and 29.9 kg/m, total cholesterol < 240 mg/dL, low-density lipoprotein (LDL) < 160 mg/dL, triglycerides < 200 mg/dL, glycemia < 126 mg/dL, systolic blood pressure (SBP) < 140 mm Hg or diastolic blood pressure (DBP) < 90 mm Hg, not smoking, not using medications with exception of oral contraceptives, normal resting and exercise electrocardiogram, and sedentary (not engaged in exercise activities lasting ≥ 30 minutes, 3 times per week during the last 3 months). This study protocol was approved by the Antonio’s Pedro University Hospital Review Board, located at the Fluminense Federal University, and conformed to the standards set by the latest revision of the Declaration of Helsinki. All subjects gave written informed consent before participation in the study. Genomic DNA was extracted from circulating leucocytes. The 4b4a genotype was identified directly after a polymerase chain reaction (PCR) amplification protocol,10Marsden P.A. Heng H.H. Scherer S.W. et al.Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene.J Biol Chem. 1993; 268: 17478-17488Abstract Full Text PDF PubMed Google Scholar and the −786T>C and 894G>T genotypes were identified by PCR, followed by restriction fragment length polymorphism. Quality control for these assays was assessed by randomly selecting samples to be re-genotyped by 2 independent researchers. No misgenotyping was observed. Haplotypes for each subject were inferred using the software PHASE version 2.1 (University of Washington, Seatle, Wash).21Stephens M. Smith N.J. Donnelly P. A new statistical method for haplotype reconstruction from population data.Am J Hum Genet. 2001; 68: 978-989Abstract Full Text Full Text PDF PubMed Scopus (6423) Google Scholar The volunteers and researchers were blinded to the genotypes and haplotypes during the study. The PCR was performed using the following primers: sense 5’-AGG CCC TAT GGT AGT GCC TTT-3’ and antisense 5’-TCT CTT AGT GCT GTG GTC AC-3’. DNA was amplified for 35 cycles consisted of denaturing at 94°C for 30 seconds, annealing at 51°C for 40 seconds, and extension at 72°C for 40 seconds. The PCR products were digested by incubation with a restriction endonuclease, NgoMIV (New England Biolabs, Ipswich, MA), at 37°C for 16 hours and visualized by gel electrophoresis. The PCR was performed using the following primers: sense 5’-AGG CCC TAT GGTAGT GCC TTT-3′ and antisense 5′-TCT CTT TAG TGC TGT GGT CAC-3′. DNA was amplified for 35 cycles, and each cycle was composed of denaturing at 94°C for 1 minute, annealing at 49°C for 40 seconds, and extension at 72°C for 40 seconds. The PCR products were visualized by gel electrophoresis. The PCR was performed using the following primers: sense 5’ AAG GCA GGA GAC AGT GGA TGG A-3’ and antisense 5’ CCC AGT CAA TCC CTT TGG TGC TCA-3’. DNA was amplified for 35 cycles consisted of denaturing at 94°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 1 minute. The PCR products were digested by incubation with Ban II (New England Biolabs, Ipswich, Mass) at 37°C for 16 hours and visualized by gel electrophoresis. Blood was drawn in the morning after 12 hours of fasting. Cholesterol and its subfractions (high-density lipoprotein [HDL] and LDL], as well as triglycerides, were determined by the dry chemistry method. Plasma glucose was measured by enzymatic in vitro test. The experimental protocol was conducted in the morning, 1 hour after a standardized light breakfast. The evaluation was performed from the first to the 12th day after the onset of menstruation. Subjects did not drink alcohol or caffeinated beverages and did not perform intense physical activities for at least 24 hours before the experimental visit. During the study, subjects were placed in a supine position in a quiet air-conditioned room (≈24°C), and they rested quietly for 10 minutes before any measurement. Then, blood pressure and vascular reactivity were assessed before (baseline) and 10, 60, and 120 minutes after a single bout of exercise. A subgroup of 8 subjects of the sample also participated in a time-control protocol, which was conducted on a different day of the experimental protocol. The order of the control and experimental protocols was randomized in this subgroup. The control protocol was composed of blood pressure and vascular reactivity assessment before (baseline) and 10, 60, and 120 minutes after standing on a treadmill for 30 minutes without exercising, which was the approximate duration of the whole exercise bout procedure described next. The exercise bout consisted of a standard maximal cardiopulmonary exercise test performed on a treadmill (Master ATL, Inbrasport, Porto Alegre, RS, Brazil). This consisted of 3 minutes of rest standing on the treadmill, 3 minutes of warm-up at 3 km/h and 0% grade, ramp protocol with linear increase in speed and grade every minute until maximal voluntary exhaustion, and 5 minutes of recovery at 4 km/h and 0% grade. The ramp protocol was individualized according to predicted maximal exercise capacity to reach volitional fatigue at approximately 10 minutes of protocol.22Wasserman K. Hansen J.E. Sue D.Y. Stringer W.W. Whipp B.J. Normal values.in: Wasserman K. Hansen J.E. Sue D.Y. Stringer W.W. Whipp B.J. Principles of Exercise Testing and Interpretation: Including Pathophysiology and Clinical Applications. Lippincott Williams & Wilkins, Baltimore2005: 160-168Google Scholar Subjects were verbally encouraged to exercise until exhaustion. All subjects met at least 2 of the following criteria to confirm that maximal effort was attained:23Midgley A.W. McNaughton L.R. Polman R. Marchant D. Criteria for determination of maximal oxygen uptake: a brief critique and recommendations for future research.Sports Med. 2007; 37: 1019-1028Crossref PubMed Scopus (318) Google Scholar (1) respiratory exchange ratio > 1.1; (2) heart rate within ± 10 beats/min−1 of the age-predicted maximum (210 – [age/0.65]); and (3) score 10 of perceived effort on Borg 0 to 10 scale. Ventilation, oxygen uptake, and carbon dioxide output were measured with each breath (CPX Ultima Gas Exchange System, Medgraphics Corp, St Paul, Minn). Electrocardiogram was monitored through 12 leads (Welch Allyn CardioPerfect Workstation, Welch Allyn, Skaneateles Falls, NY), and perceived exertion was assessed every minute. Breath-by-breath ventilation and expired gases were averaged to 20 seconds to identify peak oxygen consumption (VO2peak), which was considered the highest value of oxygen uptake during exercise. Vascular reactivity was assessed through venous occlusion plethysmography. The right arm was supported in a comfortable position, elevated above the level of the heart at a standardized height. Two cuffs were used; one (8 cm wide) was placed around the right wrist, and one (10 cm wide) was placed around the right upper arm. The arm cuff was attached to a rapid cuff inflator (EC6, Hokanson, Bellevue, Wash). A mercury in silastic strain gauge (Hokanson, Bellevue, Wash) was placed at the widest girth of the right forearm. The diameter of the strain gauge was 1 or 2 cm smaller than the widest girth of the forearm. Forearm blood flow (FBF) was measured during 3 minutes at pre- and postischemia by means of rapidly inflating the arm cuff ( T polymorphism of the endothelial nitric oxide synthase on vascular reactivity following maximal dynamic exercise.J Hypertens. 2010; 28: 764-770Crossref PubMed Scopus (8) Google Scholar, 13Rocha N.G. Neves F.J. Silva B.M. Sales A.R. Nóbrega A.C. The 894G>T endothelial nitric oxide synthase genetic polymorphism affects hemodynamic responses to mental stress performed before and after exercise.Eur J Appl Physiol. 2012; 112: 877-886Crossref PubMed Scopus (5) Google Scholar For a 2-way analysis of variance (ANOVA) (2 groups and 4 repeated measures), a total sample size of 120 subjects would be necessary to detect a difference of 35% between groups’ vascular reactivity (group main effect), considering a standard deviation within groups of 90%, P value of 0.05, and power of 0.80. Shapiro–Wilk’s test was used to verify variables’ distribution, Levene’s test was used to verify homoscedasticity, and Mauchly’s test was used to verify sphericity. Some variables were not normally distributed (ie, age, BMI, triglycerides, HDL, glycemia, VO2peak, SBP, and vascular reactivity), and thus were transformed into natural logarithms for inferential analyses. After logarithm transformation, there was no violation of the homoscedasticity assumption in any analyses. Nonetheless, vascular reactivity results deviated from the sphericity assumption, which required a correction that is described next. Three genetic models (dominant, recessive, and additive) were assessed to verify which model was better to fit the vascular reactivity data on partial correlations adjusted by all sample characteristics. In these partial correlations, eNOS gene polymorphisms were analyzed as dummy variables as follows: dominant model (heterozygous + polymorphic homozygous = 0 vs wild homozygous = 1), recessive model (polymorphic homozygous = 0 vs wild homozygous + heterozygous = 1), and additive model (polymorphic homozygous = 0 vs heterozygous = 1 vs wild homozygous = 2). Then, subjects’ characteristics according to genotypes and haplotypes were compared using independent Student t test or chi-square test. Specifically in the analyses of haplotypes, characteristics of the wild haplotype (haplotype 1 [H1]) were separately compared with each of the polymorphic haplotypes (ie, H1 vs haplotype 2 [H2], H1 vs haplotype 3 [H3], H1 vs haplotype 4 [H4]) according to the method of planned comparisons against a control group.26Rosenthal R. Rosnow R.L. Mixed sources of variance.in: Rosenthal R. Rosnow R.L. Contrast Analysis: Focused Comparisons in the Analysis of Variance. Cambridge University Press, New York1985: 66-73Google Scholar The effect of exercise or time-control (ie, no exercise) protocols on vascular reactivity was analyzed by means of a repeated-measures ANOVA, followed by the Fisher post hoc test in case of significant F values. Vascular reactivity was compared between groups using analysis of covariance (ANCOVA) models, where all subjects’ characteristics were considered as covariates. At first, a model was used to compare the baseline vascular reactivity between groups. Then, a different model was used to compare groups throughout time (ie, ANCOVA main factors: group [wild vs polymorphic] and time [baseline vs 10 minutes vs 60 minutes vs 120 minutes]). In the ANCOVA of the haplotypes, the haplotype containing only wild-type alleles (H1) was separately compared with each of the haplotypes containing polymorphic alleles (ie, H1 vs H2, H1 vs H3, H1 vs H4).26Rosenthal R. Rosnow R.L. Mixed sources of variance.in: Rosenthal R. Rosnow R.L. Contrast Analysis: Focused Comparisons in the Analysis of Variance. Cambridge University Press, New York1985: 66-73Google Scholar Greenhouse–Geisser correction was used to correct P values from ANCOVA main effects due to deviation from the sphericity assumption. In case of significant F values, Cohen’s d effect size was calculated. Results are presented as mean ± standard error of the mean. Statistical significance was considered for P ≤ .05 based on 2-tailed comparisons. All analyses were performed using STATISTICA version 8.0 software (StatSoft Inc, Tulsa, Okla). The characteristics of the entire sample were as follows: age 32 ± 1 years, BMI 25
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