Baroreflex sensitivity and variants of the renin angiotensin system genes

Subjects

Population 1.

Oulu Project Elucidating Risk of Atherosclerosis (OPERA) is a population-based, epidemiological study of cardiovascular risk factors (15). Subjects with no treatment for hypertension were randomly selected by age stratification from the register of the Social Insurance Institution covering the whole population of the City of Oulu in Northern Finland (106,500 inhabitants). Two hundred fifty-nine men and 267 women volunteered to take part, the overall participation rate being 87%.

All subjects were invited to be studied by Valsalva maneuver and 24-hour ambulatory BP measurement and 483 participated. Participants with diabetes mellitus (n = 22), coronary artery disease based on symptoms, medication or ECG (n = 35), atrial fibrillation (n = 12), frequent ectopic beats (n = 8), inadequate ambulatory BP monitoring (n = 19) or inadequate Valsalva maneuver (n = 72) were excluded, after which a total of 315 individuals (161 women and 154 men, 60% of the original cohort) remained for analyses. Of the women, 76 were classified as postmenopausal since at least 6 months had passed after their latest menstruation, but 28 of them were on estrogen or progesterone replacement therapy. Nine of the 85 premenopausal women were taking these hormones. Past and current medical history, smoking habits, alcohol consumption and physical activity were assessed with a standardized health questionnaire and leisure-time physical activity groups (1–5)were formed as described (16). A clinical examination and standard 12-lead electrocardiography were performed. Routine blood tests, including an oral glucose tolerance test, were performed on each subject.

Population 2.The reproducibility of the associations between BRS and genetic polymorphisms of CYP11B2 and ACE was tested in another population sample consisting of 89 subjects aged 36 to 37 years apparently free of cardiovascular disease. The characterization of this study group, including echocardiographic examination of the left ventricle, laboratory tests for blood lipids and assessment of habitual physical activity, smoking and ethanol consumption by two-month daily recording, has been detailed earlier (17,18). The subjects were also characterized for their heart rate and BP responses to the Valsalva maneuver. The ECG and BP recordings were analyzed for BRS by two of the authors (A.Y. and K.E.J.A.) blinded to the genotypes and other data. DNA was available for genotyping in 84 subjects, and the recordings of the Valsalva maneuver were acceptable for BRS determination in 70 subjects; 66 subjects (37 women and 29 men) had both types of data available for statistical analyses.

The design of the tests was approved by the Ethical Committees of the respective institutions, and all subjects gave their informed consent.

Baroreflex sensitivity

For the assessment of BRS, the subjects performed the Valsalva maneuver in a sitting position by blowing into a rubber tube connected to an aneroid manometer and maintaining an expiratory strain of 40 mm Hg for 15 s. The test was performed three times at 5 min intervals in Population 1 and 1 to 2 times in Population 2. Otherwise, the test method and the data analysis (19)were identical in the two study groups. In short, noninvasive arterial BP was recorded on a beat-to-beat basis, using the Finapres finger-cuff method (Ohmeda Inc.,) (20)with the monitored finger held at heart level. The ECG and continuous BP data were stored in a digital format and analyzed later using a menu-driven software package (CAFTS, Medikro Oy, Finland). A linear least-mean-squares fitting method was employed to calculate BRS from the overshoot phase after the Valsalva maneuver as the slope of the linear relationship between the RR interval (in ms) and the preceding systolic BP value (in mm Hg) (19). Only regression lines with a correlation coefficient >0.7 or p < 0.05 and an increase in BP >15 mm Hg were accepted. Baroreflex sensitivity was calculated as a mean of the results of all the accepted tests.

Genotyping of subjects

The insertion/deletion (I/D) polymorphism in intron 16 of the ACE gene (chromosome 17q23) (21)and the methionine/threonine polymorphism (M235T) in codon 235 of the angiotensinogen gene (AGT, chromosome 1q42) (22)were detected by polymerase chain reaction (PCR), using the conditions described previously (15). The subjects were also genotyped by PCR for two recently discovered polymorphisms in the aldosterone synthase gene (CYP11B2, chromosome 8q22). The details of the technique and the structure of the gene have been described elsewhere (18,23). One of the polymorphisms is located in the 5′flanking region, or promoter, of CYP11B2, 344 nucleotides before the start of the protein-coding sequence. This position can be either a cytosine (−344C) or a thymidine (−344T). The other is in the second intron of the gene. In some individuals, the usual sequence of this intron has been almost entirely replaced by a sequence typically found in the related gene, CYP11B1. Such replacement is termed gene conversion, and the alleles at this locus will be referred to as 1 (conversion) and 2 (no conversion). In Population 1, genotype data were missing in the case of two men for CYP11B2, two women for ACE and one woman for AGT polymorphism.

Blood pressure measurements

In Population 1, ambulatory BP was recorded using the fully automatic SpaceLabs 90207 oscillometric unit (SpaceLabs Inc., Redmond, Washington), which was set to take a measurement every 15 minutes from 4:00 amtill midnight and every 20 min from midnight till 4:00 am. The accuracy and reproducibility of BP readings obtained with this device have been established previously (24). In Population 2, resting brachial artery cuff BP was averaged over three measurements.

Statistical analysis

The Statistical Package for the Social Sciences for Windows, release 6.0, was used for data analysis. A chi-square test was used to assess the fit of the observed allele frequencies to the Hardy-Weinberg distribution. The coefficient of gametic linkage disequilibrium was calculated by likelihood methods, and the coefficient (D) was reported as the ratio of the unstandardized coefficient to its maximal value (25). Continuous data are given as means standard deviation (SD) according to the genotypes. Gender differences were assessed by Mann-Whitney Utest. The differences between the variables in the different genotype groups were tested with analysis of variance (ANOVA) (the Kruskal-Wallis test). A two-way ANOVA (3 × 3 factorial design) with Bonferroni’s corrections for multiple comparisons was used to test the potential synergistic effect of the ACE and AGT genes on BRS. Multiple regression analysis was used to assess the quantitative effects of age, BP, heart rate, body mass index, physical activity (log-transformed), alcohol consumption (log-transformed), smoking, serum cholesterol level and the use of estrogen or progesterone therapy on BRS. The regression analysis was made separately for men and women. The correlations between variables were evaluated by Spearman’s correlation coefficient. A value of p < 0.05 was considered statistically significant.

Population 1

Main characteristics, 24-h BP, lifestyle data and −344C/T genotype in men and women are shown in Table 1. There were no significant genotypic effects on any variables, including BP. Similarly, the ACE and AGT polymorphisms had no influence on any of these basic data (not shown). The 24-h systolic (129 ± 12 vs. 123 ± 11 mm Hg, p < 0.001) and diastolic (81 ± 8 vs. 77 ± 7 mm Hg, p < 0.001) BPs were higher in men, but there was no significant gender difference in body mass index. Men were more often smokers and their total cholesterol and triglyceride levels were higher and HDL cholesterol lower those of women.

The frequencies of the I and D alleles were 0.46 and 0.54, respectively; the frequencies of the M and T alleles were 0.53 and 0.47, respectively, and the frequencies of −344T and −344C were 0.49 and 0.51, respectively. The genotype distributions of all polymorphisms were in agreement with the Hardy-Weinberg equilibrium both in men and in women. The promoter −344C/T and intron two conversion polymorphisms occurred in linkage disequilibrium in the CYP11B2 gene (D = 0.48, χ²= 47.0, p < 0.0001). The −344CC, −344CT and −344TT genotypes were found in 23%, 48% and 28% in Population 1, respectively. The genotype distribution of the −344C/T polymorphism in 72 subjects with inadequate Valsalva maneuver was similar to that in 315 subjects with adequate tests (20 [30%] in −344TT, 39 [45%] in CT and 13 [25%] in CC genotype).

Baroreflex sensitivity was significantly lower in women than in men (8.6 ± 4.0 and 11.0 ± 4.8 ms·mm Hg⁻¹, respectively, p < 0.0001). In women, BRS was related to the CYP11B2 promoter genotype, averaging 10.1 ± 4.5, 8.7 ± 3.8 and 7.1 ± 3.2 ms·mm Hg⁻¹in the −344TT, CT and CC groups, respectively (p = 0.003) (Fig. 1); a comparable association was observed between BRS and the intron 2 polymorphism (1/1: 11.1 ± 4.4; 1/2 8.9 ± 4.1; 2/2: 7.5 ± 3.4 ms·mm Hg⁻¹, p = 0.002). In men, by contrast, BRS was unrelated to either the promoter genotype (Fig. 2)or the intron 2 genotype (data not shown) of the CYP11B2 gene. In women, the association of BRS with the CYP11B2 promoter genotype was consistent with a gene dosage effect, such that BRS increased in a linear relationship with the number of −344T alleles carried by each woman (F = 13.1, df1, p = 0.0004, R²= 0.08).

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Figure 1

Individual data on baroreflex sensitivity in relation to the −344C/T polymorphism in the promoter of CYP11B2 in women in Population 1. Horizontal linesindicate group mean values. ∗ = ANOVA; CYP11B2 = aldosterone synthase.

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Figure 2

Individual data on baroreflex sensitivity in relation to the −344C/T polymorphism in the promoter of CYP11B2 in men in Population 1. Horizontal linesindicate group mean values. ∗ = ANOVA; CYP11B2 = aldosterone synthase.

There were no significant differences in BRS between the different genotypes of ACE or AGT in women or men. When ACE and AGT were added as factors in ANOVA (3 × 3 factorial design), no significant synergistic effect of the AGT and −344C/T genotypes on BRS was observed.

In univariate analysis, BRS was associated with 24-h systolic BP in both men (r = −.22, p < 0.05) and women (r = −0.30, p < 0.0001). Baroflex sensitivity was also weakly related to age in men (r = −0.18, p < 0.05) but not in women (r = −0.13, ns). The association between BRS and the different genotypes of the CYP11B2 −344C/T promoter polymorphism in age tertiles in women is displayed in Figure 3. Although the same trends were seen in all age tertiles, the association was only significant among the women aged 41 to 46. All but two women in this tertile were premenopausal.

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Figure 3

Baroreflex sensitivity (mean ± SEM) by the −344C/T polymorphism in the promoter of CYP11B2 in age tertiles in women in Population 1. ∗ = ANOVA; CYP11B2 = aldosterone synthase.

In multiple regression analysis of the female population, BRS was related to 24-h systolic BP (beta = 0.38, p = 0.0067) and the −344C/T genotype of CYP11B2 (CC = 1, CT = 2, TT = 3; beta = 0.37, p = 0.008) but was independent of age (beta = −0.10, p = 0.45), body mass index (beta = −0.14, p = 0.33), physical activity (score 1–5; beta = 0.03, p = 0.81), daily alcohol consumption (beta = 0.03, p = 0.82), smoking (nonsmoker = 1, smokers = 2; beta = 0.07, p = 0.63), serum cholesterol (beta = −0.20, p = 0.12), heart rate (beta = −0.01, p = 0.99) and the use of estrogen or progesterone therapy (no therapy = 1, on therapy = 2; beta = −23, p = 0.07). In men, only 24-h systolic BP was an independent predictor (beta = 0.39, p = 0.013), while the other variables did not enter the model. Different assumptions about the type of the genotype effect did not alter the findings.

Population 2

The genotype distributions and baseline characteristics of the study population have been described in full detail previously (17,18). There were no significant differences in gender distribution, body mass index, heart rate, BP, physical activity, smoking, ethanol consumption of blood lipids across the −344C/T genotype groups (18). Nor did the ACE I/D polymorphisms have any influence on these basic data (17). The −344C/T genotypes of 18 subjects with inadequate Valsalva maneuver were 3 (17%) −344TT, 8 (44%) CT and 7 (39%) CC.

In the total population of 37 women and 29 men, ANOVA revealed a significant main effect of the −344C/T genotype on BRS (Fig. 4). The data were consistent with a gene dosage effect; BRS increased in a linear relationship with the number of −344T alleles carried by each subject (F = 21.1, df1, p < 0.0001, R²= 0.25). The association of BRS with the −344C/T genotype was statistically significant in both men (TT: 20.2 ± 10.8; CT: 12.6 ± 6.5; CC: 10.0 ± 2.7 ms·mm Hg⁻¹, p = 0.037, ANOVA) and women (TT: 18.1 ± 9.4; CT: 13.0 ± 4.0; CC: 9.6 ± 5.0 ms·mm Hg⁻¹, p = 0.017, ANOVA).

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Figure 4

Individual data on baroreflex sensitivity in relation to the −344C/T polymorphism in the promoter of CYP11B2 in Population 2. Horizontal linesindicate group mean values. ∗ = ANOVA; CYP11B2 = aldosterone synthase.

In women, BRS was also related to the intron two polymorphism of CYP11B2, averaging 18.9 ± 10.5, 14.6 ± 6.3 and 10.8 ± 3.9 ms·mm Hg⁻¹in the 1/1, 1/2 and 2/2 genotype groups, respectively (p = 0.036, ANOVA). The comparable trend in men was not statistically significant (data not shown). There were no differences in BRS between the I/D genotype groups of ACE in either women or men (data not shown). The sex difference in BRS did not reach statistical significance (14.7 ± 8.3 in men and 13.5 ± 6.3 ms·mm Hg⁻¹in women, p = 0.47).

In multiple regression analysis, BRS was related to the −344C/T genotype of CYP11B2 (CC = 1, CT = 2, TT = 3; beta = 0.49, p < 0.001), but was independent of sex (male = 1, female = 2, beta = 0.12, p = 0.34), body mass index (beta = 0.02, p = 0.92), serum cholesterol (beta = 0.11, p = 0.40), heart rate (beta = −0.15, p = 0.32), systolic BP (beta = −0.09, p = 0.56), left ventricular mass (beta = −0.02, p = 0.91), average daily physical activity (expenditure of metabolic equivalents; beta = 0.11, p = 0.37), smoking (nonsmoker = 0, 1–10 cigarettes/day = 1, >10 cigarettes/day = 2; beta = 0.13, p = 0.32) and average daily alcohol consumption (beta = 0.05, p = 0.69).

CYP11B2 polymorphism and cardiovascular regulation

Thus far, there is limited information available concerning the effects of CYP11B2 gene variation on cardiovascular structure and function (9,23). A recent report of studies in Population 2 suggested that the −344CC genotype is a powerful predictor of increased left ventricular mass and relatively impaired left ventricular diastolic function (18). This association was not confirmed by the study with older subjects having cardiovascular diseases, hypertension and diabetes (26). The relationship between BRS and the CYP11B2 genotypes observed in this study was, however, independent of left ventricular mass (18). Thus, the −344CC genotype may associate independently with low BRS and left ventricular hypertrophy, both of which predict increased cardiovascular mortality (1,2,27,28).

The association of BRS with CYP11B2 gene variation initially came out in a study of more than 500 randomly selected middle-aged nonhypertensive subjects (Population 1). Since multiple testing for genetic associations may notoriously lead to chance findings even in nonmixed populations, we repeated the study in a fully independent population-based sample of younger healthy subjects (Population 2). In support of our primary data, an even stronger association of BRS with the CYP11B2 genotypes was observed, this time not only in women, but also in men. Although only systolic BP and not age was related to BRS in multivariate models in Population 1, aging and BP have had independent effects on BRS in other studies (1,6,29,30), suggesting that the influence of polymorphisms on BRS may be filtered out upon aging in subjects with higher BP values (31).

Gender-related differences

The genotypic effects were analyzed separately for both genders because several previous studies have shown significant gender-related differences in the baroreceptor reflex control of heart rate in various populations (32–36). In Population 1, an association between BRS and the −344C/T genotypes was observed only among women and, furthermore, mainly in those younger than 46 years who were predominantly premenopausal. The observed gender difference in the genomic effect among the middle-aged subjects is physiologically plausible in the light of the prior studies and analogous to the well-established gender difference in BRS and BP regulation (33–36). Sex hormones may modulate the effects of circulating angiotensin II, aldosterone and cortisol at the receptor level in the central nervous system and interact with components of tissue RAS in the brain, heart, vascular bed and adrenals (37). We do not know why there was a gender difference in the association between BRS and the −344C/T genotypes in Population 1 but not in Population 2. However, the subjects in Population 1 were older, and their BP and body mass index were higher which differences may have modified the genetic influences on BRS.

Study limitations

The molecular mechanisms underlying the association between BRS and CYP11B2 polymorphism cannot be deduced from this study. Unfortunately, plasma concentrations of RAS hormones were not available for the populations we studied. However, it is reasonable to speculate that one of the CYP11B2 polymorphisms we examined, or an additional polymorphism in linkage disequilibrium with it, alters CYP11B2 gene expression in the adrenal cortex and thus influences plasma aldosterone levels which in turn affect BRS. Indeed, intravenous infusion of aldosterone impairs BRS in both animal models and man (12,13).

Prognostic information on the role of baroreflex function is based on phenylephrine-based BRS measurements (1,2). The predictive significance of BRS data obtained from a Valsalva maneuver is unknown, but the measurements are reproducible and correlate closely with indexes derived using the invasive phenylephrine method (19,38–40). Indeed, the stimulus to arterial baroreceptors obtained by the relatively high expiratory pressure (40 mm Hg) in the Valsalva maneuver is close to the stimulus obtained by a phenylephrine injection (38), and when the baroreflex slope is analyzed, as it is in this study, from the overshoot phase of the Valsalva maneuver (39), it is likely that the results of these two methods are comparable. Importantly, we only analyzed healthy subjects without apparent cardiac diseases which improves the accuracy and eliminates pitfalls of the Valsalva test (41).

Conclusions

Genetic variation in the aldosterone synthase (CYP11B2) gene is associated with interindividual variation in BRS. Because low BRS is a marker of increased risk of cardiovascular morbidity and mortality, the clinical and prognostic significance of CYP11B2 polymorphism merits further investigation.