Physical exercise and blood pressure with reference to the angiotensinogen M235T polymorphism

Rainer Rauramaa1,2, Raimo Kuhanen1, Timo A. Lakka1,3, Sari B. Väisänen1,4, Pirjo Halonen5, Markku Alén6, Tuomo Rankinen1,3 and Claude Bouchard3

1 Kuopio Research Institute of Exercise Medicine and Department of Physiology, University of Kuopio, 70100 Kuopio
2 Department of Clinical Physiology and Nuclear Medicine, Kuopio University Hospital, 70211 Kuopio
3 Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana 70808-4124
4 Department of Clinical Chemistry, Kuopio University Hospital, 70211 Kuopio
5 Computing Centre, University of Kuopio, 70211 Kuopio
6 Peurunka Rehabilitation Centre, 41340 Laukaa, Finland


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We investigated the role of the angiotensinogen (AGT) gene M235T polymorphism in determining blood pressure (BP) response to moderate intensity exercise in a 6-yr randomized controlled trial in 140 middle-aged men. Sitting, supine, and standing blood pressures were measured annually. Of the randomized men, 86% participated in the trial for 6 yr. Submaximal cardiorespiratory fitness increased by 16% in the exercise group. In the M homozygotes, sitting systolic BP decreased by 1.0 mmHg in the exercise but increased by 14.6 mmHg in the reference group (P = 0.007 for net effect). Sitting and supine diastolic BP decreased by 6.2 and 3.3 mmHg in the exercise but increased by 2.8 and 3.2 mmHg in the reference group (P = 0.026 and 0.024 for net effects), respectively. Regular moderate intensity exercise attenuates aging-related increase in systolic BP and decreases diastolic BP among the M homozygotes of the AGT gene M235T polymorphism.

physical activity; DNA polymorphism; randomized controlled trial


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
THE HIGH INCIDENCE AND PREVALENCE of elevated blood pressure, enforced by the aging of populations, indicates a need for nonpharmacological preventive measures. While a lower diastolic blood pressure confers a decreased risk of cardiovascular diseases (15), the prognostic importance of systolic blood pressure appears even more evident, particularly among elderly people (12). Recent recommendations emphasize regular moderate intensity exercise in the prevention and treatment of elevated blood pressure (18). Regular exercise has been reported to reduce blood pressure in normotensive subjects (5, 7) and in hypertensive patients (5, 7, 14) but not in all individuals (7, 13). An explanation could be that genetic factors attenuate or enhance the antihypertensive effect of exercise.

Essential hypertension is a multifactorial phenotype, and about 30% of the variance is attributable to genetic factors (3). The angiotensinogen (AGT) gene locus is a genetic determinant of high blood pressure (11). Angiotensinogen interacts with renin to produce angiotensin I, which is further cleaved by the angiotensin converting enzyme into angiotensin II, a powerful vasoconstrictor and thereby a regulator of blood pressure. The M235T polymorphism consists of a thymine-to-cytosine transition at nucleotide 704 in exon 2 of the AGT gene, leading to a Met->Thr substitution at codon 235 (1). The polymorphism alters the immunologic recognition of the angiotensinogen protein, but no differences have been found in vitro in the secretion or metabolism of the two recombinant forms of the protein. Compared with the M allele, the T allele has been associated with moderately increased plasma angiotensinogen levels and higher blood pressure, although not in all studies (3).

Our aim was to investigate the effect of regular moderate intensity physical exercise on annually measured blood pressure in a 6-yr randomized controlled trial in a population-based sample of middle-aged and elderly men. Our special interest was to investigate the role of the M235T polymorphism in the AGT gene in modifying the blood pressure response to regular exercise.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Study design.
The study is a part of The DNA Polymorphism and Carotid Atherosclerosis (DNASCO) Study, a 6-yr randomized controlled trial on the effects of a moderate intensity exercise training and genetic factors on carotid atherosclerosis and its risk factors. The study protocol was approved by the Ethical Committee of the University of Kuopio, and no violation of APS principles for research involving animals and human beings was in this study. The participants signed an informed consent. A random population sample of men was obtained by computer linkage to the national population registry using the unique personal identification code, which every Finn has. A flow-chart of the study is presented in Fig. 1. Exclusion criteria were diseases or physical conditions restricting participation in regular exercise, malignant diseases, or mental states restricting cooperation. The baseline characteristics are given in Table 1.



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Fig. 1. A flow-chart of the DNA Polymorphism and Carotid Atherosclerosis (DNASCO) study.

 

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Table 1. Characteristics of the subjects in the exercise and reference groups at the baseline

 
Exercise intervention.
The men in the exercise group were prescribed walking, jogging, cross-country skiing, swimming, and cycling. The training program was progressive: during the first 3 mo, the subjects were advised to exercise three times a week for 30–45 min per session at an intensity corresponding to brisk walking. Thereafter, the duration was increased to 45–60 min per session, and the frequency was increased to five times a week. Exercise intensity was determined individually to correspond to ventilatory aerobic threshold level, i.e., 40–60% of maximal oxygen uptake. The men in the exercise group were provided with a heart rate monitor (Polar Edge, Polar Electro) to help to adhere to the prescribed training heart rate. The subjects performed the program on their own and reported each bout in an exercise diary, which was checked by an exercise physiologist at 6-mo intervals. According to diaries, the median 6-yr energy expenditure in the exercise group was 1,300 kcal/wk. In accordance with the regulations of the Ethical Committee, the control subjects were advised to make their personal choice whether to engage in physical exercise.

Measurement of cardiorespiratory fitness.
For the assessment of cardiorespiratory fitness, maximal oxygen uptake (VO2 max) was measured using breath-by-breath respiratory gas analyses during an incremental (20 W/ min) bicycle ergometer exercise until subjective or objective (increase in oxygen consumption less than 150 ml/min, despite increase in work load) maximum level was attained. An exercise physiologist defined ventilatory aerobic threshold visually as the first nonlinear increase of ventilation during breath-by-breath respiratory gas analyses in the bicycle ergometer test. For quality control, a blinded 20% random sample of ventilatory aerobic thresholds was reanalyzed at four annual examinations showing correlation coefficient from 0.84 to 0.97 between the analyses. Body weight was measured with a digital scale in light clothing without shoes.

Measurement of blood pressure.
The same trained nurse measured blood pressure according to the MONICA protocol (20) annually throughout the trial using a random zero mercury sphygmomanometer with a cuff size of 52 x 14 cm. After the men had been lying for 45 min on an examination bed, blood pressure was measured in supine (3 measurements with 5-min interval), standing (2 measurements, immediately and after 2 min of standing), and sitting (1 measurement after 3 min of sitting) positions. For each subject, the blood pressure was measured at the same time of the day (mean individual standard deviation of 1.2 h over the 7 annual examinations). The fifth Korotkoff sound was taken as the diastolic blood pressure value. Antihypertensive medication was checked annually by a physician. The proportion of subjects receiving antihypertensive medication by genotype in each study group at the baseline and at the end of intervention is given in Table 2.


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Table 2. Antihypertensive medication among the AGT M235T genotypes at the baseline and the end of the intervention

 
DNA analysis.
Genomic DNA was isolated from white blood cells by phenol/chloroform extraction procedure followed by dialysis. The M235T polymorphism of the AGT gene was assessed with the polymerase chain reaction (PCR) followed by digestion with Tth111 I (16). The PCR conditions were slightly modified from those previously described (17). To lower the PCR temperatures, the upstream and downstream primers were shortened by seven and five base pairs (bp), respectively. The primers used were 5' -CCG-TTT-GTG-CAG-GGC-CTG-3' (upstream) and 5' -TGC-TGT-CCA-CAC-TGG-ACC-CC-3' (downstream). The PCR was performed in standard buffer (Perkin-Elmer, Norwalk, CT), and each 20-µl PCR reaction contained 100 ng genomic DNA, 0.3 µmol/l each primer, 200 µM each dNTPs, and 0.5 U Taq polymerase (Perkin-Elmer). The reactions were incubated at 95°C for 3 min, 62°C for 15 s, and 70°C for 1.0 min, followed by 40 cycles at 95°C for 15 s, annealing at 62°C for 15 s, and extension at 70°C for 1.0 min, and finally one cycle at 72°C for 10 min, using a thermal cycler (model 9600, Perkin-Elmer).

The PCR product was digested with 5 U of Tth111 I (New England BioLabs, Mississauga, ON, Canada) at 65°C for 5 h. The resulting fragments were separated on 8% acrylamide gel and visualized under UV light after ethidium bromide staining. The absence and presence of the restriction site for Tth111 I generated fragments of 165 and 141 bp, respectively. When the restriction site is not present, the nucleotide 704 in the exon 2 is T and the codon 235 encodes methionine (M allele), but when the restriction site is present, the nucleotide is C and the ensuing amino acid is threonine (T allele).

Statistical analyses.
SPSS for Windows (version 10.0) was used. The effects of exercise and the M235T polymorphism on blood pressure, adjusted for change in body weight during the intervention, were tested by repeated measures ANOVA. The differences in the seven time points of blood pressure measurements (1 baseline and 6 annual measurements) were located by Helmert contrasts, which compares each time point to the mean value of the later measurements. An independent samples, t-test was used to test for differences in the variables at the baseline and at the end of intervention between the study groups. A paired samples t-test was used to test for changes in blood pressure and other characteristics during the intervention. The independence of the results from the baseline blood pressure was tested by a linear regression analysis (study group times baseline blood pressure as an independent variable of interest). The differences in allele and genotype frequencies were analyzed with the {chi}2 test.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Characteristics of subjects.
The exercise and reference groups were similar with regard to baseline characteristics (Table 1). There were no statistically significant differences in blood pressure between the study groups at baseline (Table 1). Conformance of genotype distribution to Hardy-Weinberg equilibrium was tested by a {chi}2 test in the entire study population and in the exercise and reference group separately. At the randomization (n = 140, Fig. 1), the frequency of the M allele was similar in the exercise group (0.52; 95% CI 0.39–0.65) and the reference group (0.53; 0.40–0.66). The genotype distribution was similar between the study groups at the randomization ({chi}2 = 3.0, P = 0.219, n = 140) and at the end of the 6-yr intervention ({chi}2 = 4.8, P = 0.092, n = 120).

Except for higher sitting and supine diastolic blood pressure in the TT men of the reference group (Table 3), there were no statistically significant differences in blood pressure between the study groups in different genotypes at baseline. During the intervention, ventilatory aerobic threshold increased by 16% (P < 0.001) in the exercise group but remained unchanged in the reference group. There were no differences among genotypes in the change of ventilatory aerobic threshold in either study group. Maximal oxygen uptake was unchanged in the exercise group but decreased by 8% (P < 0.001) in the reference group. After the intervention, ventilatory aerobic threshold (P = 0.013) and maximal oxygen uptake (P = 0.050) were higher in the exercise group compared with the reference group. Body weight did not change in the exercise group but increased by 1.7 kg (P = 0.008) in the reference group. Change in body weight during the exercise intervention was used as a covariate in all models of repeated measures ANOVA. Blood pressure responses were independent of the baseline blood pressure level.


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Table 3. Sitting SBP and DBP at baseline and after 6 yr of intervention by AGT gene M235T genotype

 
Sitting blood pressure.
In the entire study population, sitting systolic blood pressure increased by 8.5 mmHg in the exercise group (P = 0.004) and by 12.0 mmHg in the reference group (P < 0.001), with the net difference being statistically nonsignificant. In a repeated measures ANOVA, an interaction effect was found between the M235T genotype and the study group. The interaction was localized by Helmert contrasts to the baseline, i.e., comparing baseline blood pressure with the mean blood pressure of the later annual measurements (F = 4.9; df = 2, 113; P = 0.009). In the M homozygotes (Table 3), sitting systolic blood pressure decreased by 1.0 mmHg in the exercise group but increased by 14.6 mmHg in the reference group (P = 0.007 for the net difference). Figure 2 shows a profile plot of systolic blood pressure level in the MM men.



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Fig. 2. Sitting systolic blood pressure (mean and SE, unadjusted) at each annual measurement in the M homozygotes. Solid line refers to the exercise group, and broken line refers to the reference group.

 
In the entire study population, sitting diastolic blood pressure remained unchanged in the exercise group but increased by 2.8 mmHg (P = 0.014) in the reference group. Consistent with systolic blood pressure, in the M homozygotes, diastolic blood pressure decreased by 6.2 mmHg in the exercise group, and increased by 2.8 mmHg in the reference group during 6 yr (Table 3, P = 0.026 for the net difference). This genotype-study group interaction effect was localized both to baseline (F = 5.0; df = 2, 113; P = 0.009) and to the first year of intervention (F = 4.5; df = 2, 113; P = 0.013). Figure 3 shows a profile plot of diastolic blood pressure level in the MM men. There were no corresponding differences or interactions in the other genotypes.



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Fig. 3. Sitting diastolic blood pressure (mean and SE, unadjusted) at each annual measurement in the M homozygotes. Solid line refers to the exercise group, and broken line refers to the reference group.

 
Supine blood pressure.
In the entire study population, supine systolic blood pressure increased by 10.0 mmHg in the exercise group, and by 10.7 mmHg (P < 0.001 for both) in the reference group. In the M homozygotes (Table 4), systolic blood pressure remained unchanged in the exercise group but increased in the reference group (P = 0.016). Systolic blood pressure increased in the MT and TT genotypes in the exercise and reference groups. In repeated measures ANOVA, no significant interaction between the genotype and study group was found.


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Table 4. Supine SBP and DBP at the baseline and after 6 yr of intervention by AGT gene M235T genotype

 
In the entire study population, supine diastolic blood pressure remained unchanged in the exercise group but increased by 4.0 mmHg (P = 0.001) in the reference group. Consistent with the sitting position, in the M homozygotes, diastolic blood pressure decreased during intervention by 3.3 mmHg in the exercise group and increased by 3.2 mmHg in the reference group (P = 0.024 for the net difference). This genotype-study group interaction effect was located to the first intervention year (F = 8.6; df = 2, 113; P < 0.001). There were no corresponding differences in the other two genotypes.

Standing blood pressure.
No statistically significant differences at baseline were found in standing systolic blood pressure between the study groups (Table 1) or by genotypes. In the entire study population, systolic blood pressure increased by 7.8 (P = 0.007) and 6.5 mmHg (P = 0.010) in the exercise and reference groups, respectively. No significant interaction between the genotype and study group was found.

In the entire study population, standing diastolic blood pressure remained unchanged. In the M homozygotes, diastolic blood pressure decreased by 3.5 mmHg in the exercise group and increased by 2.2 mmHg in the reference group. This 5.7-mmHg net difference was statistically nonsignificant. There was an interaction effect between the genotype and study group on diastolic blood pressure, and it was localized to baseline (F = 3.5; df = 2, 112; P = 0.035).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The main finding of the present 6-yr randomized controlled trial is a gene-physical activity interaction leading to an attenuation in aging-related increase in systolic blood pressure and to a decrease in diastolic blood pressure in the homozygotes of the M235T polymorphism in the AGT gene. The present data should help to explain previous contradictory findings on the blood pressure-reducing effects of regular physical exercise.

Why does regular moderate intensity exercise influence blood pressure in the M homozygotes? Angiotensinogen acts as a substrate for renin to generate angiotensin II, which elevates blood pressure primarily by promoting sodium retention and increasing vascular resistance. Plasma angiotensinogen concentration is lower in subjects carrying the M allele than in carriers of the T allele (11). This may lead to a small increase in the production of angiotensin II, thereby inducing a slight overreactivity of the renin-angiotensin-aldosterone system in the carriers of the T allele. On the other hand, in the carriers of the M allele, one could hypothesize that elevated blood pressure may depend more on higher plasma volume. In hypertensive patients with elevated plasma renin activity, the antihypertensive effect of physical exercise was lower compared with patients with normal plasma renin activity (6, 13). If the above hypotheses prove to be correct, then the implication of our study is that for subjects prone to volume-dependent hypertension, moderate intensity physical activity program should be among the first-line approaches for prevention and management. On the other hand, one should keep in mind the physiological negative feedback control in the renin-angiotensin system, which regulates plasma angiotensin II level in relation to renin activity (4). In addition, the AGT M235T polymorphism of the renin-angiotensin system may serve only as a marker for another functional polymorphism with which it is in linkage disequilibrium. One likely variant is located in the proximal promoter 6 bp upstream from the initiation site of transcription. The nucleotide substitution from adenine to guanine affects the basal transcription rate of the AGT gene, the A(-6) allele being in very strong linkage disequilibrium with the T allele of the AGT M235T polymorphism (10).

Our results do not indicate that higher intensity exercise programs would be less efficient for the primary prevention of hypertension. In fact, carriers of the T allele might respond to higher intensity exercise regimen. This idea is supported by the similar responses among AGT M235T genotypes in systolic blood pressure during a steady-state submaximal exercise (16). Compared with the DNASCO training program, the 20-wk supervised bicycle ergometer training in the HERITAGE Family Study was more intensive, corresponding to 75% of the heart rate at maximal oxygen uptake (16).

Even small reductions in blood pressure at the population level have a substantial impact on the prevalence of hypertension and the risk of coronary heart disease, stroke, and other cardiovascular diseases (2). Therefore, the present data emphasize lifestyle modification, especially physical activity, in the prevention of cardiovascular diseases, and agree with observational data (8) and current recommendations of encouraging regular physical activity for public health purpose (19). The present intervention was designed to be applicable at population level, also among elderly people. Our emphasis was on regular physical activity as an essential lifestyle component, rather than on high maximal fitness level. This was evidenced by the improved submaximal performance capacity in the exercise group. Moreover, a recent meta-analysis of randomized controlled trials of at least 4-wk duration found that increasing exercise intensity above 70% of maximal oxygen uptake does not enhance blood pressure reduction compared with lower intensity exercise (9).

Taken together with previous studies, the present results suggest that physical exercise can operate through several mechanisms to reduce blood pressure. Therefore, additional randomized controlled trials are required to identify optimal exercise programs for blood pressure control and the specific mechanisms through which regular exercise at different intensities and volumes exert their beneficial effects. Future studies on gene-exercise interactions should cover other relevant polymorphisms. Important questions are interactions between antihypertensive drugs and regular physical exercise, ideally in patients being randomized a priori by the genotype of interest. We must admit the small number of MM subjects, in whom the clinically significant exercise-induced net change in sitting systolic blood pressure of 15.6 mmHg between baseline and end of intervention was seen. Power calculations based on the present data give a sample size of 45 per group to demonstrate an intervention effect of same size in a similar study design with repeated measurements, if only M homozygotes are included as study subjects. It has to be kept in mind that the statistically significant interaction effect as seen in the present study is based on the outcomes in all genotypes. It is also important to note that the present findings concern white middle-aged men and that there may well be age, racial, and gender differences in the antihypertensive mechanisms of exercise training.

Our study design offers several strengths that increase the confidence in the results. Reliance on a random population sample, where the subjects represent a wide spectrum of physical activity and other health habits, is a crucial, although unfortunately often neglected approach in clinical trials designed to investigate the efficacy and applicability of a treatment modality. To obtain a stable baseline reference level and to alleviate the temporary changes in health behavior and in other environmental factors that potentially influence blood pressure, we applied a long lead-in period of 30 mo with three follow-up examinations before the subjects were randomized into an experimental or a reference group. The long preintervention phase strengthened the adherence of the subjects, as evidenced by the low dropout rate during the trial. Furthermore, based on the mean weekly exercise energy expenditure, the significant increase in the ventilatory aerobic threshold, and also the stabilization of body weight, the compliance of the subjects in the exercise group was very good.

We paid particular attention to the conditions of blood pressure measurements. Based on the exercise diaries, ~75% of the blood pressure measurements were done more than 24 h after last exercise session thus avoiding the acute effects of exercise. To standardize both the physiological as well as the environmental circumstances, the subjects rested in the supine position for 45 min before starting the blood pressure measurements. A unique feature is that the same trained nurse did all of the more than 3,000 blood pressure measurements. The subjects came for the examinations always at the same time of the day and year. Our study is one of the few exercise training trials in which blood pressure has been measured in supine, sitting, and standing positions. Finally, the study design allowed to use a repeated measures ANOVA, thus increasing statistical power and diminishing the risk of spurious associations. Despite the careful blood pressure measurements, there was an unexpected finding, which is difficult to explain. Subjects homozygous for the T allele had significantly lower diastolic blood pressure in the exercise group than in the reference group at the baseline. One explanation for this, as well as for the relatively large increase in systolic blood pressure during 6 yr, could be the small number of TT subjects. However, our main finding was seen among the MM subjects.

In conclusion, regular exercise training at moderate intensity reduced aging-related increase in systolic blood pressure and decreased diastolic blood pressure among the M homozygotes of the AGT gene M235T polymorphism. These data should help to define an optimal and individually adapted exercise prescription for the primary prevention and management of elevated blood pressure.


    ACKNOWLEDGMENTS
 
The study was supported by grants from the Ministry of Education in Finland (322/722/94, 80/722/95, 176/722/96, 42/ 722/97, 84/722/98, 138/722/99, 112/722/2000), from the City of Kuopio, from the Juho Vainio Foundation, from Centre de Recherche et d’Information Nutritionnelles de Paris, France, and from Fonds de la Recherche en Santé du Québec.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: R. Rauramaa, Kuopio Research Institute of Exercise Medicine, Haapaniementie 16, 70100 Kuopio, Finland (E-mail: rainer.rauramaa{at}uku.fi).

10.1152/physiolgenomics.00050.2002.


    References
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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