European Journal of Sport Science, vol. 1, issue by Human Kinetics Publishers and the European College of Sport Science

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1 European Journal of Sport Science, vol. 1, issue by Human Kinetics Publishers and the European College of Sport Science Correlations of the Renin-Angiotensin-System (RAS) Gene Polymorphisms With Cardiac Growth Factors Endothelin-1 and Angiotensin II in High Performance Athletes Christine Graf, Frank Diet, Isabel Palma-Hohmann, Nicole Mahnke, Michael Böhm, Richard Rost, and Hans-Georg Predel Background: The regulation of myocardial mass in athletes involves complex interactions between physical stress, cardiac growth factors (GF), and the individual genetic background. Studies suggest the renin-angiotensin-system (RAS) plays a key role in the development of myocardial hypertrophy. Genetic polymorphisms of the RAS, associated with an activation, have been shown to correlate with left ventricular mass (LVM) in athletes. It is not clear whether these polymorphisms influence angiotensin (A) II levels directly or only markers coprecipitating with other factors modifying LVM. Endothelin (ET)-1 acts synergistically to AII in the induction of cardiac hypertrophy. The role of these GF in an athlete s heart is not yet clarified. Objectives: To analyze the correlation of RAS-gene polymorphisms with AII and ET-1 plasma concentrations in high performance athletes. Methods: In 83 male Caucasian, mainly endurance trained, elite athletes, intensity and duration of exercise were assessed by questionnaires; LVM was determined by 2-dimensional echocardiography. Plasma concentrations of AII and ET-1 were determined by radioimmunoassay. Results: LVM/LVMi significantly above reported average levels in sedentary persons was observed. AII plasma restingconcentrations correlated with the ACE gene polymorphism (p =.039), finding the highest levels in DD-athletes. There were no further correlations between the AII levels and the angiotensinogen gene polymorphisms, and the combined polymorphisms (p =.585, p =.162). ET-1 plasma levels did not correlate with any of the investigated parameters. No GF correlates with LVM/LVMi. Conclusions: The highest AII plasma resting concentrations were found in athletes homozygous to DD. This finding suggests a functional effect of the ACE I/D genotypes, possibly influencing cardiac growth in high performance athletes. The absence of a correlation with ET-1 plasma levels does not rule out its influence; local cardiac effects remain elusive. Further longitudinal studies under controlled exercise conditions are necessary. Key Words: athlete s heart, left ventricular mass, gene polymorphism, angiotensin II, endothelin-1

2 2 Graf et al. Introduction High-intensive exercise leads to remarkable increases in cardiac mass and volume. This phenomenon, the so-called athlete s heart, has been known for more than 100 years. The precise underlying mechanisms remain poorly understood. Some authors perceive the athlete s heart as a physiological and positive adaptation to an increased volume and pressure workload, particularly in endurance trained athletes (26). This mechanistic concept was questioned by observations that athletes with similar training intensities and durations developed markedly different cardiac masses and volumes, prompting studies in homozygous and heterozygous twins and siblings (1, 2). These data implicated a modifying role of genetic determinants. Verhaaren et al. suggested that more than 60% of the variability in LVM can be explained by genetic factors (28). Exercise stimulates the sympathetic nerve system and activates the RAS. Higher levels of renin, plasma renin activity (PRA), AI, and AII are found after exercising (9, 14 16, 23). In contrast, no influence of exercise was observed for the ACE plasma levels in healthy subjects (9). AII and ET- 1 influence cardiomyocyte-growth synergistically (11, 12, 29). Recently, an association of gene polymorphisms of the RAS the angiotensin-converting enzyme (ACE) insertion/deletion and the angiotensinogen-235 methionine/threonine substitution gene polymorphisms with left ventricular mass in athletes has been reported (13, 17, 18). These polymorphisms have been associated with increased local and systemic RAS activity (4, 25). In addition, certain combined genotypes of the ACE I/D and angiotensinogen M235T gene polymorphisms have been shown to correlate with left ventricular mass in athletes (7). The objective of the present study was to elucidate the functional significance of the ACE I/D and angiotensinogen M235T gene polymorphisms in high performance athletes. Methods Study Population Eighty-three male Caucasian athletes with different endurance demanding sport disciplines (international or national level of competition), attending an annual medical routine check-up at the Institute of Cardiology and Sport Medicine at the German Sports University of Cologne, were included in the study. The individual training programs of each athlete were assessed by a questionnaire, providing that athletes had no longer detraining periods. During the last 24 hours before the study, athletes were instructed to have similar caloric, fluid, and salt intake and refrain from alcohol, tea, or coffee consumption. Blood pressure (BP) was measured in sitting position after 15 min of rest. Anthropometric data by sport type are summarized in Table 1 (mean age: 25.5 ± 0.6 years, range 16 to 43 years; mean height: ± 0.7 cm, range 173 to 193 cm; mean weight: 75.5 ± 1.0 kg, range 57 to 93 kg; mean body surface area [BSA]: 1.9 ± 0.2 m 2, range 1.68 to 2.26 m 2 ). Each athlete had performed intensive training programs for a period of 2 to 25 years (mean, 10.8 ± 0.56 years). All athletes were non smokers, and there was no history of coronary artery disease, diabetes mellitus, or renal or hepatic dysfunction. No medication was taken by any of the athletes for any reason.

3 RAS and Cardiac Growth Factors 3 Table 1 Athletic Field and Anthopometric Data of All 83 Male Caucasian Athletes Activity n Height Weight (kg) Body surface area (m 2 ) Age (years) Cycling ± ± ± ± 4 Soccer ± ± ± ± 4 Running ± ± ± ± 6 Triathlon ± ± ± ± 3 Tennis ± ± ± ± 4 Ice hockey ± ± ± ± 1 Swimming ± ± ± ± 3 Laboratory Studies To collect venous blood samples from the antecubital vein, subjects assumed a supine position. Cardiac Growth Factors AII and ET-1 plasma concentrations were determined by radioimmunoassay (RIA) techniques as has been previously described (8, 24). Echocardiographic Studies Echocardiographic studies were performed by the same investigator, according to the rules of SAHN et al. (27), using a VINGMED Sonotron (Hewlett Packard). Abbreviations Key LVM (g): left ventricular mass, using the Devereux formula (5) = 1.04 (LVDD + LVDPW + IVS) 3 LVDD 3 ) 13.6 LVMi (g/m 2 ): LVM related to the body surface area (left ventricular mass index) LVDD : left ventricular enddiastolic diameter IVS : diastolic interventricular septum thickness LVDPW : enddiastolic diameter of the posterior wall Statistics The statistical analyses were carried out using EASYSTAT (v. 3.2, applied ). The continuous variables were expressed as means ± SD according to the genotypes MM, MT, TT and II, ID, DD if not stated otherwise. The differences between variables in the different genotypes were compared with the analysis of variance (ANOVA). A 2-way ANOVA was used for the potential synergistic effect of the ACE gene and the angiotensinogen gene on AII and ET- 1. A p value <.05 was considered significant. Results Study Population The main data are presented in Table 1. There were significant differences in weight and BSA between swimmers and ice hockey players (data not shown). The mean systolic BP was ± 1.2 mmhg (range 100 to 145 mmhg), and mean diastolic BP was 78.3 ± 1.1 mmhg (range 60 to 90 mmhg). No one exceeded the mean clinical limit of 140/90 mmhg. There was no history of cardiac diseases or drug abuse.

4 4 Graf et al. Echocardiographic Findings The mean LVM of all athletes was ± 63.1 g (range to g). Related to body surface area, LVMi was ± 30.4 g/m 2 (range 89.6 to g/m_). Among the different types of sports, cyclists presented with the highest LVM and LVMi (335.7 ± 33.0 g; ± g/m 2 ; p =.009 and.002, respectively). The echocardiographic findings are summarized in Table 2. Laboratory Findings The plasma levels of the cardiac growth factors and their distribution due to the different genotypes are shown in Figures 1 and 2. AII correlates with the ACE gene polymorphisms (p =.039). The highest AII levels were found in DD athletes (p =.044 compared to ID; p =.026 compared to II). There was no correlation of AII plasma levels and the angiotensinogen gene polymorphisms. ET-1 correlated neither with ACE (p =.825) nor angiotensinogen gene polymorphisms (p =.756). No association was found for AII and ET-1, and the combined genotypes and LVM/LVMi. Table 2 Echocardiographic Data of All 83 Male Athletes in All Kinds of Sports Activity LVM (g) LVMi (g/m 2 ) IVS LVDD LVPW Cycling ± ± ± ± ± 0.13 Swimming ± ± ± ± ± 0.12 Running ± ± ± ± ± 0.00 Triathlon ± ± ± ± ± 0.09 Ice hockey ± ± ± ± ± 0.19 Tennis ± ± ± ± ± 0.00 Soccer ± ± ± ± ± 0.15 Total ± ± ± ± ± 0.13 Figure 1 The circulating cardiac growth factor levels AII and ET-1 according to the single ACE- (DD, ID, II) and angiotensinogen- (MM, MT, TT) genotypes.

5 RAS and Cardiac Growth Factors 5 Figure 2 The distribution of the ACE- and Angiotensinogen-genotypes and plasma levels of AII and ET-1 in 83 male endurance trained elite athletes. The DDTT-type was the genotypes with the significant highest LVM. There was no significance found between the combined genotypes and the GF AII and ET-1. Discussion The clinical relevance and the underlying mechanisms of an athlete s heart remain controversial. The initial concept was solely based on mechanical stimuli associated with highly intensive endurance exercise (10). To elucidate the correlation between recently disclosed gene polymorphisms of the RAS (7) and LVM in athletes, potential correlations of circulating cardiac growth factors with cardiac mass were examined in an homogenous cohort of 83 highperformance male mainly endurance trained athletes. The obtained mean LVMi value of 148 g/m 2 exceeds the upper clinical limit of 130 g/m 2 (6, 20 22), expressing the high prevalence of an athlete s heart in our study population. The greatest LVM was found in cyclists, underscoring the contribution of a combined volume and pressure work load to the development of an athlete s heart (20, 21, 26). In our population of athletes, combined polymorphisms of the ACE I/D and angiotensinogen M235T-polymorphisms were positively correlated with LVMi, with the DDTT-genotypes having the highest LVMi (7). We hypothesized that the mediators between these genotypes and increased LVM might be, at least in part, the cardiac growth factors, AII and ET-1. In accordance, in athletes homozygous to DD, the highest circulating AII plasma concentrations were found. This finding suggests a potential functional effect of the I/D genotypes, by which cardiac growth may be mediated in high performance athletes. However, the exact underlying mechanisms of the effects of these RAS gene polymorphisms in the development of an athlete s heart are not yet clarified. In persons homozygous to the DD genotype, higher systemic and local ACE levels were found (4, 25). In rat hindlimbs, local ACE gene expression and activity in vessel walls causes dose-dependent AII generation (19). Serum angiotensinogen levels correlate with the angiotensinogen gene polymorphisms, resulting in higher

6 6 Graf et al. levels in TT-type (13). Exercise increases plasma levels of renin, PRA, AI, and AII, but not ACE (9). If this mechanical effect is supported by genetic determinants, in particular higher ACE levels in DD-types, the local, especially cardiac, influences might be amplified, leading to an increase in LVM. ET-1 is also known as a cardiac growth factor, acting synergistically to AII (11, 12). Exercise did not influence ET-1 plasma levels in healthy subjects (3), but Yamazaki et al. observed an increase in ET-1 mrna levels after mechanical stress (29). Therefore, our findings of unaltered ET-1 plasma concentration do not rule out an additional local trophic effect of ET-1 in cardiac tissues. Future studies will have to include measurements of ET-1 and AII plasma concentrations and other functional components of the RAS during exercise. Conclusion In summary, in athletes homozygous to DD, the highest circulating AII plasma resting concentrations were found. This finding suggests a functional effect of the I/D genotypes, possibly modifying cardiac growth in high performance athletes. The absence of a correlation with ET-1 plasma levels does not rule out a role of ET-1 in cardiac growth regulation; local trophic cardiac effects remain elusive. Our study underscores the complexity of the regulation of cardiac mass in high performance athletes. Further longitudinal studies under controlled exercise conditions in larger cohorts of athletes are clearly warranted to elucidate the complex interplay of exercise with genetic polymorphisms and cardiac growth factors in the regulation of myocardial mass. References 1. Adams TD, Yanowitz FG, Fisher AG et al Heritability of cardiac size: an echocardiographic and electrocardiographic study of monozygotic and dizygotic twins. Circulation 71: Bouchard C, Savard R, Despres JP Body composition in adopted and biological siblings. Hum Biol 57: Cheng CP, Ukai T, Onishi K et al The role of angii and endothelin-1 in exercise-induced diastolic dysfunction in heart failure. Am J Physiol Heart Circ Physiol 280:H1853-H Danser AHJ, Schalekamp MADH, Bax WA et al Angiotensin-converting enzyme in the human heart. Circulation 92: Devereux R, Reichek N Echocardiographic determination of left ventricular mass in man. Circulation 55: Dickhuth HH, Röcker K, Nieß A et al The echocardiographic determination of volume and muscle mass of the heart. Int J Sports Med 17: Diet F, Graf C, Mahnke N et al. ACE and angiotensinogen gene genotypes and left ventricular mass in athletes. Eur J Clin Invest. In press. 8. Emanuel RL, Cain JB, Williams GH Double antibody radioimmunassay of renin activity and angiotensin II in human peripheral plasma. J Lab Clin Med 81: Fyhrquist F, Dessypris, Immonen I Marathon run: effects on plasma rennin activity, rennin substrate, angiotensin converting enzyme and cortisol. Horm Metabol Res 15: Henschen S Skilanglauf und Skiwettlauf: Eine medizinische Sportstudie. Mitt Med Klein Upsala (Jena).

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