Acta Physiologica Hungarica, Volume 101 (2), pp. 176–184 (2014) DOI: 10.1556/APhysiol.101.2014.2.6
GNB3 gene c.825C>T polymorphism and performance parameters in professional basketball players T Gülyaşar1, L Öztürk2, T Sipahi1, B Bayraktar3, G Metin3, İ Yücesir4, N Süt5 1
Faculty of Medicine, Department of Biophysics, Trakya University, Edirne, Turkey Faculty of Medicine, Department of Physiology, Trakya University, Edirne, Turkey 3 Faculty of Medicine, Department of Sports Medicine, Istanbul University, Istanbul, Turkey 4 Physical Education and Sports School, Istanbul University, Istanbul, Turkey 5 Faculty of Medicine, Department of Biostatistics, Trakya University, Edirne, Turkey 2
Received: April 10, 2013 Accepted after revision: November 18, 2013 This study has been conducted to determine whether mean values of peak oxygen consumption (VO2peak), anaerobic test parameters and knee isokinetic test measurements are different among guanine nucleotide-binding protein, beta3 (GNB3) genotype groups in a group of basketball players. Methods: Seventy-two healthy male (mean age, 22.9 ± 5.3 years) basketball players from the first division of national league participated. We studied GNB3 gene c.825C>T (rs5443) polymorphism, then divided the subjects into three groups as CC (n = 21), CT (n = 35), and TT (n = 16). Mean VO2peak, Wingate anaerobic test results, and isokinetic knee muscle strength measurements were compared among the genotype groups. Results: Mean VO2peak (60.1 ± 3.9; 56.7 ± 3.6; and 57.8 ± 3.3, respectively, p < 0.01), mean anaerobic minimum power (5.1 ± 0.4; 5.3 ± 0.5; and 4.4 ± 0.5 W/kg, respectively, p < 0.001), mean anaerobic power drop (57.0 ± 6.2; 54.2 ± 6.9; and 62.9 ± 5.3%, respectively, p < 0.001) were significantly different among the study groups, CC, CT, and TT. Individuals with TT genotype exerted lower performance in terms of isokinetic knee muscle strength. Conclusion: The presence of 825T-allele may impair athletic performance and may serve as a genetic marker of low capacity for athletic performance in male basketball players. Keywords: genetic polymorphism, GNB3, exercise capacity, VO2peak, isokinetic knee strength
TP-binding proteins (G-proteins), that composed of α- and βγ-subunits, are important signal transducers of intracellular signalling pathway. A polymorphism at position 825 (C→T) of the exon 10 of the GNB3 gene, which encodes the β3-subunit of heterotrimeric G-proteins has been described by Siffert et al. (20). The expression of 825T allele stems from alternative splicing of exon 9. This type of splicing eliminates 41 amino acids which in turn increases G-protein activation (20). This polymorphism was found to be positively associated with cardio-respiratory fitness in an obese group of women (11). There may be several explanations for this association of GNB3 gene 825T allele and cardio-respiratory fitness. First, the altered splicing may enhance Na+/H+ exchanger (NHE) activity. This ion transport system regulates electroneutral exchange of sodium and hydrogen ions (19, 21). NHE isoforms also play an important role in mediating Na+ reabsorption in the proximal tubule of nephron. Hence, an increased activity of renal NHE may result in higher Na+ reabsorption and induce a state of volume expansion. The expansion of circulatory volume has been suggested to improve both exercise performance and thermoregulation via reducing cardiovascular and heat strain occurring especially during endurance exercise. Recently, drinking high-sodium beverages
Corresponding author: Levent Öztürk, MD, Professor Department of Physiology, Trakya University, Faculty of Medicine, Edirne, Turkey Phone: +90-284-2357641 (ext. 1421); Fax: +90-284-2357652; E-mail: [email protected] 0231–424X/$ 20.00 © 2014 Akadémiai Kiadó, Budapest
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before exercise has been shown to increase plasma volume and increased exercise capacity in hot environment (22). On the other hand, exercise-associated hyponatremia, a severe and potentially life-threatening condition, is well-documented during prolonged exercise such as marathons and triathlons (9). Risk factors of exercise-associated hyponatremia include preexercise overhydration, overconsumption of fluids (> 1.5 L/h) during an athletic event, low body weight, female gender, exercise duration > 4 hours, and a hot environment (18). Second, Gβ3 subunit coded in individuals carrying 825T allele could provide some benefits on the regulation of hemodynamics (17) and on the mobilization of exercise energy substrates (4) via enhanced signal transduction through intracellular second messengers such as cAMP (11). Gβ3 subunit activates β-adrenergic receptors through phosphorylation by receptor kinase (10). Thus, activation of β-adrenergic receptors activates lipolysis from adipocytes (4). Altered regulation of substrate mobilization due to GNB3 gene polymorphism may lead to an advantage on exercise performance. Thus in the present study, we hypothesized that different genotypes encoding the GNB3 evoke phenotypes associated with altered aerobic and anaerobic exercise capacity and that the presence of c.825C>T may pose a genetic predisposition for a better exercise performance in professional male basketball players. Methods Subjects The healthy male Caucasian basketball players (mean age, 22.9 ± 5.3 years, n = 72) from the first division of national league enrolled to the study. All participants underwent a comprehensive physical examination, then maximal cardiopulmonary exercise test, Wingate anaerobic performance test and muscle strength tests with isokinetic dynamometer were planned with 3–5 day intervals. All tests were performed during pre-seasonal period. Ethical approval was obtained from local Ethics Committee before starting the study (Registration number: TUTFEK200829). This study was conducted in agreement with the Declaration of Helsinki, and written informed consent was obtained from all enrolled individuals before participating in this study. Cardiopulmonary exercise testing (CPET) The participants underwent cardiopulmonary exercise testing on a treadmill connected with a computer system. Resting data were collected for three minutes to perform baseline measurements before exercise. The device was calibrated before each study. A modified Bruce protocol (3) was used. We added a 3-minute stage 0 at 5% gradient. Continuous monitorization of oxygen uptake (VO2), ventilation (VE), and carbon dioxide production (VCO2) were performed via breath-by-breath analysis on a cardiopulmonary exercise testing system (Cortex Metalyzer 3B, Cortex Biophysik GmbH, Leipzig, Germany). Participants were forced verbally to their exercise limits by the supervising physician. Each test was terminated by subject’s voluntary termination or by achievement of two of the three following criteria: (1) a respiratory exchange ratio of 1.00 or higher, (2) heart rate reaching 85% of agepredicted maximal heart rate, and (3) subject fatigue. Peak oxygen consumption (VO2peak) was recorded. The respiratory gas exchange ratio (RER) was calculated as VCO2/VO2. Blood pressure was measured at rest, at the end of each 3-min stage, and at peak exercise by
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using a mercury sphygmomanometer. Continuous electrocardiographic recordings were also obtained. The predicted maximal heart rate (MaxHR) was derived from the formula 210 – 0.65 × age (years). Wingate anaerobic exercise test (WanT) This test (2) consists of a 30-second supra-maximal cycling against a pre-determined resistance load. All tests were performed on a cycle ergometer (Monark, Model 894-E, Sweden). The load was calculated for each subject based on their body weight (0.075 kg × kg body weight). Seat height was adjusted to each subject’s satisfaction and toe-clips were used to prevent the subject’s feet from slipping off the pedals. Before the start, 3 minutes warm-up pedalling was performed against a 30 watt (W) constant load. By the command ‘start’ the participant began pedalling as fast as possible until the end of the test period. Strong verbal motivation was given to subjects during the test. The peak power and mean power were calculated by using the data (2, 14). Isokinetic knee extension/flexion strength Strength of knee muscles was measured using an isokinetic dynamometer (Cybex Norm Lumex Inc., Ronkonkoma, NY, USA). The participating athletes were tested in a sitting position with hip flexed at 90°. Velcro-straps were used to secure the test limb to the chair. The lever arm was aligned to the axis of rotation of the knee. Data were corrected by the weight of the subject’s lower limb at 45° in order to account for the influence of the gravity effect torque. Three fixed angular velocities were used during the tests: 60°/sec, 180°/sec, 300°/sec. Isokinetic contractions were started with the knee flexed at 90° and continued through the full range of motion. Each participant completed four maximal contractions at 60°/sec, 180°/sec angular velocities, and only the best value (peak power) was retained for further statistical analysis. At 300°/sec angular velocity, subject performed 30 repeats and total work (sum of the 30 contractions, i.e. endurance) was recorded. All athletes were verbally encouraged to perform each test as maximally as possible. Visual feedback during the test was also provided. The same investigators (BB, GM) conducted the tests for all the subjects. A recovery of 1 minute was allowed between each maximal contraction, to minimize fatigue. Genotyping studies GNB3 c.825C>T (rs5443) single nucleotide polymorphism was studied. Genotyping protocol was designed according to our previous study (12) with minor modifications. In brief, DNA was extracted from blood samples of all participants by E.Z.N.A. (EaZy Nucleic Acid Isolation) blood DNA kits (Omega Bio-Tek, Doraville, GA, USA). Sense 5’-TGA CCC ACT TGC CAC CCG TGC-3’ and antisense 5’-GCA GCA GCC AGG GCT GGC-3’ primer pairs were used to enhance genomic DNAs. The 25 μl mixture prepared for polymerase chain reaction (PCR) included 250 ng of DNA, 0.5 nmol of each sense and antisense primers, 0.2 mM deoxynucleotide triphosphates (of each), 2 mM MgCl2, 75 mM Tris-HCl (pH 8.8), 20 mM (NH4)2SO4, 0.01% Tween 20, and 1.25 unit of Taq DNA polymerase (Fermentas Life Sciences). Initial denaturation step lasted 5 minutes at 94 °C, then 35 cycles of 94 °C for 1 minute, 69 °C for 45 seconds, and 72 °C for 1 minute were performed. Electrophoresis of PCR products were performed on a 2% agarose gel and read under UV light by 5 µg/mL ethidium bromide staining. In order to search for genotype of C825T polymorphism, 268 bp Acta Physiologica Hungarica 101, 2014
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PCR products were incubated with BseDI (SecI) restriction enzyme (Fermentas Life Sciences) at 55 °C for 16 hours. After incubation, the digests were electrophoresed on a 3% agarose gel and visualized under UV light by 5 µg/mL ethidium bromide staining. One band (268 bp) for homozygote TT genotype, two bands (116 and 152 bp) for homozygote CC genotype, and three bands (268, 152, and 116 bp) for heterozygote CT genotype were obtained (Fig. 1).
Fig. 1. Ethidium bromide-stained gel of digest products of C825T region of the GNB3 gene shows the CT genotype (268, 152, and 116 bp; samples 1, 4, and 5), the CC genotype (152 bp and 116 bp; sample 7), and the TT genotype (268 bp; samples 2, 3, and 6). Lane 50 bp is a size marker (GeneRuler 50 bp DNA Ladder-Fermentas Life Sciences)
Statistical analysis Data are presented as means (± standard deviation, SD) unless otherwise indicated. The following a priori null hypotheses were tested: mean values of peak oxygen consumption (VO2peak), Wingate anaerobic test parameters and knee extension/flexion isokinetic test results are not different among the genotype groups, i.e. CC, CT, and TT. The test for HardyWeinberg equilibrium was performed by comparing observed versus expected genotype frequencies using a chi-square test. Comparisons between groups were made by one-way ANOVA, and then post-hoc Tukey test was used when significantly differences were obtained due to homogeneity of variances. To control significant covariates (i.e. BMI) among the groups, we also performed Covariance Analysis (ANCOVA). Bonferroni post-hoc test was used when significant differences were obtained. Null hypotheses were rejected and significant statistical differences assumed with an alpha error of p < 0.05. Results Genotype frequencies were in Hardy-Weinberg equilibrium in the whole study group. The whole group genotype frequencies for CC, CT, and TT were 21 (29%), 35 (49%) and 16 (22%), respectively. Demographic, anthropometric characteristics of the enrolled individuals are presented in Table I. Body mass index (BMI) was significantly different (p = 0.005), whereas age was similar among the genotype groups (p = 0.593). Percent body fat, resting metabolic rate, and resting systolic and diastolic blood pressure values were also comparable
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among the genotype groups (see Table I for details). Aerobic and anaerobic exercise test parameters are given in Table II. Peak oxygen consumption was significantly lower in individuals with T allele. Post hoc analysis showed that the CC group had significantly higher VO2peak levels than the CT group (pairwise comparison p < 0.01), whereas pairwise comparisons of groups CT vs TT and CC vs TT showed no significant differences in terms of VO2peak levels (Table II). Wingate anaerobic test results varied in genotype subgroups. ANCOVA test showed that minimum power and percent power drop were significantly different among the groups (Table II). Pairwise comparisons showed that the TT group had lower minimum power values (CC vs TT, p < 0.001; CT vs TT, p < 0.001) and higher power drop levels (CC vs TT, p < 0.05; CT vs TT, p < 0.001) than the CC and CT groups. Anaerobic test results revealed that there were no significant differences between the CC and CT groups in all parameters. Peak power and average power levels obtained during Wingate test were comparable among the three groups. Table III shows isokinetic knee muscle strength test results with respect to GNB3 genotypes. In general, T allele carriers had lower isokinetic muscle strength. Test results at 60°/sec and 180°/sec velocities mainly showed peak power levels of muscles, whereas the results at 300°/sec showed endurance of muscles during knee extension and flexion. Peak power levels at 60°/sec during flexion were higher in the CC group than in the TT group (right flexion CC vs TT, p = 0.012; left flexion CC vs TT, p = 0.017). Peak power levels at 180°/sec during knee flexion were higher in the CC group than in the CT and TT groups (right flexion CC vs CT, p = 0.020; CC vs TT, p = 0.001; left flexion CC vs CT, p > 0.05; CC vs TT, p = 0.030), but muscle peak power at 180°/sec during extension failed to reach statistically significant level (Table III). In terms of muscle endurance (30 repeats at 300°/sec) all study groups were comparable except right flexion values of CC vs TT (p = 0.009). Table I. The characteristics of the basketball players according to GNB3 c.825C>T genotypes Characteristics
Genotype CC (n = 21)
CT (n = 35)
TT (n = 16)
p*
Demographic Mean age, y
0.593
22.6 ± 5.4
23.6 ± 5.6
22.1 ± 4.8
Anthropometric BMI, kg/m2
22.4 ± 1.6
23.7 ± 1.4
23.2 ± 1.4
0.005
Fat ratio, %
7.1 ± 2.0
7.5 ± 2.1
9.0 ± 3.2
0.184
RMR, kcal
1964 ± 169
1901 ± 116
1883 ± 157
0.445
Resting blood pressures
SBP, mmHg
108.8 ± 6.6
110.0 ± 8.5
110.0 ± 8.7
0.852
DBP, mmHg
68.5 ± 7.2
70.1 ± 6.3
69.0 ± 6.3
0.671
*ANOVA; data are given as mean ± SD; BMI: body mass index; RMR: resting metabolic rate; SBP: systolic blood pressure; DBP: diastolic blood pressure
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Table II. The parameters of aerobic and anaerobic exercise tests according to GNB3 c.825C>T genotypes Test parameters
Genotype
p*
CC
CT
TT
60.1 ± 3.9
56.7 ± 3.6
57.8 ± 3.3
0.007
196 ± 7
195 ± 7
196 ± 8
NS
CPET VO2peak, ml/kg/min Pulsepeak, n/min
65.2 ± 5.1
67.2 ± 5.4
67.8 ± 7.0
NS
SBPpeak, mmHg
DBPpeak, mmHg
184.7 ± 11.1
184.4 ± 8.7
181.8 ± 9.8
NS
Recovery, sec
127.3 ± 11.2
126.5 ± 13.8
130.0 ± 12.3
NS
Aerobic time, sec
620.0 ± 62.8
597.8 ± 57.1
617.9 ± 55.6
NS
Anaerobic time, sec
284.4 ± 60.4
263.6 ± 56.9
259.5 ± 42.5
NS
Total run time, sec
904.4 ± 66.1
861.4 ± 64.5
877.4 ± 49.7
NS
Peak power, W/kg
12.1 ± 1.3
11.6 ± 1.3
12.0 ± 1.1
NS
Min power, W/kg
5.1 ± 0.4
5.3 ± 0.5
4.4 ± 0.5
< 0.001
Average power, W/kg
8.3 ± 0.6
7.9 ± 0.6
7.9 ± 0.4
NS
Power drop, %
57.0 ± 6.2
54.2 ± 6.9
62.9 ± 5.3
< 0.001
WanT
*ANCOVA; data are given as mean ± SD; CPET: Cardiopulmonary exercise test; VO2peak: peak oxygen consumption; DBPpeak: peak diastolic blood pressure; SBPpeak: peak systolic blood pressure; WanT: Wingate anaerobic exercise test; NS: not significant
Table III. The parameters of isokinetic knee strength test according to GNB3 c.825C>T genotypes Test parameters
Genotype CC
CT
TT
p*
Test at 60º/sec
R extension, Nm
313.1 ± 30.5
287.3 ± 35.9
285.1 ± 49.4
0.233
L extension, Nm
309.3 ± 30.4
289.9 ± 35.8
281.9 ± 42.2
0.182
R flexion, Nm
234.3 ± 21.4
214.5 ± 33.6
204.3 ± 17.9
0.014
L flexion, Nm
230.5 ± 14.9
210.8 ± 33.2
203.1 ± 24.4
0.017
Test at 180º/sec
R extension, Nm
200.0 ± 19.1
188.7 ± 22.3
181.9 ± 29.9
0.134
L extension, Nm
186.1 ± 20.2
185.0 ± 26.8
180.1 ± 32.2
0.774
R flexion, Nm
175.1 ± 15.2
156.1 ± 19.7
146.4 ± 26.8
0.001
L flexion, Nm
162.4 ± 16.9
153.1 ± 24.4
141.3 ± 25.8
0.035
Test at 300º/sec
R extension tW, Nm
2649 ± 394
2426 ± 425
2373 ± 410
0.108
L extension tW, Nm
2275 ± 379
2382 ± 369
2350 ± 329
0.713
R flexion tW, Nm
2479 ± 488
2201 ± 400
2028 ± 331
0.011
L flexion tW, Nm
2352 ± 441
2254 ± 414
2028 ± 342
0.057
*ANCOVA; data are given as mean ± SD; Nm: Newton-metre; R: right; L: left; tW: total work
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Discussion The main finding of this study is that aerobic and anaerobic performance and muscle strength may differ with respect to GNB3 C825T polymorphism in professional basketball players. This is the first report of a detailed, i.e. both aerobic and anaerobic, performance parameters together with isokinetic knee muscle power measurement results in different GNB3 C825T genotype groups, CC, CT, and TT. However, contrary to our initial hypothesis which stated that presence of the 825T allele is associated with a better physical performance, individuals carrying the 825T allele had significantly lower VO2peak in cardiopulmonary exercise tests. Indeed, previous studies reported an association of the GNB3 825T allele with lower VO2peak in obese Korean population (8) and in healthy African-American population (13). The former study was conducted in middle aged Korean men and found an association between C825T allele and lower VO2peak in obese participants only (8). The latter study included 91 healthy normotensive African-American university students and reported a significant independent association of the 825T allele with VO2peak (13). Our study group consisted of professional first division basketball players in whom the same polymorphism, i.e. TT genotype, was also associated with a lower VO2peak. There are possible explanations for the relationship of the 825T allele and peak oxygen consumption. First, there is a strong association between the presence of the 825T allele and the occurrence of a functionally active Gβ3s splice variant (11). Thus, signal transduction is enhanced via G proteins resulting in mobilization of metabolic substrates (7, 11). As we initially hypothesized in this study, this would be advantageous to athletes, since their performance is dependent on metabolic readiness of glucose, free fatty acids and other substrates to muscles. Second, enhanced G protein activation may cause enhanced Na+/H+ exchanger activity, which in turn may lead to increased sodium reabsorption from renal tubules and induce a state of volume expansion (5). Expansion of plasma volume has been suggested to reduce cardiovascular strain observed with exercise and improve exercise performance in hot environments. Recently, pre-exercise ingestion of a high-sodium beverage has been shown to increase plasma volume before exercise and be involved less thermoregulatory and perceived strain during exercise (22). Furthermore, most of the energy generated during exercise dissipates from the body by evaporative cooling through sweating. Sweating leads to loss of both water and electrolytes including sodium, potassium, calcium and iron. Exercise induced hyponatremia is a condition characterized by cramping, weakness, and dizziness. In more serious forms, cerebral and pulmonary oedema, coma and death may occur (16). Although the evidence showing the effects of sodium regulation on performance mainly comes from researches on endurance activity like marathon or in the heat condition, it is plausible to suggest that genetic tendency to sodium loading would also be beneficial particularly for orthostatic stressful exercise activities. And finally, Na+/H+ exchanger is a ubiquitously expressed ion transporter and it may also modulate chemoreceptor activity by a more effective removal of intracellular H+ ions. Exner et al. (6) studied hypercarbic and hypoxic ventilator responses in healthy volunteers showing different GNB3 genotype (CC, CT, TT). They have concluded that there is no significant difference in short-term hypercarbic and hypoxic ventilator control in young volunteers expressing different forms of GNB3 (6). However, chemoreceptor response during exercise may still be different from short-term resting conditions. Taken together, all three explanations are in favour of better physical performance with the 825T allele. The frequency of the presence of the 825T allele presence ranges among populations from 11% in the North Brazil population to 79% in black African population (19). In a Acta Physiologica Hungarica 101, 2014
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Turkish population that consisted of 138 individuals, CC, CT, and TT genotype frequencies were reported to be 38%, 48%, and 14%, respectively. Percentage of the 825T allele was 38% (19). In another study, the frequencies of the G protein b3 subunit C825T polymorphism in Turkish hypertensive and control groups were 17.7%, 59.3%, 23.0% and 32.9%, 48.8%, 18.3% (CC, CT, TT), respectively (1). In the present study, we assessed a selective group, i.e. first division basketball players where the observed GNB3 C825T genotype distribution was as follows: CC: 21/72 (29%); CT: 35/72 (49%); TT: 16/72 (22%). Eynon et al. (7) studied C825T polymorphism in 155 elite Israeli athletes and observed a significantly higher proportion of the TT genotype among endurance athletes (19%) compared with the sprinters (5%) and the controls (8.5%). However, in a more recent study by Ruiz et al. (15), the association between the GNB3 C825T polymorphism and elite athletic status was not corroborated in a larger study group that consisted of two different (Israel and Spain) ethnic/ geographic backgrounds. In the light of above considerations on genotype frequency distributions, it is not very clear whether the 825T allele is a significant determinant of physical performance. And we believe that the present study provides substantial contribution to the hypothesis of a relationship between GNB3 TT genotype and exercise performance. Contrary to what has initially been expected, both aerobic and anaerobic aspects of physical performance were lower in participants with the 825T allele. To our knowledge, this is the first study assessing both aerobic and anaerobic performances in relation with GNB3 polymorphism. Although peak power levels were comparable among different genotypes, participants expressing GNB3 TT genotype showed significantly higher power drop in Wingate anaerobic test (Table II). This may indicate that altered G protein activation and intracellular signalling may also interact with anaerobic pathways in muscle cells. Furthermore, we evaluated isokinetic leg muscle strength at different angle velocities of knee joint. We, again found that muscle strength for knee flexion and extension were lower in participants with 825T allele when compared to the CC genotype (Table III). There are several limitations of this study. First, we did not measure plasma sodium levels. This would have enabled us to test the positive sodium balance and volume expansion hypothesis for the superior exercise performance. However, our results have showed that the presence of the 825T allele is related to inferior rather than better exercise performance. In terms of group selection, our group of participants consisted of Caucasian basketball players and their number was relatively small. Therefore, one should be careful when extrapolating these results to other sports disciplines and African-Americans. Another limitation of the study is gender distribution. All participants were males, so female athletes may show some diversity from male athletes. In summary, we report lower aerobic and anaerobic exercise performances in first division basketball players expressing GNB3 TT genotype, and suggest that TT genotype of the GNB3 gene may be disadvantageous in achieving top-level physical performance. GNB3 gene polymorphism may be a promising avenue for genetic influences on athletic performance. This study also stresses that besides genes providing genetic aptitude for better performance, there may be gene variants that may predict lower athletic performance. Conflict of Interest None of the authors reported conflict of interest.
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GNB3 gene c.825C>T polymorphism and performance parameters in professional basketball players T Gülyaşar1, L Öztürk2, T Sipahi1, B Bayraktar3, G Metin3, İ Yücesir4, N Süt5 1
Faculty of Medicine, Department of Biophysics, Trakya University, Edirne, Turkey Faculty of Medicine, Department of Physiology, Trakya University, Edirne, Turkey 3 Faculty of Medicine, Department of Sports Medicine, Istanbul University, Istanbul, Turkey 4 Physical Education and Sports School, Istanbul University, Istanbul, Turkey 5 Faculty of Medicine, Department of Biostatistics, Trakya University, Edirne, Turkey 2
Received: April 10, 2013 Accepted after revision: November 18, 2013 This study has been conducted to determine whether mean values of peak oxygen consumption (VO2peak), anaerobic test parameters and knee isokinetic test measurements are different among guanine nucleotide-binding protein, beta3 (GNB3) genotype groups in a group of basketball players. Methods: Seventy-two healthy male (mean age, 22.9 ± 5.3 years) basketball players from the first division of national league participated. We studied GNB3 gene c.825C>T (rs5443) polymorphism, then divided the subjects into three groups as CC (n = 21), CT (n = 35), and TT (n = 16). Mean VO2peak, Wingate anaerobic test results, and isokinetic knee muscle strength measurements were compared among the genotype groups. Results: Mean VO2peak (60.1 ± 3.9; 56.7 ± 3.6; and 57.8 ± 3.3, respectively, p < 0.01), mean anaerobic minimum power (5.1 ± 0.4; 5.3 ± 0.5; and 4.4 ± 0.5 W/kg, respectively, p < 0.001), mean anaerobic power drop (57.0 ± 6.2; 54.2 ± 6.9; and 62.9 ± 5.3%, respectively, p < 0.001) were significantly different among the study groups, CC, CT, and TT. Individuals with TT genotype exerted lower performance in terms of isokinetic knee muscle strength. Conclusion: The presence of 825T-allele may impair athletic performance and may serve as a genetic marker of low capacity for athletic performance in male basketball players. Keywords: genetic polymorphism, GNB3, exercise capacity, VO2peak, isokinetic knee strength
TP-binding proteins (G-proteins), that composed of α- and βγ-subunits, are important signal transducers of intracellular signalling pathway. A polymorphism at position 825 (C→T) of the exon 10 of the GNB3 gene, which encodes the β3-subunit of heterotrimeric G-proteins has been described by Siffert et al. (20). The expression of 825T allele stems from alternative splicing of exon 9. This type of splicing eliminates 41 amino acids which in turn increases G-protein activation (20). This polymorphism was found to be positively associated with cardio-respiratory fitness in an obese group of women (11). There may be several explanations for this association of GNB3 gene 825T allele and cardio-respiratory fitness. First, the altered splicing may enhance Na+/H+ exchanger (NHE) activity. This ion transport system regulates electroneutral exchange of sodium and hydrogen ions (19, 21). NHE isoforms also play an important role in mediating Na+ reabsorption in the proximal tubule of nephron. Hence, an increased activity of renal NHE may result in higher Na+ reabsorption and induce a state of volume expansion. The expansion of circulatory volume has been suggested to improve both exercise performance and thermoregulation via reducing cardiovascular and heat strain occurring especially during endurance exercise. Recently, drinking high-sodium beverages
Corresponding author: Levent Öztürk, MD, Professor Department of Physiology, Trakya University, Faculty of Medicine, Edirne, Turkey Phone: +90-284-2357641 (ext. 1421); Fax: +90-284-2357652; E-mail: [email protected] 0231–424X/$ 20.00 © 2014 Akadémiai Kiadó, Budapest
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before exercise has been shown to increase plasma volume and increased exercise capacity in hot environment (22). On the other hand, exercise-associated hyponatremia, a severe and potentially life-threatening condition, is well-documented during prolonged exercise such as marathons and triathlons (9). Risk factors of exercise-associated hyponatremia include preexercise overhydration, overconsumption of fluids (> 1.5 L/h) during an athletic event, low body weight, female gender, exercise duration > 4 hours, and a hot environment (18). Second, Gβ3 subunit coded in individuals carrying 825T allele could provide some benefits on the regulation of hemodynamics (17) and on the mobilization of exercise energy substrates (4) via enhanced signal transduction through intracellular second messengers such as cAMP (11). Gβ3 subunit activates β-adrenergic receptors through phosphorylation by receptor kinase (10). Thus, activation of β-adrenergic receptors activates lipolysis from adipocytes (4). Altered regulation of substrate mobilization due to GNB3 gene polymorphism may lead to an advantage on exercise performance. Thus in the present study, we hypothesized that different genotypes encoding the GNB3 evoke phenotypes associated with altered aerobic and anaerobic exercise capacity and that the presence of c.825C>T may pose a genetic predisposition for a better exercise performance in professional male basketball players. Methods Subjects The healthy male Caucasian basketball players (mean age, 22.9 ± 5.3 years, n = 72) from the first division of national league enrolled to the study. All participants underwent a comprehensive physical examination, then maximal cardiopulmonary exercise test, Wingate anaerobic performance test and muscle strength tests with isokinetic dynamometer were planned with 3–5 day intervals. All tests were performed during pre-seasonal period. Ethical approval was obtained from local Ethics Committee before starting the study (Registration number: TUTFEK200829). This study was conducted in agreement with the Declaration of Helsinki, and written informed consent was obtained from all enrolled individuals before participating in this study. Cardiopulmonary exercise testing (CPET) The participants underwent cardiopulmonary exercise testing on a treadmill connected with a computer system. Resting data were collected for three minutes to perform baseline measurements before exercise. The device was calibrated before each study. A modified Bruce protocol (3) was used. We added a 3-minute stage 0 at 5% gradient. Continuous monitorization of oxygen uptake (VO2), ventilation (VE), and carbon dioxide production (VCO2) were performed via breath-by-breath analysis on a cardiopulmonary exercise testing system (Cortex Metalyzer 3B, Cortex Biophysik GmbH, Leipzig, Germany). Participants were forced verbally to their exercise limits by the supervising physician. Each test was terminated by subject’s voluntary termination or by achievement of two of the three following criteria: (1) a respiratory exchange ratio of 1.00 or higher, (2) heart rate reaching 85% of agepredicted maximal heart rate, and (3) subject fatigue. Peak oxygen consumption (VO2peak) was recorded. The respiratory gas exchange ratio (RER) was calculated as VCO2/VO2. Blood pressure was measured at rest, at the end of each 3-min stage, and at peak exercise by
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using a mercury sphygmomanometer. Continuous electrocardiographic recordings were also obtained. The predicted maximal heart rate (MaxHR) was derived from the formula 210 – 0.65 × age (years). Wingate anaerobic exercise test (WanT) This test (2) consists of a 30-second supra-maximal cycling against a pre-determined resistance load. All tests were performed on a cycle ergometer (Monark, Model 894-E, Sweden). The load was calculated for each subject based on their body weight (0.075 kg × kg body weight). Seat height was adjusted to each subject’s satisfaction and toe-clips were used to prevent the subject’s feet from slipping off the pedals. Before the start, 3 minutes warm-up pedalling was performed against a 30 watt (W) constant load. By the command ‘start’ the participant began pedalling as fast as possible until the end of the test period. Strong verbal motivation was given to subjects during the test. The peak power and mean power were calculated by using the data (2, 14). Isokinetic knee extension/flexion strength Strength of knee muscles was measured using an isokinetic dynamometer (Cybex Norm Lumex Inc., Ronkonkoma, NY, USA). The participating athletes were tested in a sitting position with hip flexed at 90°. Velcro-straps were used to secure the test limb to the chair. The lever arm was aligned to the axis of rotation of the knee. Data were corrected by the weight of the subject’s lower limb at 45° in order to account for the influence of the gravity effect torque. Three fixed angular velocities were used during the tests: 60°/sec, 180°/sec, 300°/sec. Isokinetic contractions were started with the knee flexed at 90° and continued through the full range of motion. Each participant completed four maximal contractions at 60°/sec, 180°/sec angular velocities, and only the best value (peak power) was retained for further statistical analysis. At 300°/sec angular velocity, subject performed 30 repeats and total work (sum of the 30 contractions, i.e. endurance) was recorded. All athletes were verbally encouraged to perform each test as maximally as possible. Visual feedback during the test was also provided. The same investigators (BB, GM) conducted the tests for all the subjects. A recovery of 1 minute was allowed between each maximal contraction, to minimize fatigue. Genotyping studies GNB3 c.825C>T (rs5443) single nucleotide polymorphism was studied. Genotyping protocol was designed according to our previous study (12) with minor modifications. In brief, DNA was extracted from blood samples of all participants by E.Z.N.A. (EaZy Nucleic Acid Isolation) blood DNA kits (Omega Bio-Tek, Doraville, GA, USA). Sense 5’-TGA CCC ACT TGC CAC CCG TGC-3’ and antisense 5’-GCA GCA GCC AGG GCT GGC-3’ primer pairs were used to enhance genomic DNAs. The 25 μl mixture prepared for polymerase chain reaction (PCR) included 250 ng of DNA, 0.5 nmol of each sense and antisense primers, 0.2 mM deoxynucleotide triphosphates (of each), 2 mM MgCl2, 75 mM Tris-HCl (pH 8.8), 20 mM (NH4)2SO4, 0.01% Tween 20, and 1.25 unit of Taq DNA polymerase (Fermentas Life Sciences). Initial denaturation step lasted 5 minutes at 94 °C, then 35 cycles of 94 °C for 1 minute, 69 °C for 45 seconds, and 72 °C for 1 minute were performed. Electrophoresis of PCR products were performed on a 2% agarose gel and read under UV light by 5 µg/mL ethidium bromide staining. In order to search for genotype of C825T polymorphism, 268 bp Acta Physiologica Hungarica 101, 2014
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PCR products were incubated with BseDI (SecI) restriction enzyme (Fermentas Life Sciences) at 55 °C for 16 hours. After incubation, the digests were electrophoresed on a 3% agarose gel and visualized under UV light by 5 µg/mL ethidium bromide staining. One band (268 bp) for homozygote TT genotype, two bands (116 and 152 bp) for homozygote CC genotype, and three bands (268, 152, and 116 bp) for heterozygote CT genotype were obtained (Fig. 1).
Fig. 1. Ethidium bromide-stained gel of digest products of C825T region of the GNB3 gene shows the CT genotype (268, 152, and 116 bp; samples 1, 4, and 5), the CC genotype (152 bp and 116 bp; sample 7), and the TT genotype (268 bp; samples 2, 3, and 6). Lane 50 bp is a size marker (GeneRuler 50 bp DNA Ladder-Fermentas Life Sciences)
Statistical analysis Data are presented as means (± standard deviation, SD) unless otherwise indicated. The following a priori null hypotheses were tested: mean values of peak oxygen consumption (VO2peak), Wingate anaerobic test parameters and knee extension/flexion isokinetic test results are not different among the genotype groups, i.e. CC, CT, and TT. The test for HardyWeinberg equilibrium was performed by comparing observed versus expected genotype frequencies using a chi-square test. Comparisons between groups were made by one-way ANOVA, and then post-hoc Tukey test was used when significantly differences were obtained due to homogeneity of variances. To control significant covariates (i.e. BMI) among the groups, we also performed Covariance Analysis (ANCOVA). Bonferroni post-hoc test was used when significant differences were obtained. Null hypotheses were rejected and significant statistical differences assumed with an alpha error of p < 0.05. Results Genotype frequencies were in Hardy-Weinberg equilibrium in the whole study group. The whole group genotype frequencies for CC, CT, and TT were 21 (29%), 35 (49%) and 16 (22%), respectively. Demographic, anthropometric characteristics of the enrolled individuals are presented in Table I. Body mass index (BMI) was significantly different (p = 0.005), whereas age was similar among the genotype groups (p = 0.593). Percent body fat, resting metabolic rate, and resting systolic and diastolic blood pressure values were also comparable
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among the genotype groups (see Table I for details). Aerobic and anaerobic exercise test parameters are given in Table II. Peak oxygen consumption was significantly lower in individuals with T allele. Post hoc analysis showed that the CC group had significantly higher VO2peak levels than the CT group (pairwise comparison p < 0.01), whereas pairwise comparisons of groups CT vs TT and CC vs TT showed no significant differences in terms of VO2peak levels (Table II). Wingate anaerobic test results varied in genotype subgroups. ANCOVA test showed that minimum power and percent power drop were significantly different among the groups (Table II). Pairwise comparisons showed that the TT group had lower minimum power values (CC vs TT, p < 0.001; CT vs TT, p < 0.001) and higher power drop levels (CC vs TT, p < 0.05; CT vs TT, p < 0.001) than the CC and CT groups. Anaerobic test results revealed that there were no significant differences between the CC and CT groups in all parameters. Peak power and average power levels obtained during Wingate test were comparable among the three groups. Table III shows isokinetic knee muscle strength test results with respect to GNB3 genotypes. In general, T allele carriers had lower isokinetic muscle strength. Test results at 60°/sec and 180°/sec velocities mainly showed peak power levels of muscles, whereas the results at 300°/sec showed endurance of muscles during knee extension and flexion. Peak power levels at 60°/sec during flexion were higher in the CC group than in the TT group (right flexion CC vs TT, p = 0.012; left flexion CC vs TT, p = 0.017). Peak power levels at 180°/sec during knee flexion were higher in the CC group than in the CT and TT groups (right flexion CC vs CT, p = 0.020; CC vs TT, p = 0.001; left flexion CC vs CT, p > 0.05; CC vs TT, p = 0.030), but muscle peak power at 180°/sec during extension failed to reach statistically significant level (Table III). In terms of muscle endurance (30 repeats at 300°/sec) all study groups were comparable except right flexion values of CC vs TT (p = 0.009). Table I. The characteristics of the basketball players according to GNB3 c.825C>T genotypes Characteristics
Genotype CC (n = 21)
CT (n = 35)
TT (n = 16)
p*
Demographic Mean age, y
0.593
22.6 ± 5.4
23.6 ± 5.6
22.1 ± 4.8
Anthropometric BMI, kg/m2
22.4 ± 1.6
23.7 ± 1.4
23.2 ± 1.4
0.005
Fat ratio, %
7.1 ± 2.0
7.5 ± 2.1
9.0 ± 3.2
0.184
RMR, kcal
1964 ± 169
1901 ± 116
1883 ± 157
0.445
Resting blood pressures
SBP, mmHg
108.8 ± 6.6
110.0 ± 8.5
110.0 ± 8.7
0.852
DBP, mmHg
68.5 ± 7.2
70.1 ± 6.3
69.0 ± 6.3
0.671
*ANOVA; data are given as mean ± SD; BMI: body mass index; RMR: resting metabolic rate; SBP: systolic blood pressure; DBP: diastolic blood pressure
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Table II. The parameters of aerobic and anaerobic exercise tests according to GNB3 c.825C>T genotypes Test parameters
Genotype
p*
CC
CT
TT
60.1 ± 3.9
56.7 ± 3.6
57.8 ± 3.3
0.007
196 ± 7
195 ± 7
196 ± 8
NS
CPET VO2peak, ml/kg/min Pulsepeak, n/min
65.2 ± 5.1
67.2 ± 5.4
67.8 ± 7.0
NS
SBPpeak, mmHg
DBPpeak, mmHg
184.7 ± 11.1
184.4 ± 8.7
181.8 ± 9.8
NS
Recovery, sec
127.3 ± 11.2
126.5 ± 13.8
130.0 ± 12.3
NS
Aerobic time, sec
620.0 ± 62.8
597.8 ± 57.1
617.9 ± 55.6
NS
Anaerobic time, sec
284.4 ± 60.4
263.6 ± 56.9
259.5 ± 42.5
NS
Total run time, sec
904.4 ± 66.1
861.4 ± 64.5
877.4 ± 49.7
NS
Peak power, W/kg
12.1 ± 1.3
11.6 ± 1.3
12.0 ± 1.1
NS
Min power, W/kg
5.1 ± 0.4
5.3 ± 0.5
4.4 ± 0.5
< 0.001
Average power, W/kg
8.3 ± 0.6
7.9 ± 0.6
7.9 ± 0.4
NS
Power drop, %
57.0 ± 6.2
54.2 ± 6.9
62.9 ± 5.3
< 0.001
WanT
*ANCOVA; data are given as mean ± SD; CPET: Cardiopulmonary exercise test; VO2peak: peak oxygen consumption; DBPpeak: peak diastolic blood pressure; SBPpeak: peak systolic blood pressure; WanT: Wingate anaerobic exercise test; NS: not significant
Table III. The parameters of isokinetic knee strength test according to GNB3 c.825C>T genotypes Test parameters
Genotype CC
CT
TT
p*
Test at 60º/sec
R extension, Nm
313.1 ± 30.5
287.3 ± 35.9
285.1 ± 49.4
0.233
L extension, Nm
309.3 ± 30.4
289.9 ± 35.8
281.9 ± 42.2
0.182
R flexion, Nm
234.3 ± 21.4
214.5 ± 33.6
204.3 ± 17.9
0.014
L flexion, Nm
230.5 ± 14.9
210.8 ± 33.2
203.1 ± 24.4
0.017
Test at 180º/sec
R extension, Nm
200.0 ± 19.1
188.7 ± 22.3
181.9 ± 29.9
0.134
L extension, Nm
186.1 ± 20.2
185.0 ± 26.8
180.1 ± 32.2
0.774
R flexion, Nm
175.1 ± 15.2
156.1 ± 19.7
146.4 ± 26.8
0.001
L flexion, Nm
162.4 ± 16.9
153.1 ± 24.4
141.3 ± 25.8
0.035
Test at 300º/sec
R extension tW, Nm
2649 ± 394
2426 ± 425
2373 ± 410
0.108
L extension tW, Nm
2275 ± 379
2382 ± 369
2350 ± 329
0.713
R flexion tW, Nm
2479 ± 488
2201 ± 400
2028 ± 331
0.011
L flexion tW, Nm
2352 ± 441
2254 ± 414
2028 ± 342
0.057
*ANCOVA; data are given as mean ± SD; Nm: Newton-metre; R: right; L: left; tW: total work
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Discussion The main finding of this study is that aerobic and anaerobic performance and muscle strength may differ with respect to GNB3 C825T polymorphism in professional basketball players. This is the first report of a detailed, i.e. both aerobic and anaerobic, performance parameters together with isokinetic knee muscle power measurement results in different GNB3 C825T genotype groups, CC, CT, and TT. However, contrary to our initial hypothesis which stated that presence of the 825T allele is associated with a better physical performance, individuals carrying the 825T allele had significantly lower VO2peak in cardiopulmonary exercise tests. Indeed, previous studies reported an association of the GNB3 825T allele with lower VO2peak in obese Korean population (8) and in healthy African-American population (13). The former study was conducted in middle aged Korean men and found an association between C825T allele and lower VO2peak in obese participants only (8). The latter study included 91 healthy normotensive African-American university students and reported a significant independent association of the 825T allele with VO2peak (13). Our study group consisted of professional first division basketball players in whom the same polymorphism, i.e. TT genotype, was also associated with a lower VO2peak. There are possible explanations for the relationship of the 825T allele and peak oxygen consumption. First, there is a strong association between the presence of the 825T allele and the occurrence of a functionally active Gβ3s splice variant (11). Thus, signal transduction is enhanced via G proteins resulting in mobilization of metabolic substrates (7, 11). As we initially hypothesized in this study, this would be advantageous to athletes, since their performance is dependent on metabolic readiness of glucose, free fatty acids and other substrates to muscles. Second, enhanced G protein activation may cause enhanced Na+/H+ exchanger activity, which in turn may lead to increased sodium reabsorption from renal tubules and induce a state of volume expansion (5). Expansion of plasma volume has been suggested to reduce cardiovascular strain observed with exercise and improve exercise performance in hot environments. Recently, pre-exercise ingestion of a high-sodium beverage has been shown to increase plasma volume before exercise and be involved less thermoregulatory and perceived strain during exercise (22). Furthermore, most of the energy generated during exercise dissipates from the body by evaporative cooling through sweating. Sweating leads to loss of both water and electrolytes including sodium, potassium, calcium and iron. Exercise induced hyponatremia is a condition characterized by cramping, weakness, and dizziness. In more serious forms, cerebral and pulmonary oedema, coma and death may occur (16). Although the evidence showing the effects of sodium regulation on performance mainly comes from researches on endurance activity like marathon or in the heat condition, it is plausible to suggest that genetic tendency to sodium loading would also be beneficial particularly for orthostatic stressful exercise activities. And finally, Na+/H+ exchanger is a ubiquitously expressed ion transporter and it may also modulate chemoreceptor activity by a more effective removal of intracellular H+ ions. Exner et al. (6) studied hypercarbic and hypoxic ventilator responses in healthy volunteers showing different GNB3 genotype (CC, CT, TT). They have concluded that there is no significant difference in short-term hypercarbic and hypoxic ventilator control in young volunteers expressing different forms of GNB3 (6). However, chemoreceptor response during exercise may still be different from short-term resting conditions. Taken together, all three explanations are in favour of better physical performance with the 825T allele. The frequency of the presence of the 825T allele presence ranges among populations from 11% in the North Brazil population to 79% in black African population (19). In a Acta Physiologica Hungarica 101, 2014
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Turkish population that consisted of 138 individuals, CC, CT, and TT genotype frequencies were reported to be 38%, 48%, and 14%, respectively. Percentage of the 825T allele was 38% (19). In another study, the frequencies of the G protein b3 subunit C825T polymorphism in Turkish hypertensive and control groups were 17.7%, 59.3%, 23.0% and 32.9%, 48.8%, 18.3% (CC, CT, TT), respectively (1). In the present study, we assessed a selective group, i.e. first division basketball players where the observed GNB3 C825T genotype distribution was as follows: CC: 21/72 (29%); CT: 35/72 (49%); TT: 16/72 (22%). Eynon et al. (7) studied C825T polymorphism in 155 elite Israeli athletes and observed a significantly higher proportion of the TT genotype among endurance athletes (19%) compared with the sprinters (5%) and the controls (8.5%). However, in a more recent study by Ruiz et al. (15), the association between the GNB3 C825T polymorphism and elite athletic status was not corroborated in a larger study group that consisted of two different (Israel and Spain) ethnic/ geographic backgrounds. In the light of above considerations on genotype frequency distributions, it is not very clear whether the 825T allele is a significant determinant of physical performance. And we believe that the present study provides substantial contribution to the hypothesis of a relationship between GNB3 TT genotype and exercise performance. Contrary to what has initially been expected, both aerobic and anaerobic aspects of physical performance were lower in participants with the 825T allele. To our knowledge, this is the first study assessing both aerobic and anaerobic performances in relation with GNB3 polymorphism. Although peak power levels were comparable among different genotypes, participants expressing GNB3 TT genotype showed significantly higher power drop in Wingate anaerobic test (Table II). This may indicate that altered G protein activation and intracellular signalling may also interact with anaerobic pathways in muscle cells. Furthermore, we evaluated isokinetic leg muscle strength at different angle velocities of knee joint. We, again found that muscle strength for knee flexion and extension were lower in participants with 825T allele when compared to the CC genotype (Table III). There are several limitations of this study. First, we did not measure plasma sodium levels. This would have enabled us to test the positive sodium balance and volume expansion hypothesis for the superior exercise performance. However, our results have showed that the presence of the 825T allele is related to inferior rather than better exercise performance. In terms of group selection, our group of participants consisted of Caucasian basketball players and their number was relatively small. Therefore, one should be careful when extrapolating these results to other sports disciplines and African-Americans. Another limitation of the study is gender distribution. All participants were males, so female athletes may show some diversity from male athletes. In summary, we report lower aerobic and anaerobic exercise performances in first division basketball players expressing GNB3 TT genotype, and suggest that TT genotype of the GNB3 gene may be disadvantageous in achieving top-level physical performance. GNB3 gene polymorphism may be a promising avenue for genetic influences on athletic performance. This study also stresses that besides genes providing genetic aptitude for better performance, there may be gene variants that may predict lower athletic performance. Conflict of Interest None of the authors reported conflict of interest.
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