The structural arrangement and conservation of muscle contractile apparatus depend on protein complexes that bind sarcomeres together. In this context, the α-actinin is the prevailing omnipresent protein for this function (38). This protein makes up the Z-line of the sarcomere (16), which creates crossed connections to anchor actin-actin. Therefore, it can be considered an important structural element when generating and transmitting muscle contractile strength and maintaining the myofibril matrices (7,14,36,37).
North et al. (24) identified in the α-actinin-3 (ACTN3) gene the changing of the C nucleotide by T in the position/1.747 of the exon 16, resulting in a mutation in the conversion of the arginine amino acid in a premature stop codon at the 577 residue (R577X) (32). The variant R577X results in 2 alleles of the gene ACTN3 in humans, a functional R allele and a null one presenting the XX genotype, not expressing the ACTN3 in its skeletal muscle (16).
ACTN3 and the highly homologous protein α-actinin-2 (ACTN2) are the major components of mammalian skeletal muscle Z-disks and function as cross-linkers of actin thin filaments (33). In human muscle, ACTN2 is expressed in all muscle fibers, whereas ACTN3 is restricted to the fast-twitch muscle fibers (type II) responsible for rapid force generation (15–19,21,32,37). ACTN3 may confer type II fibers with a higher capacity for the absorption/transmission of force at the Z-line during rapid contractions. Furthermore, this protein promotes a higher protection against muscle damage (14,16,21,26,37).
Studies in animal models (5,6,18,19,27) reported that an ACTN3 knockout mouse model of α-actinin-3 deficiency shows a decreased activity in the anaerobic glycolytic pathway and an increased activity in the aerobic oxidative pathway. A study of athletes at the Australian Institute of Sport (40) found that those engaged in sprint or power activities had a lower incidence of ACTN3 deficiency than the general population (6% compared with 18%). In fact, among Olympic sprint athletes, there were no cases of ACTN3 deficiency.
The effect of the ACTN3 genotype has mainly been studied in elite athletes because of the fact that the influence on muscle function will be most readily observable at the extremes of human performance. In this regard, although soccer is considered as a long-duration exercise (2), it is well-known that matches are won in high short-effort durations (sprinting or jumping). Therefore, besides technical and tactical skills, muscular strength and “explosive” leg power are the most important factors contributing to successful performance during elite soccer competitions. As ACTN3 is a very important factor for producing high-power and high-velocity muscle contractions (16), the ACTN3 R577X polymorphism would be an interesting tool to explain, partly, the individual variations in soccer performance.
To examine the effect of the ACTN3 R577X polymorphism on human performance continuum, we studied the association of the ACTN3 R577X polymorphism with strength, speed, and endurance performance in a group of professional male soccer players. It must be noted that, at this point, this is the first study associating this gene polymorphism with performance in top elite soccer players. Therefore, the purpose of this study was to compare the performance of soccer players in strength, speed, and endurance tests for different genotype groups of ACTN3 (XX, RX, and RR). We hypothesized that RR soccer players would have higher scores in speed and strength tests in comparison with the XX group. Inversely, XX individuals would have the highest performance in the endurance test.
Experimental Approach to the Problem
The assessment procedures outlined below occurred over the course of 2 days, in a testing week at the early season training. All tests occurred at morning around 09:00 AM The subjects performed 4 physical tests (squat jump [SJ], countermovement jump [CMJ], 30-m sprint test, and Yo-Yo endurance test) for the assessment of their strength, speed, and endurance capabilities. Participants were refrained from taking any medication or dietary supplements with anti-inflammatory action for two weeks before the study. Each player was tested separately, instructed, and verbally encouraged to give maximal effort on all tests. The volunteers were instructed to be abstemious and for not doing any physical activity for at least 24 hours before the test and not to drink caffeine-containing beverages on the day of the tests. The subjects could withdraw from the study at any time. All the players were familiar with the tests, which were part of their routine. Blood samples were collected at the day before the first physical test.
As can be seen, muscle power was assessed in this article by means of jumping and sprinting tests, which are naturally occurring in multijoint movements in humans that involve the coordinated participation of the majority of the lower limb muscles (32,33). Indeed, we believe that this is a differential aspect of our study in comparison with previous research in the field of genetics and exercise-related phenotypes that used other tests for muscle power assessment, as maximal concentric muscle work during single-joint movements (e.g., elbow flexor contractions) at relatively low angular velocities (≤120°·s−1) (35).
Two hundred male professional players of a Brazilian first division soccer team that maintained regular training sessions and competed in official events organized by the Brazilian Soccer Federation participated in this study (Table 1). This study was approved by the Research Ethics Committee of the Federal University of Minas Gerais (ETIC-291/09) and is in compliance with all the norms established by the National Health Council (Res. 196/96) regarding research with human beings. All procedures, possible risks, and benefits of the study were explained to the volunteers before they signed the informed written consent to participate in the experiment (Table 1).
Testing was performed outdoors and on artificial turf. A standardized warm-up of 10–15 minutes was performed including jogging, shuffling, sprinting, multidirectional movements, and dynamic stretching exercises.
In a 30-m sprint test, the speed of the players was defined in 10- (V10), 20- (V20), and 30-m (V30) paths, in the field, with electronic measurements using photocells timing (placed nearly the hip of the volunteers, 1 m from the ground), with the accuracy of 0.001 second, located at 0, 10, and 30 m of the path. All the photocells were connected to a computer with the specific software (Multisprint; Hidrofit Ltda; Belo Horizonte, Brazil) for speed analysis. Each subject had 3 attempts separated by approximately 3 minutes to ensure full recovery. Subjects commenced each sprint from a standing (static) position in which their front foot was placed 50 cm behind the start line. Subjects decided themselves when to start each run with the time being recorded when the subject intercepted the photocell beam. All the volunteers were instructed to sprint as fast as possible through the distance. The trial with the best 30-m sprint time was chosen for analysis of the sprint times for 10, 20, and 30 m.
Separated by at least 10 minutes from the previous test, 2 performance tests, according to Bosco (15,16), were included to evaluate the explosive power of the leg extensor muscles on a force platform connected with a digital timer (0.001 second) (Jump Test; Brazil). This system determines flight time, which is converted to jump height using the following equation:
(where g = acceleration as a result of gravity and t = flight time). The tests used were SJ and CMJ. Subjects performed the CMJ and SJ with their hands kept on their hips throughout the jumps. During the SJ, with knees at 90° flexion, the subjects were instructed to execute a maximal vertical jump and were not allowed to use any downward movement before the maximal vertical jump. The force curves were inspected to verify no downward movements before the vertical jump. During the CMJ, the angular displacement of the knees was standardized so that the subjects were required to bend their knees to approximately 90° and then rebound upward in a maximal vertical jump. Each subject had 4 attempts interspersed with approximately a 1.5-minute rest between each jump in both the SJ and CMJ. The best jump from each subject was used in the data analysis. Performance using a timing mat can be influenced by body position during flight; therefore, the participants were instructed and carefully observed to maintain straight legs while airborne. If the knees were bent or raised, the trial was discarded, and the participant was given another attempt after a rest period. Because jumping without arm action is not common in sport, technique was demonstrated to each participant, followed by 2 submaximal attempts.
Although both tests are used to determine muscle power performance, stationary jumps and running sprints are determined by different factors. The critical aspect during running sprints (owing to the short duration of the foot contact on the ground) is the rate of force development, which in turn is determined by many factors such as muscle fiber type, synchronization of motor units, tendon stiffness, or lean mass of lower extremities (25). The elastic properties of tendons can also influence jump ability, at least in the case of CMJ. Compared with a stiffer muscle tendon complex (MTC), people with a more compliant MTC should be more efficient in using elastic strain energy during jumps (4).
Three days after the tests to ascertain vertical jump and sprint test, the players undertook an endurance test to estimate their maximal oxygen consumption (V[Combining Dot Above]O2 max) after a thorough warm-up. The V[Combining Dot Above]O2 max was evaluated by the Yo-Yo endurance test (2). This test is specific for soccer players and intermittent sports (12,13) in which the distance covered in intermittent disposition has straight relation to the aerobic ability of the athletes (3). Even though field tests are not considered gold standard to determine physiological variables, it has been suggested that relevant, specific, and operative field tests should be used in team sports (1,10,11,34). Two attempts separated by 4 hours were made by each volunteer.
Weather conditions (dry temperature, moist temperature, bulb temperature, and index of wet bulb and globe temperature [WBTG]) of the 2 days were recorded through a digital thermohygrometer (Instrutherm HT-260, Instrutherm, São Pualo, Brazil).
Venous blood samples (approximately 10 ml) were collected at the day before the first physical test from the antecubital arm vein and collected in tubes with separator gel (Venoject II; Terumo Europe, Leuven, Belgium). Thereafter, the serum was isolated through centrifugation (1,500g, 4° C for 15 minutes) and frozen at −80° C.
ACTN3 R577X Single Nucleotide Polymorphism Genotyping
The extraction of genomic DNA from the peripheral blood samples was performed according to literature protocol, using proteinase K followed by salt precipitation (20). A DNA fragment carrying the exon 16 from the ACTN3 gene was amplified from the genomic DNA, and the following initiators were used: 5′-CTGTTGCCTGTGGTAAGTGGG-3′; reverse, 5′TGGTCACAGTATGCAGGAGGG-3′, correlated to the intronic sequences adjacent. The polymerase chain reaction (PCR) reactions presented a final volume of 25 μl, with 10 mM Tris, pH 8.4, 50 mM KCl, 1.75 mM MgCl2, 0.1% Triton X-100, 0.2 mM of each deoxynucleotidetriphosphates (dNTP) (Invitrogen, Carlsbad, CA, USA), 1 U Taq DNA polymerase (Phoneutria Biotecnologia, Belo Horizonte, Brazil), and 1.0 μM of each initiator (Sinapse Biotecnologia, São Paulo, Brazil), using approximately 100 ng of genomic DNA as mold. The amplification program consisted of an initial denaturation at 94° C for 5 minutes, followed by 30 cycles, comprising 94° C for 1 minute, 64° C for 1 minute, and 72° C for 1 minute, with a final extension of 72° C for 5 minutes. The R577X alleles (codons CGA and TGA) were distinguished by the presence (577X) or absence (577R) of a restriction site of the enzyme DdeI (21). After amplification by PCR, 1 μl of the product of PCR was digested with 20 U of the enzyme DdeI in a final volume of 15 μl. The reactions were incubated overnight at 37° C. Later, the fragments digested were separated by electrophoresis with polyacrylamide gel 8%, stained with silver nitrate solution (29). The ACTN3 577R allele generates fragments in 205 and 86 base pairs (bp), whereas the ACTN3 577X allele generates fragments in 108, 97, and 86 bp (40) (Figure 1).
After verifying the regularity of the data by the Kolmogorov-Smirnov test, the 1-way analysis of variance followed by the post hoc Tukey's test was used to compare the athletes' performances in each test for the different genotype groups. The significance level adopted was p < 0.05, and the data were presented as mean and SD.
The sample size was measured with the DIMAN 1.0 software (EGK, São Paulo, Brazil). From a sample of 2,400 Brazilian professional soccer players, 95% of confidence interval was used, allowing 7% maximal error and an interest proportion of 36.7%. Therefore, based on these conditions, 170 athletes were needed in this research. Moreover, because of the possible data loss, an addition of at least 10% was done to this number. The statistical equation used to calculate the sample size was
. A statistical power of 80% and the highest coefficient of variation among the variables of the present study were used to calculate the size of the sample. Based on the highest variability of the CMJ, at least 8 volunteers per genetic group would be needed to perform this investigation.
The performances from the physical tests were used to determine the intraclass correlation coefficient (ICC) (39) and the SEM of the sample.
Figures 2–4 presents the test performances for the different genotypes evaluated.
It has been noted that RR individuals presented lower time rates for the 10-m path, compared with XX individuals (p < 0.05). The RR individuals also presented lower time rates for the 20- and 30-m paths, compared with RX and XX genotypes (p < 0.05). In jump tests, the RR and RX genotypes presented higher data compared with XX genotypes (p < 0.05). Regarding the aerobic test, the XX genotypes presented higher values for V[Combining Dot Above]O2 max, compared with the RR genotype group (p < 0.05).
The ICC and SEM of the results from each physical test were, respectively, V10 (0.98; 2.5%), V20 (0.96; 2.9%), V30 (0.96; 2.8%), SJ (0.94; 3.5%), CMJ (0.95; 3.1%), and Yo-Yo endurance test (0.92; 3.6%).
The environmental conditions during the 2 days of tests were 25.98 ± 1.91° C for dry temperature, 22.96 ± 1.11° C for moist temperature, 31.93 ± 2.36° C for bulb temperature, and 25.03 ± 1.33° C for index of WBTG.
The main objective of this study was to recognize better performance in speed and strength tests for individuals with RR and RX genotypes and better endurance performance for XX genotypes. Indeed, our results indicated that XX players presented significantly higher V[Combining Dot Above]O2 max compared with the RR genotype group. Inversely, the RR individuals presented significantly lower time rate for 10-, 20-, and 30-m paths and higher score in the jump tests, compared with the XX group.
Despite the conflicting studies in the literature, these findings were expected given the role of α-actinin-3 on skeletal muscle phenotypes (16). The structural phenotype associated with the deficiency of ACTN3 is most likely the result of functional differences between the ACTN2 and ACTN3 that, in part, are attributable to its interactions with Z-line proteins such titin. The Z-line has the function of maintenance of the integrity of the contractile apparatus during intense muscle effort in type II fibers. Titin isoforms of variable sizes are major determinants of passive stiffness or tension of the sarcomere, particularly in fast-twitch muscles. Interactions of these proteins with ACTN2 results in altered structural and elastic properties of the sarcomere compared with the ACTN3 (33). Therefore, these changes could explain, partly, the reduction of performance in XX individuals in activities in which elastic components of the stretch-shortening cycle are present, such as sprint and power.
Corroborating with our findings, Moran et al. (22) evaluated a group of 525 men and 467 women and verified that individuals with ACTN3/RR and RX genotypes were faster in speed test than the XX group. Also aiming at assessing the effect of ACTN3 on strength, Vincent et al. (36) evaluated 90 individuals grouped in ACTN3 expression (RR, RX, and XX) in different speed tests in an isokinetic machine. The authors verified that RR individuals presented better results in torque of higher angular speeds compared with XX individuals.
On the other hand, recently, Ruiz et al. (28) collected the genotypes of 66 top male and female volleyball athletes and compared them with the results of power and strength tests (SJ and CMJ). Contradicting the present study, the authors suggested that the ACTN3 expression does not directly affect power and strength values in volleyball athletes. In the study above mentioned, the author used a jump test to verify the power of the players. Differently from soccer, the ability to jump in volleyball is crucial to performance. It is present in the defensive system (block), in the offensive system, and also in the serves. Because the majority of the volleyball players must have this ability well-developed to succeed, their training is focused on this specific task. Thus, this genetic potential might not be the differential in this respective phenotype for volleyball by the assessment of jumping ability. In addition, other studies did not find any significant relationship between the ACTN3 R577X polymorphism and sprint/power or endurance performance (30,31). However, this discrepancy of the results among our present investigation and the studies above mentioned may be the result of the different types of tests used and the variability of the sample traits, ranging from professional triathlon athletes to healthy nonathletes. It must be noted that because athletes participate in specific trainings, higher capability can be identified.
Despite being considered as a long-duration exercise, soccer is also considered an intermittent and high-intensity activity (4). It is well-known that a soccer game is won in high short-effort durations, such as jumping, sprinting, and kicking. Because genetic factors may interfere in sports performance by means of the adaptability to the training (23), special attention must be given for the possible implications of our findings in the training routine of soccer athletes. Although inconclusive studies were found regarding nonathletes (7,8), the same did not occur with trained individuals. In a recent article, Norman et al. (23) performed 2 isokinetic tests with 1 week apart in moderately to well-trained subjects. They found that the RR group had a greater increase in peak torque than the XX individuals after the second evaluation, suggesting that the ACTN3 genotype could modulate the response to training and induce differences in performance between XX and RR genotype when specific exercise bouts are chronically imposed. As observed by Chan et al. (5), muscles that lack ACTN3 displays a shift from fast-twitch toward slow-twitch characteristics. In their work, the reduction of the enzyme glycogen phosphorylase in ACTN3-knockout mouses reduced the energy supply by anaerobic metabolism, minimizing the uptake of Ca2+ to the sarcoplasmic reticulum and causing a larger relaxation time, smaller diameter, and higher oxidative enzyme levels. As previously mentioned in this work, other study also found different mechanical and functional properties associated with the deficiency of ACTN3 (33). Therefore, taking all these studies together with the “principle of individuality” and, because professional soccer players already have a large training background and are at their extreme of performance, it seems appropriate to think that the same training content should not be performed by all individuals (RR, RX, and XX) at this stage. It should be noted that at the elite level, any encountered difference can be the determining factor between winning and losing, between the gold and fourth place. The major difficulty in this area is determining whether measured or observed differences in the performance of elite athletes are statistically and clinically (real world) significant. Hopkins et al. (9) argue that enhancement of performance as small as 0.3–0.4 in the coefficient of variance of performance is important for the high-level athletes. Therefore, the small but significant differences in the present study should be considered when planning the training program of soccer players. Their training program should be adjusted and be more efficient as possible, enhancing their genetic predisposal qualities, with RR and RX players performing higher sprint and power training than the XX group and the latter one performing higher aerobic training.
In summary, it is concluded that soccer players with ACTN3/RR and RX genotypes present better strength capability, compared with ACTN3/XX individuals. Inversely, XX individuals present better capability for endurance.
The present study found that ACTN3 genotype can influence the capability of soccer players. ACTN3/RR and RX individuals present better strength capability than ACTN3/XX individuals. Inversely, the latter presents better capability for endurance. As professional soccer players already have a large training background and are at peak performance, it seems appropriate to think that the same training content should not be performed by all individuals (RR, RX, and XX) at this stage. In top-level competition, the minimal difference in performance is critical to succeed. By knowing the ACTN3 genotype, the training program can be designed for an individual athlete, enhancing the sprint and power training to RR and RX players and endurance training to the XX group.
The authors thank the athletes involved with this study; to Elias, Marcilene and Luciano Cappetinne, for their proficiency and promptitude; to the São Sebastião Laboratory staff for welcoming our team. Supported by CNPQ 301074/2008-9. 475547/2007-1 CAPES/FAPEMIG APQ-5023-5.01-07/PRONEX/CRUZEIRO ESPORTE CLUBE.
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