ACTN3 GENOTYPE IS ASSOCIATED WITH TESTOSTERONE LEVELS OF ATHLETES

α-Actinin-3 (ACTN3) has been proposed to regulate skeletal muscle differentiation and hypertrophy through its interaction with the signalling protein calcineurin. Since the inhibition of calcineurin potentiates the production of testosterone, we hypothesized that α-actinin-3 deficiency (predicted from the ACTN3 XX genotype) may influence serum levels of testosterone of athletes. Objective: To investigate the association of ACTN3 gene R577X polymorphism with resting testosterone levels in athletes. Methods: A total of 209 elite Russian athletes from different sports (119 males, 90 females) were genotyped for ACTN3 gene R577X polymorphism by real-time PCR. Resting testosterone was examined in serum of athletes using enzyme immunoassay. Results: The mean testosterone levels were significantly higher in both males and females with the ACTN3 R allele than in XX homozygotes (males: RR: 24.9 (5.7), RX: 21.8 (5.5), XX: 18.6 (4.9) ng · mL-1, P = 0.0071; females: RR: 1.43 (0.6), RX: 1.21 (0.71), XX: 0.79 (0.66) ng · mL-1, P = 0.0167). Conclusions: We found that the ACTN3 R allele was associated with high levels of testosterone in athletes, and this may explain, in part, the association between the ACTN3 RR genotype, skeletal muscle hypertrophy and power athlete status.


INTRODUCTION
α-Actinins are important structural components of the Z-membrane, where they form crosslinks between the thin actin filaments and stabilize the muscle contractile apparatus [29]. Expression of α-actinin-3 (ACTN3) is limited to fast muscle fibres responsible for generating force at high velocity. A common genetic variation in the ACTN3 gene that results in the replacement of an arginine (R) with a stop codon at amino acid 577 (C-to-T transition in exon 16; rs1815739; R577X) had been identified. The X allele contains a sequence change that completely prevents the production of functional α-actinin-3 protein. α-Actinin-3 deficiency (the ACTN3 XX genotype) is common in humans (14-20%) and reduces strength, muscle mass, and fast-twitch fibre diameter, but increases the metabolic efficiency of skeletal muscle and the proportion of slow-twitch muscle fibres [2,16,17,22,26,31].
Several case-control studies have reported that ACTN3 RR genotype (or R allele) is over-represented or ACTN3 XX genotype is underrepresented in strength/sprint athletes in comparison with controls [1,2,4,9,10,19,23,30]. The hypothesis that the ACTN3 R allele may confer some advantage in power performance events has been ACTN3 GENOTYPE IS ASSOCIATED WITH TESTOSTERONE LEVELS OF ATHLETES supported by several cross-sectional studies in non-athletes [3,7,8,12,18,27].
Sarcomeric α-actinins bind to the calsarcins [11], which interact with calcineurin, a signalling factor that plays a role in the specification of muscle fibre type and myocardium hypertrophy [5,6,24].
Recently, Henesy et al. [13] found that the inhibition of calcineurin potentiates atrial natriuretic peptide induced testosterone production.
Interestingly, Pimenta et al. [20] found that immediately after eccentric training, soccer players with the ACTN3 RR and RX genotypes (n=28) presented higher levels of testosterone compared to XX homozygotes (n=9).
We therefore hypothesized that α-actinin-3 deficiency may influence resting levels of testosterone of athletes. The aim of the study was to investigate the association of ACTN3 gene polymorphism with testosterone levels in male and female athletes.

MATERIALS AND METHODS
The procedures followed in the study were conducted ethically accord- None of them had overreaching symptoms.
Statistical analyses were conducted using GraphPad InStat software. Differences in testosterone levels between different ACTN3 genotype groups were analysed using ANOVA. The squared correlation coefficient R 2 was used as a measure of explained variance. For this purpose, Spearman's correlation was used to assess the relationship (r) between the testosterone level and the ACTN3 genotypes (dummy coded as 1, 2 and 3 for XX, RX and RR, respectively). All values are means (SD). P values < 0.05 were considered statistically significant.
As expected, male athletes had significantly higher levels of testosterone than female athletes (P < 0.0001). The mean testosterone levels did not differ between athletes of different sporting disciplines of both genders (Table 1). Therefore, for the main analyses we used the combined data (i.e. combined groups of female and male athletes).
The mean testosterone levels were significantly higher in both males and females with the ACTN3 R allele than in XX homozygotes (males:

DISCUSSION
This is the first study to demonstrate that the ACTN3 genotype is associated with resting testosterone levels of both male and female athletes. We therefore confirmed in some manner the results of the previous study by Pimenta et al. [20], where ACTN3 XX genotype was shown to be associated with low testosterone levels of soccer players immediately after eccentric training.
Testosterone has an anabolic effect and stimulates the growth of muscle by increase in muscle protein synthesis and inhibition of protein breakdown via a ubiquitin-mediated pathway [25]. Several studies have confirmed that testosterone increases muscle mass, strength and endurance [25]. The possible mechanism underlying the association of the ACTN3 R577X polymorphism with testosterone levels might be explained by the findings that sarcomeric α-actinins interact with calcineurin, and its inhibition potentiates testosterone production [13]. It can be speculated that the ACTN3 RR/RX genotypes, due to their anabolic effect, might be favourable for increasing skeletal muscle mass. Indeed, Walsh et al. [27] found that carriers of the ACTN3 R allele had higher levels of both total body and lower limb fat-free mass. In addition, Zempo et al. [31] observed larger thigh muscle cross-sectional area in women with RR/RX genotypes compared with XX homozygotes.
Since the ACTN3 R allele is associated with higher testosterone levels, increased muscle mass [27,31] and increased proportion of fast-twitch muscle fibres [2,26], it may be favourable for power and strength sports. Accordingly, numerous studies have reported that the ACTN3 R allele is associated with power and strength athlete status. More specifically, Yang et al. [30] for the first time showed that the frequency of the ACTN3 XX genotype was reduced in Australian power athletes compared to controls, whereas none of the Olympians or female power athletes had an XX genotype. These findings have been supported by the independent replications in case-control studies of elite Greek track and field athletes [19], elite-level strength athletes from across the United States [23], Russian power-oriented athletes [9], Israeli sprinters [10], and Russian short-distance speed skaters [2].
There are already other genetic variants that have been reported to show associations with muscle mass [15,21], and we strongly suspect that many additional common polymorphisms, and probably rare mutations as well, will be shown to be associated with skeletal muscle hypertrophy and testosterone levels in due course.

CONCLUSIONS
In conclusion, we found that the ACTN3 R allele was associated with high levels of testosterone in athletes. These data provide some mechanistic insights into the association between α-actinin-3 deficiency, skeletal muscle hypertrophy and power athlete status.

Conflict of interest
The authors report no conflicts of interest.