ACTN3 R577X Genotype and Exercise Phenotypes in Recreational Marathon Runners

nd: Homozygosity for the X-allele in theACTN3R577X (rs1815739) polymorphism results in the complete absence of -actinin-3 in sarcomeres of fast-type muscle bers. In elite athletes, theACTN3XX genotype has been related to inferior performance in speed and power-oriented sports; however, its in uence on exercise phenotypes in recreational athletes has received less attention. We sought to determine the in uence ofACTN3genotypes on common exercise phenotypes in recreational marathon runners.Methods:A total of 136 marathoners (116 men and 20 women) were subjected to laboratory testing that included measurements of body composition, isometric muscle force, muscle exibility, ankle dorsi exion, and the energy cost of running.ACTN3genotyping was performed using TaqMan probes.Results:37 runners (27.2%) had the RR genotype, 67 (49.3%) were RX and 32 (23.5%) were XX. There was a di erence in body fat percentage between RR and XX genotype groups (15.7 5.8 vs. 18.8 5.5%; e ect size, ES,=0.5 0.4,p=0.024), whereas the distance obtained in the sit-and-reach-test was likely lower in the RX than in the XX group (15.3 7.8 vs.18.4 9.9 cm ; ES=0.4 0.4,p=0.046). Maximal dorsi exion during the weight-bearing lunge test was di erent in the RR and XX groups (54.8 5.8 vs. 57.7 5.1 degree; ES=0.5 0.5,p=0.044). Maximal isometric force was higher

ect size, ES,=0.5 0.4,p=0.024), whereas the distance obtained in the sit-and-reach-test was likely lower in the RX than in the XX group (15.3 7.8 vs.18.4 9.9 cm ; ES=0.4 0.4,p=0.046). Maximal dorsi exion during the weight-bearing lunge test was di erent in the RR and XX groups (54.8 5.8 vs. 57.7 5.1 degree; ES=0.5 0.5,p=0.044). Maximal isometric force was higher in the RR than in the XX group (16.7 4.7 vs.14.7 4.0 N/kg ;ES= 0.5 0. 3, p=0.038). There was no di erence in the energy cost of running between genotypes (~4.8 J/kg/min for all three groups, ES ~0.2 0.4).Conclusions:TheACTN3genotype might in uence several exercise phenotypes in recreational marathoners. De ciency in -actinin-3 might be accompanied by higher body fatness, lower muscle strength and higher muscle exibility and range of motion. Although there is not yet a scienti c rationale for the use of commercial genetic tests to predict sports performance, recreational marathon runners who have performed such types of testing and have theACTN3XX genotype might perhaps bene t from personalized strength training to improve their performance more than their counterparts with otherACTN3genotypes. Keywords: -actinin; exercise; performance; endurance; genetics; single nucleotide polymorphism 1. Introduction -Actinin-2 and -actinin-3 are key structural proteins in the contractile apparatus of the skeletal muscle ber, as they bind and possibly cross-link the ends of F-actin laments at the Z-line [1]. Genes2019,10, 413; doi:10.3390 /genes10060413 /journal/genes

Genes2019,10, 413 2 of 15 Whereas -actinin-2 is ubiquitously expressed in all muscle ber types, -actinin-3 expression is largely restricted to fast-type muscle bers [2]. Homozygosity for the null X-allele of the R577X polymorphism in the -actinin-3 gene,ACTN3, results in the complete absence of -actinin-3 in fast-type muscle bers [3]. Individuals with theACTN3XX genotype compensate for the de ciency of -actinin-3 through elevated expression of -actinin-2 in fast-type muscle bers [4], although several speci c muscle phenotypes have been related to -actinin-3 de ciency [5]. -Actinin-3 de ciency is believed to a ect the muscle's ability to generate rapid, forceful contractions and thus might be detrimental for the production of fast and explosive movements. This notion has been veri ed in almost 20 case-control studies, as recently reviewed by Houweling et al., (2018), with the frequency of the XX genotype being lower in elite athletes participating in sprint and power-based sports than in the general non-athletic population. By contrast, the RR genotype, which is associated with full expression of -actinin-3 in fast-type muscle bers, is highly prevalent among elite athletes in sprint/power disciplines. However, the e ect of theACTN3 XX genotype on the sports performance of recreational athletes is unexplored. The study of such a relationship might be particularly interesting given that ~20% of the world´s population is -actinin-3 de cient [6], and because genetic testing of this polymorphism has recently become a commercially available diagnostic test [7], which can inform exercise recommendations. In untrained populations,ACTN3XX individuals produce less handgrip strength and less muscle force and power than their RR counterparts [8–10], but this di erence is lost when the same genotypes are compared in active/trained individuals [11–13]. Furthermore, whereas muscle ber composition is not a ected by -actinin-3 de ciency [14]; muscle volume [8], and especially the size of fast-type muscle bers [15], is lower in XX than in RR counterparts. Finally, a higher response to strength training has been found in RR than in XX individuals [16], coupled with a lower signaling for muscle hypertrophy in XX subjects [14]. Given this information, it might be speculated that -actinin-3

ciency [14]; muscle volume [8], and especially the size of fast-type muscle bers [15], is lower in XX than in RR counterparts. Finally, a higher response to strength training has been found in RR than in XX individuals [16], coupled with a lower signaling for muscle hypertrophy in XX subjects [14]. Given this information, it might be speculated that -actinin-3 de ciency derived fromACTN3XX homozygosity might also a ect force and power production in recreational athletes and a ect sports performance. In addition to a ecting exercise performance,ACTN3genotypes might also in uence exercise-induced muscle damage, particularly after endurance events such as marathon running. Indeed, the X-allele has been associated with higher levels of several markers of muscle damage after exercise in amateur athletes [17–19]. Conversely, a higher muscle exibility and a superior range of motion has been reported in XX individuals versus their RR referents [20–22], although some authors have failed to replicate this nding [23]. Although more exible muscles are less susceptible to eccentric exercise-induced damage [24], higher muscle exibility values do not seem to attenuate marathon-induced muscle damage in XX runners [17–19]. Finally, theACTN3XX genotype has been related to lower body mass and lower fat-free mass [8,23], likely due to a reduction in muscle mass as a result of smaller fast-type ber size [15,25]. However, the e ect ofACTN3genotypes on fat mass and body composition in sedentary and clinical populations is unclear [25–27], and is unknown in recreational athletes. The methodological di erences in assessing these exercise phenotypes, the relatively small study samples in some investigations, and the wide range of age and tness levels under investigation make it di cult to ascertain whether the e ect ofACTN3genotypes is of su cient magnitude to represent a variable that a ect sports performance and training in recreational athletes, as seems to be the case in elite athlete populations. -Actinin-3 de ciency has also been related to positive phenotypes that would explain the perpetuation of theACTN3XX genotype through natural selection in human evolution. Particularly, it has been proposed that the high frequency of the X allele in some human populations

a ect sports performance and training in recreational athletes, as seems to be the case in elite athlete populations. -Actinin-3 de ciency has also been related to positive phenotypes that would explain the perpetuation of theACTN3XX genotype through natural selection in human evolution. Particularly, it has been proposed that the high frequency of the X allele in some human populations could be the result of increased metabolic e ciency, possibly enhancing the capability for endurance running [28]. This theory is supported by studies in mouse models, because a shift towards a more e cient aerobic muscle metabolism has been found inActn3knockout (KO) mice [6,29]. This has fueled the notion that the X allele might act as a thrifty allele [30], although this theory has little support in humans [31].

Genes2019,10, 413 3 of 15 Indeed, recent case-control investigations suggest that it is unlikely that theACTN3XX genotype provides an advantage in competitive endurance running performance [32,33]. The aim of the present study was to determine the in uence ofACTN3genotypes on common exercise phenotypes in recreational marathon runners. Our main hypothesis was that, compared with their RR counterparts,ACTN3XX runners would present with lower values of muscle force, but higher values of running e ciency. 2. Materials and Methods 2.1. Subjects One hundred thirty-six healthy experienced recreational marathon runners (116 men and 20 women) volunteered to participate in this study. Participants were either recruited by email from a group of runners that had participated in previous investigations or were recruited at the time of race registration. Inclusion criteria were as follows: Age 18–65 years; being free of any history of muscle, cardiac or kidney disorders; participating in the marathon at maximal possible intensity; and having a running experience of at least 3 years, with at least three marathons completed during this period. Exclusion criteria were: taking medications during the 2 weeks prior to competing or having had a musculoskeletal injury in the month prior to the competition. The ful llment of inclusion/exclusion criteria was veri ed through an ad hoc questionnaire. Age and main morphological and physical characteristics of the participants in this investigation are shown in Table. Before enrollment, each participant was informed about the risks and discomforts associated with the investigation and signed an informed consent document. The study was approved by the Camilo Jose Cela University Ethics Committee (ID ACTN3 approved 18/4/2018) in accordance with the latest version of the Declaration of Helsinki. Participants' rights and con dentiality were protected during the whole experiment, and the genetic information was used only for the purposes included in this investigation. Table 1. Age, anthropometric characteristics, running experience, and training status of marathoners with di erentACTN3R577X genotypes. Data are mean standard deviation (SD) for each genotype. Degrees of freedom=2, between-groups; 133, intra-groups; 135, total. Variable (Units) RR RX XX p 2 n(frequency) 37 (27.2%) 67 (49.3%) 32 (23.5%) - - Men/women (frequency)

used only for the purposes included in this investigation. Table 1. Age, anthropometric characteristics, running experience, and training status of marathoners with di erentACTN3R577X genotypes. Data are mean standard deviation (SD) for each genotype. Degrees of freedom=2, between-groups; 133, intra-groups; 135, total. Variable (Units) RR RX XX p 2 n(frequency) 37 (27.2%) 67 (49.3%) 32 (23.5%) - - Men/women (frequency) 31 /6 (83.8/16.2%) 58 /9 (86.6/13.4%) 27 /5 (84.4/15.6%) 0.922 - Age (years) 41.2 10.2 40.3 8.8 40.7 9.8 0.880 <0.01 Body mass (kg) 70.9 7.1 71.6 10.8 72.8 10.5 0.731 <0.01 Body height (m) 1.73 0.06 1.73 0.08 1.72 0.10 0.723 <0.01 Body mass index (kg/m 2 ) 23.7 1.7 23.8 1.2 24.5 1.5 0.26.9 0.03 Running experience (years) 9.0 7.5 8.1 7.8 8.3 6.0 0.880 <0.01 Best race time in the marathon (min) 218 27 223 38 219 39 0.881 <0.01 Completed marathons (number) 5 4 5 3 5 3 0.776 <0.01 Average training distance/week (km) 50.7 14.6 52.5 17.0 51.7 16.9 0.889 <0.01 Training sessions/week (number) 4 1 4 1 4 1 0.794 <0.01 2.2. Experimental Design All participants underwent the same testing under identical experimental conditions. Participants were registered in the 2018 edition of the Rock'n'Roll Madrid Marathon and once they had completed all the testing and nished the marathon, they were included into a common database. Subsequently, participants were divided into three groups, established according to their individualACTN3R577X genotype (RR, RX or XX groups). Because the men and women responded in the same manner when comparing the three genotypes, and the frequency of men/women was similar in all three groups (Table), we analyzed all the data without considering sex as a covariable.

Genes2019,10, 413 4 of 15 2.3. Experimental Protocol At least 1 week before the marathon, each participant received information about the bene ts and risks of the investigation and the standardization procedures. At this time, they lled out the pre-participation ad hoc questionnaire. Participants were instructed to avoid strenuous exercise, ca eine and alcohol for the 24 h before the onset of testing, which was performed the day before the race. On this day, participants signed the informed consent and anthropometric characteristics were registered by an ISAK-certi ed anthropometrist following international standards [34]. Anthropometric measurements included body mass and height ( 50 g scale; Radwag, Radom, Poland), skinfold thickness ( 0.1 mm skinfold caliper, Holtain Ltd., Crosswell, UK: triceps, subscapular, iliac crest, abdominal, anterior and posterior thigh and medial calf) and thigh circumference ( 0.5 mm ber glass measuring tape; Holtain Ltd.: above the knee, at the maximum thigh circumference and at the gluteal furrow). Three measurements were obtained on the dominant side of the body and the mean was used for data analysis. Relative adiposity (in %) was calculated from the sum of skinfolds [35]. The mean fat-free volume of the dominant thigh (in mL/kg) was measured according to the protocol described by Jones & Pearson (1969) and normalized by body mass to allow a better comparison among groups [36]. Participants underwent a standardized 10-min warm-up including low-intensity running at 8 km/h on a treadmill. Treadmill velocity was progressively increased until 10 km/h and oxygen uptake (VO2) and carbon dioxide production (VCO2) were measured at this velocity for 5 min. Expired gases were collected breath-by-breath with a metabolic cart (Metalyzer 3B, Cortex, Leipzig, Germany), and gas exchange data of the last minute was used as a representative value. Certi ed calibration gases (16% O2, 5% CO2, Cortex) and a 3-L syringe were used to calibrate the gas analyzer and the owmeter, respectively. Gas measurements were made with the clothes and shoes used during the marathon competition. The energy cost of running (in J/kg/m) was calculated using the non-protein respiratory quotient [37] and was normalized by body mass to allow

ed calibration gases (16% O2, 5% CO2, Cortex) and a 3-L syringe were used to calibrate the gas analyzer and the owmeter, respectively. Gas measurements were made with the clothes and shoes used during the marathon competition. The energy cost of running (in J/kg/m) was calculated using the non-protein respiratory quotient [37] and was normalized by body mass to allow a better between-subject comparison [38]. After 5 min of recovery, participants performed two maximal countermovement vertical jumps on a force platform (Quattrojump, Kistler, Wintherthur, Switzerland), as previously described [17]. The jumps were separated by a 1-min rest period. The jump with the highest height (in cm) was used for statistical analysis. Then, participants performed a whole-body isometric force test [39]. The isometric muscle strength was measured using a hand-held pull gauge (Isocontrol, Isometrico, Madrid, Spain) set at a frequency of 1000 Hz. For this measurement, participants were asked to stand on a50 50 cm iron base connected to a handle-bar by a non-elastic cable. The isometric gauge was inserted within the cable, and the height of the cable was individually set to provide a 135 knee exion while the back and the arms were completely extended. Participants were instructed to perform a maximal pull for 4 seconds and the peak value was used for analysis. The force obtained (in Newtons, N) was normalized to body mass (i.e., N/kg) to allow for a better comparison among genotypes. Thereafter, participants performed a maximal handgrip strength test with both hands (dominant and non-dominant) using a handgrip dynamometer (Grip-D, Takei, Japan). Performance was expressed in N and two attempts were performed with each hand; the peak value was used for statistical analysis. The lunge test was performed as a measure of dorsi exion range of motion [40]. Participants placed their foot along a measuring tape on the oor with both their big toe and heel on the centerline of the measuring tape while they leaned on a wall. The weight-bearing lunge test was performed with both limbs and the maximal dorsi exion during the test was de ned as the maximum distance of the

of motion [40]. Participants placed their foot along a measuring tape on the oor with both their big toe and heel on the centerline of the measuring tape while they leaned on a wall. The weight-bearing lunge test was performed with both limbs and the maximal dorsi exion during the test was de ned as the maximum distance of the toe from the wall while maintaining contact between the wall and knee without lifting the heel. Participants were then asked to progressively move their knee forwards while they were reclined on the wall, repeating the lunge movement until the maximum distance at which they could tolerably lunge their knee to the wall without heel lift was found [41]. At this point, dorsi exion range of motion was performed using a handheld manual goniometer (Baseline ® , The Therapy Connection Inc, Windham, NY, USA) by placing the center of the goniometer just below the lateral malleolus of the ankle, with one arm lined up through the lateral aspect of the bula and the other arm lined up with

Genes2019,10, 413 5 of 15 the fth metatarsophalangeal joint [42]. The measurement was repeated three times and the maximal ankle dorsi exion (in ) was used for analysis. On the day of the race, participants had their usual pre-competition meal at least 3 h before the race, which was not standardized among participants to avoid a ecting their individual pre-competition routine. Runners were encouraged to ingest 500 mL of water 2 h before the start of the race to increase the likelihood of being euhydrated at the start line. During the race, participants wore a race bib with a time-chip to calculate the actual amount of time that it took them from the start line of the race to the nish line (net time, in min). Participants completed the race at their own pace and drank ad libitum at the hydration stations placed at 5-km intervals with no indications about running pace or uid and food strategies. The marathon race was held in April on a sunny day with a mean dry temperature of 21.0 2.1 C (range 15–26 C, temperature readings at 30-min intervals from 0- to 5-h after the race onset) and a mean relative humidity of 43 2% (range 40–51%). 2.4. Genetic Testing Genomic DNA was isolated using an organic-based DNA extraction method adapted to Amicon ® (Sigma-Aldrich, Madrid, Spain) Ultra 0.5-mL columns, including a nal concentration step to 50 L [43]. To avoid contamination, recommendations for molecular genetics laboratories were followed, including physically-isolated work area laboratories for each process (sample manipulation and extraction). In addition, reference samples (internal controls, blank samples and negative controls) and contamination monitoring in all steps were included. Positive controls for all genotypes were obtained from the Mexican branch of the CANDELA Consortium [44]. Genotyping ofACTN3rs1815739 polymorphism (c.1858C>T; p.R577X) was conducted using a TaqMan SNP Genotyping Assay (Assay ID: C___590093_1_; Applied Biosystems, Foster City, CA, USA) and the reaction was performed in an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems). The results were analyzed using 7500 Software v2.0.5 (Applied Biosystems). 2.5. Statistical analysis The di erence in the distribution of

Consortium [44]. Genotyping ofACTN3rs1815739 polymorphism (c.1858C>T; p.R577X) was conducted using a TaqMan SNP Genotyping Assay (Assay ID: C___590093_1_; Applied Biosystems, Foster City, CA, USA) and the reaction was performed in an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems). The results were analyzed using 7500 Software v2.0.5 (Applied Biosystems). 2.5. Statistical analysis The di erence in the distribution of men/women in each genotype group was tested with crosstab and Chi square tests, including adjusted standardized residuals. The normality of the remaining variables was initially tested with the Shapiro-Wilk test and all variables showed a normal distribution. Group comparisons (RR vs. RX vs. XX) were performed using one-way analysis of variance (ANOVA). When the ANOVA showed a signi cant group-e ect, between-group di erences were assessed using the Tukey post-hoc test. The signi cance level was set at 0.05. The e ect size (ES) for each full ANOVA analysis was calculated using the Eta squared ( 2) by using between-groups sum of squares and the total sums of squares for all ES. The magnitude of 2was interpreted following the guidelines by Cohen [45] as follows: small: 0.01; medium=0.06; large: 0.14. Data are presented as mean standard deviation (SD) and all the analyses were performed with the statistical package SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). The ES was also calculated in all pairwise comparisons, by using the Hedges' g 95% con dence intervals (CI), to assess the magnitude of the between-group di erences in the phenotypes under investigation. ES were interpreted according to the following ranges:<0.2, trivial; 0.2–0.6, small; 0.6–1.2, moderate; 1.2–2.0, large; 2.0–4.0, very large; and>4.0, extremely large [46]. 3. Results The genotyping success rate was 99%. From the study sample of 136 runners, 27.2% were genotyped asACTN3RR, 49.3% were RX and 23.5% were XX. Participants had similar running experience, best race time in marathon, number of completed marathons in the three previous years, and comparable training characteristics (Table). In addition, the net race time in the investigated marathon was similar for all three genotypes (236 36, 236 44, 244 27 min, respectively;p=0.509, 2 =0.01).

Study investigates the impact of ACTN3 genotypes on exercise phenotypes in marathon runners.