Strength and Endurance Training in Older Women in Relation to ACTN3 R577X and...

[Full title: Strength and Endurance Training in Older Women in Relation to ACTN3 R577X and ACE I/D Polymorphisms]

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International Journal of Environmental Research and Public Health Article Strength and Endurance Training in Older Women in Relation to ACTN 3 R 577 X and ACE I / D Polymorphisms Cristina Romero-Blanco 1 , Mar í a Jes ú s Artiga-Gonz á lez 2 , Alba G ó mez-Cabello 3 , Sara Vila-Maldonado 4,5 , Jos é Antonio Casaj ú s 3 , Ignacio Ara 4,5 and Susana Aznar 1,5, * 1 Physical Activity and Health Promotion Research Group, Universidad de Castilla-La Mancha, 45004 Toledo, Spain; Cristina.Romero@uclm.es 2 Spanish National Cancer Center (CNIO), 28029 Madrid, Spain; mjartiga@cnio.es 3 Growth, Exercise, Nutrition and Development Research Group, Universidad de Zaragoza, 50009 Zaragoza, Spain; agomez@unizar.es (A.G.-C.); joseant@unizar.es (J.A.C.) 4 Growth, Exercise, Nutrition and Development Research Group, Universidad de Castilla-La Mancha, 45071 Toledo, Spain; sara.vila@uclm.es (S.V.-M.); ignacio.ara@uclm.es (I.A.) 5 CIBERFES Biomedical Research Networking Center on Frailty and Health Aging, 28029 Madrid, Spain * Correspondence: Susana.Aznar@uclm.es; Tel.: + 34-925-268-800 (ext. 5545) Received: 19 January 2020; Accepted: 13 February 2020; Published: 14 February 2020 Abstract: The purpose of this study is to analyze the e ff ect of two genetic polymorphisms, ACTN 3 R 577 X , and ACE I / D , on physical condition in a sample of active older women after a two-year training period. The sample was composed of 300 healthy women over the age of 60 who underwent a two-year training program. Adapted tests from the Senior Fitness Test were used. The genotyping of the polymorphisms was obtained from the participants’ DNA via buccal swabs. The analysis of the ACE polymorphism did not reveal di ff erences between genotypes. The analysis of the R 577 X polymorphism showed a favorable e ff ect for the ACTN 3 XX genotype in tests for leg strength ( p : 0.001) after training, compared to the other genotypes, and also in the analysis of the combined e ff ect of the polymorphism ( ACE II + ACTN 3 RX / XX ). The intragroup e ff ect revealed an improvement in arm strength for carriers of the X allele after 24 months of training ( p < 0.05). The endurance values significantly worsened in all study groups. Conclusions: The R 577 X polymorphism of ACTN 3 may have an important role in capacities related to muscle strength, providing a beneficial e ff ect for carriers of the X allele Keywords: ACE ; ACTN 3 ; physical fitness; genotype; women; elderly 1. Introduction Life expectancy among the world’s population is steadily increasing, and the percentage of older people in developed countries is on the rise [ 1 ]. However, ageing is not without associated problems, such as a loss of independence [ 2 ], a greater risk of falls [ 3 ], and an increase of chronic illnesses [ 4 ], all of which lead to a decline in quality of life [ 5 ]. In 2002, the World Health Organization (WHO) established a global strategy to support the active ageing of the older population as a measure to support opportunities of biopsychosocial health among this collective [ 6 ]. Since then, numerous studies have concentrated on researching measures for improving the quality of life of older people, several of which are based on the development of physical capacities as a measure of healthy ageing [ 7 – 9 ]. Furthermore, other authors have attempted to find a biological explanation [ 10 ] underlying two genetic variations which have been considered to be Int. J. Environ. Res. Public Health 2020 , 17 , 1236; doi:10.3390 / ijerph 17041236 www.mdpi.com / journal / ijerph

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Int. J. Environ. Res. Public Health 2020 , 17 , 1236 2 of 10 particularly striking: the R 577 X polymorphism (rs 1815739) of the α -actinin 3 gene ( ACTN 3 ) and the I / D polymorphism (rs 1799752) of the angiotensin-converting enzyme gene ( ACE ) ACTN 3 is a structural component protein that predominates in the Z zones of the sarcomere. A very common polymorphism of α -actinin-3 has been identified in humans in position 1747 of exon 16, wherein arginine is converted into a stop codon (R 577 X) due to a cytosine-to-thymine substitution [ 11 ]; as a result, this protein is not expressed ACTN 3 is almost exclusively expressed in type II (fast twitch) muscle fibers [ 12 ], and the deficiency of α -actinin-3 among the general population appears to be associated with a decrease of the mass, muscle strength and fragility that accompanies ageing [ 13 , 14 ]. However, among the older population, the biological e ff ect of this genetic variant is unclear. Although some studies do not show any type of relation between the loss of physical capacity and this genotype in Caucasian older populations [ 15 ], others have reported an association, especially among women; however, there is a lack of agreement on which is the favorable allele [ 16 , 17 ]. In addition, the genetic polymorphism that is best characterized in relation to exercise is the presence or absence (insertion / deletion; I / D ) of a fragment of 287 pairs of bases (pb) in intron 16 of the angiotensin-converting enzyme gene ( ACE ) [ 18 ]. This polymorphism has been strongly associated to sports. More specifically, the I allele has been related with resistance and D alleles have been related with strength / power [ 19 ]. This polymorphism has been associated both with physical condition in older people as well as with longevity [ 20 , 21 ]. The studies performed on the older population also present controversial results regarding the role of the polymorphism I / D of ACE . In several studies performed among older people, a positive correlation has been found between the presence of the D allele and speed, strength, agility and / or resistance [ 22 – 24 ]. However, other studies have not found any relation with any of the physical condition variables analyzed [ 15 , 25 , 26 ]. Considering the relation between these two polymorphisms in the field of sports [ 27 ] and the few studies available that have analyzed the e ff ect of prolonged training among older people, the purpose of this study was to assess the e ff ect of these two polymorphisms in a group of older active women after a two-year training program, as well as to study the associations between these polymorphisms and physical condition among older women 2. Materials and Methods 2.1. Participants The study sample was selected from the multi-centric EXERNET population (Research Network in Physical Exercise and Health for Special Populations) [ 28 ]. The EXERNET multi-center study is a cross-sectional study on physical fitness and body composition evaluation and its relation with healthy lifestyle among non-institutionalized elderly from six regions in Spain. The population was selected by means of a multi-step, simple random sampling, considering first the location that ensures the geographic and cultural diversity of the sample, then three di ff erent cities in each region (the capital of the region and two other cities—one with 10,000–40,000 habitants and another of 40,000–10,000 habitants) and, finally, a random assignment of the civic and sports centers. All participants were of the same Caucasian (Spanish) descent from three or more generations Prior to the recruitment of study participants, the informed consent document used in this study was approved by the Clinical Research Ethics Committee (18 / 08; 1 / 12). After signing the informed consent, a total of 300 women participated in the current study The participating subjects were selected according to the following inclusion criteria: women aged over 60 years old, who were not institutionalized, who had participated in a local physical activity program in their municipality and had signed the informed consent 2.2. Anthropometric Measures The height of participants was determined in centimeters using a portable measuring rod (SECA 711). The weight and percentage of total fat (%MG) were measured using a bioelectric impedance

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Int. J. Environ. Res. Public Health 2020 , 17 , 1236 3 of 10 device (Tanita BC 418-MA, Tanita Corp., Tokyo, Japan). Subsequently, the body mass index (BMI) was calculated using the formula BMI = kg / m 2 2.3. Active–Sedentary Behavior The validated EEPAQ (Elderly EXERNET Physical Activity Questionnaire) [ 29 ] was used to evaluate the number of hours walking per day of participants and the time dedicated to sedentary activities (number of hours sitting per day) 2.4. Assessment of Physical Condition To evaluate physical condition, adapted tests of the Senior Fitness Test battery were used [ 7 ]. We evaluated the strength of upper and lower extremities and endurance The Chair Stand Test was used to register lower extremity strength: i.e., the number of times that the participant was able to sit and stand over a 30 s period The Arm Curl Test was used to evaluate upper extremity strength: i.e., the number of flexion–extensions that the participant was able to perform in 30 s with a 2.5 kg dumbbell The Six-Minute Walk Test is an assessment of endurance, which measures the meters that the participant is able to cover walking in a circuit in six minutes The anthropometric measures, the EEPAQ questionnaire and the physical condition variables were evaluated at the beginning of the study and 24 months later. During this period, the participants followed the training plan described below 2.5. Training All the women who participated in the study received training from a professional who was qualified in physical activity and sports. This training consisted of two weekly sessions lasting one hour, and it was based on elderly physical activity guidelines in all municipalities. All programmes included a warm up based on mobility and cardiorespiratory exercises, followed by endurance, resistance training and balance training in each session. Muscle strengthening included standing and sitting on a chair (similar to a squat exercise but modified accordingly to each person’s capacity), quadriceps and calf muscles exercises to improve walking, and upper body exercises such as biceps curls and triceps and shoulder exercises—all of these with light weights. Core exercises were also included. Finally, a cool down based on low-intensity cardiorespiratory exercises followed by flexibility exercises was performed. The intensity of e ff ort during training sessions was based on the Perceived Exertion Scale (BORG scale). The scale was visible for all (displayed on a poster in the room) and used during the session All participants included in the study performed the training program for 24 months. This study only included women who, after two years of training, had a minimum of 80% adherence to the program 2.6. Genotyping The genomic DNA was obtained via buccal swabs using a standardized procedure of extraction with phenol / chloroform and subsequent precipitation in alcohol in the presence of salts [ 30 ]. The genotyping of the ACTN 3 R 577 X polymorphism was performed by amplifying a fragment of 303 pairs of bases (bp) via PCR (Polymerase Chain Reaction) using the following primers: ACTN 3 -F 5 0 -CTG TTG CCT GTG GTA AGT GGG y ACTN 3 -R 5 0 -TGG TCA CAG TAT GCA GGA GGG. Thirty-five cycles of PCR amplification were performed with an annealing temperature of 60 ◦ C The detection of the R 577 X polymorphism was performed via the enzymatic digestion of the amplicons generated by PCR with D de I (Biolabs) and subsequent visualization via electrophoresis in non-denatured 8% acrylamide gels The detection of the I / D polymorphism of ACE was performed by amplification with PCR using the following primers: 5 0 -TGGAGAGCACTCCCATCCTTTCT and 5 0 -GACGTGGCCA

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Int. J. Environ. Res. Public Health 2020 , 17 , 1236 4 of 10 TCACATTCGTCAGAT. The PCR amplification was performed by use of 35 cycles and an annealing temperature of 58 ◦ C. The result of the resulting PCR was visualized using agarose gel 1.5% with ethidium bromide To ensure the correct genotyping of the DD cases and avoid cases in which the DD genotype could be confused with ID , a new nested PCR was used [ 31 ] with the following primers: 5 0 -TGGGACCACAGGCGCCCGCCACTAC and 5 0 -TCGCCAGCCCTCCCATGCCCATAA. The PCR conditions were previously described, but with an annealing temperature of 64 ◦ C 2.7. Statistical Analysis The data obtained from the present study were analyzed using the SPSS 23.0 (SPSS Inc., Chicago, IL, USA) statistical program. Descriptive analyses were performed considering measures of central tendency, asymmetry measures and measures of shape. A normality test was performed using the Kolmogorov–Smirnov criteria A quantitative study was performed to assess the influence of the genetic variables with the remaining parameters under study. As the variables did not display normal behavior, the statistical analysis was performed via the non-parametric Kruskal–Wallis test and the Mann–Whitney U test The Wilcoxon ranges test was used for the study of related variables A 95% confidence interval was used. Likewise, the Tukey test was used to compare di ff erences between the three genotypes of ACE and ACTN 3 . To evaluate the Hardy–Weinberg equilibrium of the sample, the Chi-squared test was used 3. Results Samples of 296 women were gathered, from which the optimal DNA was obtained for amplification and ACTN 3 genotyping. However, due to technical problems, the determination of the ACE genotype could only be performed in 282 samples. The minimum age was 60 and the maximum was 91 with an average age of 73.62 ( ± 5.4) The distribution of alleles the variants was adjusted to the Hardy–Weinberg equilibrium for both genes ( ACTN 3 : χ 2 = 0.79; p = 0.37; ACE : χ 2 = 0.23; p = 0.63) The distribution of both genes was as follows: ACTN 3 : RR = 93 (31.4%); RX = 139 (47%); XX = 64 (21.6%) ACE : DD = 105 (37.2%); ID = 131 (46.5%); II = 46 (16.3%) Allelic frequencies for ACE and ACTN 3 variants are described for European population as ACE : D = 0.642, I = 0.358; and ACTN 3 : R = 0.566, X = 0.434 (1000 genomes project). In this research, the allelic frequencies were very similar ( ACE D : 0.604; I : 0.395 and ACTN 3 R : 0.549; X : 0.451) This study evaluated the e ff ect of both genotypes on the anthropometric variables under study and on the EEPAQ questionnaire. No significant di ff erences were observed in any of the analyzed variables attributable to the ACTN 3 genotype or ACE , neither at the beginning nor at the end of the training. The time dedicated to sedentary activities increased in all groups after training ACTN 3 heterozygotes significantly increased weight ( p : 0.039) and BMI ( p : 0.008) scores after training. The mean and standard deviation of the anthropometric data and EEPAQ questionnaire were as follows: BMI 28.52 ( ± 3.9) before training (BT) and 28.74 ( ± 4.2) after training (AT); % fat mass 39,1 ( ± 4.8) BT and 39.1 ( ± 4.9) AT; hours walking per day 1.83 ( ± 0.8) BT and 1.87 ( ± 0.9) AT; hours sitting per day 3.97 ( ± 1.3) AT and 4.48 ( ± 1.4) 3.1. Results Associated to ACE I / D Polymorphism Table 1 displays the values of physical activity at baseline and after training and its association with training and genotypes. The Chair Stand Test did not obtain significant results either at the

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Int. J. Environ. Res. Public Health 2020 , 17 , 1236 5 of 10 intragroup level (because of training e ff ects) or at the intergroup level (because of di ff erences between the genotypes) Table 1. Physical tests at baseline and after training and its association with training and ACE genotypes Test Genotype T 1 Mean & SD T 2 Mean & SD p p p Training E ff ect Genotype E ff ect T 1 Genotype E ff ect T 2 Chair Stand Test ACE DD ( n = 105) 14.31 ± 3.2 14.15 ± 3.2 0.71 0.409 0.665 ACE ID ( n = 131) 1495 ± 3.2 14.57 ± 2.8 0.59 ACE II ( n = 46) 14.76 ± 3.2 14.51 ± 2.7 0.897 ACE ID + II ( n = 177) 14.90 ± 3.2 14.55 ± 2.7 0.687 0.185 0.421 ACE DD + ID ( n = 136) 14.67 ± 3.2 14.38 ± 3.0 0.876 0.777 0.915 Right Arm Curl Test ACE DD 17.12 ± 3.5 17.76 ± 3.8 0.026 * 0.988 0.72 ACE ID 17.24 ± 3.5 18.08 ± 3.6 0.003 * ACE II 17.32 ± 3.5 18.03 ± 3.8 0.208 ACE ID + II 17.26 ± 3.5 18.06 ± 3.6 0.002 * 0.978 0.42 ACE DD + ID 17.19 ± 3.5 17.93 ± 3.6 < 0.001 * 0.88 0.723 Left Arm Curl Test ACE DD 17.59 ± 3.7 18.59 ± 3.9 0.006 * 0.99 0.71 ACE ID 17.67 ± 3.4 18.23 ± 3.5 0.038 * ACE II 17.63 ± 3.6 18.78 ± 3.4 0.047 * ACE ID + II 17.66 ± 3.4 18.37 ± 3.4 0.005 * 0.926 0.592 ACE DD + ID 17.64 ± 3.5 18.39 ± 3.7 0.001 * 0.892 0.683 Six-Minute Walk Test ACE DD 531.04 ± 76.1 508.17 ± 89.5 0.012 * 0.313 0.346 ACE ID 536.01 ± 64.2 498.03 ± 95.9 < 0.001 * ACE II 547.60 ± 76.4 510.99 ± 90.3 0.013 * ACE ID + II 538.97 ± 67.5 501.37 ± 94.4 < 0.001 * 0.522 0.556 ACE DD + ID 533.83 ± 69.5 502.62 ± 93.0 < 0.001 * 0.129 0.293 * p < 0.05. T 1: baseline. T 2: after training. SD: standard deviation In the case of the Arm Curl Test, all groups improved their results. In this test, after training, a significant result was obtained for the carriers of the allele D in both arms; for the genotype ACE II , this was significant in the case of the left arm In the endurance test (Six-Minute Walk Test), all genotypes worsened their values after training. When grouping the genotypes as II + ID vs DD or II vs DD + ID , no results were found attributable to the genotype, which was also the case regarding the initial moment (T 1) or after training (T 2) in any of the tests; all groups experienced improved results in the Arm Curl Test after training, whereas the Six-Minute Walk Test scores significantly worsened 3.2. Results Associated to ACTN 3 R 577 X Polymorphism The study findings are shown in Table 2 , revealing that training had a statistically significant e ff ect on the Chair Stand Test: the carriers of genotype ACTN 3 XX displayed significantly improved results compared to the other genotypes On the intragroup level, statistically significant changes were observed in all the tests. In the Arm Curl Test, after two years of training, the carriers of the allele X improved significantly with one or more copies In the Six-Minute Walk Test, all groups worsened, despite the training. When grouping by genotypes as RR vs RX + XX or XX vs RX + RR , the X allele obtained significant improvements after training in the Arm Curl Test. In endurance, significant changes were obtained and worse in both groups after training.

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Int. J. Environ. Res. Public Health 2020 , 17 , 1236 6 of 10 Table 2. Physical tests at baseline and after training and its association with training and ACTN 3 genotypes Test Genotype T 1 Mean & SD T 2 Mean & SD p p p Training E ff ect Genotype E ff ect T 1 Genotype E ff ect T 2 Chair Stand Test ACTN 3 RR ( n = 93) 14.88 ± 3.4 14.41 ± 3.4 0.890 0.853 0.024 * ACTN 3 RX ( n = 139) 14.74 ± 3.0 14.03 ± 2.9 0.061 ACTN 3 XX ( n = 64) 14.46 ± 3.1 15.26 ± 2.1 0.020 * ACTN 3 RX + XX ( n = 203) 14.66 ± 3.0 14.44 ± 2.7 0.852 0.873 0.979 ACTN 3 RR + RX ( n = 232) 14.80 ± 3.2 14.19 ± 3.1 0.289 0.574 0.011 * Right Arm Curl Test ACTN 3 RR 16.85 ± 3.5 17.39 ± 3.9 0.051 0.360 0.272 ACTN 3 RX 17.51 ± 3.3 18.22 ± 3.6 0.010 * ACTN 3 XX 17.02 ± 3.7 18.78 ± 4.0 0.005 * ACTN 3 RX + XX 17.36 ± 3.4 18.41 ± 3.7 < 0.001 * 0.309 0.165 ACTN 3 RR + RX 17.24 ± 3.4 17.87 ± 3.8 0.001 * 0.561 0.200 Left Arm Curl Test ACTN 3 RR 17.18 ± 3.6 17.75 ± 3.6 0.069 0.375 0.070 ACTN 3 RX 17.96 ± 3.3 18.52 ± 3.7 0.017 * ACTN 3 XX 17.72 ± 3.7 19.61 ± 3.9 0.004 * ACTN 3 RX + XX 17.89 ± 3.4 18.89 ± 3.8 < 0.001 * 0.235 0.079 ACTN 3 RR + RX 17.65 ± 3.5 18.20 ± 3.6 0.003 * 0.776 0.041 * Six-Minute Walk Test ACTN 3 RR 535.05 ± 67.8 492.32 ± 91.3 < 0.001 * 0.747 0.115 ACTN 3 RX 535.86 ± 76.6 510.86 ± 82.1 0.004 * ACTN 3 XX 545.32 ± 63.4 508 ± 110.8 0.003 * ACTN 3 RX + XX 538.80 ± 72.7 510.05 ± 92.3 < 0.001 * 0.640 0.049 * ACTN 3 RR + RX 535.54 ± 73.1 503.23 ± 86.3 < 0.001 * 0.466 0.172 * p < 0.05. T 1: baseline. T 2: after training. SD: standard deviation 3.3. Combined E ff ect of ACE and ACTN 3 Lastly, the genotypes of both polymorphisms were grouped, distinguishing between “POWER” ( ACE DD + ACTN 3 RR / RX ); and “NON-POWER” ( ACE II / ID + ACTN 3 XX ) [ 15 , 22 , 32 ]. The results obtained in the tests, before and after training, are displayed in Table 3 . Statistically significant di ff erences were obtained, post training, for the non-power group in the Chair Stand Test; once again, a worsening was observed in both groups in the Six-Minute Walk Test Table 3. E ff ect of training and combination of ACTN 3 and ACE genotype on physical tests Test Grouped Genotype T 1 Mean & SD T 2 Mean & SD p p p Training E ff ect Genotype E ff ect T 1 Genotype E ff ect T 2 Chair Stand Test POWER ( n = 82) 14.43 ± 3.1 13.77 ± 3.3 0.617 0.461 0.031 * NON-POWER ( n = 38) 14.86 ± 2.9 15.09 ± 1.8 0.505 Right Arm Curl Test POWER 17.19 ± 3.2 17.22 ± 3.5 0.192 0.778 0.309 NON-POWER 17.22 ± 3.3 18.14 ± 3.7 0.154 Left Arm Curl Test POWER 17.62 ± 3.6 17.92 ± 3.7 0.115 0.885 0.410 NON-POWER 17.86 ± 3.5 18.80 ± 3.4 0.124 Six-Minute Walk Test POWER 529.05 ± 79.3 504.77 ± 83.5 0.035 * 0.354 0.793 NON-POWER 546.88 ± 64.0 497.46 ± 116.0 0.018 * * p < 0.05. T 1: baseline. T 2: after training 4. Discussion This study attempts to evaluate the e ff ect of two genetic polymorphisms, ACE I / D and ACTN 3 R 577 X , among a large number of female active subjects above the age of 60 after a two-year training program. According to the literature consulted, this is the largest study performed for

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Int. J. Environ. Res. Public Health 2020 , 17 , 1236 7 of 10 both polymorphisms among this type of population, with regards to both the number of subjects and the training time The studies performed on older people have shown varied results according to multiple variables, for which age, gender and lifestyle are di ff erentiating elements. For this reason, this study was solely based on older women, aged 60 years or older, and with an active lifestyle The results obtained attribute a favorable e ff ect to the ACTN 3 XX genotype in the Chair Stand Test and the Arm Curl Test after a two-year training program; however, a generalized worsening was found for all participants in the Six-Minute Walk Test. In addition, no di ff erences were found in the I / D polymorphism of ACE among the di ff erent groups either before or after training This study has analyzed two polymorphisms that are extensively studied in the sports literature in relation to two very important parameters of physical condition in older people: strength and endurance. We found di ff erent responses according to genotype for the tests evaluating strength However, in the tests that monitored endurance, a generalized worsening was observed, despite the training, with no relation to the genotypes studied Both polymorphisms continue to be a source of study in numerous studies in the field of sports, especially in athletes [ 33 , 34 ]. However, in the case of the older population, many external factors can a ff ect a person’s physical condition, constraining di ff erent results to those obtained in professional athletes. Overall, it is striking that, despite the training and considering the participants as being active people, we found a worsening in the endurance test (Six-Minute Walk Test); nonetheless, it is important to consider that the total sedentary time increased after the two study years To our knowledge, no previous research has been undertaken on older people studying the relationship between the analyzed genotypes and the Six-Minute Walk Test after training. Giacaglia et al. [ 23 ] used the same test over an 18-month training period and assessed the relationship with I / D polymorphism ACE , without finding changes. A study by Moraes et al. [ 35 ] also failed to find significant di ff erences attributable to ACE polymorphism, with participants only improving their results after a 12-week training period. Regarding ACTN 3 R 577 X polymorphism, the results obtained indicate that, despite the poor results among all groups, the RR homozygotes significantly worsened more than the carriers of the X allele Related to the muscle strength of participants, in the Chair Stand Test, noteworthy changes were found due to training and the ACTN 3 genotype. Thus, women with the ACTN 3 XX genotype present improved results compared to the remaining groups after the 24-month training period. In the combined analysis of genes, improved results were found, once again, in the “NON-POWER” group which included this genotype ( ACE II / ID + ACTN 3 XX ) compared to the “POWER” group ( ACE DD + ACTN 3 RR / RX ). Several studies in which the training program was not performed but where the same strength test was used in older women [ 15 , 36 , 37 ] failed to find a relationship with the ACTN 3 genotype, which is similar to the results found in the first measurement performed. Kikuchi et al. [ 38 ] also performed the Chair Stand Test, without training, and failed to find a di ff erence attributable to the genotype in the group of women analyzed. In contrast to our findings, Pereira et al. [ 32 ] observed di ff erences in both genotypes of this same test favorable to the RR homozygotes of ACTN 3 and to the ACE DD group after a 12-week training period. In this case, the participants performed a shorter training, the mean age was lower, and there were significant di ff erences between genotypes and body composition, all of which may justify the discrepancies between these findings. Due to contradictory results in studies that have reviewed the e ff ects of ACTN 3 after training [ 39 ], in this study, we have tried to homogenize the characteristics of the participants in terms of the training received, age, sex and lifestyle. However, the study by Seto et al. [ 40 ] in mice could explain why better results have been found in homozygotes XX since the absence of α -actinin-3 seems to produce an increase in calcineurin activity, producing a reprogramming of the metabolic phenotype of the fast muscle fibres and causing the muscle to have a greater adaptation response to training To evaluate upper limb strength, our study used the Arm Curl Test. We were only able to find one similar study performed in older people and measuring this variable with the same test used in

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Int. J. Environ. Res. Public Health 2020 , 17 , 1236 8 of 10 this study [ 36 ]; no significant di ff erences were found which were attributable to genotype, although an improvement was found in all groups due to training. Likewise, other research groups who evaluated upper limb strength using other tests have also failed to find a relationship with ACE over time [ 26 , 41 , 42 ]. Concerning the ACTN 3 polymorphism and the Arm Curl Test, improved results were obtained after training for the XX homozygotes in the left arm. In the right arm, significant improvements were observed for all the carriers of the X allele after training. In line with these findings, Clarkson et al. [ 43 ], in a study involving a group of young women, found improved results for the group of XX homozygotes after a 12-week training. However, other authors, such as Delmonico et al. [ 44 ], obtained di ff erences over time; in this case, without any training 5. Conclusions The following conclusions may be drawn, based on the analysis of both polymorphisms in this group of the population: first, on a general level, over these two years, despite leading an active lifestyle, the women in our study increased the amount of time dedicated to sedentary activities and have worsened their endurance capacity. Second, the analysis of I / D polymorphism of ACE does not seem to have a very relevant e ff ect on the tests studied over time. Furthermore, the ACTN 3 R 577 X polymorphism could have an important role in capacities related to muscle strength, providing a beneficial e ff ect for carriers of allele X . Lastly, the combination of both polymorphisms (“POWER” vs. “NON-POWER”) does not provide additional advantages to those achieved studying the non-combined genotypes Author Contributions: C.R.-B. participated in the data collection, data analysis, the writing of the manuscript and tables, and approved the final version; M.J.A.-G. participated in the genetic data analysis, in reviewing the manuscript, and approved the final version; A.G.-C. participated in the data collection and in reviewing the manuscript; S.V.-M. participated in the data collection and in reviewing the manuscript; J.A.C. participated in data collection, the coordination of the study, and in reviewing the manuscript; I.A. participated in data collection, the coordination of the study, and in reviewing the manuscript; S.A. participated in the reviewing of the manuscript, data collection, in editing the paper, the coordination of the study, and approved the final version. All authors have read and agreed to the published version of the manuscript Funding: This study was supported by funds from the Ministerio de Educación y Ciencia (Red EXERNET DEP 2005-00046), the IMSERSO (104 / 07 and 147 / 2011), the University of Zaragoza (UZ 2008-BIO-01), the Carlos III Institute (Spanish Net on Aging and Frailty; (RETICEF)) (RD 12 / 043 / 0002), the Biomedical Research Networking Center on Frailty and Health Aging (CIBERFES) and FEDER funds from the European Union (CB 16 / 10 / 00477) Conflicts of Interest: The authors declare no conflict of interest References 1 Christensen, K.; Doblhammer, G.; Rau, R.; Vaupel, J.W. Ageing populations: The challenges ahead Lancet 2009 , 374 , 1196–1208. [ CrossRef ] 2 Mitchell, W.K.; Williams, J.; Atherton, P.; Larvin, M.; Lund, J.; Narici, M. Sarcopenia, dynapenia, and the impact of advancing age on human skeletal muscle size and strength; a quantitative review Front. Physiol 2012 , 3 , 260. [ CrossRef ] [ PubMed ] 3 Brouwer, B.; Musselman, K.; Culham, E. Physical function and health status among seniors with and without a fear of falling Gerontology 2004 , 50 , 135–141. [ CrossRef ] 4 MacNee, W.; Rabinovich, R.A.; Choudhury, G. Ageing and the order between health and disease Eur. Respir J 2014 , 44 , 1332–1352. [ CrossRef ] [ PubMed ] 5 Giles, L.C.; Hawthorne, G.; Crotty, M. Health-related quality of life among hospitalized older people awaiting residential aged care Health Qual. Life Outcomes 2009 , 7 , 71. [ CrossRef ] [ PubMed ] 6 WHO. Report of the World Health Organization. Active ageing: A policy framework Aging Male 2002 , 5 , 1–37. [ CrossRef ] 7 Rikli, R.E.; Jones, C.J. Development and validation of criterion-referenced clinically relevant fitness standards for maintaining physical independence in later years Gerontologist 2013 , 53 , 255–267. [ CrossRef ]

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