Conceived and designed the experiments: PR EP VK SS. Performed the experiments: PR EP KT. Analyzed the data: PR EP TT. Contributed reagents/materials/analysis tools: PR VK. Wrote the paper: PR. Other: Designed the original study: JK MK TR. Commented on the manuscript: EP TT KT JK MK TR SS VK.
The authors have declared that no competing interests exist.
Muscle strength declines on average by one percent annually from midlife on. In postmenopausal women this decrement coincides with a rapid decline in estrogen production. The genetics underlying the effects of estrogen on skeletal muscle remains unclear. In the present study, we examined whether polymorphisms within
A cross-sectional data analysis was conducted with 434 63-76-year-old women from the population-based Finnish Twin Study on Aging. Body anthropometry, muscle cross-sectional area (mCSA), isometric hand grip and knee extension strengths, and leg extension power were measured. COMT Val158Met and ESR1 PvuII genotypes were determined by the RFLP method. mCSA differed by COMT genotypes (p = 0.014) being significantly larger in LL than HL individuals in unadjusted (p = 0.001) and age- and height-adjusted model (p = 0.004). When physical activity and age were entered into GEE model, COMT genotype had a significant main effect (p = 0.038) on mCSA. Furthermore, sedentary individuals with the HH genotype had lower muscle mass, strength and power, but they also appeared to benefit the most from physical activity. No association of ESR1 PvuII polymorphism with any of the muscle outcomes was observed.
The present study suggests that the COMT polymorphism, affecting the activity of the enzyme, is associated with muscle mass. Furthermore, sedentary individuals with potential high enzyme activity were the weakest group, but they may potentially benefit the most from physical activity. This observation elucidates the importance of both environmental and genetic factors in muscle properties.
One of the most important functions of skeletal muscle is voluntary movement. Muscle also serves as an amino acid reservoir and a site for various metabolic activities participating e.g. to the glucose metabolism of the whole body
In women, menopause is characterized by rapid decline in the production of estrogen, an anabolic female sex hormone, and coincides with an accelerated deterioration in muscle performance
The synthesis and degradation of estrogens are mediated by several enzymes involved in multiple and complex metabolic pathways. After initial hydroxylation of estrogens by isoenzymes belonging to the cytochrome P450 family, they are further metabolized by catechol-
ESR1 was recently shown to be expressed in human skeletal muscle
Theoretically, polymorphisms residing in genes related to estradiol metabolism and action, in this case
This study is a part of the Finnish Twin Study on Aging (FITSA), which investigates the genetic and environmental effects on the disablement process in older female twins. The detailed study design, including selection procedures, determination of zygosity and description of the participants, has been reported elsewhere
In the whole study sample the prevalence of various diseases potentially effecting muscle properties were: 12 % for coronary heart disease, 8 % for asthma, 7 % for cerebrovascular disease, 6 % for type 2 diabetes, 4 % for rheumatoid arthritis, 29 % for knee, 14 % for hip and 12 % for foot and ankle osteoarthritis. 67 % of all the subjects had never used HRT, whereas 14 % were former users and 19 % current users. Moreover, 3 % of the subjects in the entire population were current cortisol users and 12 % former users, whereas 85 % had never been under long-term cortisol treatment. Subjects with the above-mentioned diseases and different HRT or cortisol status were equally distributed between genotypes.
Nonfasting blood samples were collected between 0830 and 0930 hours and sera stored at −70°C for later analysis. Total serum 17β-estradiol was determined by competitive immunoenzymatic colorimetric assay (NovaTec Immunodiagnostica GmbH, Dietzenbach, Germany) and serum sex hormone binding globulin (SHBG) levels using solid-phase, chemiluminescent immunometric assay (Immulite® 1000 SHBG, DPC [Diagnostic Products Corporation], Los Angeles, USA). Free estradiol levels were calculated from 17β-estradiol and SHBG levels according to a previously presented method
Body mass and body height were measured using standard procedures. Lean body mass and total body fat were assessed by bioelectrical impedance (Spectrum II; RJL Systems, Detroit, MI, USA). The CV in our laboratory has been less than 2 % for lean body mass and less than 3 % for body fat
Maximal isometric strength, defined as the maximum voluntary contraction performed at a specific joint ankle against unyielding resistance, was measured for knee extension and hand grip. Maximal isometric hand grip and knee extension strengths were measured on the dominant side in a sitting position using an adjustable dynamometer chair (Good Strength, Metitur, Jyväskylä, Finland). After familiarization with the test, three to five maximal efforts separated by a one-minute interval were conducted. Mid-life maximal isometric hand grip strength, strength of a non-bearing limb, has been reported to correlate with strength of other muscle groups thus being a good indicator of overall strength
Information concerning physical activity was collected using the scale of Grimby
Genomic DNA was extracted from EDTA-anticoagulated whole blood according to standard procedures (PUREGENE® Kit, Gentra Systems Inc., Minneapolis, USA). Genotyping for Val158Met and PvuII polymorphisms was performed using PCR (thermal cycler: Eppendorf® Mastercycler® gradient, Eppendorf, Boulder, CO, USA) followed by restriction fragment length polymorphism (RFLP) analysis
The G to A transition at the 158th codon in the
In ESR1 PvuII genotyping a 373-bp PCR fragment was produced using a primer pair (Oligomer Oy, Helsinki, Finland) consisting of forward (
RFLP identification was carried out by two independent investigators from whom data on phenotypes was concealed. Genotyping was successfully performed in 423 for COMT Val158Met and 421 for ESR1 PvuII site out of 434 subjects. Reasons for missing determinations include insufficient amount of DNA or contamination of the blood sample.
Hardy-Weinberg equilibrium was tested using the likelihood ratio test. Allele frequencies were determined by gene counting. All statistical models were constructed in SAS, version 9.1 using the generalized estimating equations approach (GEE), which allows taking into account the correlation between sisters within a twin pair. All outcome variables were normally distributed except for estradiol concentrations, which were skewed towards low concentrations and were considered to follow the gamma-distribution. Two types of single genotype models were constructed, one including the unadjusted main effects of the genotypes, and the other adjusted for age and height. To assess genotype-genotype and genotype-physical activity interactions a reference category was selected for the categorical predictor variables of physical activity (sedentary level), COMT (the HH genotype) and ESR1 Pvull (the pp genotype). Planned contrasts were used in comparing mean levels of each outcome variable between the predictor variable levels and their interactions against the reference category. Test-wise type I error rate was set at 0.05 in all analyses and partial correlation coefficients from the GEE model contrasts
We hypothesized that subjects with assumed lower amount of circulating estradiol (HH genotype) and potential lower levels of ESR1 transcript (pp genotype) would be less responsive to estradiol and thus have worse muscle properties in comparison with other combinations. In the models including physical activity, subjects with potential low amount of circulating estradiol (HH genotype) or suggested low amount of ESR1 transcript (pp genotype) combined with sedentary life-style, were assumed to be weaker and have smaller muscles than other combinations. In our approach, reference groups were chosen according to these initial hypothesis and the mean values of other groups compared to that of the reference groups. The reference groups for the interaction effect were formed based on the combination of the main effect reference categories. The main effects of the two components of interest are always presented in contrast to the reference group.
In our study population 79 subjects (18.0 %) were homozygous for the high activity allele (HH), 208 (47.9 %) heterozygotes (HL) and 137 (31.6 %) homozygous for the low active allele (LL). The allele frequencies were 0.43 for the H and 0.57 for the L allele. The genotype distribution of the entire cohort was in Hardy-Weinberg equilibrium (χ2 = 0.004, p = 0.95) suggesting that the subjects represented a homogeneous genetic background. Subject characteristics according to Val158Met genotypes are presented in
Variable | COMT genotypes | p for trend | ||
HH (n = 79) | HL (n = 208) | LL (n = 136–137) | ||
Weight (kg) | 70.0 (1.7) | 70.3 (1.0) | 70.0 (1.2) | 0.972 |
Height (cm) | 157.5 (0.87) | 158.2 (0.53) | 159.7 (0.66) | 0.085 |
Body fat (kg) | 24.0 (1.1) | 24.5 (0.8) | 23.6 (0.87) | 0.763 |
Lean body mass (kg) | 46.1 (0.68) | 45.6 (0.36) | 46.5 (0.47) | 0.194 |
BMI (kg/m2) | 28.2 (0.7) | 28.2 (0.4) | 27.6 (0.5) | 0.565 |
Estradiol (nmol/l) | 0.29 (0.051) | 0.35 (0.054) | 0.39 (0.069) | 0.462 |
Free estradiol (nmol/l) | 0.0059 (0.0008) | 0.0073 (0.0010) | 0.0083 (0.0015) | 0.242 |
Muscle CSA (mm2) | 5950.9 (109.8) | 5880.6 (73.7) | 6199.8 (91.5) | 0.014 |
Hand grip strength (N) | 190.6 (6.0) | 189.3 (3.8) | 194.0 (5.7) | 0.763 |
Knee extension strength (N) | 352.5 (17.2) | 322.5 (14.7) | 343.4 (16.7) | 0.092 |
Leg extension power (W) | 101.0 (4.2) | 99.1 (2.6) | 100.9 (3.4) | 0.858 |
Data are mean (SE).
Adjusted for age and height
Contrasts: COMTLL vs. COMTHL (p = 0.004); COMTLL vs. COMTHH (p = 0.078); COMTHL vs. COMTHH (p = 0.569)
The most common genotype was Pp (n = 187, 43.1 %), whereas pp genotype was more frequent (n = 144, 33.2 %) than PP (n = 90, 20.7 %). The allele frequencies were 0.44 and 0.56 for the P and p alleles, respectively. The genotypes were slightly out of Hardy-Weinberg equilibrium (χ2 = 3.943, p = 0.047) suggesting that our study sample may not be representative of the target population. Physical characteristics, including hormone levels, were similar in all PvuII genotypes. Furthermore, PvuII polymorphism was not associated with any of the measured muscle variables (
Variable | ESR1 genotypes | p for trend | ||
PP (n = 90) | Pp (n = 187) | pp (n = 144) | ||
Weight (kg) | 70.3 (1.4) | 69.6 (1.1) | 70.7 (1.3) | 0.797 |
Height (cm) | 159.2 (0.88) | 158.3 (0.58) | 158.5 (0.63) | 0.669 |
Body fat (kg) | 24.3 (1.1) | 23.7 (0.7) | 24.6 (1.0) | 0.664 |
Lean body mass (kg) | 46.3 (0.52) | 45.7 (0.44) | 46.2 (0.44) | 0.573 |
BMI (kg/m2) | 27.8 (0.6) | 27.9 (0.4) | 28.3 (0.5) | 0.813 |
Estradiol (nmol/l) | 0.36 (0.08) | 0.31 (0.04) | 0.40 (0.08) | 0.584 |
Free estradiol (nmol/l) | 0.0075 (0.0017) | 0.0062 (0.0006) | 0.0086 (0.0017) | 0.375 |
Muscle CSA (mm2) | 6124.6 (121.5) | 5927.3 (81.8) | 6002.5 (97.4) | 0.393 |
Hand grip strength (N) | 192.0 (6.3) | 190.3 (4.4) | 192.3 (5.2) | 0.950 |
Knee extension strength (N) | 347.7 (17.8) | 337.2 (14.8) | 323.2 (15.1) | 0.416 |
Leg extension power (W) | 99.2 (3.5) | 98.3 (2.5) | 103.5 (3.7) | 0.494 |
Data are mean (SE).
Adjusted for age and height
We further studied whether ESR1 modified the effects of COMT. The results of age-adjusted models are shown in
Effect (reference group) | Muscle CSA | Hand grip strength | Knee extension strength | Leg extension power | |||||||||||||
Mdf | SE | p value | rY⋅e | Mdf | SE | p value | rY⋅e | Mdf | SE | p value | rY⋅e | Mdf | SE | p value | rY⋅e | ||
Val158Met main | HL | −64.46 | 136.44 | 0.637 | −0.034 | 0.76 | 7.37 | 0.918 | 0.007 | −18.89 | 11.16 | 0.090 | −0.124 | −1.39 | 4.34 | 0.749 | −0.023 |
effect (HH) | LL | 307.15 | 147.78 | 0.148 | 6.94 | 9.11 | 0.446 | 0.054 | −6.80 | 12.11 | 0.574 | −0.041 | 1.86 | 5.16 | 0.718 | 0.026 | |
PvuII main effect | Pp | −60.34 | 125.72 | 0.631 | −0.034 | −7.92 | 7.83 | 0.312 | −0.072 | −15.32 | 11.74 | 0.192 | −0.096 | −4.48 | 4.79 | 0.349 | −0.067 |
(pp) | PP | 165.45 | 151.92 | 0.276 | 0.078 | −4.81 | 9.17 | 0.600 | −0.037 | −2.78 | 11.01 | 0.801 | −0.019 | −2.97 | 5.37 | 0.581 | −0.040 |
Val158Met*PvuII | HLPp | −256.27 | 318.00 | 0.420 | −0.058 | 16.13 | 18.57 | 0.385 | 0.062 | 14.90 | 28.24 | 0.598 | 0.039 | −0.59 | 10.86 | 0.957 | −0.004 |
interaction effect | HLPP | −304.91 | 381.91 | 0.425 | −0.057 | 4.09 | 19.41 | 0.833 | 0.015 | −15.94 | 26.63 | 0.549 | −0.044 | −6.98 | 11.41 | 0.540 | −0.044 |
(HHpp) | LLPp | −572.52 | 324.83 | 0.078 | −0.126 | −17.25 | 20.40 | 0.398 | −0.060 | −9.03 | 31.12 | 0.772 | −0.021 | 0.70 | 12.39 | 0.955 | 0.004 |
LLPP | −302.04 | 380.25 | 0.427 | −0.057 | −44.14 | 25.18 | 0.080 | −0.124 | −61.44 | 28.44 | −0.158 | −18.20 | 13.14 | 0.166 | −0.099 |
In further analyses we examined whether physical activity level modulates the effects of COMTVal158Met (
Diagram presents the mean values (+SE) for CSA, hand grip strength, knee extension strength and leg extension power from GEE model according to COMT genotypes (HH, HL and LL) and physical activity (sed for sedentary, mod for moderately active and act for active). The model is adjusted with age. Results from statistical testing are shown in
Effect (reference group) | Muscle CSA | Hand grip strength | Knee extension strength | Leg extension power | |||||||||||||
Mdf | SE | p value | rY⋅e | Mdf | SE | p value | rY⋅e | Mdf | SE | p value | rY⋅e | Mdf | SE | p value | rY⋅e | ||
Val158Met main | HL | 47.79 | 123.75 | 0.699 | 0.028 | 4.41 | 7.16 | 0.538 | 0.044 | −4.87 | 10.62 | 0.646 | −0.034 | 0.01 | 4.34 | 0.997 | 0.000 |
effect (HH) | LL | 340.29 | 147.30 | 0.164 | 12.31 | 8.69 | 0.157 | 0.101 | 5.42 | 11.93 | 0.649 | 0.034 | 1.99 | 5.18 | 0.701 | 0.028 | |
Physical activity | mod | 135.35 | 105.35 | 0.199 | 0.092 | 17.58 | 6.05 | 0.204 | 48.19 | 9.97 | < | 0.337 | 15.31 | 3.25 | < | 0.323 | |
main effect (sed) | act | 223.79 | 127.19 | 0.078 | 0.126 | 24.22 | 7.12 | 0.237 | 60.91 | 10.67 | < | 0.390 | 16.34 | 4.25 | < | 0.268 | |
Val158Met*physical | HLmod | −588.22 | 246.92 | −0.169 | −36.76 | 14.52 | −0.178 | −65.69 | 23.74 | −0.201 | −23.76 | 7.82 | −0.215 | ||||
activity interaction | HLact | −260.30 | 316.71 | 0.411 | −0.059 | −25.35 | 16.40 | 0.122 | −0.110 | −61.37 | 26.53 | −0.169 | −27.57 | 10.24 | −0.191 | ||
effect (HHsed) | LLmod | −849.69 | 260.99 | −0.228 | −25.24 | 16.59 | 0.128 | −0.108 | −53.31 | 26.58 | −0.147 | −17.35 | 8.65 | −0.144 | |||
LLact | −644.78 | 331.07 | −0.139 | −21.59 | 17.69 | 0.222 | −0.087 | −62.35 | 28.02 | −0.163 | −25.42 | 11.26 | −0.161 |
sed = sedentary
mod = moderately active
act = active
Effect (reference group) | Muscle CSA | Hand grip strength | Knee extension strength | Leg extension power | |||||||||||||
Mdf | SE | p value | rY⋅e | Mdf | SE | p value | rY⋅e | Mdf | SE | p value | rY⋅e | Mdf | SE | p value | rY⋅e | ||
PvuII main effect | Pp | −13.08 | 127.74 | 0.918 | −0.007 | −4.90 | 7.41 | 0.508 | 0.047 | −10.16 | 10.02 | 0.311 | −0.075 | −5.63 | 4.49 | 0.210 | −0.090 |
(pp) | PP | 170.25 | 162.36 | 0.294 | 0.075 | −2.81 | 8.78 | 0.749 | 0.023 | −2.82 | 10.45 | 0.787 | −0.020 | −3.23 | 5.23 | 0.537 | −0.045 |
Physical activity | mod | 27.06 | 116.03 | 0.816 | 0.017 | 17.05 | 5.91 | 0.202 | 37.90 | 9.40 | < | 0.286 | 11.95 | 3.32 | < | 0.252 | |
main effect (sed) | act | 115.03 | 132.59 | 0.386 | 0.062 | 26.27 | 6.87 | < | 0.264 | 50.63 | 9.67 | < | 0.362 | 12.53 | 3.80 | 0.232 | |
PvuII*physical | Ppmod | −214.46 | 217.09 | 0.323 | −0.071 | −8.96 | 13.05 | 0.493 | 0.049 | −13.22 | 19.57 | 0.499 | −0.050 | −9.99 | 7.44 | 0.179 | −0.097 |
activity interaction | Ppact | 133.93 | 234.63 | 0.568 | 0.041 | −19.71 | 15.79 | 0.212 | 0.089 | 8.30 | 23.57 | 0.725 | 0.026 | −7.58 | 9.25 | 0.412 | −0.059 |
effect (ppsed) | PPmod | −270.12 | 316.90 | 0.394 | −0.061 | 20.86 | 16.28 | 0.200 | 0.091 | −22.52 | 23.62 | 0.340 | −0.070 | −8.07 | 8.90 | 0.364 | −0.065 |
PPact | −129.81 | 369.17 | 0.725 | −0.025 | 12.89 | 18.20 | 0.479 | 0.051 | −0.90 | 23.36 | 0.969 | −0.003 | 1.32 | 10.05 | 0.896 | 0.010 |
sed = sedentary
mod = moderately active
act = activ
In the model including ESR1 genotype, physical activity and age as explanatory variables, physical activity had a main effect on muscle strength and power (sedentary subjects were weaker than moderately active or active individuals, p≤0.004,
In the present study we examined the contribution of inter-individual variation in two candidate genes involved in estrogen metabolism and action,
Since the
Our results suggest that Val158Met polymorphism affects muscle size such that LL genotype favors larger muscle cross-sectional area. This result is supported by a similar outcome in a study with early pubertal girls
In our study sample, no association between PvuII polymorphism in
In theory, polymorphisms within
In the GEE model dissecting the interaction effects of COMT Val158Met polymorphism and physical activity on muscle properties a more clear gradient of the effects was observed. The HH subjects showed more variation in relation to physically active life-style compared to other genotypes as measured by knee extension strength and leg extension power. For example, the adjusted mean values in moderately active subjects with the HH genotype were 36.7 % higher in knee extension strength than in sedentary subjects with the same genotype, whereas within the HL genotype this difference was only 7.9 % in the favor of the moderately active subjects (
Physical activity had a significant main effect on muscle strength and power measures, in the models investigating the interaction effects between COMT or ESR1 genotype and physically active life-style. Here, both moderately active and active individuals were stronger than sedentary subjects. In muscle mass, however, the effect was less clear or absent. This observation shows that our assessment of physical activity level with the modified scale of Grimby was in accordance with our expectations in muscle performance variables, but not in muscle mass. This notion seems reasonable taken into account the general mode of physical activity in older subjects; physical activity in the ages around 60 and 70 in general is not hypertrophying that would be evident as an increased muscle mass, but rather includes various types of aerobic everyday activities affecting the properties of muscle performance. Our assessment was clearly able to differentiate sedentary individuals from more active ones in the model investigating the interaction of physical activity with the COMT genotype.
A limitation of the present study is the relatively small sample size, which may have also been selected towards rather healthy women creating a possible healthy population bias. Moreover, we present results from various measurements describing muscle strength. Our test battery includes variables presenting both isometric (hand grip and knee extension strength) and dynamic (leg extension power) muscle performance as well as measures from both lower and upper limbs. Furthermore, during isometric testing, the speed of muscle contraction is not as essential as in muscle power measurements. On the other hand, these data provide a multifaceted estimate of the effects of the chosen genotypes on whole body musculature. The results provided by our cross-sectional data set should be further confirmed in a follow-up study and, if possible, with a larger sample and an intervention trial to see, if the response to training is actually genotype-dependent.
In conclusion, the identification of the genetic susceptibility factors predisposing the elderly to impaired muscle performance could provide novel insights into the etiology of sarcopenia and would enable the recognition of those people at high risk of disability. Analysis of genetic variants, such as SNPs, represents a powerful approach to examine the role of candidate genes in the progression of this obviously multifactorial state. In the present study, we found an association between a polymorphism in the
The authors want to thank all the women participating in this study and Kaisa-Leena Tulla, Erkki Helkala and Tuovi Nykänen for technical assistance in the lab.