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Hormone Replacement Therapy and Messenger RNA Expression of Estrogen Receptor Coregulators after Exercise in Postmenopausal Women

DIELI-CONWRIGHT, CHRISTINA M.1; SPEKTOR, TANYA M.2; RICE, JUDD C.2; SATTLER, FRED R.3; SCHROEDER, E. TODD1

Medicine & Science in Sports & Exercise: March 2010 - Volume 42 - Issue 3 - p 422-429
doi: 10.1249/MSS.0b013e3181b7193f
CLINICAL SCIENCES
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The use of hormone replacement therapy (HRT) is a potential treatment to relieve symptoms of menopause in postmenopausal women; however, the effects on skeletal muscle are unclear. Specifically, it is unknown if HRT enhances estrogen receptor (ER) transcriptional activation in skeletal muscle at rest and after resistance exercise.

Purpose: To evaluate changes in the messenger RNA (mRNA) expression of ER coregulators (steroid receptor coactivator-1 (SRC-1) and silencing mediator of retinoid and thyroid receptors (SMRT)) in postmenopausal women after a maximal eccentric exercise bout.

Methods: Fourteen postmenopausal women were divided into two groups: Control, women not using HRT (n = 6, 59.2 ± 4.2 yr, 63.1 ± 17.4 kg); or HRT, women using traditional HRT (n = 8, 58.5 ± 3.7 yr, 89.5 ± 23.7 kg). Participants performed 10 sets of 10 maximal eccentric repetitions of single-leg extension on the Cybex dynamometer at 60°·s−1 with 20-s rest periods between sets. Muscle biopsies of the vastus lateralis were obtained from the exercised leg at baseline and 4 h after the exercise bout. mRNA expression was determined using real-time polymerase chain reaction for SRC-1 and SMRT.

Results: mRNA expression of SRC-1 significantly increased (P ≤ 0.01; 2.4- to 5.2-fold change) and mRNA expression of SMRT significantly decreased (P ≤ 0.01; −1.3- to −4.3-fold change) after the exercise bout in both groups. We observed significantly greater changes in mRNA expression of SRC-1 and SMRT (P ≤ 0.01) in the HRT group compared with controls after exercise.

Conclusions: A single bout of maximal eccentric exercise enhances ER transcriptional activity with a greater response present in postmenopausal women using HRT.

1Division of Biokinesiology and Physical Therapy, Clinical Exercise Research Center, University of Southern California, Los Angeles, CA; and 2Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA; 3Keck School of Medicine, University of Southern California, Los Angeles, CA

Address for correspondence: E. Todd Schroeder, Ph.D., 1540 E Alcazar St CHP-155, Los Angeles, CA 90033; E-mail: eschroed@usc.edu.

Submitted for publication March 2009.

Accepted for publication July 2009.

The effects of estrogen and hormone replacement therapy (HRT) are widespread throughout the human body, and they are commonly prescribed to treat symptoms of menopause. However, the effects of HRT on skeletal muscle are unknown. Estrogen receptors (ER) are present in many tissues and are found in the greatest amounts in the target tissues with reproductive functions such as the mammary glands, ovaries, vagina, and uterus. Most recently, ER have been identified in human skeletal muscle (23,44-46), yet their role has not been identified. The majority of estrogen's actions occur through the ER protein, a member of the nuclear hormone superfamily. ER directly stimulate messenger RNA (mRNA) expression by interacting with coactivators and corepressors. ER activation occurs as follows: estrogen binds to the ER and induces a conformational change in the receptor and then the ER bound with estrogen binds to its estrogen response element in the promoter of target genes and recruits coregulator proteins that physically bridge the receptor and the RNA polymerase complex (24).

Coactivators and corepressors interact with and modulate ER function. Coactivators including steroid receptor coactivator-1 (SRC-1), glutamate receptor-interacting protein (GRIP), and amplified in breast cancer (AIB), preferentially interact with an agonist-activated ER to potentiate transcriptional activity. Coactivators promote coupling of the receptor to the transcriptional apparatus, causing an alteration in the architecture of the target promoter to further facilitate transcription (27). Corepressors, such as nuclear receptor corepressor (NCor) and silencing mediator of retinoid and thyroid receptors (SMRT), interact with antagonistic-activated ER to suppress ER activation by altering the structure of the responsive promoter to decrease its ability to be transcribed (27). SRC-1 and SMRT are extensively studied in breast cancer cells to indicate ER activity and expression (13,22). Therefore, examining these proteins in skeletal muscle in postmenopausal women would provide an indication of ER transcriptional activity after a stimulus such as resistance exercise. By directly extracting muscle tissue, ER transcriptional activity can be measured by examining coactivator and corepressor mRNA expressions. Examining changes in ER coregulator mRNA expression after maximal eccentric exercise will provide novel data in the areas of skeletal muscle physiology and endocrinology. Further, investigating the effects of resistance training on ER coregulators may provide new information on the mechanisms of estrogen in skeletal muscle.

The effects of estrogen on skeletal muscle have been studied in animal and in vitro models. In animal models, estrogen influences MyoD (2) to attenuate leukocyte infiltration and influence satellite cell activation. Estrogen may play a role in skeletal myoblast growth as indicated by an increase in myogenic mRNA expression in estrogen-treated myoblasts (11,12). Our laboratory recently demonstrated the effects of estradiol-based therapies on MyoD and found that estradiol increases MyoD mRNA expression in human skeletal muscle cells (10). Therefore, a potential effect of HRT use in postmenopausal women includes up-regulation of MyoD to enhance satellite cell activation and hypertrophic responses, leading to prevention of sarcopenia. Previous studies have examined the effects of HRT use on skeletal muscle in postmenopausal women. Some studies have indicated that the use of HRT does not influence muscle strength, muscle mass, or protection against sarcopenia (3,19,25,30,40), whereas others have indicated that estrogen and synthetic HRT may provide an anabolic effect on skeletal muscle (7,16,32,35-37). Nonetheless, these studies have not involved muscle biopsies or muscle protein analysis.

Recently, our laboratory investigated the effects of estradiol on ER coregulators in cultured human myoblasts. Our study provides the first evidence demonstrating that estradiol treatment increases SRC-1 mRNA expression and decreases SMRT mRNA expression, indicating that coactivator recruitment is present, which may ultimately activate the ER (10). On the basis of the results from our in vitro study, we hypothesized that in postmenopausal women, after a single bout of maximal eccentric resistance exercise, HRT will induce a greater increase in the mRNA expression level of SRC-1 (coactivator) and a greater decrease in the mRNA expression level of SMRT (corepressor) when compared with controls. The aim of this study was to determine the effects of HRT on ER coregulators in human skeletal muscle after a maximal eccentric exercise bout in postmenopausal women.

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METHODS

Study participants.

We studied 14 healthy untrained (no participation in consistent, structured weight, or cardiovascular training) postmenopausal women, aged 55-65 yr. Participants provided written informed consent approved by the University of Southern California (USC) Institutional Review Board. The participants had to be without spontaneous menstrual bleeding for at least 6 months. Participants were recruited to either a control group (not taking any form of HRT) or a HRT group. Participants not using a particular hormone therapy must have been free from drug therapy for at least 3 months.

Participants were recruited to the HRT group if they were using one of the following types of oral HRT for at least 3 months: Premarin (0.3, 0.45, or 0.625 mg tablet per day), Prempro (0.3, 0.45, or 0.625 mg tablet per day), Estrace (0.5 or 1.0 mg tablet per day), FemHRT (0.5 or 1.0 mg tablet per day), or Menest (0.3 or 0.625 mg tablet per day). Premarin, Menest, and Estrace are synthetic forms of HRT containing estrogen only. Prempro and FemHRT are synthetic forms of HRT containing estrogen and progestin. Oral forms and doses were chosen on the basis of popularity of usage and common doses prescribed determined by a university gynecologist. Because of a lack of research in HRT use and ER coactivators, there was no justification to limit recruitment of HRT users to either estrogen alone or estrogen/progestin forms. In addition, we did not expect the different types of HRT to affect the outcomes of the study.

Screening of subjects was performed, with clinical history, physical examination, and laboratory tests, including complete blood count with differential, coagulation profile, fasting blood glucose, liver and kidney function tests, thyroid-stimulating hormone, lipid profile, urinalysis, ECG, and chest x-ray. Exclusion criteria included cancer within the last 5 yr, diabetes or fasting blood sugar >126 mg·dL−1, active cirrhosis or hepatitis, uncontrolled hypothyroidism or hyperthyroidism, lung disease limiting or requiring O2, creatinine clearance <30 mL·min−1, alanine aminotransferase (ALT) > 1.5× upper limit of normal (ULN), or cardiovascular abnormalities (myocardial infarction, heart failure, active angina, or ischemia within the last 6 months), musculoskeletal disease, or injury (rheumatoid arthritis, neuropathy, back injury, etc.) that would prevent resistance training, use of testosterone or other anabolic therapies within the last 6 months, and use of heparin or coumadin within the last 6 months.

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Experimental design.

All participants were familiarized with the procedures and equipment and had anthropometric measures made before the acute exercise bout. Dietary analysis and physical activity history were assessed. The control and HRT groups performed 10 sets of 10 repetitions of maximal eccentric knee extensions. Muscle biopsies were obtained at baseline and 4 h after exercise. Participants fasted at least 6 h before each biopsy.

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Blood draws.

Venous blood was collected from the antecubital vein by standard sterile procedures at the USC General Clinical Research Center (GCRC) and was analyzed by the Los Angeles County-University of Southern California Clinical Laboratory for chemistries and blood counts. Two blood draws were performed. Blood draw 1 was performed during pre-entry visit 1 and analyzed for complete blood count with differential, coagulation profile, fasting blood glucose, liver and kidney function tests, thyroid-stimulating hormone, lipid profile, and circulating estrogen. Blood draw 2 was performed during visit 5 and was analyzed for circulating estrogen. Circulating estrogen was measured using radioimmunoassays (Quest Diagnostics, San Juan Capistrano, CA). Participants fasted for 6-8 h before each blood draw.

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Anthropometric and body composition testing.

Total body mass, fat mass, fat-free mass, percent fat, and bone mineral density were determined using a total-body dual-energy x-ray absorptiometry (Model DPX-IQ 2288 with Smart Scan version 4.7e; Lunar Radiation Corporation, Madison, WI). Quality assurance was performed using a single acrylic block three times per week to confirm the accuracy and precision of the DEXA system. The same experienced investigator was responsible for performing and analyzing all scans. Body composition variables were necessary to analyze because they may serve as potential confounders for the outcome variables. The main storage site for estrogen is adipose tissue; therefore, it is necessary to measure body composition.

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Physical activity assessment.

All potential participants were prescreened over the phone by the principal investigator to ensure that they were not participating in regular strenuous physical activity. Physical activity levels were assessed for each participant during pre-entry visit 1 using the entry questionnaire and physical activity scale (1). The purpose of the physical activity assessment was to confirm that the participants were not participating in regular activities that would interfere with out study design. In addition, if the participants were at different fitness levels, the assessments served as a means to control for within-subject and between-subject variabilities.

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Dietary analysis.

The participants completed a 3-d dietary food record provided by the USC GCRC Bionutrition Department. The participants were instructed to record their dietary intake during two weekdays and one weekend day. Participants discussed their completed food record with a registered dietitian from the Bionutrition Department to determine accuracy in portion size estimates and detail of food intake. Analysis of the dietary record was performed by the registered dietician using Nutritionist Pro (Nutrition Analysis Software Version 1.3; Jones and Bartlett Publishers, Inc., Sudbury, MA). The dietary assessment served as a means to control for within-subject and between-subject variabilities on the basis of dietary intake of different nutrients that may act as potential confounders.

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Strength testing.

Strength testing was preceded by 5 min of warm-up on a cycle ergometer. Strength testing was performed using the Cybex Norm dynamometer (Cybex International, Inc., Ronkonkoma, NY). Peak concentric and eccentric torque of the knee extensors was performed with the dominant leg using the Cybex Norm dynamometer at 60°·s−1. The Cybex was calibrated immediately before the exercise bout. Participants were positioned on the Cybex by visually aligning the placement of the lateral femoral condyle with the axis of rotation for the Cybex. A strap was placed distally on the dominant leg at the level of the load cell. The load cell was positioned 3 cm proximal to the talocrural joint. During the maximal loading, shoulder harness, hip restraint, and thigh strap (exercised leg) were used to limit excessive movement and secure the participant to the device. The settings were recorded to ensure exact placement for each exercise bout.

The tester determined the participants' peak eccentric and concentric torque. This was determined to assess whether the participants were generating maximal effort during each set of the acute training bout. Participants received detailed instructions on the eccentric and concentric leg extension exercises, and performed no more than five repetitions of each exercise to achieve peak torque. The strength testing only occurred once because of recent indications that repeat strength testing is not warranted (39).

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Muscle biopsies.

Percutaneous muscle biopsies (150-200 mg) were obtained from the vastus lateralis of the exercised leg 1 wk before the acute exercise bout and 4 h after exercise. Four hours after exercise is an optimal time point to sample for changes in skeletal muscle mRNA expression stimulated by exercise (47). Biopsy specimens were collected using sterile conditions and local anesthesia (1% lidocaine) with a 5-mm Stille biopsy needle (Micrins Surgical, Lake Forest, IL) from the midportion of the vastus lateralis muscle. The postexercise biopsy was performed at a distance of 2-4 cm proximal to the first site. Muscle tissue samples were immediately flash-frozen in liquid nitrogen and stored at −80°C until being processed for analysis.

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Acute eccentric resistance exercise bout.

The participants completed one acute bout of single-leg knee extension of maximal eccentric isokinetic loading on the dominant leg using the Cybex Norm dynamometer (Cybex International, Inc.) at 60°·s−1. The Cybex was calibrated immediately before the exercise bout. Participants were positioned on the Cybex using the settings recorded from the strength testing visit. The exercise protocol consisted of 10 sets of 10 maximal eccentric repetitions with the participants performing the eccentric component while the investigator performed the concentric component by moving the limb back to the starting position (15° before full extension). Each repetition was separated by the time it took the testing investigator to manually return the lever arm back to 75° of knee flexion (i.e., starting position). Each of the 10 sets was separated by 20 s. Biofeedback was provided on the computer screen, and verbal encouragement was provided by the tester during each maximal contraction. The acute exercise bout occurred approximately 2 wk after strength testing and 1 wk after the baseline muscle biopsy to allow recovery from soreness. The exercise stimulus used in this study was designed to induce muscle damage to stimulate muscle protein synthesis. High-intensity eccentric resistance training induces muscle damage that stimulates protein synthesis and ultimately muscle hypertrophy to a greater magnitude than concentric resistance training (38).

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Use of positive control genes.

Positive controls genes were analyzed to confirm that the basic conditions of the study design experiment produced a positive result. The mRNA expression of MyoD, a positive regulator of muscle differentiation and growth, is commonly quantified in skeletal muscle biopsies after resistance exercise (4,8,9,28,29,43,47). Previous data indicate that MyoD expression increases after resistance exercise (8,28,29,43,47). In addition, glucose transporter 4 (GLUT4) mRNA expression, a glucose transporter that facilitates the passive diffusion of glucose into muscle cells, is examined in skeletal muscle after resistance exercise bouts, and levels of GLUT4 mRNA expression increase after resistance exercise (14,15,20,31). Owing to convincing evidence of the effects of exercise on mRNA expression of MyoD and GLUT4, these genes served as positive controls to determine the effects of the acute exercise bout on skeletal muscle mRNA expression analyses.

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RNA extraction.

Total RNA was isolated after homogenization (Polytron PT1200C; Kinematica, Lucerne, Switzerland) of 30-40 mg of muscle tissue with a monophasic solution containing guanidine-isothiocyanate-containing lysis buffer and β-mercaptoethanol. The concentration and purity of the RNA was determined using a UV spectrophotometer (NanoDrop ND-1000; Thermo Scientific, Waltham, MA) by measuring absorbance at 260 and 280 nm.

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Reverse transcription and complementary DNA synthesis.

Five hundred nanograms of total skeletal muscle RNA was reverse-transcribed to synthesize complementary DNA (cDNA) using TaqMan reverse transcription reagents according to the manufacturer's instructions (Applied Biosystems, Branchburg, NJ). Briefly, a 50-μL reaction mixture was constructed consisting of 0.5 μg of cellular RNA, 5 μL of 10 × reverse transcription buffer, 11 μL of MgCl2, 10 μL of deoxynucleotide 5'-triphosphate, 2.5 μL of random hexamer, 1.0 μL of RNase inhibitor, 1.25 μL of reverse transcriptase, and nuclease-free water. RNA was reverse-transcribed into cDNA with the following temperature-time protocol: 25°C for 10 min, 48°C for 30 min, 95°C for 5 min, and 4°C infinite (Mycycler; Bio-Rad, Hercules, CA).

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Oligonucleotide primers for polymerase chain reaction.

Oligonucleotide primers were used to amplify the mRNA expression of SRC-1, SMRT, MyoD, and GLUT4. Primer sequences were designed using Primer3 program (33). The primer sequences for the specific target mRNA and melt curve temperatures are shown in Table 1. Melt curves were determined for each primer set to ensure amplification of pure polymerase chain reaction (PCR) products. GAPDH was used as an internal control for detecting changes in mRNA expression using quantitative real-time PCR (qRT-PCR), which was previously used as an internal control to examine myogenic mRNA expression after resistance exercise (5,18,21,26,29,41,42).

TABLE 1

TABLE 1

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qRT-PCR.

A qRT-PCR method was applied to determine the relative expression levels of mRNA for SRC-1, SMRT, MyoD, and GLUT4. A total of 10 ng of cDNA were added to each of the 20-μL PCR for SRC-1, SMRT, MyoD, GLUT4, and GAPDH. Specifically, each PCR contained the following mixture: 10 μL of 2.5× iQSYBRGreen Supermix (Bio-Rad), 7 μL of RNase-free water, 10 ng of cDNA, and 10 pmol of each primer (forward and reverse) of interest. All samples were run in quadruplicate. Each PCR was amplified using Bio-Rad iCycler iQ thermal cycler (Bio-Rad). Thermal cycling conditions were as specified by the manufacturer: the amplification profile involved the initial denaturation step at 95°C for 3 min, followed by 60 cycles consisting of denaturation at 95°C for 15 s, and primer annealing and extension at 56°C for 1 min.

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mRNA quantification.

All data were determined by normalizing the cDNA measured in eight replicates (four replicates repeated) for each participant sample to GAPDH (internal control). The data were averaged to account for the change in mRNA expression as a result of the exercise stimulus. The data were normalized to biopsy 1 (before exercise) for each participant to determine the fold change of mRNA expression after exercising. All participant samples were then averaged to determine the average mRNA expression before and after exercise for each group. mRNA expression was evaluated by a relative quantification method (47). Briefly, this method is based on the fact that the difference in threshold cycles (ΔCT) between the genes of interest and the internal control genes is proportional to the relative expression level of the genes of interest. The data were analyzed using the 2−ΔCT method (34) to compare the relative mRNA expression (arbitrary units) between preexercise and postexercise biopsies and is called the fold change in mRNA expression.

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Statistical methods.

Statistical analyses were performed using SPSS version 16.0 (SPSS, Inc., Chicago, IL). Descriptive statistics and Pearson correlations were used to analyze the control and HRT groups. Independent t-tests were performed to test group differences (control vs HRT) with Bonferroni adjustments. A probability level of P ≤ 0.05 was used to determine statistical significance. Group differences in mRNA expression were determined by a 2 × 2 ANCOVA, with factors of time (before and 4 h after exercise) and group (control and HRT). Weight, body mass index, bone mineral density, bone mineral content (BMC), percent fat, fat mass, lean mass, protein intake, and carbohydrate intake were included as covariates. Simple linear regression techniques were used to determine the best individual predictor for changes in mRNA expression of SRC-1, SMRT, MyoD, and GLUT4.

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RESULTS

Baseline characteristics of the study participants are displayed in Table 2. Body weight and lean body mass were significantly greater in the HRT group (P < 0.05). There were no differences between groups in dietary intake, physical activity, and muscle strength. Although there were group differences in weight and lean mass, our results from ANCOVA indicated that participant characteristics were not significant covariates. Therefore, the data presented reflect results from independent t-tests. There was a significant increase in SRC-1 mRNA expression in the control and HRT groups after exercise (P < 0.01; Fig. 1). SRC-1 mRNA expression increased to a greater extent in the HRT group when compared with the control group (P < 0.01; Fig. 1). There was a significant decrease in SMRT mRNA expression in the control and HRT groups after exercise (P < 0.01; Fig. 2). SMRT mRNA expression decreased to a greater extent in the HRT group when compared with the control group (P < 0.01; Fig. 2). Regression analyses indicated that the best predictor of changes in mRNA expression of SRC-1 and SMRT was lean body mass (R2 = 0.37, P = 0.02 and R2 = 0.40, P = 0.01, respectively).

TABLE 2

TABLE 2

FIGURE 1

FIGURE 1

FIGURE 2

FIGURE 2

There was a significant increase in MyoD mRNA expression in the control and HRT groups after exercise (P < 0.01; Fig. 3). MyoD mRNA expression increased to a greater extent in the HRT group when compared with the control group (P < 0.01; Fig. 3). There was a significant increase in GLUT4 mRNA expression in the control and HRT groups after exercise (P < 0.01; Fig. 4). There were no differences in GLUT4 mRNA expression between the two groups (P > 0.05; Fig. 4). Regression analyses indicated that the best predictor of changes in mRNA expression of MyoD was lean body mass (R2 = 0.37, P = 0.02).

FIGURE 3

FIGURE 3

FIGURE 4

FIGURE 4

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DISCUSSION

The primary finding from our study was that an acute bout of maximal eccentric exercise alters ER coregulator gene expression in postmenopausal women. In addition, ER coregulator gene expression was further enhanced in postmenopausal women using HRT. This is the first study to examine ER coregulators in skeletal muscle in postmenopausal women. Our findings were in support of our hypothesis: after a maximal bout of eccentric resistance exercise, postmenopausal women using HRT demonstrated a greater increase in the mRNA expression levels of SRC-1 and a greater decrease in the mRNA expression level of SMRT when compared with the postmenopausal women not using HRT. Previous studies have indicated that use of HRT does not influence muscle strength, muscle mass, or protection against sarcopenia (3,19,25,30,40). However, estrogen and synthetic HRT may provide an anabolic effect on skeletal muscle as demonstrated with improved muscle performance and lean body mass (7,16,32,35-37). Nonetheless, these studies have not involved muscle biopsies or muscle protein analysis. Our study provides unique evidence that HRT use affects skeletal muscle by altering mRNA expression levels of ER coregulators combined with resistance exercise. Potential pathways altered by the ER include muscle growth and hypertrophy, inflammation and repair, and metabolism.

As indicated by increased expression of SRC-1, both the control and HRT groups demonstrated increases in ER activity after exercise. These results demonstrate that despite differences in estrogen levels, maximal eccentric exercise stimulates the ER coactivator and the receptor itself. However, the greater expression of SRC-1 is a direct result of HRT use combined with maximal eccentric exercise in that group, causing a larger effect on the ER. Exact benefits of increased coactivator recruitment to the ER are unclear, yet may lead to the activation of myogenic pathways to promote muscle protein synthesis and hypertrophy. Possible effects on other pathways in the muscle may result, and thus, further studies are warranted.

Decreased expression of SMRT in both the control and HRT groups indicated that this corepressor has a reduced ability to alter ER transcriptional activity after exercise. These results reveal that despite differences in estrogen levels, maximal eccentric exercise decreases mRNA expression of SMRT. However, the greater decrease in the expression of SMRT in the HRT group is a direct result of HRT use combined with maximal eccentric exercise, causing a larger effect on the ER. Specific benefits of decreased corepressor recruitment to the ER are unclear, yet results imply that SMRT had a reduced ability to suppress ER transcription and to promote enhanced transcriptional ability.

In addition, our results indicated little between-subject variability within the two groups for mRNA expression of SRC-1 and SMRT. The data were consistent in each group without the presence of statistical outliers, indicating a strong treatment effect from the acute exercise and HRT use. Despite group differences in body composition with the HRT group having significantly greater mass, group differences in mRNA expression of SRC-1 and SMRT were not affected by weight or other covariates. Importantly, baseline gene expression of SRC-1 and SMRT did not differ between the two groups. Fold changes seen in both groups resulted from the exercise stimulus in the control group and HRT combined with exercise in the HRT group.

The exercise stimulus used in this study was designed to induce muscle damage to stimulate muscle protein synthesis. A plausible explanation for our findings may be a direct result of the type of exercise stimulus. High-intensity eccentric resistance training induces muscle damage that stimulates protein synthesis and ultimately muscle hypertrophy to a greater magnitude than concentric resistance training (38). Maximal eccentric resistance exercise, such as the protocol used here, also produces muscle inflammation and breakdown. Elevated markers of muscle inflammation and breakdown were demonstrated in our study (data not shown), which may result in altered ER coregulator gene expression. This type of exercise increased coactivator mRNA expression to potentially influence transcription at that ER in postmenopausal women. The present study emphasizes the use of eccentric resistance exercise as an appropriate means to alter ER coregulator expression in postmenopausal women, which is further enhanced with HRT use.

In addition to ER coregulators, we examined changes in mRNA expression of MyoD and GLUT4. These genes were included to serve as positive controls in an effort to confirm that the basic conditions of this study were able to produce a positive result. Further, results from our analyses of MyoD and GLUT4 provide insight into the effects of HRT on skeletal muscle after exercise once the ER is activated or the coactivator expression is enhanced. MyoD, a protein that plays a key role in regulating muscle differentiation and hypertrophic responses of skeletal muscle (17), is commonly quantified in skeletal muscle biopsies after resistance exercise (4). In our study, after a maximal bout of eccentric resistance exercise, mRNA expression levels of MyoD significantly increased in both groups and to a greater magnitude in the HRT group. Results from our study are similar to those from previous studies that have reported increased MyoD expression after resistance exercise (8,28).

Increased MyoD mRNA expression in the HRT group indicates that HRT use enhances MyoD expression in skeletal muscle after resistance exercise. In animal models, estrogen influences MyoD (2) to attenuate leukocyte infiltration and influence satellite cell activation. Estrogen may play a role in skeletal myoblast growth as indicated by an increase in myogenic mRNA expression in estrogen-treated myoblasts (11,12). Our data also indicate an influence of estradiol-based therapies on MyoD. Therefore, a potential effect of HRT use in postmenopausal women may include up-regulation of MyoD to enhance satellite cell activation and hypertrophic responses, leading to prevention of sarcopenia. Attenuation of sarcopenia can result in improved muscle strength and mass, balance, and decreased risk of fractures. Further studies using skeletal muscle gene analyses are necessary to determine the long-term effects of HRT on sarcopenia.

Possible inferences can be made with our results from ER coregulator mRNA expression and myogenic mRNA expression. ER coactivator expression was increased after exercise to increase transcriptional activity at the ER. In addition, SRC-1 expression was enhanced in the HRT group as was MyoD expression. Because of estrogen's ability to up-regulate MyoD, a relationship may exist between MyoD and HRT use in skeletal muscle. Therefore, increased coactivator expression from HRT use may result in up-regulation of MyoD to promote muscle hypertrophy. This study provides insight into potential mechanisms of action of estradiol in skeletal muscle, an area that has received minimal investigation. Further, our results lay the basis for future studies on the benefits and risks of HRT use on skeletal muscle with exercise in postmenopausal women. Levels of GLUT4 mRNA expression increase after resistance exercise (15). In our study, mRNA expression levels of GLUT4 significantly increased in both groups, similar to previous studies. In animal models, GLUT4 is positively regulated by estradiol in skeletal muscle (6). However, our study demonstrated no significant differences between groups in GLUT4 mRNA expression, indicating no relationship between HRT and regulation of GLUT4 expression in skeletal muscle.

In summary, we demonstrated that a single bout of maximal eccentric resistance exercise increases ER coactivator expression and decreases ER corepressor expression in postmenopausal women. A greater magnitude of change in mRNA expression of SRC-1 and SMRT was indicated in the HRT group. Therefore, maximal eccentric exercise combined with HRT alters ER transcriptional activity in skeletal muscle. Overall, we present novel findings that provide insight into the effects of eccentric resistance exercise on ER coregulators in postmenopausal women with and without HRT use.

This study was supported by the Clinical Exercise Research Center at the University of Southern California and local NCRR GCRC M0I RR000043. No funding for this work was received from any of the following institutions: National Institutes of Health, Wellcome Trust, Howard Hughes Medical Institute, and others.

The authors thank the nurses and personnel of the general clinical research center of the University of Southern California for their help with the clinical portion of this study. The results of the present study do not constitute endorsement by American College of Sports Medicine.

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Keywords:

SRC-1; SMRT; MyoD; ECCENTRIC EXERCISE

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