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Original Research

Hormone Therapy and Maximal Eccentric Exercise Alters Myostatin-Related Gene Expression in Postmenopausal Women

Dieli-Conwright, Christina M.1; Spektor, Tanya M.2; Rice, Judd C.2; Sattler, Fred R.3; Schroeder, E. Todd1

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Journal of Strength and Conditioning Research: May 2012 - Volume 26 - Issue 5 - p 1374-1382
doi: 10.1519/JSC.0b013e318251083f
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Abstract

Introduction

Hormone therapy (HT) is widely used by postmenopausal women to alleviate symptoms of menopause, prevent osteoporosis, and more recently, to prevent breast cancer recurrence (30). Potential effects of HT on skeletal muscle are not well established, yet our recent findings indicate that after an acute maximal eccentric exercise bout, postmenopausal women using HT experience less skeletal muscle damage indicated by a lower interleukin (IL)-6, IL-8, IL-15, and TNF-α gene expression (8) while experiencing a heightened myogenic gene response shown by an increase in gene expression levels of MyoD, myogenin, follistatin, Myf5, MRF4) (6). Previous studies have investigated the myogenic gene expression response in young and older women to resistance exercise (24,25) yet have not examined potential effects of HT use on this response, with the exception of our recent findings noted above (6,8). There is evidence to support the anabolic effect of estrogen and synthetic HT on skeletal muscle (27,32), yet it is important to investigate the effects of HT use specifically on myostatin-related gene expression. Understanding the relationship between estrogen and skeletal muscle by quantifying changes in myostatin-related gene expression is critical in understanding potential benefits of HT and endogenous estrogen on hypertrophic mechanisms, particularly in older women at a risk of developing sarcopenia.

Factors that affect the growth of skeletal muscle can have monumental effects on overall health and viability. One such factor regulating skeletal muscle growth is myostatin. Myostatin is a member of the activin-transforming growth factor-β (TGF-β) superfamily, which binds to the activin receptor type II family members, most notably activin receptor IIb (ActRIIb) (17). Myostatin inhibits skeletal muscle growth through repression of proliferation, differentiation, and protein synthesis (26). Myostatin is inhibited in part by the follistatin-related gene (FLRG) protein (12). Whether the use of HT influences myostatin levels in skeletal muscle is yet to be determined and may potentially impact muscle strength and mass ultimately influencing one's ability to carry out activities of daily living.

In addition to ActRIIb and FLRG, myostatin signaling is regulated by proteins that may antagonize its biological actions. Follistatin, for example, antagonizes numerous members of the TGF-β superfamily, including myostatin (3), by binding to the ActRIIb myostatin receptor site (17). The most potent regulator of myostatin is follistatin-like-3 (FSTL3), which shares a similar structural and functional homology with follistatin and serves as the primary binding-inhibiting protein of myostatin (2). The FSTL3 binds to myostatin to promote the disassociation between myostatin and the ActRIIb, resulting in the inhibition of myostatin activity (12). Another myostatin inhibitory protein referred to as GDF (growth and differentiation)–associated serum protein-1 (GASP-1) has recently been examined in rat skeletal muscle in response to prolonged stretching (4). However, to our knowledge, studies in human skeletal muscle are lacking and warrant further investigations to determine mechanisms of action of GASP-1. The inactivating mutations to the myostatin gene result in a hypermuscular phenotype in mice, cows, and humans (11,23,31). Collectively, ActRIIb, FLRG, FSTL3, follistatin, and GASP-1 are key components of the myostatin signaling pathway and may be targeted through resistance training, and further with HT use, to improve muscle strength, and hypertrophy in older adults.

Myogenic gene expression levels such as myostatin, myogenin, myoD, and follistatin have been shown to be responsive to a single bout of resistance exercise (8,20,24) and to long-term resistance exercise in humans (15,16,28). However, few studies have investigated changes in gene expression of ActRIIb, FLRG, FSTL3, and GASP-1. In particular, the combined effects of resistance exercise and HT on myostatin-related gene expression have received minimal investigation despite the protective and anabolic properties of estrogen on skeletal muscle (7,8). The purpose of this investigation was twofold: (a) to determine the effects of an acute eccentric exercise bout on myostatin-related gene expression and (b) to determine whether HT use potentiates a myogenic response to maximal acute eccentric exercise in postmenopausal women. We hypothesized that in postmenopausal women, after a single bout of maximal eccentric exercise, (a) myostatin-related gene expression will be significantly downregulated in women using and not using HT and (b) estrogen-based HT will enhance myostatin-related gene expression compared with women not taking HT. From a practical standpoint, this investigation was designed to determine whether HT use modifies the effects of eccentric exercise on the regulation of myostatin, which is necessary to determine possible musculoskeletal benefits of HT use in postmenopausal women.

Methods

Experimental Approach to the Problem

Data from this study are generated from muscle samples collected from a previous investigation (7). This follow-up approach was completed to provide data on gene targets, which were previously not investigated in postmenopausal women using HT combined with an acute bout of resistance exercise, to determine the possible effects of the use of synthetic estrogen on muscle growth pathways, specifically related to the myostatin protein which inhibits muscle growth. All research subjects completed an acute eccentric resistance exercise bout (10 sets of 10 maximal knee extension contractions), which has been shown to previously stimulate changes in muscle gene expression. Muscle samples were collected before and 4 hours after the exercise bout. Within each muscle sample, gene expression of myostatin, ActRIIb, follistatin, FLRG, FSTL3, and GASP-1 was determined using previously published gene primer sequences and SYBR green technology using reverse transcriptase polymerase chain reaction (RT-PCR).

Subjects

To our knowledge, this exercise protocol was not used by other investigators; thus, we powered this study from prior literature investigating myogenic factors via muscle biopsy (37). A priori analysis determined that a sample size of 6 participants in each group would have 80% power to detect a difference in means for myostatin mRNA expression of 1.5 (the difference between group 1 mean [μ1] of 2.8 and group 2 mean [μ2] of 1.3) assuming that the common SD is 0.8 with an effect size of 1.88 using a 2 group t-test with a 0.05 two-sided significance level.

Eligibility inclusion criteria included the following: (a) the subjects were postmenopausal women, 55–65 years of age, (b) the subjects were apparently healthy but could not have participated in >60 minutes of total exercise per week, and (c) the subjects may or may not be using oral HT for a minimum of 3 months before participating in the study. This study was submitted to the University of Southern California Institutional Review Board for approval, and all the subjects completed a written informed consent form.

Fourteen healthy, postmenopausal women, 55–65 years of age who were sedentary were enrolled in the study. All the participants, including the control group (no HT use), were sedentary (participation in <60 minutes of exercise per week) at the time of study enrollment that was determined by phone interview conducted by the PI (Dieli-Conwright) during the summer of 2008, before study enrollment. A complete physical activity history was not obtained. The participants were classified as postmenopausal if they had not experienced spontaneous menstrual bleeding for at least 6 months. Six participants were in the control group because they were not taking any form of HT, and 8 participants were in the HT group because they were taking HT. The participants in the control group must have not taken HT therapy for at least 3 months. The participants were recruited to the HT group if they were using oral HT for at least 3 months. Doses were previously prescribed (unrelated to the study) by a university gynecologist. Because of lack of data on effects of HT on skeletal muscle regulators, recruitment of the HT group was not limited to the participants using either estrogen-alone or estrogen plus progestin combination formulations; therefore, type of HT use differed between participants.

The participants underwent an initial screening for eligibility with a history and physical examination performed by the study physician at the general clinical research center at the USC. After the physician's examination, a nurse performed a single blood draw of the antecubital vein under fasting conditions from each subject, which was analyzed by the Endocrinology Laboratory in the general clinical research center for the following measures: estrogen, glucose, insulin, thyroid hormone, and blood cell counts. Exclusion criteria included cancer within the last 5 years, diabetes or fasting blood glucose ≥126 mg·dl−1, cirrhosis or active hepatitis, uncontrolled thyroid function, clearance <30 ml·min−1, alanine aminotransferase > 1.5× upper limit of normal, or chronic lung problems or cardiovascular disease (myocardial infarction, heart failure, or active angina within the prior 6 months before enrollment), musculoskeletal disease or injury (rheumatoid arthritis, neuropathy, back injury, etc.) that would prevent resistance training, use of testosterone or other anabolic therapies, heparin, or coumadin within the last 6 months.

Procedures

Body Composition

At baseline, total body mass, fat mass, lean mass, and percent fat were measured using a total body dual-energy x-ray absorptiometer (model DPX-IQ 2288 with Smart Scan version 4.7e; Lunar Corporation, Madison, WI, USA). The same experienced investigator (Dieli-Conwright) was responsible for performing and analyzing all scans.

Physical Activity Assessment

All potential participants were prescreened over the phone by the principal investigator (Dieli-Conwright) to ensure that they were sedentary and not participating in regular strenuous physical activity. Baseline, including current and past year, physical activity levels were assessed for each participant using the Entry Questionnaire and Physical Activity Scale (1). Lifetime physical activity patterns were not obtained.

Dietary Analysis

All the participants completed a 3-day dietary food record and were instructed to record their dietary intake over the course of 2 typical weekdays and 1 weekend day. Dietary entries were reviewed with a registered dietician to determine accuracy in portion size estimates and detail of food intake. Nutritionist ProTM (Nutrition Analysis Software Version 1.3, Jones and Bartlett Publishers, Inc., Sudbury, MA, USA) was used for dietary analysis of intake data.

Strength Testing

Strength testing was performed at baseline using the Cybex NormTM dynamometer (Cybex International Inc. Ronkonkoma, NY, USA). A 5-minute warm-up session on a cycle ergometer preceded the strength testing for all the participants. The participants received detailed instructions on the eccentric and concentric leg extension exercises and performed 10 repetitions of each exercise at maximal effort. The maximum torque (effort measured) of the knee extensors for eccentric and concentric leg extension was performed with the dominant leg at 60°·s−1, a common setting used in clinical exercise physiology research studies (15,16). The participants were positioned on the Cybex by visually aligning the placement of the lateral femoral condyle with the axis of rotation of the lever arm. 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 talocural joint. During maximal loading, a 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 acute eccentric exercise bout.

The tester determined the participants' eccentric and concentric maximum torques as the highest produced torque. The maximum torque was determined to assess whether the participants were generating maximal effort during each set of the acute training bout. Because of the high reproducibility of strength testing, which has been previously shown (33), all the participants performed 1 session of strength testing before the acute training bout. The participants filled out and orally confirmed a modified Borg soreness scale (5) 2–3 days after the strength testing.

Eccentric Resistance Exercise

All the participants completed 1 acute bout of maximal single-leg eccentric knee extension exercise on the dominant leg using the Cybex NormTM dynamometer (Cybex International Inc) at 60°·s−1 1 week after strength testing. All the participants including the Control and HT groups performed the exercise bout under similar conditions including an 8-hour fast before the bout and initiation of testing at approximately 8 AM. Information of sleep patterns, arousal states, and hydration levels was not assessed before the exercise bout. The 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 assisted the participant during the concentric component by moving the limb back to the starting position (15° before full extension). The exercise protocol was designed to initiate skeletal muscle damage necessary to induce myogenic gene expression changes and as demonstrated in previous research (9,16). 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). A 20-second rest period was given between each of the sets. Biofeedback was provided on the computer screen, and verbal encouragement was provided by the tester during each repetition. The acute eccentric exercise bout occurred approximately 2 weeks after strength testing and 1 week after the baseline muscle biopsy to allow recovery from soreness. The participants filled out and orally confirmed a modified Borg soreness scale (5) 2–3 days after the acute eccentric exercise bout.

Muscle Biopsies

Percutaneous muscle biopsies (150–200 mg) were obtained from the vastus lateralis of the exercised leg, 1 week before the acute eccentric exercise bout and 4 hours postexercise. Muscle biopsies were not obtained before the initiation of HT use in the HT group. Four hours after exercise is an optimal time point to assess changes in myogenic gene expression stimulated by exercise (20). Biopsy specimens were collected using sterile conditions and local anesthesia (1% lidocaine) with a 5-mm Bergstrom muscle biopsy needle (Micrins Surgical, Lake Forest, IL, USA) 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 processed for analysis.

RNA Extraction and cDNA Synthesis

Total RNA was isolated after homogenization (Kinematica Polytron PT1200C, Lucerne, Switzerland) of 30–40 mg of muscle tissue with a monophasic solution containing guanidine isothiocyanate–containing lysis buffer and β-Mercaptoethanol (Qiagen RNeasy Tissue Kit, Valencia, CA, USA). After thorough homogenization, the samples were centrifuged at 12,000 rpm at 4° C for 10 minutes, and the resulting supernatant (free of insoluble protein and high molecular weight DNA) was transferred to a new microcentrifuge tube. Approximately 600 μl of Buffer RLT was added to these samples; the samples were homogenized by passing the lysate 5 times though a 20-gauge needle fitted to an RNase-free syringe. Approximately 600 μl of 70% ethanol was added to these samples. The samples were centrifuged for 15 seconds at 12,000 rpm. The supernatant was transferred to a new microcentrifuge tube. The RNA pellet was then exposed to subsequent washes including steps one wash of 700 μl Buffer RW1 and 2 washes of Buffer RPE. Finally, the resultant air-dried RNA pellet was dissolved in 40 μl RNase-free water. The diluted RNA samples were then stored at −80° C until later analyses.

The concentration and purity of the RNA was determined using a UV spectrophotometer (NanoDrop ND-1000, Thermo Scientific, Waltham, MA, USA) by measuring absorbance at 260 and 280 nm. On average, the yield of total RNA from 30 to 40 mg of muscle tissue was 586.29 ng·μl−1 (SE ± 12.61) for the Control group and 589.14 ng·μl−1 (SE ± 15.98) for the HT group.

After total RNA concentration determination, 500 ng of total skeletal muscle RNA was reverse transcribed to synthesize cDNA using Taqman reverse transcription reagents, according to the manufacturer's instructions (Applied Biosystems, Branchburg, NJ, USA). The following reverse transcription reaction mixture was prepared (50 μl total): (a) 10× RT buffer, (b) MgCl2, (c) dNTPs, (d) random hexamers, (e) RNase inhibitor, (f) reverse transcriptase, (g) RNase-free water, and (h) 500 ng of total cellular RNA. The RNA was reverse transcribed into cDNA with the following temperature-time protocol: 25° C for 10 minutes, 48° C for 30 minutes, 95° C for 5 minutes, and 4° C infinite (Mycycler, BioRad, Hercules, CA, USA).

Oligonucleotide Primers for Polymerase Chain Reaction

Forward and reverse oligonucleotide primers were used to amplify the gene expression of myostatin, ActRIIb, follistatin, FSTL3, FLRG, and GASP-1. Primer sequences were designed using Primer3 program (29). The primer sequences for the specific target genes and melt curve temperatures are shown in Table 1. Melt curves were determined for each primer set to ensure amplification of pure PCR products. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for detecting changes in gene expression using quantitative real-time PCR (qRT-PCR), which has previously been used as an internal control to examine myogenic gene expression after resistance exercise (20,21,25). In addition, we repeated all PCR analyses using 18S rRNA as an alternative internal control gene. Results using 18S rRNA (data not shown) and GAPDH did not differ and we chose to present data pertaining to GAPDH here.

Table 1
Table 1:
Primer sequences used for qRT-PCR.*

Quantitative Real-Time Polymerase Chain Reaction

A qRT-PCR method was applied to determine relative gene expression levels for myostatin, ActRIIb, follistatin, FSTL3, FLRG, and GASP-1. A total of 10 ng of cDNA was added to each of the 20-μl PCR reaction for myostatin, ActRIIb, follistatin, FSTL3, FLRG, and GASP-1, and GAPDH. In each PCR reaction, the following mixture was used: 10 μl 2.5× iQSYBR green supermix (BioRad, Hercules, CA, USA); 7 μl RNase-free water, 10 ng of cDNA and 10 PMOL of each primer (Forward and Reverse) of interest. All the samples were run in quadruplicate. Each PCR reaction was amplified using BioRad iCycler iQ thermal cycler (BioRad). Thermal cycling conditions were as specified by the manufacturer: The amplification profile involved the initial denaturation step at 95° C for 3 minutes, followed by 60 cycles consisting of denaturation at 95° C for 15 seconds, and primer annealing and extension at 56° C for 1 minute.

mRNA Quantification

All data were determined by normalizing the cDNA measured in 8 replicates (4 replicates repeated) for each participant sample to GAPDH (internal control) and then averaging the data to account for the change in gene (mRNA) expression as a result of the exercise stimulus. The data were then normalized to biopsy #1 (preexercise) for each participant to determine the fold change of gene expression after exercising. All the participant samples were then averaged to determine the average gene expression before and after exercise for each group. Gene expression was evaluated by a relative quantification method (24). The 2^ − ΔΔCT method (19) was used to calculate the changes (in fold) in gene expression as a result of the eccentric exercise bout. In this method, gene of interest (GOI) expression was normalized to ICG (internal control gene) expression and normalized to preexercise value [ΔCT = (CT GOI POSTEXERCISECT ICG POSTEXERCISE) − (CT GOI PREEXERCISE − CT ICG PREEXERCISE)] within each group.

Statistical Analyses

Statistical analyses were performed using SPSS, version 16.0 (SPSS Inc., Chicago, IL, USA).

Descriptive statistics and Pearson correlations were used to analyze the control and HT groups. An independent t-test was used to compare participant characteristics. Data were normally distributed, and parametric analyses were used to compare gene expression. A 2 (group: HT, Control) × 2 (biopsy: baseline, posttesting) repeated-measures analysis of variance was used to compare changes in mRNA gene expression between biopsy time points and for each group (HT and Control). The alpha level of significance used was p ≤ 0.05 with Bonferroni adjustments. Data were normalized to biopsy #1 (preexercise) for each participant to determine the fold change of mRNA gene expression after exercising. All participant samples were then averaged to determine the average mRNA expression before and after exercise. Weight and lean mass served as covariates. If an overall significant F value was obtained, Tukey's Honestly Significant Difference analysis was used to isolate the significant mean values. A probability level of p ≤ 0.05 was used to determine statistical significance for all analyses. Intraclass correlation coefficients (ICCs) were calculated to assess the test-retest reliability of gene expression variables.

Results

Fourteen healthy postmenopausal women 55–65 years of age volunteered to participate in the study. Baseline study participant characteristics are shown in Table 2. Weight and lean body mass were significantly greater in the HT group than in the control group (p < 0.05), potentially because of significantly higher levels of circulating estrogen in the HT group. Despite group differences in weight and lean mass, differences in myostatin-related mRNA expression were not affected by these variables when they were included as covariates.

Table 2
Table 2:
Participant characteristics.*†

The HT group included 4 estrogen-alone users and 4 estrogen-progesterone users. The HT containing estrogen alone was used by four women Premarin, n = 2; Menest, n = 2) or a combination of estrogen and progesterone by 4 other women (Prempro, n = 2; FemHRT, n = 2). Results did not differ within the HT group for all gene expression measures. The participants in both groups were not using another concomitant drugs or therapies that would interfere with our outcome measures such as additional endocrine therapies.

To control for potential group differences, dietary intake (3-day diet analysis) and physical activity levels were assessed. There were no group differences in caloric consumption and physical activity levels, measured by self-reported questionnaires. All the participants had low activity levels with an average of 2.5 ± 0.5 hours of low-intensity exercise per week, consisting of mainly household chores and leisure walking. There were no significant group differences in perceived muscle soreness or 1-repetition maximum (1RM) between groups (Table 2). Average ratings of perceived exertion for both groups was 5.0 ± 0.5 (strong perceived soreness) out of 10 for the 1RM testing and 8.0 ± 0.5 (very strong perceived exertion) out of 10 for the acute exercise bout (p > 0.05).

Relative Change in Gene Expression

Myostatin and ActRIIb gene expression decreased postexercise for both groups, and the exercise bout resulted in a significant change in mRNA levels when compared with preexercise levels (p < 0.01; Figure 1). In addition, the relative reductions in myostatin and ActRIIb gene expression were significantly greater for the HT group (−19.6-fold; −9.4-fold, respectively) than for the controls (−4.1-fold, −3.8-fold, respectively; p < 0.05). Follistatin, FSTL3, FLRG, and GASP-1 gene expression increased postexercise for both groups which was significantly greater than preexercise expression values (p < 0.01; Figure 2). Follistatin gene expression was significantly greater in the HT group (5.8-fold) than in the controls (2.1-fold; p < 0.05). The increase in FSTL3 gene expression was significantly greater in the HT group (10.9-fold) than in the controls (3.4-fold; p < 0.05). The largest significant group difference was present for FLRG gene expression with a 14.4-fold change measured in the HT group compared with a 2.5-fold change measured in the control group (p < 0.05). The increase in GASP-1 gene expression was significantly greater in the HT group (12.5-fold) than in the controls (4.1-fold; p < 0.05). There were no significant group differences in preexercise mRNA levels between the HT and Control groups (data not shown; p < 0.05). Test-retest correlation coefficients were high for all gene expression variables (ICC 0.90, 95% Confidence Interval 0.84–0.94), indicating the presence of substantial reliability.

Figure 1
Figure 1:
Changes (in fold) in stimulatory myostatin-related gene expression after a bout of maximal eccentric resistance exercise normalized to glyceraldehyde 3-phosphate dehydrogenase and relative to preexercise levels. Values are mean ±SE. *Significantly different from control group (p < 0.05). ActRIIb = activin receptor IIb.
Figure 2
Figure 2:
Changes (in fold) in inhibitory myostatin-related gene expression after a bout of maximal eccentric resistance exercise normalized to glyceraldehyde 3-phosphate dehydrogenase and relative to preexercise levels. Values are mean ±SE. *Significantly different from control group (p < 0.05). FLRG = follistatin-related gene; FSTL3 = follistatin-like-3; GASP-1 = GDF-serum associated protein-1.

Discussion

Our investigation examined changes in myostatin-related gene expression at rest and after maximal eccentric resistance exercise in postmenopausal women with and without HT use. Major findings from our study include (a) after eccentric exercise, postmenopausal women expressed lower levels of myostatin and ActRIIb gene expression and higher levels of follistatin, FSTL3, FLRG, and GASP-1; and (b) after eccentric exercise, postmenopausal women using HT showed a heightened response of all genes of interest including myostatin, ActRIIb, follistatin, FSTL3, FLRG, and GASP-1. These findings indicate that HT use combined with eccentric exercise elicits a greater response in myostatin-related gene expression involved in skeletal muscle growth and hypertrophy. The current analyses expands on our previous reports, which indicated that these postmenopausal women using HT demonstrated a greater protective effect against exercise-induced skeletal muscle damage (8) and a greater hypertrophic gene expression response to eccentric exercise (6). Investigating the impact of HT use on skeletal muscle gene expression in postmenopausal women is critical in beginning to understand whether synthetic estrogens affect muscle hypertrophy and possibly sarcopenia.

Stimulatory myostatin-related gene expression (myostatin and ActRIIb) decreased postexercise. Myostatin, a negative regulator of myogenesis, inhibits satellite cell proliferation and differentiation (22). Decreased expression of myostatin in older women after an acute bout of eccentric exercise has been previously indicated (8), yet a novel finding in this study is the decrease in ActRIIb levels in both groups. ActRIIb, which binds myostatin to initiate myostatin signaling pathways, has not been studied in women. In 1 study, ActRIIb gene expression declined in older men after an acute resistance exercise bout (14), in accordance with our results. A unique aspect of our study is the greater suppression of the ActRIIb response in the HT group, which may aid in the prevention of age-related skeletal muscle mass declines. Based on these findings, we speculate, HT combined with maximal eccentric resistance exercise could prevent age-related skeletal muscle loss through the down regulation of stimulatory myostatin-related genes. This hypothesis will need to be tested in future clinical investigations.

Increased follistatin gene expression after eccentric resistance exercise in postmenopausal women has been previously indicated (8); thus, to further investigate the combined effects and interaction of maximal eccentric exercise and HT use, we examined additional inhibitory myostatin-related genes. Expression levels of FSTL3, FLRG, and GASP-1, which possess inhibitory effects on myostatin similar to follistatin, increased after exercise in both groups. To our knowledge, few human and animal studies (2,4) investigated these particular inhibitory myostatin-related genes. Aoki et al. (4) examined changes in myostatin signaling after muscle stretching in male rats. In agreement with our findings, Aoki et al. (4) found increased gene expression levels of FSTL3 and GASP-1 after the muscle stretching protocol. From the available literature, this study provides the most applicable supportive data using an animal model to examine changes in skeletal muscle gene expression of FSTL3 and GASP-1. The FSTL3, which binds myostatin, and GASP-1, which inhibits myostatin-ActRIIb binding, are critical components of the myostatin signaling pathway and further investigations are needed to understand the effects of exercise and HT on alterations in gene expression.

The FLRG, a protein that inhibits the binding of myostatin to ActRIIb (12), has received minimal investigation. A study by Willoughby (36) provides the only human evidence to document changes in FLRG gene expression in human skeletal muscle. Willoughby (36) found an increase in FLRG gene expression after 6 and 12 weeks of resistance training in young men, which supports our finding. Similar to follistatin, FSTL3, FLRG, and GASP-1 levels were greater in women using HT in our study. This adds to the unique contribution of our study, which suggests that HT use, combined with eccentric resistance exercise, promotes upregulation of follistatin and its related genes, which may aid in the antagonization of myostatin activity to preserve age-related muscle loss. In general, as with our myostatin findings, further exercise interventions are necessary to examine the potential anabolic benefits of estrogen on follistatin, FSTL3, FLRG, and GASP-1 in older women.

Collectively, our work emphasizes the important role of estrogen in human skeletal muscle and its potential to enhance the benefits of resistance exercise, specifically with the use of synthetic forms of estrogen such as HT. Our data adds to the growing body of literature that supports the function of estrogen in skeletal muscle to possibly attenuate muscle growth through the upregulation of MyoD, lessen exercise-induced muscle damage, and inhibit myostatin signaling pathways. Because of the presence of estrogen receptors in human skeletal muscle in men and women (18), it is possible that estrogen may directly influence myostatin signaling pathways and therefore mechanisms of muscle hypertrophy; however, the exact role and function of the estrogen receptors in skeletal muscle has not been determined. The changes in myostatin-related genes in our study may directly or indirectly relate to the activation of the estrogen receptors yet evidence to support this is lacking, and we are currently conducting experiments to determine the relation between myogenic gene expression and estrogen receptor activation.

Although a number of skeletal muscle gene expression profiling analyses have compared young and older adults (10,28,34,35), we chose to focus our investigation on older women in an effort to compare myostatin-related gene expression of HT and non-HT users and to provide mechanistic insight into the role of estrogen in skeletal muscle in women. Our data support previous reports that suggest estrogen and synthetic HT are anabolic agents (6,27,32) showing increased expression of inhibitory myostatin-related genes and decreased expression of stimulatory myostatin-related genes in the HT users. The current analysis expands on our prior observations that HT users express greater myogenic regulatory, proteolytic, and skeletal muscle growth factor gene expression, which is expected to preserve muscle mass (8). However, larger randomized clinical trials are necessary to determine effects of HT on baseline skeletal muscle gene expression and whether progestational components of HT contribute to the effects of estrogen.

A potential limitation of our study is the significant differences in body weight and lean mass among the Control and HT groups. In regards to body weight and body mass index (BMI), a probable relationship may exist between obesity and myostatin serum and protein levels. The most pertinent study by Hittel et al. (13) measured significantly elevated serum and protein levels of myostatin in extremely obese women (BMI 48.9 ± 4.9 kg·m−2; 45.8 ± 3.9 years) when compared with lean (BMI 25.7 ± 1.3 kg·m−2; 41.2 ± 5.0 years) and obese (BMI 32.3 ± 1.1 kg·m−2; 46.8 ± 4.2 years) women. Although our study did not include extremely obese women and did not measure serum and protein levels of myostatin, the investigation by Hittel et al. (13). emphasizes the importance of potential differences in myostatin as a function of body weight. Using analysis of covariance analyses, we were able to include weight and BMI as covariates in our statistical model, which ultimately did not affect our results. However, future investigations should preferably include specific body mass inclusion-exclusion criteria, to prevent confounding results from obese individuals. Regarding lean mass differences, it is important to acknowledge that functional strength differences may exist as the HT group had significantly greater muscle mass, despite similar 1RM values. Future studies may need to consider body mass- and lean mass-matching participants particularly when strength and skeletal muscle gene expression is of interest. In doing so, a possible relation between functional strength and altered myogenic gene expression may be concluded. In addition, it is important to note that although this study examined significant changes in skeletal muscle gene expression, this may not result in similar changes in protein synthesis. Further experiments are warranted to confirm the presence of posttranslational modifications, which may differ from gene expression levels.

In summary, the findings of this study demonstrate that without vigorous exercising, ambulatory, free living postmenopausal women using HT expressed greater levels of myostatin-related genes. A maximal bout of eccentric resistance exercise combined with HT further induced changes in gene expression of myostatin, ActRIIb, follistatin, FSTL3, FLRG, and GASP-1, the major factors in myostatin signaling. The use of HT combined with resistance exercise in postmenopausal women appears to promote muscle hypertrophy by increasing levels of genes responsible for muscle growth and inhibiting genes responsible for muscle breakdown. Whether these important new findings in postmenopausal women can be used to attenuate or prevent sarcopenia in older women remains to be determined.

Practical Applications

Resistance exercise is known to greatly influence signaling pathways involved in muscle hypertrophy. During the aging process, these signaling pathways may become impaired thus contributing to a loss in lean muscle mass and a decline in functional capacity. Possible training and treatments options, such as eccentric exercise and hormone therapy (HT), are continually being developed to prevent a loss in lean muscle mass; however, it is important to recognize possible sex difference in these options as men and women undergo different hormonal changes throughout the aging process. For example, the use of HT or synthetic female sex steroids may impact muscle hypertrophy and strength in postmenopausal women who are using HT to alleviate menopausal symptoms. Possible benefits of HT use to prevent and minimize age-related muscle loss may result when combined with participation in regular bouts of resistance exercise; however, larger studies are necessary to determine conclusive musculoskeletal benefits of HT use. The present information can be used by strength coaches and fitness professionals, who regularly advise older adults on exercise prescription, to broaden their understanding of muscle wasting mechanisms and training and treatment regimens to possibly alleviate age-related muscle loss.

Acknowledgments

The authors would like to thank the subjects who participated in this study. They wish to 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 Clinical Exercise Research Center at the University of Southern California and NIH NCRR GCRC M0I RR000043 provided funds for this project.

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Keywords:

skeletal muscle; estrogen; resistance exercise

© 2012 National Strength and Conditioning Association