Unaccustomed eccentric exercise damages skeletal muscle tissue, activating mechanisms of recovery and remodeling that may be influenced by the female sex hormone 17β-estradiol (E2). Using high density oligonucleotide based microarrays, we screened for differences in mRNA expression caused by E2 and eccentric exercise. After random assignment to 8 days of either placebo (CON) or E2 (EXP), eighteen men performed 150 single-leg eccentric contractions. Muscle biopsies were collected at baseline (BL), following supplementation (PS), +3 hours (3H) and +48 hours (48H) after exercise. Serum E2 concentrations increased significantly with supplementation (P<0.001) but did not affect microarray results. Exercise led to early transcriptional changes in striated muscle activator of Rho signaling (STARS), Rho family GTPase 3 (RND3), mitogen activated protein kinase (MAPK) regulation and the downstream transcription factor FOS. Targeted RT-PCR analysis identified concurrent induction of negative regulators of calcineurin signaling RCAN (P<0.001) and HMOX1 (P = 0.009). Protein contents were elevated for RND3 at 3H (P = 0.02) and FOS at 48H (P<0.05). These findings indicate that early RhoA and NFAT signaling and regulation are altered following exercise for muscle remodeling and repair, but are not affected by E2.
Citation: MacNeil LG, Melov S, Hubbard AE, Baker SK, Tarnopolsky MA (2010) Eccentric Exercise Activates Novel Transcriptional Regulation of Hypertrophic Signaling Pathways Not Affected by Hormone Changes. PLoS ONE 5(5): e10695. https://doi.org/10.1371/journal.pone.0010695
Editor: Alejandro Lucia, Universidad Europea de Madrid, Spain
Received: January 26, 2010; Accepted: April 16, 2010; Published: May 18, 2010
Copyright: © 2010 MacNeil et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by a grant in aid from the Natural Sciences and Engineering Council of Canada (NSERC) to M.T.(183817-07). S.M. was supported by financial assistance from the Glenn foundation for Medical Research. L.M. was supported by a graduate scholarship from NSERC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Myofibres have the capacity to be remodeled to best meet their functional and metabolic demands. Changes in physical activity can initiate a remodeling process toward increased (hypertrophy) or decreased (atrophy) muscle mass , , . Through protein signaling pathways, integration of chemical, mechanical and bioenergetic signals change the genetic expression patterns for cell size, function and metabolic processes . Physical activities that incorporate unaccustomed eccentric contractions are typically associated with high levels of muscle damage, inflammation and delayed onset muscle soreness (DOMS) , , . Eccentric contractions, identified by a lengthening while under tension, create an insult to myofibres which may be characterized by: damage to the sarcoplasmic reticulum , t-tubules  and structural proteins , , , the presence of muscle protein in blood , , Z-line streaming , , soreness and fatigue , .
Murine and rodent research has often indicated an attenuation of exercise induced membrane damage , , , , , structural proteins , and inflammation , , ,  along with enhanced satellite cell activation , ,  with exposure to the sex hormone 17β-estradiol (E2). This reduced muscle damage and improved recovery may result from potential antioxidant, membrane stabilizing, or gene regulation properties of E2 , , , , . As the most abundant estrogen, E2 exerts estrogenic properties that affect the differentiation, growth and function of reproductive, skeletal, neural and muscular tissues , . However, human studies do not consistently support the effectiveness of E2 to attenuate exercise-induced muscle damage, mostly reporting similar values for CK efflux, inflammation and loss of muscle function when comparing men and women , .
Recent microarray analyses have uncovered novel transcriptional programs that coordinate the regeneration and repair of damaged muscle following eccentric exercise , , . These studies have indentified clusters of genes representing important mechanisms for recovery and adaptation that include the regulation of inflammation , , growth , , stress response proteins ,  and membrane biosynthesis . Similar analyses have also described sex differences in the expression levels of genes involved in metabolism and growth inhibition that may result from variations in body composition and hormone content , , . Specifically, women express a higher abundance of mRNA for several genes involved in fat metabolism that include trifunctional protein β and lipoprotein lipase , and greater expression of the negative regulators of the anabolism growth factor receptor-bound 10 and activin A receptor IIB .
The effect of E2 administration on the transcriptome expression profile of skeletal muscle following a single bout of intense eccentric exercise has not yet been evaluated. In this study we used microarray analysis to identify how global mRNA abundance is altered by E2 supplementation in men after 150 eccentric contractions. We hypothesized that the anti-oxidant and membrane stabilizing properties of E2 would attenuate the amount of muscle damage experienced, thereby modifying the expression of mRNA species involved in membrane homeostasis, growth and stress management. Furthermore, we hypothesized that the use of gene array analysis would allow us to detect novel genes relevant to the hypertrophic growth signaling stimulated by intense exercise.
Materials and Methods
All participants were given an information sheet describing all of the testing procedures before providing written consent to participate. The study conformed to the standards outlined in the Declaration of Helsinki and was given approval by the Research Ethics Board of McMaster University (05–438).
Subjects and anthropometrics
Eighteen young healthy men volunteered as participants in this study. All subjects were pre-screened to ensure that they were healthy, fit and had not regularly participated in resistance exercise in the preceding 6 months. Body composition was measured using dual energy x-ray absorptiometry (DEXA) scans (GE Lunar Prodigy, Fairfield, CT). Thigh muscle cross-sectional area was calculated using anthropomorphic measurements of mid-thigh circumference and skinfold thickness  to control for potential differences in total work completed. The subject demographics were (mean ± SD): age, 21±2 y; height, 181±5 cm; weight, 76.9±12.8 kg.
Subjects were assigned in a randomized, double-blind manner to either a control (CON, N = 9) or experimental (EXP, N = 9) group. CON subjects consumed 400 mg glucose polymer (Polycose; Abbott Laboratories, Ross Division, St. Laurent, Quebec, Canada) for 10 days. EXP subjects consumed ∼300 mg glucose with 1 mg E2 (Estrace; Shire BioChem, Inc., St. Laurent, Quebec, Canada) for 2 days followed by 2 mg E2 for 8 days. We have previously used this protocol to increase serum E2 concentrations to levels seen during the luteal phase of the menstrual cycle . Glucose and E2 tablets were concealed in gelatin capsules. On the morning of the ninth day, subjects reported to the laboratory and performed the exercise protocol. Supplementation continued until the day of the final biopsy and blood collection to maintain serum E2 concentrations throughout the collection protocol. Subjects in both groups were instructed to take one pill at the same time each day and return any unused pills. All subjects reported 100% compliance.
Exercise protocol and tissue collection
Muscle damage was induced with a previously developed eccentric exercise protocol . Approximately 2 weeks before the exercise protocol, subjects were given a familiarization session with a Biodex isokinetic dynamometer (System 3, Biodex Medical Systems Inc., Ronkonkoma, NY). On the testing day, following a short warm-up (10 min of light cycling), subjects were seated in the dynamometer with their right leg strapped to a lever arm. The lever arm was programmed to extend their leg to 150° of flexion (where 180° is full extension) at a moderate speed (30°/s), then flex their leg to 90° of flexion at a faster speed (120°/s). Subjects did not have to contract maximally during the extension phase. During the flexion phase, subjects were instructed to attempt to maximally resist flexion of the knee (i.e. voluntary ‘maximal’ contraction) against the descending lever arm throughout the entire range of motion. The complete test consisted of 15 sets of 10 repetitions, each set separated by 1 minute of rest.
Prior to each tissue collection, subjects abstained from any other form of physical exertion (within 72 h), avoided alcohol (within 48 h), ate their habitual diet (within 48 h), and abstained from caffeine (within 12 h). Each subject consumed a 350 Kcal defined formula diet (57% carbohydrates, 15% protein and 28% fat) two hours before each muscle biopsy and did not eat again until after the final biopsy of each session was taken. These nutritional and activity controls were taken to ensure that the muscle damage would be the only variable to differentially affect the outcomes between biopsies .
Muscle biopsies were taken from the vastus lateralis of the control (left) leg during the familiarization session (baseline, BL) and after 8 days of supplementation (post supplementation, PS) and the exercised (right) leg 3 hours (3H) and 48 hours (48H) after exercise, in anatomically distinct sites approximately 6 cm apart . The post exercise collection times were chosen because they represent two distinct phases of recovery from muscle damage . The muscle biopsies were quickly dissected of fat and connective tissue, sectioned into RNase-free cryovials (∼30 mg/piece), flash frozen with liquid nitrogen and stored at −86°C until analysis. Blood samples were drawn from the antecubital vein into heparinized tubes at the same collection times, placed on ice, centrifuged at 1750 g at 4°C for 10 min and stored at −20°C future analyses.
Blood hormone and enzyme concentrations
Serum E2 (Fertigenix-E2-EASIA, Biosource Europe S.A, Nivelles, Belgium) and testosterone (Fertigenix-TESTO-EASIA, Biosource Europe S.A, Nivelles, Belgium) concentrations were measured by enzyme amplified-sensitivity immunosorbent assays (EASIA) according to manufacturer's specifications using BL and PS blood collections. Serum lactate dehydrogenase (LDH) activities were measured in BL, 3H and 48H blood collections with a colourimetric LDH quantification assay (K726-500, Biovision Research Products, Mountain View, CA) according to manufacturer's specifications. All hormone and enzyme measurements were done in duplicate.
The total RNA was extracted from the frozen skeletal muscle biopsy as described previously in detail by our group . Briefly, ∼30 mg of skeletal muscle was homogenized on ice in 2 mL of Trizol Reagent (Life Technologies, Cat. No. 15596, Gaithersburg, MD). The homogenate was incubated for 10 min at room temperature, followed by phase separation using 200 µL of chloroform and precipitation of the total RNA from the aqueous phase using 500 µL of isopropyl alcohol. The RNA pellet was then washed three times in 75% ethanol and re-suspended in 15 µL DEPC-treated water, aliquoted, and stored at −86°C. The concentration and purity of the RNA was determined using a UV spectrophotometer (Shimadzu UV-1201; Mandel Scientific, Guelph, Ontario) at the absorbance of 260/280 nm. Measurements were done in duplicate and had an average coefficient of variation (CV) of <10%. The average purity (OD260/OD280) of the samples was 1.7 before DNase treatment. RNA integrity was assessed in a randomly chosen subset of samples using agarose gel electrophoresis, and the OD ratio of 28S to 18S rRNA was consistently greater than 1 for each sample.
Prior to microarray chipping and real time quantitative RT-PCR analysis, the isolated RNA samples were treated with DNA-free™ recombinant DNase I (Ambion Inc, Austin, TX) according to the manufacturer's instructions to remove any potential genomic DNA contamination.
The resulting total RNA samples were further assessed for integrity prior to chipping using a Nanodrop Spectrophotometer and the Agilent Bioanalyzer Nano Chip System. Samples which passed this initial quality control assurance step were then amplified one round, using an Illumina TotalPrep Kit (Ambion) to generate cDNA then cRNA according to the manufacturer's instructions. This was again assessed for quality by using the Nanodrop and Bioanalyzer as described above. Labeled cRNA samples that passed this second round of quality control were then hybridized to Human Ref-8 BeadChips (Illumina) according to the manufacturer's instructions (approximately 23,000 genes), using equipment specified by the manufacturer (Illumina). Briefly, 850 ng biotin-labeled cRNA in 11.3 µl nuclease-free water was adjusted to 34 µl through the addition of 22.7 µl of 5∶3 HybE1 buffer/formamide. The sample was heated at 65°C for 5 min, allowed to cool to room temperature, and then immediately added to a single array of an 8-array Human Ref-8 BeadChip. Once all 8 samples were added to each BeadChip, it was sealed in a Hyb Cartridge and incubated for 16 h at 55°C with rotation in an Illumina hybridization oven (rotation setting 5). Following overnight hybridization, BeadChips were moved to a slide rack and serially washed using gentle rotation in glass staining dishes filled with a) 250 ml Illumina Wash Buffer×5 min, b) 250 ml 100% ethanol×10 min, c) 250 ml Illumina Wash Buffer×2 min. BeadChips were then blocked for 10 min in 4 ml Block E1 buffer (Illumina), followed by staining for 10 min in 1 µg/ml Streptavidin-Cy3 conjugate (GE Healthcare) in Block E1 buffer. Stained BeadChips were finally washed using gentle rotation in a glass staining dish filled with 250 ml Illumina Wash Buffer×5 min. BeadChips were dried by centrifugation at 280 g for 4 min and stored in a light-tight box until reading.
Processed arrays were read using a BeadStation array reader (Illumina) according to the manufacturer's instructions.
Gene ontology analysis
In the lists of genes that were significantly differentially expressed with exercise in our study, we carried out gene ontology (GO) analysis to determine the relative enrichment of genes with common or related functionalities to gain insight into biological processes mediated by E2 or exercise. This was carried out using the web interface driven GoMiner tool using an FDR of 5% . Genes were also referenced to their biological functions and canonical pathways with Ingenuity Pathway Analysis (IPA) software. This software identifies functions and pathways most significant to the data set in two ways: the number of differentially expressed genes included in a pathway or function and calculation of a p-value using a Fisher's Exact Test to determine the probability of the association of the data set.
Real-time RT-PCR analysis
Changes in gene expression relative to baseline values were measured using real-time reverse transcription-polymerase chain reaction (RT-PCR). Regulator of calcineurin 1 (RCAN1) and capping protein (actin filament) muscle Z-line, alpha 1 (CAPZA1) were selected for analysis because of their roles in growth and sarcomerogenesis . Hemeoxygenase 1 (HMOX1) was chosen for analysis because of its role in stress management . The selected housekeeping gene was β2-microglobulin. Its constant expression following eccentric exercise has been shown in previous work , and was confirmed for the current study. The efficiencies of all primers were tested and determined to be greater than 98%. The primer and probe sequences for these genes can be found in Table 1.
RT-PCR was completed using a TaqMan® real-time method. The primers and a probe to each target gene were designed based on the cDNA sequence in GenBank (http://www.ncbi.nlm.nih.gov/sites/entrez/?db=gene) with primer 3 designer (http://frodo.wi.mit.edu/primer3-0.4.0/input.htm). All target gene probes were labeled with FAM at their 5′ ends and BHQ-1 at their 3′ ends. Duplex RT-PCR was performed on an iCycler real-time PCR system (Bio-Rad Laboratories, Hercules, CA) in the One-step TaqMan® RT-PCR Master Mix Reagents (Roche, Branchburg, New Jersey) according to the manufacture's instruction with target gene primers, target probe, housekeeping gene primers and housekeeping gene probe in the same reaction . Determination of significant gene expression change was done as previously described . The genes of interest were normalized to the housekeeping gene, β2-microglobulin by following the standard method. Briefly, CT values of the housekeeping gene were subtracted from the CT values of the gene of interest giving a δCT. This is equivalent to the log2 difference between endogenous control and target gene . Values were then normalized to baseline, δδCT. All samples were run in triplicate, fluorescence emission was detected using FAM and Tamra filters, and CT was automatically calculated.
Muscle biopsy samples were homogenized and prepared for polyacrylamide gel electrophoresis using methods previously described . Briefly, frozen skeletal muscle tissue samples (25–35 mg) were hand homogenized in 25 µl of phosphate buffer (50 mM Kpi, 5 mM EDTA, 0.5 mM DTT, 1.15%KCl (w/v)) per milligram of tissue. A protease inhibitor cocktail (Sigma, St. Louis, Missouri) was added to the phosphate buffer immediately prior to use at a ratio of 1∶1,000. Samples were centrifuged at 600 g for 10 min at 4°C and the supernatant aliquoted for analyses. Protein concentrations of each sample were determined using the method described by Lowry et al .
Samples were loaded on 10% SDS-polyacrylamide gels and transferred to a PVDF membrane. Membranes were blocked with 5% BSA (wt/vol) in Tris-buffered saline with 0.1% Tween (vol/vol) (TBST) and incubated in primary antibody: RND3 (Abcam, Cambridge, MA; ab50316, 1∶1000); FOS (Abcam; ab16902, 1∶1000); total p38MAPK (Cell Signaling Technology, Danvers, MA; no. 9212, 1∶1000); p38MAPK (Thr180/Tyr182) (Cell Signaling Technology; no. 9215, 1∶1000); total GSK-3β (Cell Signaling Technology; no. 9315, 1∶1000); GSK-3β (Ser 9) (Cell Signaling Technology; no. 9323, 1∶1000), β-actin (BD Biosciences, Mississauga, ON; no. 612657, 1∶10000). After washing in TBST, membranes were incubated in either HRP-linked anti-rabbit or anti-mouse IgG secondary antibody (Amersham Biosciences, Piscataway, NJ; no. NA934V, 1∶6000), washed with TBST and developed using ECL (Amersham Biosciences; model no. RPN2106). Membranes were exposed to x-ray film (Biomax XAR; Kodak, Rochester, New York) which were then scanned with a Dell 920 scanner at 300 DPI and saved in TIF file format. Using Image J v1.40 g software (National Institutes of Health, Bethesda, Maryland), background noise was removed and bands in the region of interest were selected for analysis. Individual profile plots were generated and area under the curve measured in arbitrary units (AU).
Statistical and bioinformatics analysis
Student's unpaired t-tests were used to determine differences in subject characteristics and total work. A 2-way repeated measures ANOVA (supplementation protocol × time) was used to assess differences in LDH, E2 concentration, testosterone concentration, protein levels and the linear 2−δδCT data set for gene expression measured with RT-PCR using computerized software (Statistica, Statsoft). When statistical significance was achieved, Tukey's honestly significance difference post-hoc test was used to determine the significance among the means. STATISTICA for Windows 5.0 (Statsoft, Tulsa OK) was used to perform t-tests and ANOVAs. The threshold for significance was set at P≤0.05. Data are presented as mean ± SEM unless otherwise indicated.
Gene array data analyses were done comparing baseline, post supplementation, 3 and 48 hours post exercise using simple paired t-test on log2 expression ratios. Genes were ranked by p-value and the inference reported following adjustment for multiple testing using the FDR and the Benjamini and Hochberg method. Among those genes with an adjusted q-value (based on FDR) of <0.05, we used hierarchical clustering (based on the HOPACH algorithm) to find groups of genes with similar profiles across the subjects.
Subject and work characteristics
CON and EXP groups were not different in age, weight, height, body fat percentage, average thigh cross-sectional area or total work completed (Table 2). All subjects completed the required 150 eccentric contractions.
E2 and testosterone concentration were affected by supplementation with E2
Following 8 days of supplementation serum E2 concentration increased by 146% (P<0.001) and testosterone concentration was reduced by 26% (P = 0.01) in the EXP group (Table 3). Both hormone concentrations remained unchanged in the CON group.
Eccentric exercise induced muscle damage
The appearance of the muscle protein LDH in serum is an indirect indicator of muscle membrane damage. LDH activity was elevated 13.8% (P<0.05) 48 hours after exercise (Table 4). The EXP values did not differ from the CON values at any time.
Microarray data identifies altered mRNA expression during recovery from eccentric exercise that is not affected by E2
Eccentric exercise significantly increased the early mRNA expression of 310 genes at 3H. DNA microarray analyses did not identify differential mRNA expression of any gene as a result of E2 at any time (we could not reject the global null that E2 was independent of mRNA expression of all genes represented on the chip). For this reason, microarray data at each timepoint was collapsed between groups, increasing the sample size to 18 subjects. Mean fold change for genes with ratios greater than 1 at 3H was 2.3±0.1. In addition, 301 genes had ratios less than 1 at 3H with a mean fold change of 0.8±0.01. By 48H, all genes had expression values that were not different from baseline. The complete data set is freely available at GEO (accession no. GSE19062).
Of the genes differentially expressed at 3H, we identified 25 that participate in two signaling cascades for muscle growth and adaptation: ras homologue gene family, member A (RHOA) and nuclear factor of activated T-cells (NFAT) (Table 5). Other regulators of muscle growth and remodeling not directly involved in RHOA or NFAT signaling that were also highly induced included: ATF3, MYC, XIRP1, HBEGF and DNAJB4 (Table 5).
IPA identified several biological functions related to muscle growth and remodeling identified by the number of differentially expressed genes included in the function and the calculation of a P-value using a Fisher's Exact Test with a threshold for significance set at P≤0.05. They included: cancer (P<0.05, 233 molecules), gene expression (P<0.05, 146 molecules), cell assembly and organization (P<0.05, 69 molecules), cell morphology (P<0.05, 59 molecules) and skeletal and muscular system development and function (P<0.05, 50 molecules). In the same manner, IPA also identified three canonical pathways related to gene expression and growth/proliferation: ILK signaling (P = 0.005, 16/186 molecules), RAN signaling (P = 0.005, 4/23 molecules) and PI3K/AKT signaling (P = 0.006, 12/136 molecules).
RT-PCR analysis provides additional genes involved in actin dynamics and regulation of RhoA and NFAT signalling
Targeted real time RT-PCR was conducted on several genes selected a priori for their involvement in recovery from skeletal muscle damage. Previous work using microarray analysis and RT-PCR identified the expression of novel genes following a similar eccentric protocol that are likely involved in the recovery and adaptation to damaging exercise . Confirming the identification of two of its transcript variants in the microarray, RCAN1 mRNA content was highly elevated at 3H (16.6-fold, P<0.001) (Fig. 1A). Also induced was HMOX1 at both 3H (3.9-fold, P = 0.009) and 48H (3.5-fold, P = 0.002) (Fig. 1B).
Graph A – RCAN1 (N = 18). Graph B – HMOX1 (N = 18). 3H = 3 hours post exercise, 48H = 48 hours post exercise. Values are mean ± SEM. **Significant difference vs. baseline when collapsed across supplementation (P<0.01). ***Significant difference vs. baseline when collapsed across supplementation (P<0.001).
Expression of CAPZA1, a regulator of the growth of actin filaments, was increased at 48H (1.8-fold, P = 0.04) (Fig. 2).
Signaling proteins and protein content are affected by eccentric exercise
Phosphorylated p38MAPK and GSK-3β negatively regulate NFAT by promoting its export from the nucleus. Phosphorylation (Thr180/Tyr182) of p38MAPK was significantly lower at 3H (0.77-fold, P = 0.07) and 48H (0.73-fold, P = 0.005) as a result of exercise with no effect of E2 (Fig. 3A). Phosphorylation (Ser9) of GSK-3β was not affected by either exercise or E2 (Fig. 3B).
BL = baseline, 3H = 3 hours post exercise, 48H = 48 hours post exercise. Graph A – p38MAPK (Thr 180/Tyr 182) (N = 18). Graph B – GSK-3β (Ser9) (N = 18). Values are mean ± SEM. **Significant difference vs. baseline when collapsed across supplementation (P<0.01).
Two species highly expressed at 3H by the microarray were selected for western blot analysis. RND3 was significantly higher at both 3H (1.34-fold, P = 0.02) and 48H (1.39-fold, P<0.01) (Fig. 4A). FOS was significantly higher at 48 hours following exercise (1.16-fold, P<0.05) (Fig. 4B).
The sex hormone E2 displays anti-oxidant and membrane stabilizing properties that could protect skeletal muscle from the effects of exercise induced muscle damage and influence genetic expression patterns , . Using microarray, real time RT-PCR and protein analyses, we have identified that 8 days of E2 supplementation did not affect the myofibre transcriptome in men. However, a single bout of eccentric exercise did induce differential mRNA transcription in the hypertrophic signaling pathways RhoA and nuclear factor of activated T-cells (NFAT) (Fig. 5), changes in the phosphorylation status of related signaling proteins and the protein quantities of two of the upregulated genes.
Eccentric exercise promoted greater expression of targets within the STARS/RhoA/AP1 and NFAT/AP1 signaling pathways for hypertrophy and actin biogenesis and organization.
Of the genes affected early after exercise, one of the greatest inductions was observed in the novel actin binding protein striated muscle activator of Rho signaling (STARS). This protein is a muscle-specific transducer of cytoskeletal signaling in cardiac and skeletal muscle that responds to calcineurin activation and biomechanical stress , , , . STARS stimulates growth through a mechanism requiring actin polymerization and Rho GTPase activation, increasing serum response factor (SRF)-mediated gene transcription (Fig. 6) , , , . Originally identified in cardiac muscle, STARS mRNA content increases more than 3-fold following the hypertrophic signaling of pressure overload , . More recently, Lamon et al. identified a 3.4-fold increase in STARS mRNA in human skeletal muscle following 8 weeks of resistance training . Our measurement of more than a 10-fold increase suggests that this gene is very important for the early signaling for growth and remodeling following eccentric exercise.
RCAN – regulator of calcineurin; HMOX1 – hemeoxygenase 1; ARHGEF7 and ARHGEF12 – Rho guanine nucleotide exchange factor 7 and 12; ARHGAP24 – Rho GTPase activating protein 24; RND3 – Rho family GTPase 3; DIAPH1 – diaphanous homologue 1; CORO1C – Coronin, actin binding protein 1; FLNB – Filamin B, beta; CAPZA1 – capping protein (actin filament) muscle Z-line alpha 1; ACTA2 – actin, alpha 2, smooth muscle, aorta; ACTN1 – actinin, alpha 1; AP1 – activator protein 1; FOS – FBJ murine osteosarcoma viral oncogene homologue; FOSB – FBJ murine osteosarcoma viral oncogene homologue B; JUND – Jun D proto-oncogene.
A downstream target of STARS associated with skeletal muscle hypertrophy and adaptation is the Rho GTPase, RhoA , . Rho GTPases are a family of small signaling G proteins that interact with effector proteins to regulate actin cytoskeleton, cell cycle progression and gene transcription , , , . These molecular signals switch to an active GTP bound state under the control of Rho GEFs (guanidine exchange factors), and return to their inactive GDP bound state by Rho GAPs (GTPase-activating proteins) , , . For the first time, our array analysis has identified increased expression of two GEFs (ARHGEF7 and ARHGEF12) and reduced expression of a GAP (ARHGAP24) that specifically regulate RhoA , . These gene expression modifications, if translated into altered protein quantities, could increase the potential for RhoA activation.
Although increased activity of RhoA protein is necessary for myogenesis induction, it must be downregulated before myotube formation can proceed , . This is achieved by RND3, another negative regulator of RhoA activity whose upregulation is an essential step of myoblast fusion , , . Our damaging exercise protocol resulted in an early induction of this gene at 3H and elevated protein content by 48H. Cell culture experiments have identified that in the presence of growth factors, RND3 mRNA increases of ∼1.7-fold result in greater protein content within 30 h . This in turn inhibits RhoA activity and promotes myotube formation and elongation .
Once activated, RhoA signaling is associated with myogenesis and actin remodeling in various cell types through regulation of genes that include DIAPH1 , , CORO1C , FLNB  and CAPZA1 . Through SRF activation, RhoA and STARS also mediate the induction of actin proteins ACTA2 , ACTN1  and members of the AP1 transcription factor complex: FOS , , FOSB  and JUND . Each of these genes was induced by eccentric exercise at 3H, as identified by our gene array and targeted real time RT-PCR analysis and our data indicate that the increased transcription of FOS was effectively translated into protein, increasing levels significantly by 48H. Although the number of studies investigating these genes after exercise is few, some support can be found for the upregulation of select downstream targets. Following eccentric exercise, the largest induction occurs with the transcription factor FOS 2–8 hours post exercise (23 to 38-fold increases) , . Resistance training results in a 2.7-fold increase in ACTN1 after 8 weeks . Thirty minutes of high intensity running increased the expression of FOS (7.0-fold) FOSB (17.8-fold) and JUND (7.6-fold) . Given that the mRNA levels for STARS, associated regulatory and transcription factors and downstream targeted genes were all significantly elevated 3 h after exercise, it appears that STARS signaling through a RhoA/SRF pathway is important for early skeletal muscle remodeling following damaging exercise.
A second calcineurin influenced signaling pathway identified in our microarray analysis to be transcriptionally active was nuclear factor of activated T-cells (NFAT). NFAT proteins exist in the cytoplasm of cells in a phosphorylated and inactive state . The influx of calcium following sustained contraction or damage increases the binding of calcineurin to NFAT, dephosphorylating conserved serine residues and promoting translocation of NFAT into the nucleus , , , . Once inside the nucleus, NFAT cooperatively binds to DNA with transcription factors AP1 and MAF initiating the transcription of prohypertrophic genes (Fig. 7) . Our microarray analysis also identified a significant induction of the genes NFATc1 and MAF at 3H. Along with the greater expression of the AP1 complex components FOS, FOSB and JUND, an increased abundance of NFATc1 and MAF could improve signaling by NFAT.
p38MAPK – p38 mitogen activated protein kinase; GSK-3β – glycogen synthase kinase 3 beta; MAF – v-maf musculoaponeurotic fibrosarcoma oncogene homologue (avian).
The NFAT pathway interacts with mitogen activated protein kinases (MAPK) and glycogen synthase kinase-3β (GSK-3β) for coordination of the hypertrophic response , . p38MAPK and GSK-3β act as a negative regulators of cardiac hypertrophy, rephosphorylating NFAT and promoting its export from the nucleus , , . Although GSK-3β mRNA content was higher in the microarray, its activity did not change and would not have affected NFAT nuclear residence. GoMiner analysis identified MAPK regulation as a transcriptionally active biological process through the induction of four related kinases (MAPKAPK2, MAPK6, MAP3K6 and MAP3K8), three related phosphatases (PP2CA, DUSP8 and DUSP16) and one surface receptor (ADRB2). Western blotting confirmed that p38MAPK phosphorylation status was lower after exercise, reaching significance by 48H. Lower activity of p38MAPK would assist in the transcriptional activity of NFAT, and may also occur to inhibit the induction of apoptosis and necrosis .
It should be noted that molecules that function in the recovery and repair of skeletal muscle through other mechanisms were also induced by our exercise protocol. Similar to other reports, ATF3 , MYC , , XIRP1 , HBEGF  and DNAJB4 , ,  were highly up-regulated (6.1 to 28.4-fold) early after exercise. In addition, the altered mRNA content of members of the signaling pathways ILK, RAN and PI3K/AKT identified them as being transcriptionally active. Their respective functions in actin cytoskeleton remodeling , transport across the nuclear envelope for gene expression  and protein synthesis  relate to the top biological functions returned by gene ontology analysis. Interestingly, the biological function that contained the greatest number of molecules was cancer (233 molecules), likely due to similar alterations to the regulation of cellular growth experienced with both exercise and cancer.
Maintained muscle contractions and exercise-induced damage to the sarcoplasmic reticulum and sarcolemma may result in the accumulation of excess calcium, known as Ca2+ overload , . As a signaling molecule, Ca2+ binds to calcineurin, activating both STARS  and NFAT , , , , . Unrestrained calcineurin activity can be regulated in skeletal and cardiac muscle by the inhibitors regulator of calcineurin 1 (RCAN1, aka MCIP1, DSCR1) , , , ,  and hemeoxygenase 1 (HMOX1) . Increased mRNA content of RCAN1 was identified in microarray and targeted real time RT-PCR, confirming a previous gene expression profile that also identified an increase following exercise (3.8-fold) . RT-PCR also identified significant increases in HMOX1 at both time points after exercise, similar to the 8-11-fold induction following 5 days of resistance training . Together, the upregulation of these two calcineurin inhibitors identifies the importance of regulating the elevated calcineurin activity that occurred following unaccustomed eccentric exercise .
The primary mechanism through which estrogens influence the growth, differentiation and function of tissues occurs via the estrogen receptors ERα and ERβ , , . As transcription factors, active homodimers and heterodimers of ERα and ERβ bind to estrogen response elements (EREs) in nuclear and mitochondrial DNA, increasing the transcription of target genes . In a nongenomic manner, E2 also interacts with a number of proteins that include endothelial nitric oxide synthase (eNOS) and MAPK, altering the signals that modulate cellular differentiation, migration and survival , , . Our observation that the myofibre transcriptome was unaffected by E2 is surprising. Although women have higher E2 concentrations than men, protein quantities of ERα and ERβ are similar and our supplementation protocol successfully increased circulating E2 to the level seen during the luteal phase of the menstrual cycle in healthy women , . This suggests that the differential expression between men and women of genes involved in metabolism and growth regulation , ,  may result from factors beyond E2 alone; factors that may include variations in body composition, X-chromosome genes, and/or other sex hormones (i.e., progesterone).
These results indicate that E2 supplementation does not affect the transcriptional pattern in skeletal muscle following eccentric exercise in men. However, the stress of a single bout of exercise induced a transcriptional response in two signaling pathways, STARS/RhoA/AP1 and NFAT/AP1, providing important insights for future research into the early hypertrophic response.
Conceived and designed the experiments: LM MAT. Performed the experiments: LM SB. Analyzed the data: LM SM AH. Contributed reagents/materials/analysis tools: SM MAT. Wrote the paper: LM. Biopsy collection: MAT. Collected muscle biopsies: SB.
- 1. Favier FB, Benoit H, Freyssenet D (2008) Cellular and molecular events controlling skeletal muscle mass in response to altered use. Pflugers Arch 456: 587–600.FB FavierH. BenoitD. Freyssenet2008Cellular and molecular events controlling skeletal muscle mass in response to altered use.Pflugers Arch456587600
- 2. Heineke J, Molkentin JD (2006) Regulation of cardiac hypertrophy by intracellular signalling pathways. Nat Rev Mol Cell Biol 7: 589–600.J. HeinekeJD Molkentin2006Regulation of cardiac hypertrophy by intracellular signalling pathways.Nat Rev Mol Cell Biol7589600
- 3. Wennerberg K, Rossman KL, Der CJ (2005) The Ras superfamily at a glance. J Cell Sci 118: 843–846.K. WennerbergKL RossmanCJ Der2005The Ras superfamily at a glance.J Cell Sci118843846
- 4. Bassel-Duby R, Olson EN (2006) Signaling pathways in skeletal muscle remodeling. Annu Rev Biochem 75: 19–37.R. Bassel-DubyEN Olson2006Signaling pathways in skeletal muscle remodeling.Annu Rev Biochem751937
- 5. Armstrong RB (1990) Initial events in exercise-induced muscular injury. Med Sci Sports Exerc 22: 429–435.RB Armstrong1990Initial events in exercise-induced muscular injury.Med Sci Sports Exerc22429435
- 6. Clarkson PM, Hubal MJ (2002) Exercise-induced muscle damage in humans. Am J Phys Med Rehabil 81: S52–69.PM ClarksonMJ Hubal2002Exercise-induced muscle damage in humans.Am J Phys Med Rehabil81S5269
- 7. Newham DJ, Jones DA, Clarkson PM (1987) Repeated high-force eccentric exercise: effects on muscle pain and damage. J Appl Physiol 63: 1381–1386.DJ NewhamDA JonesPM Clarkson1987Repeated high-force eccentric exercise: effects on muscle pain and damage.J Appl Physiol6313811386
- 8. Jones DA, Newham DJ, Torgan C (1989) Mechanical influences on long-lasting human muscle fatigue and delayed-onset pain. J Physiol 412: 415–427.DA JonesDJ NewhamC. Torgan1989Mechanical influences on long-lasting human muscle fatigue and delayed-onset pain.J Physiol412415427
- 9. Friden J, Lieber RL (2001) Eccentric exercise-induced injuries to contractile and cytoskeletal muscle fibre components. Acta Physiol Scand 171: 321–326.J. FridenRL Lieber2001Eccentric exercise-induced injuries to contractile and cytoskeletal muscle fibre components.Acta Physiol Scand171321326
- 10. Chen YW, Hubal MJ, Hoffman EP, Thompson PD, Clarkson PM (2003) Molecular responses of human muscle to eccentric exercise. J Appl Physiol 95: 2485–2494.YW ChenMJ HubalEP HoffmanPD ThompsonPM Clarkson2003Molecular responses of human muscle to eccentric exercise.J Appl Physiol9524852494
- 11. Arnett MG, Hyslop R, Dennehy CA, Schneider CM (2000) Age-related variations of serum CK and CK MB response in females. Can J Appl Physiol 25: 419–429.MG ArnettR. HyslopCA DennehyCM Schneider2000Age-related variations of serum CK and CK MB response in females.Can J Appl Physiol25419429
- 12. Beaton LJ, Tarnopolsky MA, Phillips SM (2002) Contraction-induced muscle damage in humans following calcium channel blocker administration. J Physiol 544: 849–859.LJ BeatonMA TarnopolskySM Phillips2002Contraction-induced muscle damage in humans following calcium channel blocker administration.J Physiol544849859
- 13. Friden J, Kjorell U, Thornell LE (1984) Delayed muscle soreness and cytoskeletal alterations: an immunocytological study in man. Int J Sports Med 5: 15–18.J. FridenU. KjorellLE Thornell1984Delayed muscle soreness and cytoskeletal alterations: an immunocytological study in man.Int J Sports Med51518
- 14. Newham DJ, McPhail G, Mills KR, Edwards RH (1983) Ultrastructural changes after concentric and eccentric contractions of human muscle. J Neurol Sci 61: 109–122.DJ NewhamG. McPhailKR MillsRH Edwards1983Ultrastructural changes after concentric and eccentric contractions of human muscle.J Neurol Sci61109122
- 15. Amelink GJ, Kamp HH, Bar PR (1988) Creatine kinase isoenzyme profiles after exercise in the rat: sex-linked differences in leakage of CK-MM. Pflugers Arch 412: 417–421.GJ AmelinkHH KampPR Bar1988Creatine kinase isoenzyme profiles after exercise in the rat: sex-linked differences in leakage of CK-MM.Pflugers Arch412417421
- 16. Stupka N, Tarnopolsky MA, Yardley NJ, Phillips SM (2001) Cellular adaptation to repeated eccentric exercise-induced muscle damage. J Appl Physiol 91: 1669–1678.N. StupkaMA TarnopolskyNJ YardleySM Phillips2001Cellular adaptation to repeated eccentric exercise-induced muscle damage.J Appl Physiol9116691678
- 17. Amelink GJ, Koot RW, Erich WB, Van Gijn J, Bar PR (1990) Sex-linked variation in creatine kinase release, and its dependence on oestradiol, can be demonstrated in an in-vitro rat skeletal muscle preparation. Acta Physiol Scand 138: 115–124.GJ AmelinkRW KootWB ErichJ. Van GijnPR Bar1990Sex-linked variation in creatine kinase release, and its dependence on oestradiol, can be demonstrated in an in-vitro rat skeletal muscle preparation.Acta Physiol Scand138115124
- 18. Amelink GJ, Bar PR (1986) Exercise-induced muscle protein leakage in the rat. Effects of hormonal manipulation. J Neurol Sci 76: 61–68.GJ AmelinkPR Bar1986Exercise-induced muscle protein leakage in the rat. Effects of hormonal manipulation.J Neurol Sci766168
- 19. Stupka N, Tiidus PM (2001) Effects of ovariectomy and estrogen on ischemia-reperfusion injury in hindlimbs of female rats. J Appl Physiol 91: 1828–1835.N. StupkaPM Tiidus2001Effects of ovariectomy and estrogen on ischemia-reperfusion injury in hindlimbs of female rats.J Appl Physiol9118281835
- 20. Komulainen J, Koskinen SO, Kalliokoski R, Takala TE, Vihko V (1999) Gender differences in skeletal muscle fibre damage after eccentrically biased downhill running in rats. Acta Physiol Scand 165: 57–63.J. KomulainenSO KoskinenR. KalliokoskiTE TakalaV. Vihko1999Gender differences in skeletal muscle fibre damage after eccentrically biased downhill running in rats.Acta Physiol Scand1655763
- 21. Enns DL, Iqbal S, Tiidus PM (2008) Oestrogen receptors mediate oestrogen-induced increases in post-exercise rat skeletal muscle satellite cells. Acta Physiol (Oxf) 194: 81–93.DL EnnsS. IqbalPM Tiidus2008Oestrogen receptors mediate oestrogen-induced increases in post-exercise rat skeletal muscle satellite cells.Acta Physiol (Oxf)1948193
- 22. Tiidus PM, Holden D, Bombardier E, Zajchowski S, Enns D, et al. (2001) Estrogen effect on post-exercise skeletal muscle neutrophil infiltration and calpain activity. Can J Physiol Pharmacol 79: 400–406.PM TiidusD. HoldenE. BombardierS. ZajchowskiD. Enns2001Estrogen effect on post-exercise skeletal muscle neutrophil infiltration and calpain activity.Can J Physiol Pharmacol79400406
- 23. Stupka N, Lowther S, Chorneyko K, Bourgeois JM, Hogben C, et al. (2000) Gender differences in muscle inflammation after eccentric exercise. J Appl Physiol 89: 2325–2332.N. StupkaS. LowtherK. ChorneykoJM BourgeoisC. Hogben2000Gender differences in muscle inflammation after eccentric exercise.J Appl Physiol8923252332
- 24. St Pierre Schneider B, Correia LA, Cannon JG (1999) Sex differences in leukocyte invasion in injured murine skeletal muscle. Res Nurs Health 22: 243–250.B. St Pierre SchneiderLA CorreiaJG Cannon1999Sex differences in leukocyte invasion in injured murine skeletal muscle.Res Nurs Health22243250
- 25. Tiidus PM, Deller M, Liu XL (2005) Oestrogen influence on myogenic satellite cells following downhill running in male rats: a preliminary study. Acta Physiol Scand 184: 67–72.PM TiidusM. DellerXL Liu2005Oestrogen influence on myogenic satellite cells following downhill running in male rats: a preliminary study.Acta Physiol Scand1846772
- 26. Enns DL, Tiidus PM (2008) Estrogen influences satellite cell activation and proliferation following downhill running in rats. J Appl Physiol 104: 347–353.DL EnnsPM Tiidus2008Estrogen influences satellite cell activation and proliferation following downhill running in rats.J Appl Physiol104347353
- 27. Persky AM, Green PS, Stubley L, Howell CO, Zaulyanov L, et al. (2000) Protective effect of estrogens against oxidative damage to heart and skeletal muscle in vivo and in vitro. Proc Soc Exp Biol Med 223: 59–66.AM PerskyPS GreenL. StubleyCO HowellL. Zaulyanov2000Protective effect of estrogens against oxidative damage to heart and skeletal muscle in vivo and in vitro.Proc Soc Exp Biol Med2235966
- 28. Sribnick EA, Ray SK, Banik NL (2004) Estrogen as a multi-active neuroprotective agent in traumatic injuries. Neurochem Res 29: 2007–2014.EA SribnickSK RayNL Banik2004Estrogen as a multi-active neuroprotective agent in traumatic injuries.Neurochem Res2920072014
- 29. Tiidus PM (1995) Can estrogens diminish exercise induced muscle damage? Can J Appl Physiol 20: 26–38.PM Tiidus1995Can estrogens diminish exercise induced muscle damage?Can J Appl Physiol202638
- 30. Kahlert S, Grohe C, Karas RH, Lobbert K, Neyses L, et al. (1997) Effects of estrogen on skeletal myoblast growth. Biochem Biophys Res Commun 232: 373–378.S. KahlertC. GroheRH KarasK. LobbertL. Neyses1997Effects of estrogen on skeletal myoblast growth.Biochem Biophys Res Commun232373378
- 31. Gruber CJ, Tschugguel W, Schneeberger C, Huber JC (2002) Production and actions of estrogens. N Engl J Med 346: 340–352.CJ GruberW. TschugguelC. SchneebergerJC Huber2002Production and actions of estrogens.N Engl J Med346340352
- 32. Katzenellenbogen BS, Choi I, Delage-Mourroux R, Ediger TR, Martini PG, et al. (2000) Molecular mechanisms of estrogen action: selective ligands and receptor pharmacology. J Steroid Biochem Mol Biol 74: 279–285.BS KatzenellenbogenI. ChoiR. Delage-MourrouxTR EdigerPG Martini2000Molecular mechanisms of estrogen action: selective ligands and receptor pharmacology.J Steroid Biochem Mol Biol74279285
- 33. Clarkson PM, Hubal MJ (2001) Are women less susceptible to exercise-induced muscle damage? Curr Opin Clin Nutr Metab Care 4: 527–531.PM ClarksonMJ Hubal2001Are women less susceptible to exercise-induced muscle damage?Curr Opin Clin Nutr Metab Care4527531
- 34. Mahoney DJ, Safdar A, Parise G, Melov S, Fu M, et al. (2008) Gene expression profiling in human skeletal muscle during recovery from eccentric exercise. Am J Physiol Regul Integr Comp Physiol 294: R1901–1910.DJ MahoneyA. SafdarG. PariseS. MelovM. Fu2008Gene expression profiling in human skeletal muscle during recovery from eccentric exercise.Am J Physiol Regul Integr Comp Physiol294R19011910
- 35. Zambon AC, McDearmon EL, Salomonis N, Vranizan KM, Johansen KL, et al. (2003) Time- and exercise-dependent gene regulation in human skeletal muscle. Genome Biol 4: R61.AC ZambonEL McDearmonN. SalomonisKM VranizanKL Johansen2003Time- and exercise-dependent gene regulation in human skeletal muscle.Genome Biol4R61
- 36. Maher AC, Fu MH, Isfort RJ, Varbanov AR, Qu XA, et al. (2009) Sex differences in global mRNA content of human skeletal muscle. PLoS One 4: e6335.AC MaherMH FuRJ IsfortAR VarbanovXA Qu2009Sex differences in global mRNA content of human skeletal muscle.PLoS One4e6335
- 37. Welle S, Tawil R, Thornton CA (2008) Sex-related differences in gene expression in human skeletal muscle. PLoS One 3: e1385.S. WelleR. TawilCA Thornton2008Sex-related differences in gene expression in human skeletal muscle.PLoS One3e1385
- 38. Roth SM, Ferrell RE, Peters DG, Metter EJ, Hurley BF, et al. (2002) Influence of age, sex, and strength training on human muscle gene expression determined by microarray. Physiol Genomics 10: 181–190.SM RothRE FerrellDG PetersEJ MetterBF Hurley2002Influence of age, sex, and strength training on human muscle gene expression determined by microarray.Physiol Genomics10181190
- 39. Housh DJ, Housh TJ, Weir JP, Weir LL, Johnson GO, et al. (1995) Anthropometric estimation of thigh muscle cross-sectional area. Med Sci Sports Exerc 27: 784–791.DJ HoushTJ HoushJP WeirLL WeirGO Johnson1995Anthropometric estimation of thigh muscle cross-sectional area.Med Sci Sports Exerc27784791
- 40. Devries MC, Hamadeh MJ, Graham TE, Tarnopolsky MA (2005) 17beta-estradiol supplementation decreases glucose rate of appearance and disappearance with no effect on glycogen utilization during moderate intensity exercise in men. J Clin Endocrinol Metab 90: 6218–6225.MC DevriesMJ HamadehTE GrahamMA Tarnopolsky200517beta-estradiol supplementation decreases glucose rate of appearance and disappearance with no effect on glycogen utilization during moderate intensity exercise in men.J Clin Endocrinol Metab9062186225
- 41. Beaton LJ, Allan DA, Tarnopolsky MA, Tiidus PM, Phillips SM (2002) Contraction-induced muscle damage is unaffected by vitamin E supplementation. Med Sci Sports Exerc 34: 798–805.LJ BeatonDA AllanMA TarnopolskyPM TiidusSM Phillips2002Contraction-induced muscle damage is unaffected by vitamin E supplementation.Med Sci Sports Exerc34798805
- 42. Vissing K, Andersen JL, Schjerling P (2005) Are exercise-induced genes induced by exercise? Faseb J 19: 94–96.K. VissingJL AndersenP. Schjerling2005Are exercise-induced genes induced by exercise?Faseb J199496
- 43. Mahoney DJ, Carey K, Fu MH, Snow R, Cameron-Smith D, et al. (2004) Real-time RT-PCR analysis of housekeeping genes in human skeletal muscle following acute exercise. Physiol Genomics 18: 226–231.DJ MahoneyK. CareyMH FuR. SnowD. Cameron-Smith2004Real-time RT-PCR analysis of housekeeping genes in human skeletal muscle following acute exercise.Physiol Genomics18226231
- 44. Zeeberg BR, Feng W, Wang G, Wang MD, Fojo AT, et al. (2003) GoMiner: a resource for biological interpretation of genomic and proteomic data. Genome Biol 4: R28.BR ZeebergW. FengG. WangMD WangAT Fojo2003GoMiner: a resource for biological interpretation of genomic and proteomic data.Genome Biol4R28
- 45. Liu XM, Peyton KJ, Ensenat D, Wang H, Schafer AI, et al. (2005) Endoplasmic reticulum stress stimulates heme oxygenase-1 gene expression in vascular smooth muscle. Role in cell survival. J Biol Chem 280: 872–877.XM LiuKJ PeytonD. EnsenatH. WangAI Schafer2005Endoplasmic reticulum stress stimulates heme oxygenase-1 gene expression in vascular smooth muscle. Role in cell survival.J Biol Chem280872877
- 46. Melov S, Tarnopolsky MA, Beckman K, Felkey K, Hubbard A (2007) Resistance exercise reverses aging in human skeletal muscle. PLoS ONE 2: e465.S. MelovMA TarnopolskyK. BeckmanK. FelkeyA. Hubbard2007Resistance exercise reverses aging in human skeletal muscle.PLoS ONE2e465
- 47. Canales RD, Luo Y, Willey JC, Austermiller B, Barbacioru CC, et al. (2006) Evaluation of DNA microarray results with quantitative gene expression platforms. Nat Biotechnol 24: 1115–1122.RD CanalesY. LuoJC WilleyB. AustermillerCC Barbacioru2006Evaluation of DNA microarray results with quantitative gene expression platforms.Nat Biotechnol2411151122
- 48. Tarnopolsky MA, Parshad A, Walzel B, Schlattner U, Wallimann T (2001) Creatine transporter and mitochondrial creatine kinase protein content in myopathies. Muscle Nerve 24: 682–688.MA TarnopolskyA. ParshadB. WalzelU. SchlattnerT. Wallimann2001Creatine transporter and mitochondrial creatine kinase protein content in myopathies.Muscle Nerve24682688
- 49. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275.OH LowryNJ RosebroughAL FarrRJ Randall1951Protein measurement with the Folin phenol reagent.J Biol Chem193265275
- 50. Tiidus PM, Enns DL (2009) Point:Counterpoint: Estrogen and sex do/do not influence post-exercise indexes of muscle damage, inflammation, and repair. J Appl Physiol 106: 1010–1012; discussion 1014–1015, 1021.PM TiidusDL Enns2009Point:Counterpoint: Estrogen and sex do/do not influence post-exercise indexes of muscle damage, inflammation, and repair.J Appl Physiol10610101012; discussion 1014–1015, 1021
- 51. Tiidus PM (2005) Can oestrogen influence skeletal muscle damage, inflammation, and repair? Br J Sports Med 39: 251–253.PM Tiidus2005Can oestrogen influence skeletal muscle damage, inflammation, and repair?Br J Sports Med39251253
- 52. Kuwahara K, Teg Pipes GC, McAnally J, Richardson JA, Hill JA, et al. (2007) Modulation of adverse cardiac remodeling by STARS, a mediator of MEF2 signaling and SRF activity. J Clin Invest 117: 1324–1334.K. KuwaharaGC Teg PipesJ. McAnallyJA RichardsonJA Hill2007Modulation of adverse cardiac remodeling by STARS, a mediator of MEF2 signaling and SRF activity.J Clin Invest11713241334
- 53. Mahadeva H, Brooks G, Lodwick D, Chong NW, Samani NJ (2002) ms1, a novel stress-responsive, muscle-specific gene that is up-regulated in the early stages of pressure overload-induced left ventricular hypertrophy. FEBS Lett 521: 100–104.H. MahadevaG. BrooksD. LodwickNW ChongNJ Samani2002ms1, a novel stress-responsive, muscle-specific gene that is up-regulated in the early stages of pressure overload-induced left ventricular hypertrophy.FEBS Lett521100104
- 54. Arai A, Spencer JA, Olson EN (2002) STARS, a striated muscle activator of Rho signaling and serum response factor-dependent transcription. J Biol Chem 277: 24453–24459.A. AraiJA SpencerEN Olson2002STARS, a striated muscle activator of Rho signaling and serum response factor-dependent transcription.J Biol Chem2772445324459
- 55. Kuwahara K, Barrientos T, Pipes GC, Li S, Olson EN (2005) Muscle-specific signaling mechanism that links actin dynamics to serum response factor. Mol Cell Biol 25: 3173–3181.K. KuwaharaT. BarrientosGC PipesS. LiEN Olson2005Muscle-specific signaling mechanism that links actin dynamics to serum response factor.Mol Cell Biol2531733181
- 56. Barrientos T, Frank D, Kuwahara K, Bezprozvannaya S, Pipes GC, et al. (2007) Two novel members of the ABLIM protein family, ABLIM-2 and -3, associate with STARS and directly bind F-actin. J Biol Chem 282: 8393–8403.T. BarrientosD. FrankK. KuwaharaS. BezprozvannayaGC Pipes2007Two novel members of the ABLIM protein family, ABLIM-2 and -3, associate with STARS and directly bind F-actin.J Biol Chem28283938403
- 57. Wei L, Zhou W, Croissant JD, Johansen FE, Prywes R, et al. (1998) RhoA signaling via serum response factor plays an obligatory role in myogenic differentiation. J Biol Chem 273: 30287–30294.L. WeiW. ZhouJD CroissantFE JohansenR. Prywes1998RhoA signaling via serum response factor plays an obligatory role in myogenic differentiation.J Biol Chem2733028730294
- 58. Lamon S, Wallace MA, Leger B, Russell AP (2009) Regulation of STARS and its downstream targets suggest a novel pathway involved in human skeletal muscle hypertrophy and atrophy. J Physiol 587: 1795–1803.S. LamonMA WallaceB. LegerAP Russell2009Regulation of STARS and its downstream targets suggest a novel pathway involved in human skeletal muscle hypertrophy and atrophy.J Physiol58717951803
- 59. Takano H, Komuro I, Oka T, Shiojima I, Hiroi Y, et al. (1998) The Rho family G proteins play a critical role in muscle differentiation. Mol Cell Biol 18: 1580–1589.H. TakanoI. KomuroT. OkaI. ShiojimaY. Hiroi1998The Rho family G proteins play a critical role in muscle differentiation.Mol Cell Biol1815801589
- 60. Rossman KL, Der CJ, Sondek J (2005) GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol 6: 167–180.KL RossmanCJ DerJ. Sondek2005GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors.Nat Rev Mol Cell Biol6167180
- 61. Jaffe AB, Hall A (2005) Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol 21: 247–269.AB JaffeA. Hall2005Rho GTPases: biochemistry and biology.Annu Rev Cell Dev Biol21247269
- 62. Fortier M, Comunale F, Kucharczak J, Blangy A, Charrasse S, et al. (2008) RhoE controls myoblast alignment prior fusion through RhoA and ROCK. Cell Death Differ 15: 1221–1231.M. FortierF. ComunaleJ. KucharczakA. BlangyS. Charrasse2008RhoE controls myoblast alignment prior fusion through RhoA and ROCK.Cell Death Differ1512211231
- 63. Van Aelst L, D'Souza-Schorey C (1997) Rho GTPases and signaling networks. Genes Dev 11: 2295–2322.L. Van AelstC. D'Souza-Schorey1997Rho GTPases and signaling networks.Genes Dev1122952322
- 64. Etienne-Manneville S, Hall A (2002) Rho GTPases in cell biology. Nature 420: 629–635.S. Etienne-MannevilleA. Hall2002Rho GTPases in cell biology.Nature420629635
- 65. Tcherkezian J, Lamarche-Vane N (2007) Current knowledge of the large RhoGAP family of proteins. Biol Cell 99: 67–86.J. TcherkezianN. Lamarche-Vane2007Current knowledge of the large RhoGAP family of proteins.Biol Cell996786
- 66. Charrasse S, Comunale F, Grumbach Y, Poulat F, Blangy A, et al. (2006) RhoA GTPase regulates M-cadherin activity and myoblast fusion. Mol Biol Cell 17: 749–759.S. CharrasseF. ComunaleY. GrumbachF. PoulatA. Blangy2006RhoA GTPase regulates M-cadherin activity and myoblast fusion.Mol Biol Cell17749759
- 67. Castellani L, Salvati E, Alema S, Falcone G (2006) Fine regulation of RhoA and Rock is required for skeletal muscle differentiation. J Biol Chem 281: 15249–15257.L. CastellaniE. SalvatiS. AlemaG. Falcone2006Fine regulation of RhoA and Rock is required for skeletal muscle differentiation.J Biol Chem2811524915257
- 68. Wennerberg K, Forget MA, Ellerbroek SM, Arthur WT, Burridge K, et al. (2003) Rnd proteins function as RhoA antagonists by activating p190 RhoGAP. Curr Biol 13: 1106–1115.K. WennerbergMA ForgetSM EllerbroekWT ArthurK. Burridge2003Rnd proteins function as RhoA antagonists by activating p190 RhoGAP.Curr Biol1311061115
- 69. Guasch RM, Scambler P, Jones GE, Ridley AJ (1998) RhoE regulates actin cytoskeleton organization and cell migration. Mol Cell Biol 18: 4761–4771.RM GuaschP. ScamblerGE JonesAJ Ridley1998RhoE regulates actin cytoskeleton organization and cell migration.Mol Cell Biol1847614771
- 70. Xie Y, Tan EJ, Wee S, Manser E, Lim L, et al. (2008) Functional interactions between phosphatase POPX2 and mDia modulate RhoA pathways. J Cell Sci 121: 514–521.Y. XieEJ TanS. WeeE. ManserL. Lim2008Functional interactions between phosphatase POPX2 and mDia modulate RhoA pathways.J Cell Sci121514521
- 71. Gao G, Chen L, Dong B, Gu H, Dong H, et al. (2009) RhoA effector mDia1 is required for PI 3-kinase-dependent actin remodeling and spreading by thrombin in platelets. Biochem Biophys Res Commun 385: 439–444.G. GaoL. ChenB. DongH. GuH. Dong2009RhoA effector mDia1 is required for PI 3-kinase-dependent actin remodeling and spreading by thrombin in platelets.Biochem Biophys Res Commun385439444
- 72. Xavier CP, Rastetter RH, Stumpf M, Rosentreter A, Muller R, et al. (2009) Structural and Functional Diversity of Novel Coronin 1C (CRN2) Isoforms in Muscle. J Mol Biol. CP XavierRH RastetterM. StumpfA. RosentreterR. Muller2009Structural and Functional Diversity of Novel Coronin 1C (CRN2) Isoforms in Muscle.J Mol Biol
- 73. Bello NF, Lamsoul I, Heuze ML, Metais A, Moreaux G, et al. (2009) The E3 ubiquitin ligase specificity subunit ASB2beta is a novel regulator of muscle differentiation that targets filamin B to proteasomal degradation. Cell Death Differ 16: 921–932.NF BelloI. LamsoulML HeuzeA. MetaisG. Moreaux2009The E3 ubiquitin ligase specificity subunit ASB2beta is a novel regulator of muscle differentiation that targets filamin B to proteasomal degradation.Cell Death Differ16921932
- 74. Papa I, Astier C, Kwiatek O, Raynaud F, Bonnal C, et al. (1999) Alpha actinin-CapZ, an anchoring complex for thin filaments in Z-line. J Muscle Res Cell Motil 20: 187–197.I. PapaC. AstierO. KwiatekF. RaynaudC. Bonnal1999Alpha actinin-CapZ, an anchoring complex for thin filaments in Z-line.J Muscle Res Cell Motil20187197
- 75. Niu Z, Iyer D, Conway SJ, Martin JF, Ivey K, et al. (2008) Serum response factor orchestrates nascent sarcomerogenesis and silences the biomineralization gene program in the heart. Proc Natl Acad Sci U S A 105: 17824–17829.Z. NiuD. IyerSJ ConwayJF MartinK. Ivey2008Serum response factor orchestrates nascent sarcomerogenesis and silences the biomineralization gene program in the heart.Proc Natl Acad Sci U S A1051782417829
- 76. Nelson TJ, Balza R Jr, Xiao Q, Misra RP (2005) SRF-dependent gene expression in isolated cardiomyocytes: regulation of genes involved in cardiac hypertrophy. J Mol Cell Cardiol 39: 479–489.TJ NelsonR. Balza JrQ. XiaoRP Misra2005SRF-dependent gene expression in isolated cardiomyocytes: regulation of genes involved in cardiac hypertrophy.J Mol Cell Cardiol39479489
- 77. Santalucia T, Christmann M, Yacoub MH, Brand NJ (2003) Hypertrophic agonists induce the binding of c-Fos to an AP-1 site in cardiac myocytes: implications for the expression of GLUT1. Cardiovasc Res 59: 639–648.T. SantaluciaM. ChristmannMH YacoubNJ Brand2003Hypertrophic agonists induce the binding of c-Fos to an AP-1 site in cardiac myocytes: implications for the expression of GLUT1.Cardiovasc Res59639648
- 78. Macian F, Lopez-Rodriguez C, Rao A (2001) Partners in transcription: NFAT and AP-1. Oncogene 20: 2476–2489.F. MacianC. Lopez-RodriguezA. Rao2001Partners in transcription: NFAT and AP-1.Oncogene2024762489
- 79. Trenerry MK, Carey KA, Ward AC, Cameron-Smith D (2007) STAT3 signaling is activated in human skeletal muscle following acute resistance exercise. J Appl Physiol 102: 1483–1489.MK TrenerryKA CareyAC WardD. Cameron-Smith2007STAT3 signaling is activated in human skeletal muscle following acute resistance exercise.J Appl Physiol10214831489
- 80. Puntschart A, Wey E, Jostarndt K, Vogt M, Wittwer M, et al. (1998) Expression of fos and jun genes in human skeletal muscle after exercise. Am J Physiol 274: C129–137.A. PuntschartE. WeyK. JostarndtM. VogtM. Wittwer1998Expression of fos and jun genes in human skeletal muscle after exercise.Am J Physiol274C129137
- 81. Hogan PG, Chen L, Nardone J, Rao A (2003) Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev 17: 2205–2232.PG HoganL. ChenJ. NardoneA. Rao2003Transcriptional regulation by calcium, calcineurin, and NFAT.Genes Dev1722052232
- 82. Wilkins BJ, Molkentin JD (2004) Calcium-calcineurin signaling in the regulation of cardiac hypertrophy. Biochem Biophys Res Commun 322: 1178–1191.BJ WilkinsJD Molkentin2004Calcium-calcineurin signaling in the regulation of cardiac hypertrophy.Biochem Biophys Res Commun32211781191
- 83. Molkentin JD (2004) Calcineurin-NFAT signaling regulates the cardiac hypertrophic response in coordination with the MAPKs. Cardiovasc Res 63: 467–475.JD Molkentin2004Calcineurin-NFAT signaling regulates the cardiac hypertrophic response in coordination with the MAPKs.Cardiovasc Res63467475
- 84. Fiedler B, Wollert KC (2004) Interference of antihypertrophic molecules and signaling pathways with the Ca2+-calcineurin-NFAT cascade in cardiac myocytes. Cardiovasc Res 63: 450–457.B. FiedlerKC Wollert2004Interference of antihypertrophic molecules and signaling pathways with the Ca2+-calcineurin-NFAT cascade in cardiac myocytes.Cardiovasc Res63450457
- 85. Crabtree GR (1999) Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT. Cell 96: 611–614.GR Crabtree1999Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT.Cell96611614
- 86. Beals CR, Sheridan CM, Turck CW, Gardner P, Crabtree GR (1997) Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3. Science 275: 1930–1934.CR BealsCM SheridanCW TurckP. GardnerGR Crabtree1997Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3.Science27519301934
- 87. Braz JC, Bueno OF, Liang Q, Wilkins BJ, Dai YS, et al. (2003) Targeted inhibition of p38 MAPK promotes hypertrophic cardiomyopathy through upregulation of calcineurin-NFAT signaling. J Clin Invest 111: 1475–1486.JC BrazOF BuenoQ. LiangBJ WilkinsYS Dai2003Targeted inhibition of p38 MAPK promotes hypertrophic cardiomyopathy through upregulation of calcineurin-NFAT signaling.J Clin Invest11114751486
- 88. Hawke TJ, Atkinson DJ, Kanatous SB, Van der Ven PF, Goetsch SC, et al. (2007) Xin, an actin binding protein, is expressed within muscle satellite cells and newly regenerated skeletal muscle fibers. Am J Physiol Cell Physiol 293: C1636–1644.TJ HawkeDJ AtkinsonSB KanatousPF Van der VenSC Goetsch2007Xin, an actin binding protein, is expressed within muscle satellite cells and newly regenerated skeletal muscle fibers.Am J Physiol Cell Physiol293C16361644
- 89. Postel R, Vakeel P, Topczewski J, Knoll R, Bakkers J (2008) Zebrafish integrin-linked kinase is required in skeletal muscles for strengthening the integrin-ECM adhesion complex. Dev Biol 318: 92–101.R. PostelP. VakeelJ. TopczewskiR. KnollJ. Bakkers2008Zebrafish integrin-linked kinase is required in skeletal muscles for strengthening the integrin-ECM adhesion complex.Dev Biol31892101
- 90. Kardassis D, Murphy C, Fotsis T, Moustakas A, Stournaras C (2009) Control of transforming growth factor beta signal transduction by small GTPases. Febs J 276: 2947–2965.D. KardassisC. MurphyT. FotsisA. MoustakasC. Stournaras2009Control of transforming growth factor beta signal transduction by small GTPases.Febs J27629472965
- 91. Sasai N, Agata N, Inoue-Miyazu M, Kawakami K, Kobayashi K, et al. (2009) Involvement of PI3K/Akt/TOR pathway in stretch-induced hypertrophy of myotubes. Muscle Nerve. N. SasaiN. AgataM. Inoue-MiyazuK. KawakamiK. Kobayashi2009Involvement of PI3K/Akt/TOR pathway in stretch-induced hypertrophy of myotubes.Muscle Nerve
- 92. Wright DC (2007) Mechanisms of calcium-induced mitochondrial biogenesis and GLUT4 synthesis. Appl Physiol Nutr Metab 32: 840–845.DC Wright2007Mechanisms of calcium-induced mitochondrial biogenesis and GLUT4 synthesis.Appl Physiol Nutr Metab32840845
- 93. Berchtold MW, Brinkmeier H, Muntener M (2000) Calcium ion in skeletal muscle: its crucial role for muscle function, plasticity, and disease. Physiol Rev 80: 1215–1265.MW BerchtoldH. BrinkmeierM. Muntener2000Calcium ion in skeletal muscle: its crucial role for muscle function, plasticity, and disease.Physiol Rev8012151265
- 94. Rothermel BA, Vega RB, Williams RS (2003) The role of modulatory calcineurin-interacting proteins in calcineurin signaling. Trends Cardiovasc Med 13: 15–21.BA RothermelRB VegaRS Williams2003The role of modulatory calcineurin-interacting proteins in calcineurin signaling.Trends Cardiovasc Med131521
- 95. Oh M, Rybkin II, Copeland V, Czubryt MP, Shelton JM, et al. (2005) Calcineurin is necessary for the maintenance but not embryonic development of slow muscle fibers. Mol Cell Biol 25: 6629–6638.M. OhII RybkinV. CopelandMP CzubrytJM Shelton2005Calcineurin is necessary for the maintenance but not embryonic development of slow muscle fibers.Mol Cell Biol2566296638
- 96. Leinwand LA (2001) Calcineurin inhibition and cardiac hypertrophy: a matter of balance. Proc Natl Acad Sci U S A 98: 2947–2949.LA Leinwand2001Calcineurin inhibition and cardiac hypertrophy: a matter of balance.Proc Natl Acad Sci U S A9829472949
- 97. Yang J, Rothermel B, Vega RB, Frey N, McKinsey TA, et al. (2000) Independent signals control expression of the calcineurin inhibitory proteins MCIP1 and MCIP2 in striated muscles. Circ Res 87: E61–68.J. YangB. RothermelRB VegaN. FreyTA McKinsey2000Independent signals control expression of the calcineurin inhibitory proteins MCIP1 and MCIP2 in striated muscles.Circ Res87E6168
- 98. Tongers J, Fiedler B, Konig D, Kempf T, Klein G, et al. (2004) Heme oxygenase-1 inhibition of MAP kinases, calcineurin/NFAT signaling, and hypertrophy in cardiac myocytes. Cardiovasc Res 63: 545–552.J. TongersB. FiedlerD. KonigT. KempfG. Klein2004Heme oxygenase-1 inhibition of MAP kinases, calcineurin/NFAT signaling, and hypertrophy in cardiac myocytes.Cardiovasc Res63545552
- 99. Pilegaard H, Ordway GA, Saltin B, Neufer PD (2000) Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise. Am J Physiol Endocrinol Metab 279: E806–814.H. PilegaardGA OrdwayB. SaltinPD Neufer2000Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise.Am J Physiol Endocrinol Metab279E806814
- 100. Mendez P, Azcoitia I, Garcia-Segura LM (2005) Interdependence of oestrogen and insulin-like growth factor-I in the brain: potential for analysing neuroprotective mechanisms. J Endocrinol 185: 11–17.P. MendezI. AzcoitiaLM Garcia-Segura2005Interdependence of oestrogen and insulin-like growth factor-I in the brain: potential for analysing neuroprotective mechanisms.J Endocrinol1851117
- 101. Klinge CM (2008) Estrogenic control of mitochondrial function and biogenesis. J Cell Biochem 105: 1342–1351.CM Klinge2008Estrogenic control of mitochondrial function and biogenesis.J Cell Biochem10513421351
- 102. Levin ER (2009) Plasma membrane estrogen receptors. Trends Endocrinol Metab 20: 477–482.ER Levin2009Plasma membrane estrogen receptors.Trends Endocrinol Metab20477482
- 103. Kim HP, Lee JY, Jeong JK, Bae SW, Lee HK, et al. (1999) Nongenomic stimulation of nitric oxide release by estrogen is mediated by estrogen receptor alpha localized in caveolae. Biochem Biophys Res Commun 263: 257–262.HP KimJY LeeJK JeongSW BaeHK Lee1999Nongenomic stimulation of nitric oxide release by estrogen is mediated by estrogen receptor alpha localized in caveolae.Biochem Biophys Res Commun263257262
- 104. Watters JJ, Campbell JS, Cunningham MJ, Krebs EG, Dorsa DM (1997) Rapid membrane effects of steroids in neuroblastoma cells: effects of estrogen on mitogen activated protein kinase signalling cascade and c-fos immediate early gene transcription. Endocrinology 138: 4030–4033.JJ WattersJS CampbellMJ CunninghamEG KrebsDM Dorsa1997Rapid membrane effects of steroids in neuroblastoma cells: effects of estrogen on mitogen activated protein kinase signalling cascade and c-fos immediate early gene transcription.Endocrinology13840304033
- 105. Wiik A, Glenmark B, Ekman M, Esbjornsson-Liljedahl M, Johansson O, et al. (2003) Oestrogen receptor beta is expressed in adult human skeletal muscle both at the mRNA and protein level. Acta Physiol Scand 179: 381–387.A. WiikB. GlenmarkM. EkmanM. Esbjornsson-LiljedahlO. Johansson2003Oestrogen receptor beta is expressed in adult human skeletal muscle both at the mRNA and protein level.Acta Physiol Scand179381387
- 106. Wiik A, Ekman M, Johansson O, Jansson E, Esbjornsson M (2009) Expression of both oestrogen receptor alpha and beta in human skeletal muscle tissue. Histochem Cell Biol 131: 181–189.A. WiikM. EkmanO. JohanssonE. JanssonM. Esbjornsson2009Expression of both oestrogen receptor alpha and beta in human skeletal muscle tissue.Histochem Cell Biol131181189