Gene expression profiling in skeletal muscle 3 hours following the performance of an eccentric exercise
ABSTRACT: Eccentric exercise (ECC) can result in ultra-structural and histological damage to skeletal muscle. The damage incurred following ECC is typically followed by a subsequent regenerative and adaptive response. The specific mechanisms that drive this response, particularly in human muscle, are not well understood. The objective of this study was to characterize the early molecular response in skeletal muscle following ECC in humans. We used an Agilent whole human genome microarray to assess global gene expression in male subjects (N=35) at 3 hours post-100 eccentric contractions of the knee extensors. ANCOVA (age and BMI covariates) was used to compare mRNA expression between the ECC and non-exercised (CON) legs of each subject. Novel transcripts from IPA identified networks were confirmed with quantitative real-time (qRT)-PCR. qRT-PCR analysis of 3 of these transcripts (IkBα, TNFRSF1A and ICAM-1) confirmed changes observed in the microarray analysis. 35 male subjects performed an eccentric exercise protocol consisting of 100 maximal eccentric contrations of the knee extensors. 3 hours after the completion of the exercise regimen, a muscle biopsy was taken from the vastus lateralis of both legs. The non-exercised leg served as the control. Gene expresssion was analyzed using an ANCOVA, with covariates for age and BMI.
Project description:Eccentric exercise (ECC) can result in ultra-structural and histological damage to skeletal muscle. The damage incurred following ECC is typically followed by a subsequent regenerative and adaptive response. The specific mechanisms that drive this response, particularly in human muscle, are not well understood. The objective of this study was to characterize the early molecular response in skeletal muscle following ECC in humans. We used an Agilent whole human genome microarray to assess global gene expression in male subjects (N=35) at 3 hours post-100 eccentric contractions of the knee extensors. ANCOVA (age and BMI covariates) was used to compare mRNA expression between the ECC and non-exercised (CON) legs of each subject. Novel transcripts from IPA identified networks were confirmed with quantitative real-time (qRT)-PCR. qRT-PCR analysis of 3 of these transcripts (IkBα, TNFRSF1A and ICAM-1) confirmed changes observed in the microarray analysis. Overall design: 35 male subjects performed an eccentric exercise protocol consisting of 100 maximal eccentric contrations of the knee extensors. 3 hours after the completion of the exercise regimen, a muscle biopsy was taken from the vastus lateralis of both legs. The non-exercised leg served as the control. Gene expresssion was analyzed using an ANCOVA, with covariates for age and BMI.
Project description:The adult skeletal muscle is a plastic tissue with a remarkable ability to adapt to different levels of activity by altering its excitability, its contractile and metabolic phenotype and its mass. Knowledge on the mechanisms responsible for muscle mass comes primarily from models of muscle inactivity or denervation or from genetic models of muscle diseases. Given that the underlying exercise-induced transcriptional mechanisms regulating muscle mass are not fully understood, here we investigated the cellular and molecular adaptive mechanisms taking place in fast skeletal muscle of adult zebrafish in response to swimming. Fish were trained at low swimming speed (0.1 m/s; non-exercised) or at their optimal swimming speed (0.4 m/s; exercised). A significant increase in fibre cross-sectional area (1,290 ± 88 vs. 1,665 ± 106 μm2) and vascularization (298 ± 23 vs. 458 ± 38 capillaries/mm2) was found in exercised over non-exercised fish. Gene expression profiling evidenced the transcriptional activation of a series of complex networks of extracellular and intracellular signaling molecules and pathways involved in the regulation of muscle mass, myogenesis and angiogenesis, many (e.g. BMP, TGF, FGF, Notch, Wnt, MEF2, Shh, EphrinB2) not previously associated with exercise-induced contractile activity, and that recapitulate in part the transcriptional events occurring during skeletal muscle regeneration. These results demonstrate that fibre hypertrophy is responsible for the growth-promoting effects of exercise accompanied by a switch to a more oxidative capacity of white muscle fibres to fuel the increased energy demands. Importantly, our study identified novel molecular mechanisms regulating muscle mass and function in vertebrates. Adult zebrafish were subjected or not to a swim training regime consisting of swimming at the optimal swimming speed for this species for 6 h/day, 5 days/week for a total of 4 weeks (20 experimental days). Total RNA of fast muscle from individual non-exercised (n = 8) and exercised (n = 8) zebrafish was analyzed.
Project description:Mice were subjected to 50 eccentric contractions (EC) or 50 isometric contractions (IC) using a non-invasive model, and then sacrificed 48 hours later. RNA from the tibialis anterior of 4 animals were pooled and then split into two groups for hybridization onto two separate Affymetrix MGU74Av2 chips. Control samples were contralateral to the exercised legs, and were only subjected to enough contractions to measure isometric torque. Eccentric contractions (ECs), in which a muscle is forced to lengthen while activated, result in muscle injury and, eventually, muscle strengthening and prevention of further injury. Although the mechanical basis of eccentric contraction-induced injury has been studied in detail, muscle's biological response is less well characterized. This study presents the development of a minimally-invasive model of EC injury in the mouse, follows the time course of torque recovery after an injurious bout of ECs, and uses Affymetrix microarrays to compare the gene expression profile 48 hours after ECs to both isometrically stimulated muscles and contralateral muscles. Torque dropped by about 55% immediately after the exercise bout, and recovered to initial levels 7 days later. 36 known genes were upregulated after ECs compared to contralateral and isometrically stimulated muscles, including five muscle specific genes: muscle LIM protein (MLP), Muscle Ankyrin Repeat Proteins (MARP 1 and 2; also known as cardiac ankyrin repeat protein and Arpp/Ankrd2, respectively), Xin, and Myosin Binding Protein H. The time courses of MLP and MARP expression after the injury bout (determined by quantitative real-time polymerase chain reaction) indicate that these genes are rapidly induced, reaching a peak expression level of 6-11 times contralateral values 12-24 hours after the EC bout and returning to baseline within 72 hours. Very little gene induction was seen after either isometric activation or passive stretch, indicating that the MLP and MARP genes may play an important and specific role in the biological response of muscle to EC-induced injury. Keywords = mouse tibialis anterior eccentric contraction muscle
Project description:Consequence of physical exercise in skeletal muscle was investigated in C57BL/6 mice after 4 weeks of exercise training and compared to sedentary controls. Exercised mice received four 4 weeks of regular exercise training on a motorized treadmill and were compared to sedentary controls. 6 mice of each Treatment were used to extract RNA from the quadriceps muscle three hours after the last training bout
Project description:Dahl salt-sensitive (DS) rats were obtained from Harlan Sprague Dawley Laboratory at 5 weeks of age. At 6 weeks of age, physiologic cardiac hypertrophy was generated by a; vigorous daily exercise regimen for 6 weeks (e group). The exercise protocol is based on those described previously with modifications (Wisloff U et al., 2001; Jin H et al., 1994). Rats were exercised daily for 6 weeks on a rodent treadmill (Exer-6M; Columbus Instruments). The exercise program consisted of three weeks of progressively strenuous exercise regimens; followed by three weeks of maintenance period, during which the rats were exercised at 16 m/min at a 5o incline for 90 minutes/day. All rats completed the exercise protocol. Pathological cardiac hypertrophy was generated by feeding a 6% NaCl diet to DS rats at 6 weeks of age (h group) (Inoko M et al., 1994). Control rats (c group) were age matched and sedentary DS rats fed normal rat chow. Read more at http://cardiogenomics.med.harvard.edu/groups/proj1/pages/rat_home.html<br><br>Note that files GSM11886.txt and GSM12308.txt, and files GSM11887.txt and GSM12309.txt as downloaded from GEO contain identical data.
Project description:Skeletal muscle adapts to exercise training of various modes, intensities and durations with a programmed gene expression response. This study dissects the independent and combined effects of exercise mode, intensity and duration to identify which exercise has the most positive effects on skeletal muscle health. Full details on exercise groups can be found in: Kraus et al Med Sci Sports Exerc. 2001 Oct;33(10):1774-84 and Bateman et al Am J Cardiol. 2011 Sep 15;108(6):838-44. This study uses a middle aged group of subjects that have 3+ markers of metabolic syndrome. One group remains an inactive control, while 5 groups undergo 9 mo supervised exercise training. Exercise groups are as follows: Inactive control (group B); Mild aerobic exercise - low amount/mod intensity (group A); Moderate aerobic exercise - low amt/vig intensity (group D); High aerobic exercise - high amt/vig intensity (group C); resistance training only (group F); and mod aerobic + resistance training (group E). Each group has 10 subjects (5 men and 5 women), however 3 subjects failed array QC, leaving 8 subjects in group E and 9 subjects in group F. Data were all analyzed pre to post training in a RM ANCOVA, covaried for age and sex or regression to determine genotype/phenotype interactions.
Project description:The molecular mechanisms underlying the sex differences in human muscle morphology and function remain to be elucidated. The purpose of the study was to detect the sex differences in the skeletal muscle transcriptome in both the resting state and following anabolic stimuli, resistance exercise. We used microarrays to profile the transcriptome of the biceps brachii of young men and women who underwent an acute unilateral RE session following 12 weeks of progressive training. Bilateral muscle biopsies were obtained either at an early (4h post-exercise) or late recovery (24h post-exercise) time point. Muscle transcription profiles were compared in the resting state between men (n=6) and women (n=8), and in response to acute RE in trained exercised vs. untrained non-exercised control muscle for each sex and time point separately (4h post-exercise, n=3 males, n=4 females; 24h post-exercise, n=3 males, n=4 females). A logistic regression-based method (LRpath), following Bayesian moderated t-statistic (IMBT), was used to test gene functional groups and biological pathways enriched with differentially expressed genes.
Project description:Four healthy human volunteers underwent an acute bout of resistance exercise with the right leg at 2 pm. Biopsies were removed from the Vastus Lateralis muscle 6 h (8 pm) and 18 h (8 am) after exericise
Project description:Full title: Eccentric exercise activates novel transcriptional regulation of hypertrophic signaling pathways not affected by hormone changes. Unaccustomed eccentric exercise damages muscle tissue stimulating mechanisms of recovery and remodeling that may be affected by cellular protection by the sex hormone 17β-estradiol (E2). Using cDNA microarrays, we screened for differences in mRNA expression caused by E2 and eccentric exercise. After randomly 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. 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 (Hologic QDR 1000W, Waltham, MA). Thigh muscle cross-sectional area was calculated using anthropomorphic measurements of mid-thigh circumference and skinfold thickness. 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. 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. On the testing day, following a short warm-up (10 min of light cycling), subjects were seated in a Biodex isokinetic dynamometer (System 3, Biodex Medical Systems Inc., Ronkonkoma, NY) with their right leg strapped to a lever arm. The lever arm was programmed to extend their leg to 150º of flexion (where 180º is full extension) at a moderate speed (30º/s), then flex their leg to 90°of flexion at a faster speed (120°/s). 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. 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. Blood was drawn from the antecubital vein at the same collection times. Muscle and blood samples were processed and stored for future measurements.
Project description:A single bout of exercise followed by intake of carbohydrates leads to glycogen supercompensation in the prior exercised muscle. The molecular mechanisms underlying this well-known phenomenon remain elusive. Here we report that a single bout of exercise induces marked activation of glycogen synthase (GS) and AMP-activated protein kinase (AMPK) for several days beyond normalized muscle glycogen content in man. Acute muscle specific deletion of AMPK activity in mouse muscle abrogated the ability for glycogen supercompensation, providing genetic evidence that AMPK serves as essential driver for glycogen supercompensation. Muscle proteomic analyses revealed elevated glucose uptake capacity in the prior exercised muscle while key proteins in fat oxidation and glycolysis largely remained unchanged. The temporal order of these sustained cellular alterations induced by a single bout of exercise provide a mechanism to offset the otherwise tight feedback inhibition of glycogen synthesis and glucose uptake by glycogen, ultimately leading to muscle glycogen supercompensation.