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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 14, 2004 as doi:10.1096/fj.03-1228fje. |
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Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences,
* The Medical School, Queen’s Medical Centre, University of Nottingham, Nottingham, UK; and
Department of Cardiovascular & Metabolic Diseases, Pfizer Global Research and Development, Groton, Connecticut, USA
2Correspondence: Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, The Medical School, Queen’s Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK. E-mail: simonwynjones2003{at}yahoo.co.uk
SPECIFIC AIMS
After injury such as head trauma or stroke and during illness such as cancer and AIDS there is significant loss of skeletal muscle mass, paralleled by a loss of muscle function. The aim of this study was to provide a comprehensive profiling of a number of candidate genes associated with atrophy and anabolism during immobilization and subsequent exercise rehabilitation and relate these changes to measurements of muscle mass and function in humans, thereby gaining clearer insights into the pathways responsible for skeletal muscle atrophy and subsequent rehabilitation in humans.
PRINCIPAL FINDINGS
After medical screening, nine untrained, healthy men (18-30 y) participated in this study, which was approved by the University of Nottingham Medical School Ethics Committee. Measurements of lean mass, muscle function, and gene expression (from needle muscle biopsies) were determined before and after immobilization and at various points throughout the exercise rehabilitation, as illustrated in Fig. 1
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We profiled the expression of 14 genes from multiple pathways previously implicated by animal studies in regulating muscle protein synthesis and degradation. We examined the role of the calpain proteolytic system by measuring the expression of calpain 1, calpain 2, and calpain 3 proteases and expression of the specific endogenous calpain inhibitor calpastatin. Exercise-induced muscle damage and remodeling have been linked to changes in the calpain system, and overexpression of calpastatin in a transgenic mouse model has been found to reduce the extent of wasting during muscle unloading. We assessed the involvement of the ubiquitin-dependent proteolytic system, which has been linked to muscle atrophy in multiple clinical settings, by monitoring expression of the 20S proteasome 
7 subunit and of ubiquitin E3 ligases: E3, muscle-specific muscle ring finger 1 (MuRF1), and muscle atrophy box factor (MAFbx). The two latter ligases have been shown to be intimately linked to muscle wasting with animal models in multiple catabolic conditions. To determine the role of the IGF-1 signaling pathway in disuse atrophy and subsequent exercise-stimulated hypertrophy, we assessed the phosphatidylinositol-3-OH kinase PI(3)K/Akt pathway by measuring the expression of glycogen synthase kinase 3 (GSK3-) deactivated by Akt and the calcineurin NFAT pathway by measuring casein kinase-1 (CK1-) and p38 Map kinase (p38 MAPK), both of which have been reported to affect calcineurin-NFAT-mediated hypertrophy. We also examined the role of nuclear factor kappaB (NF-
B) survival pathways by measuring the expression of inhibitory B kinase ß (IKK-ß), since atrophy induced by rat hind limb unloading has been shown to increase NF-B p50 subunit activity. Finally, we measured the expression of myostatin, a member of the TGF-ß superfamily, and the TGF-ß binding receptor, activin receptor IIB (ActRIIB). Myostatin is a purported negative mediator of muscle mass, since myostatin knockout mice exhibit gross hypertrophy.
1. Identification of candidate genes that mediate human skeletal muscle atrophy
Four days before immobilization, a needle biopsy sample was taken at rest from the vastus lateralis muscle and immediately frozen in liquid nitrogen for subsequent basal gene expression analysis. Basal measurements of lean mass of the left thigh region were determined by Dual X-Ray Absortiometry. Maximal isometric strength (kg) of the knee extensor muscles was determined during a static voluntary contraction with subjects securely seated with the knee flexed at 90°, using an isometric strain gauge coupled to a digitized recorder. Isokinetic work by the knee extensors was measured by subjects performing 30 consecutive maximal knee extensor contractions at an angular velocity of 90°/s while securely seated in a Cybex dynamometer. To induce muscle atrophy, the left leg of each subject was immobilized at a knee angle of 
160° for 2 wk by the fitting of a light polyester cast, reaching from groin to the toes. Immediately after removal of the cast, a second muscle biopsy was obtained at rest, then further measurements of lean mass and muscle function were made.
Immobilization for 2 wk caused a reduction in quadriceps lean mass (skeletal muscle mass) of 4.7 ± 0.9% (P<0.001), reduced isometric strength by 27 ± 3% (P<0.001), and reduced peak and total isokinetic work by 22 ± 3% (P<0.01) and 21 ± 3% (P<0.01), respectively, compared with basal. Immobilization caused a number of profound changes in gene expression, most notably in the ubiquitin proteasome system with increased expression of the 20S proteasome 7-subunit by 26% (P<0.05) and the muscle-specific E3 ligase MAFbx by 62% (P<0.01). There was a trend for an up-regulation in the muscle-specific ligase, MuRF1, which was increased on average by 34% (P=0.1). There was no change in expression of the ubiquitously-expressed E3 ligase. There was a 28% increase in expression of GSK3- (P<0.01) and a reduction in expression of the muscle-specific calpain 3 (p94; 36%, P<0.001) compared with basal.
2. Muscle contraction performed immediately after immobilization instigates rapid and marked changes in anabolic and catabolic genes associated with the suppression of muscle catabolism and the instigation of muscle hypertrophy and remodeling
After removal of the polyester cast, subjects performed 5 bouts of 30 supervised and consecutive maximal intensity isokinetic knee extensions (each bout separated by 1 min rest) from a position of 90–180° knee flexion and at an angular velocity of 180°/s while securely seated in a Cybex isokinetic dynamometer. The next day, 24 h after the removal of the cast (and the first bout of rehabilitation training), another resting needle muscle biopsy was obtained to determine the effect of contraction performed immediately after a period of immobilization on gene expression. Compared with immediate postimmobilization, there was a significant reduction in muscle-specific ligases MAFbx (39%, P<0.01) and MuRF1, (33%, P<0.05). There was a significant reduction in myostatin expression (48%, P<0.05) but no change in the expression of the myostatin binding receptor ActRIIB. Expression of calpain 1 (32%, P<0.05), calpain 2 (109%, P<0.001), calpastatin (87%, P<0.001), IKK-ß (37%, P<0.05), and CK1- (68%, P<0.01) was significantly increased compared with immediately postimmobilization.
3. Exercise rehabilitation has a chronic affect on gene expression associated with training induced muscle remodeling and restoration of mass
After removal of the polyester cast, subjects entered a 6 wk program of rehabilitation training that involved reporting to the laboratory on three occasions each week and performing 5 bouts of 30 maximal isokinetic knee extensions (each separated by 1 min) at a velocity 180°/s while supervised. After 6 wk of exercise training, lean mass was significantly greater than immediately postimmobilization (P<0.001) and had returned to basal (P=0.81). In line with this restoration of muscle mass, exercise rehabilitation led to a return in muscle function. After 4 wk of exercise rehabilitation, isometric strength increased by 23% compared with immediately postimmobilization (P<0.01) and was no different from basal. After 6 wk rehabilitation, isometric strength was further improved and was on average 10% greater than basal (P=0.17). Plotting isometric strength (kg) against lean mass (g) showed a significant positive correlation over the course of the 8 wk experiment (r=0.46, P<0.01). Peak and total isokinetic work were increased by 36% and 28%, respectively, after 4 wk of exercise rehabilitation compared with postimmobilization values (both P<0.001) and returned to basal preimmobilization values. After 6 wk of rehabilitation, peak (P<0.001) and total (P<0.001) isokinetic work was significantly greater than basal values.
To determine the effect of chronic exercise rehabilitation on gene expression, needle biopsies were taken after 1 and 6 wk of exercise rehabilitation. With exercise rehabilitation, expression of the 20S proteasome 7 subunit, calpain 2, calpain 3, calpastatin, IKK-ß, and CK1- all returned to basal levels. In contrast, after 1 wk of rehabilitation, calpain 1 expression increased another 18% from the 24 h exercise point (P=0.056) and remained significantly greater than basal expression after 6 wk of exercise rehabilitation (P<0.05). The continuation of rehabilitation training for 6 wk maintained myostatin expression at a significantly lower level than basal (P<0.05), again with no change in expression of the ActRIIB receptor.
CONCLUSIONS AND SIGNIFICANCE
For the first time we have provided a comprehensive profiling of genes recently associated with atrophy and anabolism during immobilization and subsequent rehabilitation in humans, together with functional outcomes. We report compelling evidence to indicate that human skeletal muscle disuse atrophy is linked to altered expression of the 20S proteasome and the muscle-specific proteolytic genes MAFbx, MuRF1, and calpain 3. There was no change in the expression of genes associated with muscle hypertrophy and remodeling during immobilization, with the exception of the purported dominant negative IGF-1 mediator, GSK3-, which increased.
We also show that muscle contraction performed immediately after immobilization rapidly instigates profound changes in gene expression collectively linked to the suppression of muscle catabolism and the instigation of muscle hypertrophy and remodeling, with a decline in MAFbx, MuRF1, and myostatin expression and increased expression of calpain 1, calpain 2, calpastatin, IKK-ß, and CK1- observed within 24 h. Finally, we report that after immobilization, exercise training has a chronic effect on gene expression, with myostatin down-regulated and calpain 1 expression up-regulated throughout the 6 wk of rehabilitation, possibly linked to training-induced remodeling of muscle after the restoration of mass. These changes in gene expression and associated changes to muscle mass and function reported here provide critical insight into the pathways responsible for skeletal muscle atrophy and subsequent rehabilitation in humans and should be of considerable value when attempting to develop future therapeutics and biomarkers of clinical efficacy.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1228fje; 
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) were taken before and immediately after immobilization and at 24 h, 1 and 6 wk of rehabilitation training. Biopsies were obtained immediately after an acute bout of exercise (
) and at 24 h and 6 wk of rehabilitation training. DEXA scans (
) were obtained before and immediately after immobilization and at 1 and 6 wk of rehabilitation training. Isokinetic and isometric muscle function (
) was measured before and immediately after immobilization and at 1, 4, and 6 wk of training.