Immobilization of muscle caused by injury and/or surgery results in marked muscle atrophy. Given that the major determinant of strength is muscle size, this loss of muscle mass is paralleled by a decrease in muscle function, which impacts appreciably on health care costs and patients' quality of life, particularly in the absence of appropriate rehabilitation. Conversely, exercise training alone or combined with dietary supplementation has been shown to induce skeletal muscle hypertrophy. Until recently, however, little information has been available concerning the molecular signaling events that regulate the loss of skeletal muscle mass during immobilization in humans and even less concerning the molecular regulation of muscle rehabilitation after atrophy, particularly in the context of prescribed exercise and/or nutritional interventions. Increasing our understanding of this regulation, together with the development of approaches to reduce atrophy and/or stimulate hypertrophy in a clinical setting, is highly desirable because it could markedly reduce health care costs and accelerate the rate of rehabilitation from injury/illness. This short review sets out to identify some of the molecular signaling events that accompany muscle atrophy during limb immobilization in humans and how these events are modulated during prescribed exercise rehabilitation aimed at restoring muscle mass and function. The novelty of the work described is that it has examined, for the first time, whether purported regulators of skeletal muscle atrophy and hypertrophy from cell- and animal-based research are affected by skeletal muscle atrophy and exercise-mediated rehabilitation in humans. Many of the genes determined have not been measured before in humans and certainly not during muscle wasting and subsequent exercise-mediated rehabilitation, irrespective of the experimental model used. Finally, the potential for nutritional intervention to influence anabolic and catabolic signaling in muscle during immobilization and rehabilitation will be considered in the light of recent observations, leading to speculation about the development of therapeutic interventions aimed at combating muscle wasting and facilitating muscle rehabilitation in humans.
GENE EXPRESSION IN IMMOBILIZATION-INDUCED HUMAN MUSCLE ATROPHY
Adult skeletal muscle mass is determined by the relative rates of protein synthesis and protein degradation. Therefore, skeletal muscle atrophy during immobilization is likely to be a consequence of a reduction in muscle protein synthesis and/or an increase in protein degradation, although neither has been studied to any great extent in immobilized humans or during rehabilitation training. Furthermore, despite the existence of several robust candidate pathways (5), the molecular mechanisms responsible for regulating skeletal muscle atrophy in humans during immobilization and the subsequent restoration of muscle mass are presently unclear.
The author's laboratory was the first to show that immobilization-induced atrophy in healthy human skeletal muscle is linked to altered expression of several specific candidate genes (9). In this study, the authors profiled the expression of 14 genes from several pathways, which, based on animal studies, had been implicated in regulating muscle protein synthesis and degradation. In brief, the authors examined the role of the calpain proteolytic system and the expression of the specific endogenous calpain inhibitor, calpastatin. Calpains are a family of Ca2+-activated cysteine proteases that process specific cytoskeletal/membrane-associated or membrane-targeted proteins. With respect to skeletal muscle, elevated calpain activity is present in areas of macrostructure anchorage. In line with this observation, it seems that calpains do not degrade actin and myosin directly but rather degrade proteins involved in the assembly of myofibrillar proteins, such as titin, vinculin, dystrophin, and desmin ((8); Fig. 1). Exercise-induced muscle damage and remodeling have been linked to the changes in the calpain system in human skeletal muscle (4), and the overexpression of calpastatin in a transgenic mouse model has been found to reduce the extent of wasting during muscle unloading (15). In addition, we assessed the involvement of the ubiquitin-dependent proteolytic system, which has been linked to muscle atrophy in numerous catabolic settings (11,14), by monitoring the expression of the 20S proteasome α7 subunit and also the expression of ubiquitin E3 ligases, namely E3α, muscle-specific muscle ring finger 1 (MuRF1), and muscle atrophy box factor (MAFbx; Fig. 1). These two latter ligases have been shown to be intimately linked to muscle wasting in animal models (2) and are known to be under transcriptional regulation in multiple catabolic conditions (11,14). There is an opinion that protein degradation can occur in a sequential manner via the calpain- and the ubiquitin-dependent proteolytic systems (Fig. 1).
From the perspective of protein synthesis in atrophy, we measured the expression of genes involved in the insulin-like growth factor 1 (IGF-1) signaling pathway, the calcineurin-nuclear factor of activated T cells pathway, and myostatin. Myostatin is a purported negative mediator of muscle mass because myostatin-knockout mice exhibit gross hypertrophy (12).
We were able to show that immobilization produced, on average, a 5% decrease in quadriceps mass that was linked to altered expression of the 20S proteasome and the muscle-specific proteolytic genes, MAFbx, MuRF1, and calpain 3 (Fig. 2). There was little 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, glycogen synthase kinase 3α (GSK3α), which increased. This is perhaps not too surprising, given that it is becoming increasingly recognized that the regulation of the muscle protein synthesis is profoundly influenced by posttranslational modification of anabolic signaling proteins (e.g., protein kinase B (PKB), mammalian target of rapamycin (mTOR), and p70 S6 kinase) rather than transcriptional regulation (3,10). This makes some sense because altering the rate of muscle protein translation via the phosphorylation status of anabolic proteins means that protein synthesis can be up- or down-regulated rapidly depending on the activity, hormonal, and/or nutritional status of an individual. Recent research has demonstrated that the phosphorylation state of the PKB/mTOR/p70 S6 kinase anabolic signaling axis is central to the regulation of protein synthesis in human (3) and animal (10) skeletal muscle. Thus, the increase in muscle protein synthesis that accompanies exercise and/or protein ingestion in vivo has been shown to be directly linked to the magnitude of phosphorylation of the component parts of this pathway. Similarly, insensitivity of muscle protein synthesis to nutrition and exercise in the elderly has been shown to be associated with defects in this pathway, which may be mediated by inflammatory cytokines (3).
GENE EXPRESSION IN HUMAN MUSCLE DURING EXERCISE-INDUCED REHABILITATION AFTER ATROPHY
A major finding from the study of Jones et al. (9) was that a short bout of muscle contraction performed immediately after immobilization rapidly instigated a number of profound changes in gene expression collectively linked to the suppression of muscle catabolism and the instigation of muscle hypertrophy and remodeling. In particular, a decline in MAFbx, MuRF1, and myostatin expression and an increase in the expression of calpain 1, calpain 2, calpastatin, inhibitory κB kinase, and casein kinase 1α were observed within 24 h of this initial single bout of rehabilitation exercise (Fig. 2). This clearly points to a powerful role of exercise in rapidly switching a muscle from a catabolic to anabolic state, particularly in the case of catabolic gene transcription. After 6 wk of prescribed rehabilitation exercise (three times per week, maximal voluntary isokinetic knee extension exercise), muscle mass and gene expression had returned to basal, with the exception of myostatin (down-regulated) and calpain 1 (up-regulated), which were sustained throughout the 6 wk of rehabilitation, suggesting important roles for these genes in long-term exercise-induced remodeling of muscle, additional to their role in the restoration of mass (Fig. 2). This also points to calpain 1 and myostatin as being potential targets for therapeutic intervention.
Tantalizingly, these data indicate that the recruitment of proteolytic systems can be blunted by the introduction of prescribed muscle contraction, which, together with contraction-mediated stimulation of protein synthesis via posttranslational modification of anabolic signaling pathways, will offset muscle atrophy and stimulate anabolism. This points to the introduction of muscle contraction as early as possible after immobilization as being highly desirable or even maintenance of low-grade contraction during immobilization, if possible.
A POTENTIAL ROLE FOR NUTRITIONAL INTERVENTION
Dietary carbohydrate and/or protein intake is known to acutely stimulate net muscle protein synthesis, particularly when combined with exercise training. This effect is achieved by dietary-mediated increases in plasma insulin and amino acid concentrations altering anabolic and catabolic signaling pathways in skeletal muscle. These observations raise the distinct possibility that the targeting of muscle-specific signaling pathways via nutritional and/or exercise interventions may be of significant therapeutic benefit during catabolic conditions and may facilitate recovery during rehabilitation in humans. For example, it is known that physiological concentrations of insulin can almost completely suppress muscle protein degradation in human skeletal muscle (1), at least in the noncatabolic state, which may be achieved by insulin-mediated inhibition of MAFbx and MuRF1 (see below). Furthermore, such insulin concentrations could easily be achieved by dietary carbohydrate ingestion.
Recent animal- and cell-based evidence point to a functional coupling of catabolic and anabolic signaling in muscle via the PKB/mTOR/forkhead transcription factor (FOXO) signaling axis, such that the stimulation of muscle protein synthesis by exercise and/or amino acids and insulin and the concomitant inhibition of protein degradation by FOXO-mediated suppression of MAFbx and MuRF1 occur in a coordinated manner, and vice versa in catabolic states ((7); Fig. 3). By way of example, if PKB activity (phosphorylation) is increased by amino acids or insulin, protein synthesis will be increased via activation of mTOR signaling; but, furthermore, FOXO will become transcriptionally inactive (phosphorylated) and will be unable to bind to the promoter regions of MAFbx and MuRF-1, thereby inhibiting protein degradation. It is tantalizing to hypothesize that this series of events will be reversed in immobilization, resulting in the concomitant up-regulation of muscle protein breakdown via MAFbx and MuRF1 transcription through FOXO and the down-regulation of PKB/mTOR-dependent muscle protein synthesis, resulting in atrophy (Fig. 3). It is not yet known whether this functional coupling of muscle protein synthesis and degradation prevails in human skeletal muscle, but this scenario clearly suggests that the development of nutritional strategies to target the PKB/mTOR/FOXO signaling axis may have therapeutic benefit during immobilization and rehabilitation. As outlined previously, the acute increase in muscle protein synthesis that accompanies exercise and/or protein ingestion in vivo has been shown to be directly linked to the magnitude of phosphorylation of the component parts of this signaling axis. Whether this effect persists over the time frame necessary to attenuate immobilization-induced wasting and/or the restoration of mass during rehabilitation in humans is unknown.
Finally, the ingestion of creatine, which can be consumed in the form of large quantities of meat and fish or more commonly and effectively as a dietary supplement, has been shown to facilitate the restoration of muscle mass and function during rehabilitation after immobilization-induced muscle wasting (6). As creatine ingestion per se has been found to have no effect on muscle protein synthesis in humans, the precise mechanism by which this is achieved is currently unclear. Importantly, the combination of creatine ingestion and resistance training does seem to increase muscle hypertrophy above that seen with training alone, which may be the consequence of individuals being able to perform more work during training as a result of a creatine-mediated maintenance of muscle ATP turnover during fatiguing exercise. From a molecular perspective, increased expression of muscle growth transcription factors and the involvement of the IGF-1 pathway have been implicated with this hypertrophic effect of creatine. Very recent evidence suggests that creatine supplementation augments satellite cell number and myonuclei incorporation in muscle during resistance training (13).
This short review has illustrated that the messenger RNA of the muscle-specific E3 ligases, MAFbx and MuRF1, are up-regulated in human skeletal muscle after immobilization-induced wasting, which confirms published research from animal models, involving severe muscle wasting, indicating that these are regulators of muscle atrophy. Furthermore, the novel finding that the MAFbx and MuRF1 messenger RNA is rapidly down-regulated (within 24 h) after a single bout of rehabilitation exercise in human skeletal muscle points to muscle contraction as being a potential potent therapeutic intervention in a number of clinical conditions. This latter suggestion is bolstered by the finding that the myostatin gene expression, which was unaffected by immobilization, was consistently down-regulated throughout the 6 wk of rehabilitation, confirming myostatin's purported role in inhibiting hypertrophy. This review has also identified a number of responses in the calpain family at a transcriptional level that will be useful in elucidating differential roles for calpains during muscle atrophy and exercise-induced rehabilitation of muscle.
From the perspective of muscle protein synthesis, it seems that altering the rate of muscle protein translation via the phosphorylation status of anabolic proteins means that protein synthesis can be up- or down-regulated rapidly depending on the activity, hormonal, and/or nutritional status of an individual. Recent research has demonstrated that the phosphorylation state of the PKB/mTOR/p70 S6 kinase anabolic signaling axis is central to the regulation of protein synthesis in human and animal skeletal muscle.
Finally, the potential for nutritional interventions, alone or in combination with exercise, to modulate anabolic and catabolic signaling in skeletal muscle is becoming increasingly recognized but requires further research because the extent of direct comparability between cell- and animal-based muscle research and humans is currently unknown. Nevertheless, research of this nature could be of major importance in the development of therapeutic interventions aimed at combating muscle wasting in humans, particularly given that very recent findings point toward a functional coupling of anabolic and catabolic signaling in muscle.
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