Physiological control of muscle mass in humans during resistance exercise, disuse and rehabilitation Andrew J. Murton and Paul L.

Physiological control of muscle mass in humans during resistance exercise, disuse and rehabilitation Andrew J. Murton and Paul L. Greenhaff School of Biomedical Sciences, The University of Nottingham, Queen s Medical Centre, Nottingham, UK Correspondence to Dr Andrew Murton, School of Biomedical Sciences, The University of Nottingham, Queen s Medical Centre, Nottingham, UK Tel: +44 115 823 0129; fax: +44 115 823 0142; e-mail: andrew.murton@nottingham.ac.uk Current Opinion in Clinical Nutrition and Metabolic Care 2010, 13:249 254 Purpose of review The preservation of skeletal muscle mass is central to maintaining mobility and quality of life with aging and also impacts on our capacity to recover from illness. However, our understanding of the processes that regulate muscle mass in humans during exercise, chronic disuse and rehabilitation remains unclear. This brief review aims to highlight some of the more recent and important findings concerning these physiological stimuli. Recent findings Although several studies have detailed the molecular events that occur following an acute bout of resistance exercise, a paucity of data appears to remain concerning the molecular and signaling events that underpin resistance exercise training. Reports of increased transcripts for inflammatory proteins following eccentric but not concentric exercise could represent the stimulus for the instigation of structural adaptations that occur following intense muscle lengthening contractions. Studies investigating processes underlying disuse-induced muscle atrophy provide initial evidence to support the notion that transient increases in muscle protein degradation occur following the onset of muscle disuse in humans. Summary The need for further studies to improve our basic understanding of muscle-associated processes in humans remains, particularly in relation to the temporal changes in muscle processes that occur during resistance training. Keywords muscle disuse atrophy, muscle protein turnover, nutrition, resistance exercise Curr Opin Clin Nutr Metab Care 13:249 254 ß 2010 Wolters Kluwer Health Lippincott Williams & Wilkins 1363-1950 Introduction The maintenance of skeletal muscle mass is largely dependent on the balance between rates of muscle protein synthesis and muscle protein breakdown. It is known that both processes are responsive to food ingestion and changes in muscle contractile activity, including inactivity, but our understanding of the mechanisms regulating muscle protein synthesis and degradation in humans remains unclear. Here, we report on recent developments in the study of processes thought responsible for governing human skeletal muscle protein turnover in response to altered demand. Specifically, the effects of acute and chronic resistance training, inactivity and remobilization will be discussed, with inconsistencies and gaps in our understanding highlighted. Resistance exercise Resistance exercise is well established as an effective strategy for the enhancement of muscle mass, but the adaptation of muscle protein metabolism to acute bouts of resistance exercise and chronic resistance training programs is only starting to become clear. Adaptations to acute bouts of exercise Translation initiation of muscle protein synthesis, ubiquitin proteasome-mediated muscle proteolysis and changes in the expression levels of myostatin (a negative regulator of muscle growth) are areas of current interest in the study of skeletal muscle adaptation to acute resistance exercise. In a recent publication by Deldicque et al. [1], the mrna levels of the ubiquitin ligase MAFbx/ atrogin-1, thought important in the induction of muscle atrophy [2], were elevated in muscle thigh samples immediately following high intensity leg extension exercise, but were subsequently suppressed below basal levels when examined 24 and 72 h later. In light of the elevated muscle protein synthesis known to occur following resistance exercise [3], these findings could be indicative of early protein breakdown to aid muscle-remodeling processes followed by a phase of reduced proteolysis, presumably to support overall muscle hypertrophy. 1363-1950 ß 2010 Wolters Kluwer Health Lippincott Williams & Wilkins DOI:10.1097/MCO.0b013e3283374d19

250 Anabolic and catabolic signals However, although these and other findings highlight the dynamic transcriptional response elicited by exercise, they also illustrate the inherent difficulty of interpreting true physiological meaning from precious snapshots of data, particularly in the absence of target protein expression measurements and determination of muscle protein turnover. Nevertheless, the concomitant reduction in myostatin mrna expression levels following resistance exercise [1,4,5,6,7] provides some level of credence to the suggestion of an elevated hypertrophic response 24 h after exercise, although this has not been universally observed [8]. Intriguingly, it has recently been demonstrated that the mrna expression levels of key proteins thought to underpin ubiqutin proteasome-mediated muscle protein degradation differ between eccentric and concentric muscle contractions performed in the same individual [9]. Specifically, the authors demonstrated increased mrna levels for proteasome subunit a-1, ubiquitin B and ubiquitin C occurred exclusively following eccentric exercise; in contrast, elevations in mrna levels for Foxo1 and an additional ubiquitin ligase linked to muscle atrophy conditions, MuRF1 [2], were limited to concentric exercise. mrna levels of MAFbx/atrogin-1 remained at basal levels following both eccentric and concentric exercise. These observations led the authors to conclude that the disparity between the two modes of exercise was due to the increased drive for muscle remodeling as a consequence of purported myofibrillar damage following eccentric exercise and the increased energy demands of concentric versus eccentric exercise at the same relative workload. In support of the latter suggestion, Foxo1 is known to co-ordinate processes involved in carbohydrate oxidation [10], in addition to acting as a transcription factor to MAFbx/atrogin-1 and MuRF1 [11,12]. The authors argue that the contradiction of the measured changes in MAFbx/atrogin-1 and MuRF1 mrna levels to their perceived role in muscle protein breakdown is indicative of MAFbx/atrogin-1 and MuRF1 being instrumental to muscle atrophy and not muscle proteolysis per se. Indeed, the observation that MAFbx/ atrogin-1 and MuRF1 protein levels do not necessarily reflect limb protein breakdown in humans [13] and the purported regulatory role of MuRF1 in energy metabolism via modulation of creatine kinase activity [14] provides weight to the authors assertions. It is acknowledged that eccentric training, when performed at high intensities, is associated with greater improvements in muscle strength, muscle girth and a trend toward increased muscle cross-sectional area compared with concentric exercise alone [15 ]. A study profiling transcripts significantly modulated by either concentric or eccentric contraction demonstrated increases in transcripts associated with the inflammatory response occurred following eccentric exercise only [16]. A more recent study demonstrated that components of the inflammatory response remained elevated following a subsequent bout of eccentric exercise and observed the increased protein levels of the monocyte chemoattractant protein-1 (MCP1) chemokine, important in the recruitment of macrophages, localized within cells associated with muscle repair [17]. These observations, in part, have led to the concept that inflammation following eccentric exercise may act as a signal to elicit skeletal muscle remodeling, presumably in an attempt to limit future exercise-induced damage. The consumption of protein is firmly established as a stimulator of muscle protein synthesis in healthy young individuals, particularly when combined with resistance exercise [3]. However, the most effective protein composition, dosing strategy and timing of consumption to maximize muscle protein synthesis, remain elusive and are areas of active research. Tang et al. [18 ], investigating the effect of origin of the dietary proteins on mixed muscle protein synthesis (as opposed to whole body protein kinetics) observed whey hydrolysate to stimulate mixed muscle protein synthesis more than casein both at rest and following resistance exercise. In contrast, enhanced mixed muscle protein synthesis following whey ingestion versus soy protein isolate was observed following resistance exercise only. The differences observed between protein sources on mixed muscle protein synthesis occurred despite all three eliciting a similar rise in circulatory essential amino acids, leaving the differences in the rate of digestion between protein sources as a likely candidate for the response described. Separately, the same group demonstrated the greatest increases in myofibrillar protein synthesis to occur following feeding used in conjunction with resistance exercise, as opposed to feeding alone [19 ]. In contrast, sarcoplasmic protein synthesis, though elevated by feeding at 1, 3 and 5 h postingestion, was not further enhanced in participants performing resistance exercise. The authors observation that myofibrillar and sarcoplasmic protein synthesis are similarly and transiently elevated over a 5 h period postfeeding appears at odds with previous reports of reduced sensitivity of sarcoplasmic protein synthesis rates following protein ingestion [20,21]. The authors assert that this may be the consequence of other studies utilizing constant amino acid availability, potentially resulting in a sustained, but nonphysiological hyperaminoacidemia, and subsequently resulting in increased sarcoplasmic protein synthesis. A recent study examining the effect of feeding and acute resistance exercise on muscle signaling changes in trained individuals made several interesting observations [22]. First, resistance exercise resulted in decreased phosphorylation of eukaryotic initiation factor-2be (eif2be), indicative of an increased drive toward preinitiation complex formation. eif2be phosphorylation occurred

Physiological control of muscle mass Murton and Greenhaff 251 independently of feeding status and in the absence of changes in phosphorylation, and presumably activity, of the upstream regulator glycogen synthase kinase-3ß (GSK3ß). Second, the greatest increases in phosphorylation for the kinases involved in promoting ribosomal biosynthesis, p70s6k and S6K, occurred when exercise was performed with the consumption of a mixed meal. Intriguingly, no recorded change in the phosphorylation state of the upstream kinase, mammalian target of rapamycin (mtor), was observed. This could reflect temporal changes in the signaling pathways and the timing of muscle biopsy sampling; conversely, it could represent another example of mtor-independent activation of translation initiation processes [23]. These observations, in conjunction with evidence of exercise-dependent increases in myofibrillar protein synthesis occurring independently of changes in AKT-mTOR signaling [24] and the observed disassociation between anabolic signaling and muscle protein synthesis in response to insulin and amino acid administration [13], strongly suggest a critical reappraisal of the signaling mechanisms that regulate muscle protein synthesis is warranted. Adaptations to resistance exercise training Although resistance exercise training is known to induce skeletal muscle hypertrophy, there is a paucity of studies investigating the temporal changes in muscle protein turnover and muscle molecular events that would accompany adaptations to resistance exercise training. As a consequence, it has become commonplace to extrapolate the data generated from studies of acute resistance exercise to explain the chronic adaptations that occur in response to training. However, no evidence exists to validate this practice and the need for research on the temporal changes to resistance training remains. In an attempt to delineate the response to chronic resistance exercise training, Tang et al. [25] had participants undergo an 8-week-training program that was restricted to one leg only, thereby utilizing the contralateral leg as an untrained control. Immediately following a bout of resistance exercise performed by both legs in the postprandial state, mixed muscle protein synthesis was found to be greater in the trained than untrained limb, but the relationship was reversed when the protein synthesis response was examined over a 28 h postexercise period. With no apparent difference in mixed muscle protein synthesis at rest, these observations suggest that resistance training elicits a temporal shift in mixed muscle protein synthesis response to exercise. Muscle disuse Chronic periods of reduced muscular contraction occur during bedrest, spaceflight or limb immobilization. As a consequence, various models of human muscle disuse have been developed and can be broadly divided into models eliciting regional muscle disuse, typically via the immobilization/suspension of a limb, or models that embody a systemic approach by enforcing a period of bedrest. Regardless of the methodological approach adopted, significant muscle atrophy and reduced muscular strength ensue [26,27,28 33,34 ]. Recent studies have reaffirmed suggestions that a shift from a slow to a fast phenotype occurs in muscle fibers subjected to disuse [35 ], with slow-twitch type-i fibers generally observed to undergo the greatest decline in crosssectional area. Observations of reduced vastus lateralis and gastrocnemius muscle thickness following 5-week bedrest, but no significant change in the size of other muscles examined [32], and with fiber-type specific reduction in cross-sectional area differing between muscles after 60 days of bedrest [36], suggest that the response to disuse varies between muscles and is not necessarily due to the original fiber-type composition. Recent observations of 35 days of bedrest culminating in a decline in myosin concentration of type I and IIa muscle fibers and a purported parallel decline in actin protein [37 ] would suggest the preferential reduction in myofibrillar proteins, probably mainly as a consequence of reduced synthesis. However, this is in contradiction with other reports of unchanged concentrations of myosin, actin and myofibrillar proteins, following bedrest for both 60-day and 90-day periods or after 35 days of limb suspension [38,39 ]. An explanation for the contradictory results is difficult to ascertain due to the methodological variability between studies, most notable of which include the method utilized to induce muscle disuse, the ratio of male and female participants and the period of disuse. However, wherein declines in myosin and actin concentration have been reported, the consistent observation of a co-ordinated reduction in both filamentous proteins, thereby maintaining the ratio of the two contractile proteins, suggests the presence of a tightly regulated system, presumably to retain adequate muscle contractile function despite mass losses [37 ]. The suggestion of sexual dimorphism in the response of muscle protein synthesis to disuse is an interesting concept. Although muscle protein synthesis in the postabsorptive and fed state of middle-aged individuals is known to be equivalent between the sexes [40 ], by old age, anabolic resistance ensues with female muscle protein synthesis appearing particularly refractory to feeding compared with men [41]. Given that muscle immobilization shares similar hallmarks of anabolic resistance [28], it appears judicious to investigate the effect of sex on muscle protein kinetics during disuse. In support of the potential for sexual dimorphism, a recent study of 14-day limb immobilization showed fiber-type

252 Anabolic and catabolic signals specific reductions in cross-sectional area differed between men and women [42 ]. Likewise, differences in the recovery of muscle strength between the sexes following a period of muscle immobilization have previously been reported [43 ]. Over recent years, several studies have reported on the declines in muscle protein synthesis that occur following either limb immobilization [44] or bedrest [45,46]. Despite mixed amino acids being a potent stimulator of muscle protein synthesis under normal conditions, nutritional strategies have largely failed to prevent disuse-induced muscle atrophy [31,47]. These observations corroborate the findings by Glover et al. [28] of anabolic resistance in skeletal muscle following 14 days of limb suspension. In particular, they noted a delayed and overall smaller response of myofibrillar muscle protein synthesis to constant amino acid provision in the immobilized leg versus the nonimmobilized contralateral leg. This was evident at both low and high dose amino acid administration; however, robust changes in the AKT-mTOR signaling pathway largely failed to reflect changes in myofibrillar protein synthesis rates [28]. The presence and significance of enhanced muscle protein degradation during human muscle disuse is debatable [44,48], but several recent observations have helped to give credence toward the argument of increased proteolysis. The report of increased levels of ubiquitinated proteins concomitant to elevated mrna levels of MuRF1 and MAFbx/atrogin-1 following 48 h, but not 14 days, of limb immobilization [42 ], in conjunction with elevated intramuscular interstitial levels of 3-methylhistidine following 72 h limb suspension [49] and unchanged rates of mixed muscle protein breakdown following 21-day bedrest [50], suggests that elevated muscle protein breakdown following disuse may be transient and limited to the first few days following onset. If true, reasons for why protein breakdown rates return to basal levels, whereas muscle mass continues to decline as a consequence of suppressed muscle protein synthesis are unknown, but would represent a natural area for further investigation. Rehabilitation The prescribing of resistance exercise as a therapy for the reversal of injury or illness-induced muscle loss appears particularly effective. The daily use of human centrifugation at speeds sufficient to produce an artificial gravity of 2.5G for 1-h periods prevented declines in muscle torque, fiber cross-sectional area, myosin heavy chain mrna levels [35 ] and maintained mixed muscle protein synthesis during a period of bedrest [50]. Likewise, resistance exercise during 60-day bedrest maintained and elevated the cross-sectional area of the soleus and vastus lateralis leg muscles, respectively [47]. It also prevented changes in type I and IIa fiber diameters, maintained the proportion of hybrid fibers [47] and prevented increases in MuRF1 protein levels [36]. These recent findings highlight the effectiveness of exercise countermeasures to prevent muscle atrophy; indeed, observations of increased calf muscle cross-sectional area compared to baseline in patients 3, 6 and 12 months following 90-day bedrest [51 ] would suggest that the adaptational response of the muscle to remobilization may not be in proportion to the size of the stimulus. Although the effectiveness of muscle contraction as a countermeasure to prevent muscle loss during muscle disuse and to increase muscle mass during rehabilitative therapy is unequivocal, the molecular and signaling mechanisms by which this occurs remain unknown. Conclusion Only by understanding both the physiological response of skeletal muscle to nutrition and exercise, and the associated mechanisms that underpin these processes, can we hope to refine strategies for the prevention of muscle loss as a consequence of disease or disuse. Future work focusing on ratifying the signaling and molecular events that modulate muscle protein synthesis and protein degradation would greatly improve our understanding of the muscle response to changing environmental stimuli. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 346 347). 1 Deldicque L, Atherton P, Patel R, et al. 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The study demonstrates protein ingestion to increase cdk2 mrna expression and prevent postresistance exercise decreases in myostatin and myogenin mrna in human muscle. These observations are consistent with muscle remodeling. 6 Dennis RA, Przybyla B, Gurley C, et al. Aging alters gene expression of growth and remodeling factors in human skeletal muscle both at rest and in response to acute resistance exercise. Physiol Genomics 2008; 32:393 400. 7 Drummond MJ, Fujita S, Abe T, et al. Human muscle gene expression following resistance exercise and blood flow restriction. Med Sci Sports Exerc 2008; 40:691 698. 8 Jensky NE, Sims JK, Rice JC, et al. The influence of eccentric exercise on mrna expression of skeletal muscle regulators. Eur J Appl Physiol 2007; 101:473 480.

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