Regulation of Protein Synthesis in Skeletal Muscle

Ziel:  Zusammenfassung  relevanter  Studienergebnisse  zur  Regulation  der  Proteinsynthese  in  der  Skelettmuskulatur.  Ergebnisse:  Prozesse  der  Proteinsynthese verändern  sich  entsprechend  unterschiedlicher  physiologischen  Belastungen. Muskuläre  Belastung,  Energiebedarf  und  verfügbare  Nährstoffe  beeinflussen die  Proteinsynthese  der  Skelettmuskulatur  durch  die  Regulation  ribosomaler Aktivität. Die tägliche Variation der Skelettmuskelmasse beträgt etwa 0,5% und hängt von der Proteinsyntheserate ab. Die Proteinsynthese ist in der arbeitenden Muskulatur während der Belastung inhibiert und steigt während der Nachbelastungsphase an. Krafttraining steigert speziell die myofibrilläre Proteinsynthese. Im Gegensatz dazu werden mitochondriale Proteine bevorzugt durch Ausdauertraining synthetisiert. Diese Ergebnisse zeigen, dass Prozesse der Proteinsynthese den Umbauprozess  der  Skelettmuskulatur  durch  Training  fördern.  Unterschiedliche Signalwege kontrollieren die ribosomale Aktivität der Muskelzelle in Abhängigkeit von externen Signalen. Die Phosphorylierung von Proteinen durch den mTOR/p70S6K Signalweg und die Modulation des Energiesensors AMPK während muskulärer Arbeit sind wichtige Faktoren für die ribosomale Regulation der Proteinsynthese. Zusätzlich ist auch die transkriptionelle Regulation ein entscheidender Faktor, der die Synthese bestimmter Proteine nach Training reguliert. Schlussfolgerung: Die Proteinsynthese in der Skelettmuskulatur ist ein dynamischer Prozess, der unter anderem durch energetische Faktoren beeinflusst wird. Der Einfluss der Trainingsintensität auf Prozesse des Proteinabbaus sollte in zukünftigen Studien genauer untersucht werden um zu einem tieferen Verständnis der Plastizität der Skelettmuskulatur beizutragen.

Schlüsselwörter: Muskelplastizität, Belastung, Aminosäure, Ribosom, Hypertrophie.

Purpose: Overview of relevant studies of the regulation of protein synthesis in human  skeletal  muscle.  Findings:  Muscle  protein  synthesis  is  altered  in  a  number of physiological situations. Muscle loading, energetic requirements and nutrients exert  a  pronounced  effect  on  protein  synthesis  in  skeletal  muscle  by  regulating ribosomal  activity.  Skeletal  muscle  mass  varies  daily  by  about  0.5%,  depending on  the  rate  of  protein  synthesis.  Protein  synthesis  is  specifically  suppressed  in working muscle but shows a sustained increase post-exercise. This response reflects  the  protein  pool  demonstrating  adaptation  after  the  repeated  impact  of the exercise stimulus. There is a specific increase in myofibrillar protein synthesis with strength training. By contrast mitochondrial proteins are predominantly synthesized after endurance training. The findings support the view that cumulative increases in myocellular protein synthesis allow the gradual remodelling of muscle ultrastructure with training. Distinct signalling pathways emerge which control  ribosomal  activity  in  function  of  environmental  cues.  Phosphorylation of  accessory  factors  by  the  mTOR/p70S6K  pathway  and  its  modulation  by  the intracellular energy sensor, AMPK, during muscle work evolve as core elements of  ribosomal  regulation.  In  humans,  additional  phenomena  such  as  changes  in transcript expression are suggested to specify the spectrum of proteins that are synthesized post-exercise. Conclusion: Protein synthesis is a dynamic read-out of anabolic stimuli to skeletal muscle that is dictated by energetic requirements. The intensity-dependent contribution of protein breakdown remains to be addressed to reinforce the current understanding of the control of muscle plasticity.

Key Words: Muscle plasticity, load, amino acid, ribosome hypertrophy.

Skeletal  muscle  tissue  is  subjected  to  considerable  metabolic demand with physical activity (19, 34). It has been presumed for more  than  a  century  that  such  elevations  in  energy  expenditure  pronouncedly  affect  muscle  mass  and  composition  (41).  The biological processes that govern anabolic and catabolic reactions in  skeletal  muscle  have  been  described  only  more  recently.  The present  data  document  that  skeletal  muscle  demonstrates  important changes in protein synthesis and breakdown in response to changes in demand.
This  article  will  review  the  evidence  for  control  of  muscle protein synthesis with particular regard to its regulation by exercise and amino acids. Due to restrictions in space the reviewed material does not include all relevant literature and may be considered as a selective view of the author. The interested reader is referred to further reviews on the larger topic (27, 43, 52).

The  effects  of  exercise  on  the  muscle  phenotype  (22)  emphasize that mechanical factors exert a main influence on muscle anabolism (Fig. 1). For instance, the systematic increase of muscle loading with resistance type training leads to a significant enlargement of muscle  fibre  cross-section  after  a  few  training  sessions  (45).  The consequent anatomical changes and associated alterations of the muscle-tendon  unit  improve  muscle  strength  (37).  On  the  other hand, exposure to real or simulated microgravity with spaceflight or  immobilisation  pronouncedly  reduces  muscle  mass  within  a dozen days (3). The counter effect of stretch and resistive forms of exercise (7, 38) indicates that primary mechanical strain of skeletal muscle and not gravity governs muscle mass (38).

The time course of adjustments in muscle mass with altered muscle loading illustrates the important regulation of protein synthesis by mechanical cues. Heavy resistance training sessions involving 10 knee extensions at 80% of 1RM over 10 sessions in 3 weeks can  produce  a  net  gain  in  cross  sectional  area  of  vastus  lateralis muscle of approximately 4% (8). Hypertrophy continues to manifest at a similar rate up to 27 sessions when a net gain in protein mass between 0.4- 0.5% per exercise session is calculated (Fig. 2) (46) This hypertrophy is related to a doubling of protein synthesis in the first 4 hours of recovery from resistance exercise. Fractional synthesis rate has reported to increase to 0.1% of total myofibrillar protein per hour after comparable resistance type exercise (54). The response in protein synthesis is maintained with repetition of the resistance stimulus in the trained state. These observations imply that a rapid up-regulation of protein synthesis post exercise contributes to the accretion of muscle mass with regimens increasing muscle loading.
The importance of regulated protein synthesis is supported by studies on muscle atrophy with unloading. In this situation the loss of muscle mass per day (- 0.5%) mirrors the values calculated for resistance training. This is paralleled by a fall in fractional synthesis rate of myofibrillar protein from ~0.45 to 0.2% h-1 (11). Protein degradation probably plays an important part to this muscle remodelling as proteolytic processes are known to be affected by changes in muscle activity. The quantitative (i.e. absolute) contribution of this regulation does not appear to be reported (51).
A main observation regarding muscle’s adjustment to work is that  exercise  also  alters  muscle  composition.  Thereby  important differences  exist  between  the  specific  remodelling  of  the  cellular components  constituting  the  muscle  motor,  myofibrils,  and  the main  powerhouse  of  the  cell,  mitochondria  (26).  Whereas  ‘high load-low  repetition’  types  exercise,  increase  volume  content  of myofibrils,  ‘low  load-high  repetition’  regimens  are  known  to  elevate  mitochondria  content  of  muscle  fibres  (26).  These  opposite adjustments  invoke  the  existence  of  control  mechanisms  which specifically regulate muscle makeup in function of mechanical and energetic demand. A main observation in this regard is that peak mechanical load and not work dictates the response of myofibrillar protein synthesis (8).

Studies  with  amino  acid  tracers  explain  the  effect  of  mechanical and  metabolic  events  resulting  from  neuromuscular  activity  in terms of regulatory influences on protein metabolism (8, 16). This becomes manifest in an increase in mixed muscle protein synthesis (i.e.  the  weighted  average  change  of  all  proteins  in  muscle)  following different forms of exercise (54). This response of muscle protein metabolism can last for 24- 48 hours as shown for resistance type exercise (42, 53). Depending on the training state the adjustments in protein synthesis may concern both the mitochondrial and myofibrillar protein pools of muscle (54). For instance myofibrillar protein synthesis in untrained subjects is elevated after resistance type but not endurance type of exercise. Conversely, synthesis of mitochondrial  proteins  is  seen  after  endurance  type  exercise  in  both untrained and trained subjects. Cumulative effects of this transient elevation in protein synthesis following each bout of exercise seem likely to induce the above-mentioned muscle adaptation with training (17, 26).
In  order  to  understand  the  control  of  protein  synthesis  it  is best to describe it from the angle of the organelle which is responsible for the synthesis of protein molecules: the ribosome. Ribosomes  are  large  protein  factories  which  mediate  the  translation  of genetic  information  encoded  on  messenger  ribonucleic  (mRNA) templates in a corresponding peptide strand (Fig. 3A). Ribosomes are composed of two subunits each of which contains a complex of ribonucleic acids and basic proteins. The smaller subunit binds the mRNA, whereas the larger subunit dynamically incorporates specific transfer RNAs with their bound amino acid. After initial binding to mRNA the ribosome scans the mRNA for triplets of nucleotides that encode amino acids and synthesizes a corresponding peptide strand. This is achieved through joining amino acids to the carboxyl end of the growing chain as they derive from the transfer RNAs. The genetic code is based on 64 combinations of triplets that arise from the four nucleobases adenine (A), guanine (G), uracil (U) or cytosine (C). Translation typically starts at an ‘ATG’ sequence and stops at one of three stop sequences (i.e. UAG, UAA or UGA). The activity of the ribosome is subtly regulated by a number of accessory factors that promote or inhibit translation initiation (Fig. 3B).

While  resistance  exercise  enhances  muscle  protein  synthesis, the net protein balance remains negative (i.e. catabolic) in the absence of food intake (50). In fact, skeletal muscle protein synthesis is regulated by a number of dietary factors, including essential  amino  acids  (31, 40).  Thereby  amino  acids  and  the branched-chain amino acid leucine in particular, constitute the main active ingredient (4, 55). In this regard, it is important to emphasize that the availability of amino acids up-regulates muscle  protein  synthesis  (11).  This  effect  -  for  which  anatomical endpoints are not defined - has been related to the presence of essential amino acids valine, leucine, and phenylalanine but not nonessential amino acids serine, alanine, or proline.
In  consequence  nutritional  measures  are  increasingly  important to avoid a negative nitrogen balance in clinical situation such as inactivity, injury, aging and disease which enhance catabolic drive (2, 6, 10).
Based on culture studies deprivation of even a single essential amino acid is expected to causes a decrease in the synthesis of  essentially  all  cellular  proteins  through  an  inhibition  of  the initiation  phase  of  mRNA  translation  (29).  The  explicit  influence of the lack in a single amino acid for protein synthesis in exercised skeletal muscle has to the best of our knowledge not been assessed. Based on supplementation studies, it is known however, that the ingestion of a high-quality protein meal (25-30g of protein per meal) maximally stimulates muscle protein synthesis in both young and older individuals (27). This points out that amino acid intake and physical activity act synergistically to counteract the decline in muscle protein synthesis with age (36).

In  the  past  15  years  molecular  mechanisms  have  been  identified which explain mechano- and amino acid-regulated protein synthesis in mammalian cells (27). A central theme of these cellular responses is that they integrate extra-cellular stimuli through coupled biochemical  processes  which  perpetuate  the  post-translational modification of protein phosphorylation (16). Regarding the control of ribosomal activity by these signalling cascades a key role has been assigned to phosphorylation of the ribosomal S6 protein and the translational repressor, eukaryotic initiation factor 4E binding protein 1 (4EBP1) (1, 31). S6 phosphorylation is carried out by the 70-kDa ribosomal protein S6 kinase (p70S6K). This modification is particularly important for the translation of mRNAs containing a 5'-terminal oligopyrimidine motif, many of which encode proteins involved  in  mRNA  translation  (29).  By  contrast,  phosphorylation of 4EBP-1 relieves the translation factor eIF4E from inhibition allowing the activation of the ribosome (1). Together, p70S6K and 4EBP1 coordinate the behaviour of both eukaryotic initiation factors and the ribosome (47). For instance the fall in muscle protein synthesis during muscle work is probably been related to reduced phosphorylation of 4EBP1 (31). Accordingly, p70S6K activity is robustly  induced  after  phosphorylation  of  regulatory  sites  Thr389, pS411 and Thr421/Ser424 during the acute response to stressful resistance exercise in men (13, 30) in relation to training volume (49) and gains in muscle mass (48). Thus the measurement of p70S6K phosphorylation allows conclusions about the degree of activation of protein synthesis.
p70S6K  and  4EBP-1  situate  distal  to  the  phosphotransferase  mammalian  target  of  rapamycin  (mTOR)  (1, 31).  This  enzyme is central for the integration of various effects, including those of exercise, hormones and nutritional strategies (14). The activity of mTOR is acutely blunted during exercise by a mechanism that in the  heart  involves  the  sensor  of  energy  charge,  AMP-dependent protein kinase (AMPK) (56). Correspondingly, protein synthesis is inhibited thus enhancing amino acid availability for energy metabolism (14). During recovery from exercise this inhibition is suppressed. In this regard it is important to note that insulin signalling via  AKT  to  mTOR  and  downstream  muscle  protein  synthesis,  as identified in small animals, is not necessarily associated with muscle protein synthesis during feeding, exercise, and immobilization in humans (32). The study of the activation of the mTOR/p70S6K signalling pathway alone provides insight into the specificity and temporal sequence of muscle plasticity in different species.

Regarding  the  regulation  of  muscle  hypertrophy  in  response to resistance training the integrin-associated focal adhesion kinase (FAK) meet the requirements of mechano-dependent ribosome regulation. FAK is part of sarcolemmal focal adhesion complexes (costameres)  involved  in  force  transmission  between  muscle  fibres  and  the  initiation  of  mechanical  signal  transduction  towards activation of the ribosomal p70S6K by muscle loading (23, 30). The connection of FAK to acute regulation of ribosomal activity is established through the FAK-dependent promotion of p70S6K activation after muscle loading (30) and increased FAK activation status after resistance training (54). In this regard it is important to note that the phosphorylation of the downstream target of FAK, c-jun N-terminal kinase ( JNK) (18, 28) in skeletal muscle is quantitatively related to muscle tension (33).

The assessment of the acute effects of exercise reveals important information on the specific control of protein synthesis in skeletal muscle. Firstly, it is striking that protein synthesis is depressed during intense exercise before it rises again during the recovery phase from a workout (31, 42). This possibly reflects the energetic requirements of protein synthesis which are not met during intense muscle work. In rodent muscle this is supported by the implication of AMPK in the inhibition of mTOR and downstream protein synthesis with endurance exercise (1) (Fig. 3B). The degree to which such a mechanism is involved in the modulation of muscle’s response to endurance and resistance type training in non idealized situation in men where type and duration of exercise, and diet are not specifically controlled, is not established.
In this regard it is important to consider that that the spectrum of proteins being synthesized after exercise is refined after a repeated impact with training. For instance, in untrained subjects the synthesis rate of mitochondrial proteins is elevated after both resistance and endurance type exercise (54). After training for resistance  or  endurance,  however,  up-regulated  synthesis  of  mitochondrial  proteins  is  confined  to  endurance  exercise.  A  definite explanation of this observation is not readily available. Probably it is related to increased amounts of mitochondrial gene transcripts after endurance training (44) because these serve as templates for protein  translation.  Gene  profiling  studies  point  out  that  altered transcript  expression  with  endurance  training  on  a  bicycle  involves  the  down-regulation  of  messages  for  the  main  myofibrillar components of fast type muscle fibres (Fig. 4). Transcript profiling also identifies a marked reduction in the expression of mitochondrial factors in quadriceps muscle during recovery from eccentric vs. concentric type bicycle exercise at matched power output (20). The  lengthening  of  contracting  muscle  during  the  eccentric  but not concentric type bicycle exercise implies the important role of mechanical  factors  for  muscle’s  expression  response  to  exercise. This relates to the well understood influence of ‘muscle stretch’ for myofibrillar protein deposition (34). These findings highlight a potentially  important  relation  of  mRNA  expression  and  the  protein synthetic  response  of  exercised  muscle  (5).  The  findings  support the hypothesis that quantitative changes in expressed genes define the set of transcripts that can be translated by the ribosome.
In  line  with  this  suggestion,  exercise-induced  myogenesis  is now implied in the recovery of muscle mass during rehabilitation (32). This possibly reflects the replenishment of synthetic capacity for the production of encoded proteins.

A common observation with endurance type training on a bicycle is the maintained increase in protein turnover of mitochondria despite a flattening of the net response in muscle mass or composition (17). The underlying mechanisms are not understood but likely reflect a concomitant increase in protein synthesis and breakdown in frequently recruited muscle fibres. This possibly reflects wear-and-tear of molecules that necessitates replacement to rebuild destroyed biological structures (9). The liberated amino acid could therefore also serve metabolic purposes such as suspected from the historic experiments of Liebig (14, 41, 42). Balanced increases in anabolic and catabolic processes could explain why muscle mass remains unchanged after endurance training despite the increased  synthesis  of  myofibrillar  and  mitochondrial  proteins. Similar relationships are expected for load bearing exercise from the observation that hypertrophy of muscle fibres with downhill skiing is inversely related to fibre cross sectional area the before the intervention (21).
A  mechanistic  understanding  of  the  underlying  processes  is not available. In this regard, observations on the effects of muscle loading  on  mechano-sensitive  FAK  are  of  interest.  FAK  has  been shown to amplify the effects of muscle loading on gene expression (15). Under normal loading increased amount of FAK has been shown to promote the expression of both, protein translation and degradation factors (15). This role relates to the enhanced presence of FAK with the sarcolemma of muscle fibre types which demonstrate elevated protein turnover and expression of ribosomal subunits due to frequent recruitment for contraction (25, 35). This notion is in line with reduced activation of FAK in the situation of muscle unloading (24). Thus quantitative changes in this upstream signal regulator would offer an explanation for elevated protein turnover with sustained increases in muscle recruitment.

The review of the literature underscores that protein synthesis is pronouncedly regulated by mechanical load, energy charge and supplementation of essential amino acids. Differential sensitivity of the ribosomal  translation  capacity  for  contractile  and  mitochondrial factors allows controlling macroscopic effects by tailored exercise interventions. A number of molecular concepts offer to maximize the therapeutic effects of exercise interventions although relevant aspects of molecular fine tuning are insufficiently understood.

Thanks
The  financial  support  of  the  Région  Rhône-Alpes  (France),  the Swiss National Science Foundation and the EU FP7 grant Myoage (contract No:223576) is acknowledged.