Progressive high-resistance exercise with 8–12 repetitions per set to near failure for beginners and 1–12 repetitions for athletes will increase muscle protein synthesis for up to 72 h; approx. 20 g of protein, especially when ingested directly after exercise, will promote high growth by elevating protein synthesis above breakdown. Muscle growth is regulated by signal transduction pathways that sense and compute local and systemic signals and regulate various cellular functions. The main signalling mechanisms are the phosphorylation of serine, threonine and tyrosine residues by kinases and their dephosphorylation by phosphatases. Muscle growth is stimulated by the mTOR (mammalian target of rapamycin) system, which senses (i) IGF-1 (insulin-like growth factor 1)/MGF (mechano-growth factor)/insulin and/or (ii) mechanical signals, (iii) amino acids and (iv) the energetic state of the muscle, and regulates protein synthesis accordingly. The action of the mTOR system is opposed by myostatin-Smad signalling which inhibits muscle growth via gene transcription.

Introduction

The aim of this chapter is to introduce readers with limited insight into signal transduction to the control of skeletal muscle growth; we have previously covered this topic with an emphasis on human muscle growth [1].

Environmental stimuli, notably exercise and nutrition, have profound effects on muscle mass. Progressive high-resistance exercise is the key exercise form that increases muscle mass [2]. Applying the loading in the range of 60–85% of one repetition maximum, 8–12 repetitions per set to near failure are recommended for beginners and 1–12 repetitions for athletes. The number of sets per muscle group per training session is controversial; there is a ‘one set per muscle is enough’ school of thought but others advocate up to 8 sets per muscle group for trained athletes. A split routine system is often used by athletes; ‘split routine’ means that each muscle group is typically only trained twice per week rather than daily. This is supported by research showing that high-intensity exercise can increase the synthesis of myofibrillar proteins and muscle collagen for at least 72 h after exercise [3]. Thus a daily re-stimulation of protein synthesis might not be needed for high growth effects. Protein breakdown also increases post-exercise and it will exceed protein synthesis in starved individuals. Thus feeding before, during or after exercise is crucial to promote protein accretion and net muscle growth. There is some evidence that protein should be taken directly after exercise if growth stimulation is the key objective because ingestion of the same meal 2 h later will not stimulate muscle growth [4]. About 10 g of essential amino acids (approx. 20 g of protein; a bit more than a pint of milk) stimulates near-maximal muscle protein synthesis in young and old subjects [5]. It is thus prudent to recommend ingesting this amount of protein at the end of resistance exercise to achieve high growth effects.

It is evident from body-building magazines and websites that many ergogenic aids and some bizarre treatments such as the abortion pill RU486 are used by at least some athletes but we are not able to review the safety, effectiveness and mechanism of action of these treatments in the present chapter. Also the available evidence suggests that anabolic steroids are still used to increase muscle mass beyond what is achievable with legal methods (www.steroid.com).

Introduction to muscle-specific signal transduction

In this section we will discuss the regulation of muscle function by signal transduction pathways. During development, mononuclear precursor cells fuse to form muscle fibres that can be up to 20 cm long in humans. Muscle fibres differ from most other cells because they are multinucleated and there are tens of thousands of nuclei in long human fibres. Most nuclei will be post-mitotic (i.e. they do not divide anymore). Thus if fibres grow due to exercise or nutritional stimulation, will nuclear numbers increase and match fibre growth? The answer is yes: nuclear or DNA synthesis is ‘outsourced’ to the so-called satellite cells. Satellite cells are mononucleated cells that respond to growth stimuli and can fuse with muscle fibres to increase fibre numbers in the latter. Satellite cells appear to be necessary for muscle growth because if satellite cells are blocked by mild γ-irradiation then synergist ablation (a radical growth-stimulating intervention) will not cause hypertrophy as it does in control rat muscles [6].

The number of fibres within one muscle differs considerably in the human population. For example, between 393000 and 903000 fibres were counted in the vastus lateralis muscles of nine young subjects [7]. Such differences in fibre numbers can explain the large differences in muscle size seen in untrained adults. Subjects with low fibre numbers will never be able to develop muscles of the size seen in elite body-builders. The number of muscle fibres does not increase significantly in human muscles after resistance training but there is evidence for multiplication of muscle fibres in animal muscles in response to dramatic growth-inducing interventions.

We will now introduce mechanisms of signal transduction in order to prepare readers with a little background in this area for the subsequent discussion of muscle growth-regulating mechanisms. The growth, metabolism, proliferation and most other functions of a muscle fibre depend on signals such as nutrient availability, temperature, pH, light, partial pressure of oxygen, ROS (reactive oxygen species) and mechanical stimuli. In multicellular organisms, nervous and endocrine systems are further inputs. Cells detect this ever-changing mix of signals via specific sensor proteins. Examples of sensor proteins include cell-membrane receptors and amino acid-, calcium- or AMP-sensing proteins. Activation of sensor proteins will trigger signal transduction cascades that make the link between input signals and cellular functions. The main mechanism of signal transduction is the phosphorylation of either a serine, threonine or tyrosine residue of a protein. These three amino acids share a hydroxy group whose phosphorylation will increase the negative charge and induce a conformational change in the protein. This in turn will affect the protein’s activity, interaction with binding partners, localization or will mark it for degradation. It has been estimated that a third of all proteins contain covalently bound phosphate which demonstrates the importance of this signalling mechanism. Enzymes that phosphorylate proteins are termed protein kinases and those that dephosphorylate proteins are termed protein phosphatases. There are genes for 518 protein kinases in the human genome which is probably matched by hundreds of phosphatases. Protein kinases are typically part of a kinase cascade which functions like a ‘fire bucket brigade’: signal→sensor protein→kinase A→kinase B→kinase C→regulatory protein→changed cellular function. This model is oversimplified because many signal transduction pathways converge or branch out, allowing the integration of several inputs which is similar to other information-processing systems such as the brain or computers. The following processes are under the control of signal transduction pathways: (i) transcription of genes into mRNA; (ii) translation of mRNA into protein; (iii) protein modification altering catalytic activity; (iv) regulation of protein breakdown; and (v) regulation of cell division, proliferation and cell fusion. When it comes to adult muscle growth regulation then the mTOR (mammalian target of rapamycin) and myostatin pathways are the most important players; we will now review these pathways.

mTOR system

Over the last decade the mTOR system has emerged as a key regulator of protein synthesis and growth in many types of cell. At its core lies a sort of analogue computer which integrates inputs from several signalling pathways, such as IGF-1 (insulin-like growth factor 1)/MGF (mechano-growth factor)/insulin [8,9], mechanotransduction [10], amino acid sensing [1,11] and energy charge [12]. A diagram of the mTOR signalling system is shown in Figure 1.

Regulation of muscle protein synthesis (6) in response to IGF-1 and insulin (1), energy levels (3), resistance training (4) and amino acids (5) via the mTOR pathway (2)

Figure 1
Regulation of muscle protein synthesis (6) in response to IGF-1 and insulin (1), energy levels (3), resistance training (4) and amino acids (5) via the mTOR pathway (2)

See the main text for a detailed explanation of this Figure. See text for abbreviations.

Figure 1
Regulation of muscle protein synthesis (6) in response to IGF-1 and insulin (1), energy levels (3), resistance training (4) and amino acids (5) via the mTOR pathway (2)

See the main text for a detailed explanation of this Figure. See text for abbreviations.

IGF-1 is a systemic factor that is produced mainly by the liver in response to GH (growth hormone) [13]. Blood levels of GH increase during exercise, as exercise is a powerful stimulator of the hypothalamus, the part of the brain that controls secretion of GH from the pituitary gland. However, resistance exercise induces adaptations primarily in the overloaded muscles and it is unlikely that systemic IGF-1 is of major importance for exercise-induced muscle hypertrophy. IGF-1 and its splice variant MGF (a factor that increases in response to muscle stretch) are expressed in skeletal muscle [8]. Earlier experiments with chronic muscle stretching demonstrated a correlation between IGF-1 expression and hypertrophy of rodent skeletal muscles but an increase of IGF-1 splice variant expression is not always observed in human muscles after resistance exercise [14,15]. Thus it is still unclear whether the increased expression of IGF-1 splice variants is crucial for stimulation of muscle growth after exercise in humans.

IGF-1 can associate with six binding proteins and binds to specific receptors on the muscle membrane [13]. Once IGF-1 has bound to its receptor it will activate a signalling cascade that includes PI3K (phosphoinositide 3-kinase), PKB (protein kinase B), mTOR and p70 S6K (ribosomal S6 kinase) [9]. Activation of this cascade will eventually lead to activation of translation and increased protein synthesis.

We will now present an overview of the mTOR signalling cascade. IGF-1 and insulin binding to their receptors leads to receptor autophosphorylation and conformational changes followed by recruitment and phosphorylation of IRS (insulin receptor substrate) (see 1 in Figure 1). IRS then binds and activates PI3K. PI3K is a powerful stimulator of cell differentiation and growth in many tissues. PI3K forms PIP3 [phosphatidylinositol (3,4,5)-trisphosphate] which binds to the so-called PH (pleckstrin homology) domain of PDK1 {PIP3 [phosphatidylinositol (3,4,5) triphosphate]-dependent protein kinase 1} and PKB (see 2 in Figure 1). PIP3 is a sort of matchmaker which allows phosphorylation of Thr308 by PDK1 after the Ser473 site has been phosphorylated by the mTOR-rictor complex (note that this mTOR complex cannot be inhibited with rapamycin and is different from the mTOR-raptor complex).

There is now a large body of evidence to suggest that PKB activation (note that there are three isoforms of PKB) induces hypertrophy of muscle cells. Experiments with intramuscular injection of an activated PKB DNA construct into regenerating fibres show that muscle fibres taking up this construct undergo hypertrophy [16]. In vitro and in vivo studies demonstrate prior phosphorylation of PKB at Ser473 after high-intensity muscle contraction when protein synthesis increases [17,18]. PKB phosphorylates TSC2 (tuberous sclerosis complex protein 2) at Ser939 and Thr1462 which, via the GTP-binding protein Rheb, leads to an activation of mTOR.

TSC2 can also be phosphorylated on Ser1345 and Tyr1227 (note that these sites are different from the PKB sites) by the AMP and glycogen-dependent AMPK (AMP-activated protein kinase) [12] (see 3 in Figure 1). This input inhibits mTOR and protein synthesis preventing cellular growth when energy turnover is high and/or glycogen is low [1,17].

It appears that mTOR can be activated in response to muscle loading independently of IGF-1 and PI3K (see 4 in Figure 1). A previous study on ex vivo stretched rodent muscle suggests that PLD (phospholipase D) can activate mTOR through generation of phosphatidic acid [9]. Isoforms of PLD (PLD1 and PLD2) are localized to the Z-disc that is a site for mechanical force transmission in skeletal muscles.

In general, mTOR has emerged as a key integrator of signalling in skeletal muscles and other cells. As already mentioned, protein synthesis in skeletal muscles is stimulated by the supply of essential amino acids [5] (see 5 in Figure 1). The exact nature of the nutrient-sensing mechanism is unknown but recent data suggest that a specific PI3K pathway might be involved (IGF-1 and insulin signal via a class 1 PI3K pathway) [11].

Activation of mTOR leads to stimulation of translation via several factors. mTOR phosphorylates 4E-BP1 [eIF-4E (eukaryotic initiation factor 4E)-binding protein] which detaches from eIF-4E resulting in the formation of the 43S pre-initiation complex. The 43S pre-initiation complex then binds mRNA and translation can begin (see 6 in Figure 1). Another important factor is p70 S6K (also known as S6K1) whose phosphorylation increases for a long period after high-intensity muscle contraction [17,19]. Phosphorylation of p70 S6K causes its detachment from eIF3 (note the similarity between 4E-BP1 and p70 S6K in this respect) and this event also promotes translation initiation.

To summarize, the mTOR system is a signalling system that integrates inputs from IGF-1/insulin, mechanical stimulation, amino acids and energy turnover and regulates protein synthesis accordingly.

Myostatin pathway

The accelerator and brake are for the speed of a car what the mTOR and myostatin pathway are for the growth of a muscle: mTOR accelerates growth whereas myostatin brakes it. An overview of myostatin signalling is given in Figure 2. Myostatin was discovered as a new, secreted member of the TGF-Β (transforming growth factor-Β) family by degenerative PCR (a technique by which new members of gene families can be identified). A myostatin knockout construct was then introduced into mice to investigate the function of myostatin [20,21]. The resultant transgenic mice had muscles that were 2–3 times the size of those found in the wild-type due to both hyperplasia and hypertrophy, suggesting that myostatin signalling inhibits these processes. Soon after, extreme muscling in cattle and previously in humans [22] was shown to be due to genetic variations causing a lack of functional myostatin.

Myostatin-Smad signalling in skeletal muscle

Figure 2
Myostatin-Smad signalling in skeletal muscle

See the main text for a detailed explanation of this Figure.

Figure 2
Myostatin-Smad signalling in skeletal muscle

See the main text for a detailed explanation of this Figure.

Myostatin signalling is regulated on many levels before it is produced and binds to its receptor. The transcription of the myostatin gene (see 1 in Figure 2) is activated by glucocorticoids [23] and most studies show a decline of myostatin mRNA or circulating myostatin after resistance exercise [24] but some studies do not [25].

Myostatin is expressed as a precursor protein which is twice cleaved by proteases to first remove the N-terminal signal sequence (which is important for targeting it for secretion out of the muscle fibre) and then a second time to yield an N-terminal fragment (named pro-peptide) and a C-terminal fragment [the ∼12 kDa C-terminal fragment usually occurs as a ∼24 kDa dimer (see 2 in Figure 2); only this dimer is the biologically active myostatin] [20]. Active myostatin binds to a variety of physiological binding proteins which include the aforementioned pro-petide, gasp 1 (growth and differentiation factor-associated serum protein-1), follistatin and FLRG (follistatin-related gene) [20] (see 4 in Figure 2). Binding of myostatin to these proteins will render myostatin inactive. In an elegant two-hybrid screen study (a technique to find novel binding partners for proteins), Titin-cap (or telethonin; see 3 in Figure 2), a Z-disc protein, has been discovered as an intracellular protein that binds myostatin and controls its release [26]. Myostatin activity does thus depend on the presence and availability of binding partners and on myostatin expression. Myostatin is an ideal therapeutic target and monoclonal anti-myostatin antibodies have been developed to this end; these antibodies have been shown to be effective in dystrophic mice [27]; patients with muscular dystrophies are currently recruited for trials with monoclonal anti-myostatin antibodies produced by Wyeth. Myostatin is an ideal therapeutic target for several reasons: myostatin signalling is muscle specific; myostatin is secreted and can be targeted extracellularly; there are physiological inhibitor molecules of myostatin signalling (for example, follistatin); and a lack of myostatin causes hypertrophy in human beings with no apparent side effects [22]. It seems likely that body-builders and other athletes will try to obtain anti-myostatin antibodies and thus doping testers should develop tests for such antibodies sooner rather than later.

Active myostatin binds to TGF-Β IIA and IIB receptors which then recruit TGF-Β type I receptors (also known as activin receptor-like kinases or ALKs; see 5 in Figure 2). The type II receptor then phosphorylates and activates the kinase that is part of type I receptors [20]. The type I receptor then phosphorylates the so-called Smad2 and Smad3 at serine residues [27,28].

Consistent with the role of Smads in the regulation of muscle mass is the finding that overexpression of Ski, a protein that inhibits Smad, causes hypertrophy of fast muscle fibres [29]. Smad phosphorylation causes the dissociation of the Smad from the receptor and it promotes nuclear import by importin-dependent and -independent pathways [28]. Smad2/3 forms complexes with Smad4 and these complexes bind to stretches of DNA which are termed SBE (Smad-binding elements) [28] (see 6 in Figure 2). The muscle growth inhibitory effect of myostatin is presumably transcriptionally regulated but the exact mechanisms are currently unclear. Myostatin inhibits the proliferation and differentiation of mononucleated muscle cells which are precursors of myotubes or muscle fibres [20]. This could, via two mechanisms, affect muscle size, first via the determination of muscle cell/fibre numbers and secondly via the control of satellite cell proliferation. In a different study it was shown that myostatin inhibited myoblast differentiation by down-regulating the muscle development control factor MyoD [20].

Given the dramatic effects of myostatin on muscle mass we find it hard to comprehend that only one study shows an inhibition of protein synthesis [30]. Protein synthesis measurements in muscles of animals and humans with myostatin mutations or myostatin-inhibiting treatments should be made and compared with controls.

To summarize, myostatin is the opponent of mTOR signalling when it comes to control of muscle growth. Myostatin is regulated by its expression and by the presence of various binding partners. Myostatin signals via activin receptors and Smad transcription factors to control the proliferation and differentiation of muscle cells.

What has not been covered

Much that is relevant for muscle growth regulation has not been covered and many references have had to be omitted due to the lack of space. Notably, we have omitted the regulation of protein breakdown by the proteasome, cathepsin and calpain systems; the regulation of these systems is likely to be important for both muscle atrophy and growth situations. We have not covered the links from the forkhead (FKHR, Foxo) and NF-κB (nuclear factor κB) signalling pathways to the protein breakdown systems. Titin kinase has been linked to muscle growth and, together with integrin signalling and other sarcolemma-spanning complexes, these structures are candidates for sensing the as yet unknown mechanical signals during resistance exercise. Calcineurin has been suggested to be a muscle growth-regulating pathway but transgenic mouse muscles suggest otherwise.

Summary

  • Progressive high-resistance exercise with 8–12 repetitions per set to near fatigue for beginners and 1–12 repetitions for athletes followed by ingestion of approx. 20 g of protein directly after exercise are recommended for high muscle growth stimulation; such intervention will increase protein synthesis rather than protein breakdown for up to three days resulting in muscle hypertrophy.

  • Muscle growth is regulated by signal transduction pathways that sense multiple signals and regulate cellular functions such as gene expression, translation and proliferation. The main mechanism of signal transduction is the phosphorylation of serine, threonine and tyrosine residues by kinases and the dephosphorylation by phosphatases.

  • The mTOR system senses (i) IGF-1/MGF/insulin and/or (ii) mechanical signals, (iii) amino acids and (iv) the energetic state of the muscle, and regulates protein synthesis accordingly. Activation of the mTOR system increases protein synthesis and causes hypertrophy.

  • Myostatin-Smad signalling inhibits muscle growth by affecting gene transcription; inhibition of this pathway leads to muscle hypertrophy and is a potential treatment for muscle wasting.

We are funded by the BBSRC (Biotechnology and Biological Sciences Research Council), EPSRC (Engineering and Physical Sciences Research Council), NHS Grampian and the Bone Group, University of Aberdeen, Aberdeen, Scotland, U.K.

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