and human beings with specific epinephrine-derived compounds (i.e. b2- adrenoceptor agonists) leads to muscle hypertrophy via stimulation of muscle protein synthesis and depression of muscle protein degradation. Moreover, infusions of epinephrine to humans suppresses release of phenylalanine from muscle suggesting an anti-catabolic e ect. Indeed, epinephrine decreases expression of ubiquitin-conjugating enzymes, thus suggesting the mechanism by which epinephrine inhibits degradation in muscle (i.e. via decreasing proteasome-based degradation). This di culty in classification highlights the complexity of assigning single actions to molecules which may act via several distinct mechanisms and may do so with di erent kinetics. Nevertheless, because of its role as a stress hormone, the actions of epinephrine are generally associated with the catabolic response to major illness. Epinephrine has been shown to bind to G-coupled receptors in the plasma membrane. In response to epinephrine binding G-proteins activate adenylate cyclase, which produces cAMP to activate protein kinase A (PKA). PKA is then able to phosphorylate and e ect changes within muscle. One target of PKA is the cAMP response element binding protein (CREB), which stimulates the transcription of target genes. PKA can inhibit myogenic regulatory factors (MRFs) MyoD and Myf5, thus potentially accounting for the belief that epinephrine largely inhibits muscle protein synthesis. cAMP may also activate other transcription factors such as activator protein 2 (AP2) and NFkB, which can have potent catabolic e ect on muscle (see Section 5.2.2.4).

5.2.2Local Factors (Autocrine/Paracrine)

A second class of signals are ‘‘local’’ factors, produced in muscle. Local factors, like hormones, tend to be peptides but unlike hormones have a propensity to act in a paracrine or autocrine manner. However, it remains unclear if some or all of these factors are produced by other local cells (for example nerve and macrophages) to act upon muscle. Moreover, recently some ‘‘muscle’’ growth factors have been shown to be present also in the systemic circulation (for example Nerve Growth Factor (NGF)), suggesting that actions may not be exclusively local. This also supports the notion that contrary to popular belief, muscle is an endocrine organ and, as such, probably the largest in the body.

MechanoGrowth Factor (MGF) is a splice variant of IGF-1. Both MGF and IGF-1 act to promote synthesis and inhibit degradation and can be thought of as anabolic signals akin to the hormone insulin. MGF, as the name implies, is an autocrine signal released in response to mechanical load. Fibroblast growth factor (FGF) appears to be autocrine, and to promote both synthesis and degradation. Presumably the fact that both processes are promoted creates a situation similar to TH. Therefore, FGF may act as a catabolic mediator like TH when degradation is more strongly promoted, a condition shown in C. elegans mutants and possibly accounting for extreme wasting in a case report of a patient with facioscapulohumeral muscular dystrophy. Alternatively FGF may act as an anabolic factor, as the name implies, when synthesis is more

strongly promoted (i.e. similar to the anabolic e ects of epinephrine-derived b2 agonists) and this may account for the notion that FGF largely works on muscle stem cells. Presumably, the fact that both synthesis and degradation are promoted by an autocrine signal creates a situation in which muscle poises itself to respond to other changes in the extramuscular environment via inhibition of either synthesis or degradation, a case of classical negative feedback regulation, which is widespread throughout physiologic systems. Indeed, at least for C. elegans, this is the case and insulin is the negative regulator.

Myostatin is an autocrine member of the Transforming Growth Factor beta (TGFb) family of growth factors. Currently, myostatin is thought predominantly to inhibit protein synthesis. NGF and several other growth factors of the Neurotrophin family are also autocrine signals. NGF, cilliary neurotrophic factor (CNF) and neuregulin all appear to act to promote synthesis and inhibit degradation. Neurotrophins, akin to IGF/MGF, have been shown to be released by muscle in response to contractile activity. Unlike IGF/MGF, neurotrophins have also been shown to be released by neurons, thus raising the possibility that the neurotrophins act similarly to MGF in balancing against other autocrine signals such as FGF but also act distinctly to allow cross-tissue coordination, for example acetylcholine receptor (AChR) clustering between nerves and muscle.

Cytokines act in both an autocrine and a paracrine fashion between muscles but also allow for muscle-macrophage communication. The prevailing view is that tumor necrosis factor alpha (TNFa) inhibits synthesis and promotes degradation, thus acting similarly to catabolic hormones. Likewise, Interferon gamma (IFNg) acts in a catabolic fashion by promoting degradation and also synthesis (e.g. akin to TH).

The remaining extramuscular signals can be broadly thought of as neurotransmitters (i.e. ACh), metabolites (i.e. amino acids), ions (i.e. Ca21) and extracellular matrix proteins. ACh has been shown to inhibit protein degradation in C. elegans and has a well-known myogenic e ect in animals. Ca21 acts to inhibit protein synthesis and increase breakdown but may also do the opposite. Diet-derived amino acids also strongly promote synthesis while inhibiting degradation. Lastly, attachment to the extracellular matrix has been linked to both modulation of synthesis and degradation. The current model is that stronger attachment leads to increased synthesis while weaker attachment leads to increased degradation.

5.2.2.1Mechanisms of Action: Insulin/IGF Spliceoforms

IGF-1 is a hormone similar in molecular structure to insulin. Insulin and IGF-1 are growth factors generated at a distance from muscle (for example IGF-1Ea at the liver and insulin at the pancreas), and within muscle itself, for example MGF.13,14 Insulin deficiency leads to muscle wasting when insulin-dependent diabetes is left untreated or is poorly controlled. Moreover, GH deficiency reduces circulating IGF-1 through its action as a secretagogue, and consequently attenuates both muscle and whole body growth. Insulin suppresses

protein breakdown (i.e. during postprandial periods; see Section 5.3.1) and stimulates protein synthesis, at least in animals. Although using di erent receptors, in general terms these hormones work in the same fashion by binding to insulin or IGF receptors at the plasma membrane. Since these receptors are themselves tyrosine kinases, growth-factor binding stimulates kinase activity, which permits the generation of phosphorylated binding sites on the receptor itself intracellularly. Various proteins are known to bind to these phosphorylated sites; however, there are currently two widely appreciated signaling systems that function downstream of insulin/IGF receptors. Both pathways involve the binding of an adaptor protein to the receptor followed by activation of the pathway. The first pathway utilizes Shc as an adaptor to recruit SOS and then the oncogenic guanosine triphosphate hydrolase (GTPase), Ras. Ras allows dimerization and thus activation of the protein kinase Raf, which subsequently activates other protein kinases such as mitogen-activated protein kinases (MAPKs) via a phosphorylation cascade. The second pathway utilizes insulin receptor substrate (IRS) as an adaptor, which recruits the oncogenic phosphoinositide 3-kinase (PI3K). PI3K similarly acts via phosphorylation cascades, which result in the activation of protein kinase B (Akt) and mammalian target of rapamycin complex 1 (mTORc1) among others (see Section 5.3.1.1 for more detailed discussion). Activation of MAPK downstream of Raf/Akt allows activation of transcription factors, which then translocate to the nucleus and upregulate the synthesis of specific transcripts, including initiation and elongation factors, thereby increasing translational capacity. At the same time activation of Akt allows activation of mTORc1, which activates ribosomal protein S6K1 (P70S6K1) and inhibits 4E-BP1, thereby driving global mRNA translation (i.e. increasing e ciency). Activation of Akt likewise inhibits the transcription factor Forkhead in human rhabdomyosarcoma (FKHR) thereby downregulating the synthesis of specific transcripts, including ubiquitin ligases. With E3 ligase enzymes such as muscle atrophy F-box (MAFbx) and muscle-ring finger 1 (MuRF1) repressed, ubiquitin proteasome based degradation goes down. While not yet demonstrated in humans, it has also recently been shown that mTORc1, which acts downstream of Akt also has the ability to inhibit autophagy, thereby driving down lysosome-based degradation. Thus, insulin/IGF appears able to coordinately regulate both synthesis and degradation and to do so via multiple mechanisms.

5.2.2.2Mechanisms of Action: Fibroblast Growth Factor (FGF)

There are currently no definitive data on FGF being a causative/preventative factor of muscle disease in man, however we include it as it is a growth factor specifically identified for fibroblasts.3 FGF, like insulin, binds to its receptor at the plasma membrane. Like the insulin receptor, the FGF receptor is itself a kinase. FGF has been shown to signal through the Ras kinase cascade to e ect gene transcription in various, non-human, muscles. FGF has also been shown to signal through this pathway in C. elegans to stimulate lysosomal-based degradation. FGF has also been shown to signal via FGFR receptor in rodent

muscle to stimulate both muscle protein synthesis and proteasome-based degradation. While these signals may go through Ras/MAPK, the intracellular pathway(s) remain to be elucidated. Additionally, loss of FGF has been linked to worsening of muscular dystrophies in mice, e ects theorized to be due to decreased muscle stem cells migration.

5.2.2.3Mechanisms of Action: Myostatin

The circulating concentrations of myostatin protein are higher in patients with acquired immunodeficiency (HIV) syndrome than in healthy young men15 and systemic administration of myostatin to rodents causes atrophy. On the other hand myostatin knockout mice are enormously muscular, due to both hypertrophy and hyperplasia.

Myostatin is a peptide hormone of the transforming growth factor beta (TGFb) family. Myostatin binds to activin type II receptors, which recruits a type I TGFb receptor to form a fully active receptor complex. Following activation the transcription factors small and mothers against decapentaplegic homolog 2/3 (Smad2/3) are phosphorylated and are then able to translocate to the nucleus to e ect mRNA transcription. Myostatin induces cachexia by activating the ubiquitin proteolytic system through an NFkB-independent, FKHR-dependent mechanism. The decrease in Akt activity in response to myostatin treatment and resultant nuclear localization of the FKHR transcription factors, which regulate the expression of atrophy-related genes may explain this observation. Furthermore, the downregulation of Akt likely reduces translational e ciency through its diminished capacity to regulate downstream anabolic signaling (discussed in Section 5.3.1.1). As such myostatin is a potent negative regulator of muscle mass.

5.2.2.4Mechanisms of Action: Cytokines

Cytokines have diverse functions in muscle but when present in excess they seem to have catabolic e ects.16 The two main cytokines that have been shown to regulate proteostasis in muscle are TNFa and IL-6. For example, TNFa has also been named cachectin because its administration causes cachexia in rodents. Similarly IL-6 can also induce protein degradation in myotubes in vitro and in muscles in vivo.

TNFa and IL-6 have been shown to be produced and secreted by muscle cells and as such have been termed ‘‘myokines’’. TNFa acts via TNF receptors (p55TNFR and p75TNFR) expressed on the surface of muscle fibers. Upon TNFa binding the receptors act to form signalosomes, which have the net e ect of activating NFkB. One e ect of the activation of NFkB is that it translocates to the nucleus where it can activate muscle gene expression. One set of genes that is upregulated in response to NFkB action are proteasome subunits and ubiquitin ligases, thus increasing the cellular proteolytic capacity. Activation of NFkB is also a major signaling hub for signaling pro-apoptotic events.