What is muscle fibre phenotype?

Post-natal muscle is a highly heterogeneous syncytial tissue, comprising muscle fibres and extracellular matrix, with the ability to rapidly undergo biochemical and physical fibre changes in response to appropriate external stimuli, such as nervous and hormonal stimulations, to adapt to the accompanying functional demands imposed on it (Caiozzo, Reference Caiozzo2004). The feature of functional plasticity indicates that skeletal muscle is highly amenable to changes in coordinated gene expression. Although the extracellular matrix of muscle, as exemplified by the orderly arrangement of endomycium, perimycium and epimycium, is a key determinant of meat quality (Fang et al., Reference Fang, Nishimura and Takahashi1999; McCormick, Reference McCormick1999), this review is focussed on the regulation of muscle fibre phenotype. The physical and biochemical characteristics of muscle fibres can be conveniently defined under three distinct but overlapping categories: fibre number, fibre size and fibre type (Figure 1). Fibre types reflect differences in their biochemical (metabolic) and biophysical properties that arise from differences in coordinated expression of muscle gene isoforms (Schiaffino and Reggiani, Reference Schiaffino and Reggiani1996).

Figure 1

Categories of skeletal muscle fibre phenotype.

A feature of muscle fibre phenotype regulation that will be apparent is that the same signalling factor or pathway (such as IGF-1) can often have multiple phenotypic effects with regard to its influence on fibre number, size and type.

Muscle formation and regeneration

In vertebrates, most skeletal muscles are derived from muscle progenitor or precursor cells present in somites, which arise by segmentation of the paraxial mesoderm, located on either side of the notochord and neural tube in the early embryo. As it develops, each somite can be divided into the epaxial dermomyotome, which gives rise to the epaxial muscles, and into the hypaxial dermomyotome, from which derives the rest of the body and limb muscles. The first muscle mass to form, under the dermomyotome, is the myotome, which contributes to the trunk muscles. The early developmental events surrounding myogenesis are under intense molecular scrutiny (Buckingham, Reference Buckingham2001). Myogenic cell fate specification is acquired from the activation of myogenic determination genes, namely the basic helix-loop-helix (bHLH) proteins encoded by Myf-5 and MyoD. The other two members of the myogenic bHLH gene family, myogenin and Mrf4, are implicated, along with MyoD, in the subsequent activation of muscle-specific genes during myogenic differentiation. MEF-2 family, characterised by the presence of a MADS-box motif, also plays an important role in muscle differentiation (Buckingham et al., Reference Buckingham, Bajard, Chang, Daubas, Hadchouel, Meilhac, Montarras, Rocancourt and Relaix2003). Prior to the activation of Myf-5 or MyoD, signallings by Wnts, produced by the dorsal neural tube and surface ectoderm, and hedgehog (Hh) proteins, from the notochord and floor plate of the neural tube, are needed to serve as early triggers or inductive signals of myogenesis (Tajbakhsh and Buckingham, Reference Tajbakhsh and Buckingham2000). Hh signalling plays an additional role in committing progenitor cells to the slow-twitch lineage, a process specified by Blimp-1, originally identified as a factor that promotes B-cell maturation (Baxendale et al., Reference Baxendale, Davison, Muxworthy, Wolff, Ingham and Roy2004).

In limb muscle development, muscle progenitor cells opposite the limb buds must first delaminate from the hypaxial dermomyotome and migrate into the limb region. A number of homeobox proteins have been found to be essential for this migrational process (e.g. Pax3, c-met and its ligand hepatocyte growth factor (HGF)/scatter factor, and Lbx1) (Buckingham et al., Reference Buckingham, Bajard, Chang, Daubas, Hadchouel, Meilhac, Montarras, Rocancourt and Relaix2003). Upon arriving at their destination, they begin to express MyoD and Myf-5 to mark the onset of myogenesis.

After appropriate rounds of cell proliferation, myoblasts leave the cell cycle and fuse to form multinucleated myotubes. The homeobox proteins of Mox2 and Msx1, and several growth factors, including insulin-like growth factor (IGF)-1 and -2 (Morali et al., Reference Morali, Jouneau, McLaughlin, Thiery and Larue2000), fibroblast growth factors (FGFs), bone morphogenic proteins (BMPs) and platelet-derived growth factors, variously contribute to the regulation of proliferation and differentiation (Zorzano et al., Reference Zorzano, Kaliman, Guma and Palacin2003). The first muscle fibres that appear are known as primary fibres (around gestation day 35 in the pig and 12.5 days in the mouse), around which subsequent secondary fast fibres form at the time when innervation begins to be established (beginning at day 50 to day 87 in the pig and day 15 in the mouse) (Picard et al., Reference Picard, Lefaucheur, Berri and Duclos2002; Buckingham et al., Reference Buckingham, Bajard, Chang, Daubas, Hadchouel, Meilhac, Montarras, Rocancourt and Relaix2003; Da Costa et al., Reference Da Costa, McGillivray and Chang2003; Caiozzo, Reference Caiozzo2004).

As post-mitotic cells, skeletal muscle fibres are unable to undergo self-replication. Damaged fibres are replaced with newly synthesised fibres through the proliferation, differentiation and subsequent fusion of muscle satellite cells (myoblasts). Satellite cells constitute a reservoir of undifferentiated muscle precursor cells, located between the basal lamina and the sarcolemma, that are activated in response to muscle injury or growth stimuli to proliferate and ultimately fuse to generate new fibres. Muscle satellite cells are characterised by the expression of myostatin, a transforming growth factor (TGF)-β like factor implicated in limiting muscle growth, c-met, Myf-5 and Pax-7 (Buckingham, Reference Buckingham2001; Buckingham et al., Reference Buckingham, Bajard, Chang, Daubas, Hadchouel, Meilhac, Montarras, Rocancourt and Relaix2003). It appears that not all muscle satellite cells are derived from somitic cells. There exists other population(s) of muscle precursor cells in skeletal muscle, the adult muscle stem cells, with haematopoietic and myogenic potentials (Asakura, Reference Asakura2003; Relaix, Reference Relaix2006). These adult muscle stem cells express haematopoietic markers, such as c-kit, CD45, CD34 and Sca1 (markers that are absent on satellite cells) but not myogenic markers like Pax7 and Myf-5 (present in satellite cells) (Asakura, Reference Asakura2003; Polesskaya et al., Reference Polesskaya, Seale and Rudnicki2003). Recent work suggests that during growth and regeneration, adult stem cells proliferate and undergo phenotypic conversion, which involves Wnt signalling, into myogenic satellite cells (Asakura, Reference Asakura2003; Polesskaya et al., Reference Polesskaya, Seale and Rudnicki2003). It is increasingly apparent that many of the regulatory genes involved in embryonic myogenesis are also required in post-natal muscle fibre formation by satellite cells.

Fibre number: mediators of muscle hyperplasia

It is generally regarded that by birth, an animal, such as the pig, would have nearly the same number of muscle fibres as in adulthood (Wigmore and Stickland, Reference Wigmore and Stickland1983). Therefore, the extent of fibre number formation or hyperplasia during foetal development will have a major bearing on muscle growth potential. In post-natal muscle, fibre number may not necessarily be constant. Periodic repair and replacement of damaged fibres are necessary to maintain functional integrity, a process performed by satellite cells that can proliferate and fuse with damaged fibres or fuse to form new fibres (Goldring et al., Reference Goldring, Partridge and Watt2002). The relative contribution of satellite cells to the formation of new fibres (hyperplastic growth) and to existing fibres (hypertrophic growth) is not clear, but is likely to be dependent on the nature of the inductive signals. Fibre number determination can be affected by any factor that plays a role in embryonic or post-natal myogenesis through myoblast specification, proliferation and/or differentiation. The main signalling factors to be considered that affect fibre number are IGF-1 and -2, myostatin and nutrition.

IGF-1 and IGF-2 activate Erk-MAPK pathway of cell proliferation (hyperplasia)

Growth factors such as IGFs and FGF by virtue of their ability to stimulate cell proliferation are regarded as potent agents that can affect fibre number, pre- and post-natally (Bass et al., Reference Bass, Oldham, Sharma and Kambadur1999). Indeed, daily injections of growth hormone (GH) in the sow during early pregnancy has been shown to enhance foetal fibre number (Rehfeldt et al., Reference Rehfeldt, Kuhn, Vanselow, Fürbass, Fiedler, Nürnberg, Clelland, Stickland and Ender2001), an effect likely to be mediated by IGF-1. IGF-1 and -2 stimulate both muscle cell proliferation and differentiation through the interaction with the type 1 IGF receptor (IGF1R), insulin receptor exon 11- (IR-A) and insulin receptor exon11+ (IR-B), all of which are transmembrane tyrosine kinase receptors (Denley et al., Reference Denley, Cosgrove, Booker, Wallace and Forbes2005). These activated receptors initiate two major signalling cascades (Figures 2 and 3). The type 2 IGF receptor (IGF-2R) (known also as mannose-6-phosphate receptor) and a family of high affinity IGF binding proteins (IGFBPs) 1 to 6 modulate the availability of IGF-1 and IGF2 to bind to receptors. IGF-2R has no intrinsic signalling transduction capability and serves to sequester IGF-2 from potential receptor interactions and to internalise and degrade IGF-2 (Denley et al., Reference Denley, Cosgrove, Booker, Wallace and Forbes2005). Liver is the main endocrine source of IGF-1 (IGF-1Ea), which is induced by GH. Additionally, damaged, stretched or load-bearing muscles are local sources IGF-1 and IGF-2 which induce proliferation and hypertrophy in an autocrine / paracrine manner (Adams, Reference Adams2002). Besides the major IGF-1Ea isoform, a spliced variant with a carboxyl terminus different from IGF-1Ea, named MGF (mechano growth factor), is rapidly inducible in skeletal muscle and appears to be an early trigger for satellite cell proliferation (Yang and Goldspink, Reference Yang and Goldspink2002). The autocrine / paracrine muscle production of IGF-2 during muscle differentiation and regeneration was found to participate in a positive feedback loop to further enhance muscle differentiation (Erbay et al., Reference Erbay, Park, Nuzzi, Schoenherr and Chen2003; Wilson et al., Reference Wilson, Hsieh and Rotwein2003) and regeneration (Kirk et al., Reference Kirk, Oldham, Jeanplong and Bass2003). Although both IGF-1 and -2 are clearly important to muscle growth, their relative contribution to cell proliferation, differentiation and hypertrophy is uncertain.

Figure 2

The IGF-induced Erk-MAPK proliferation signalling pathway of hyperplasia. IGF-1, IGF-2 or insulin binding to tyrosine kinase receptor triggers a phosphorylation cascade that leads to transcriptional and protein modifications, culminating in increase cell proliferation. Integrin can also activate the Erk-MAPK pathway independent of tyrosine kinase receptor binding. Note that activated Ras can also activate the PI3K pathway.

Figure 3

The IGF-induced PI3K-Akt1 signalling pathway of muscle differentiation and hypertrophy. It affects a range of cellular processes, including the promotion of protein synthesis, glycogen synthesis, and cell proliferation and differentiation, that culminate in muscle hypertrophy. mTOR can be activated by amino acids (nutrients) and induce local IGF-2 production which, in turn, acts in a positive feedback manner. The PI3K cascade can be independently activated by exercise and by integrin stimulation (see also integrin stimulation of Erk-MAPK pathway, Figure 2). Activated Akt1 via inhibiting Raf and MEK phosphorylation is involved in inhibitory crosstalk with the Erk-MAPK pathway. Selected abbreviations: PI3K =  phosphatidylinositol 3′-kinase, PDK1 =  phosphatidylinositol-dependent protein kinase 1, GSK3β =  glycogen synthase kinase 3 β, mTOR =  mammalian target of rapamycin, GS =  glycogen synthase, and p70S6K =  ribosomal protein S6 kinase. Note that Akt, GSK3β, mTOR, p70S6k and PHAS-1 are serine/threonine kinases.

IGF-1 (as well as insulin and IGF-2) binding activates the receptor tyrosine kinase (IGF1R or insulin receptor), which subsequently recruits insulin receptor substrate 1 (IRS-1), a non-enzymatic docking protein that propagates the signal to two crucial signalling pathways, the mitogen-activated protein kinase (Erk-MAPK) pathway, via Ras-Raf-MEK-Erk (Rommel et al., Reference Rommel, Clarke, Zimmermann, Nuñez, Rossman, Reid, Moelling, Yancopoulos and Glass1999; Zimmermann and Moelling, Reference Zimmermann and Moelling1999; Glass, Reference Glass2003a) and the phosphatidylinositol 3’-kinase (PI3K)-Akt1 pathway (Glass, Reference Glass2003b; Vollenweider, Reference Vollenweider2003). The Ras-Erk MAP kinase (Erk-MAPK) pathway, a serine/threonine phosphorylation cascade, is responsible for cell proliferation and plays an important role in hyperplastic growth (Figure 2). The PI3K-Akt1 pathway is a major route to muscle differentiation and hypertrophy (see later section). Ras is a membrane bound GTPase, and cycles between an inactive Ras-GDP to an active Ras-GTP. Expression of oncogenic Ras inhibits myogenic differentiation (Mitin et al., Reference Mitin, Kudla, Konieczny and Taparowsky2001) in part by promoting cell proliferation via the MAP kinase pathway. Interestingly, activated Ras was also found to induce the expression of slow myosin heavy chain (MyHC), although the downstream mechanism that leads to this fibre type-specific effect is not understood (Murgia et al., Reference Murgia, Serrano, Calabria, Pallafacchina, Lømo and Schiaffino2000). Both Erk-MAPK and PI3K-Akt1 pathways are necessary and complement each other in mediating post-natal muscle growth (Haddad and Adams, Reference Haddad and Adams2004).

Myostatin inhibits cell proliferation

Myostatin (Mst), also known as growth and differentiation factor 8 (GDF-8), is a secreted negative regulator of muscle mass that belongs to the transforming growth factor (TGF)-β superfamily (Kocamis and Killefer, Reference Kocamis and Killefer2002). Its biological significance is demonstrated in several breeds of double-muscling cattle (e.g. Belgian Blue and Piedmontese) and Mst-null mice as dramatic increase of muscularity through fibre hypertrophy and hyperplasia (Grobet et al., Reference Grobet, Martin, Poncelet, Pirottin, Brouwers, Riquet, Schoeberlein, Dunner, Ménissier, Massabanda, Fries, Hanset and Georges1997; McPherron and Lee, Reference McPherron and Lee1997; Marchitelli et al., Reference Marchitelli, Savarese, Crisa, Nardone, Marsan and Valentini2003). The Belgian Blue carries a naturally occurring homozygous 11-bp deletion in the coding region of Mst whereas the Piedmontese possesses a homozygous G → A point mutation that changes the cysteine residue in Mst to a tyrosine (Kambadur et al., Reference Kambadur, Sharma, Smith and Bass1997). The porcine Mst gene has been molecularly cloned and characterised, but a mutation that confers the enlarged muscle phenotype has not been described (Ji et al., Reference Ji, Losinski, Cornelius, Frank, Willis, Gerrard, Depreux and Spurlock1998). Mst arrests muscle cells in the G₁ and G₂ phases of the cell cycle, through the up-regulation of cyclin-dependent kinase (cdk) inhibitor p21 and down-regulation of cdk-2, thereby inhibiting cell proliferation (Thomas et al., Reference Thomas, Langley, Berry, Sharma, Kirk, Bass and Kambadur2000). This inhibition is mediated, at least in part, through the p38 MAPK stress response pathway (Philip et al., Reference Philip, Lu and Gao2005) (Figure 4). Mst also appears to directly inhibit muscle differentiation by interfering with the activity of MyoD (Langley et al., Reference Langley, Thomas, Bishop, Sharma, Gilmour and Kambadur2002). Muscle wasting conditions, such through disease or disuse atrophy, are associated with elevated Mst in affected muscles (McCroskery et al., Reference McCroskery, Thomas, Maxwell, Sharma and Kambadur2003). A primary effect of Mst inactivation in post-natal growth is an increase of satellite cell proliferation and differentiation (McCroskery et al., Reference McCroskery, Thomas, Maxwell, Sharma and Kambadur2003). GH, mediated through IGF-1, was shown to inhibit Mst expression in skeletal muscles and C2C12 myotubes (Liu et al., Reference Liu, Thomas, Asa, Gonzalez-Cadavid, Bhasin and Ezzat2003). Conversely, Mst expression in porcine embryonic myogenic cells up-regulates the production of IGF binding protein-3 (IGFBP-3), which reduces the activities of IGF-1 and -2 (Kamanga-Sollo et al., Reference Kamanga-Sollo, Pampusch, White and Dayton2003). These results suggest that the Mst and IGF signalling pathways are closely connected in an antagonistic manner.

Figure 4

The p38 MAPK stress response pathway of cell arrest and atrophy. It is activated by a variety of stimuli, including exercise and activated Ras, and through different receptor families. It mediates a range of effects: cell arrest, differentiation, atrophy as well as mitochondrial biogenesis. Additionally, TGFβ receptors readily signal through Smad proteins which also lead to cell arrest. Ski functions to inhibit Smad signalling thus relieving cell arrest and promoting proliferation. Selected abbreviations: IL-1 =  interleukin 1, TLRs =  toll-like receptors, TNF α =  tumour necrosis factor α, and TAK =  TGFβ-activated kinase.

Recent micro-array investigations further found that Mst-null mice exhibited raised Wnt4 expression which stimulated satellite cell proliferation (Steelman et al., Reference Steelman, Recknor, Nettleton and Reecy2006). The use of both micro-array and proteomic analyses showed a clear fibre phenotype switch in Mst-null muscles from slow to fast-twitch fibres (Bouley et al., Reference Bouley, Meunier, Chambon, De Smet, Hocquette and Picard2006; Steelman et al., Reference Steelman, Recknor, Nettleton and Reecy2006) (see section IGF-1, β₂-agonist and myostatin-null signal fast fibre phenotype). Mst has been recently found to promote adipogenesis in C3H 10T(1/2) cells which appears to be associated with adipocyte lineage commitment (Artaza et al., Reference Artaza, Bhasin, Magee, Reisz-Porszasz, Shen, Groome, Fareez and Gonzalez-Cadavid2005). However, in committed bovine preadipocytes, Mst clearly suppresses differentiation of preadipocytes into adipocytes (Hirai et al., Reference Hirai, Matsumoto, Hino, Kawachi, Matsui and Yano2007).

Fibre size: mediators of muscle hypertrophy/atrophy

Post-natal muscle growth is primarily a function of enlargement and elongation of existing fibres as a result of net protein synthesis, known as muscle hypertrophy (Figure 1) (Glass, Reference Glass2003b). Conversely, loss of muscle mass as a consequence of disease or muscle inactivity is primarily due to a reduction in fibre size, described as muscle atrophy (Glass, Reference Glass2003b). Muscle differentiation is a prerequisite to hypertrophy and, as detailed below, the same factors are often involved in both processes (differentiation and hypertrophy). In this section, we consider some well known signalling factors and pathways that mediate muscle hypertrophy or atrophy (IGF-PI3K pathway, contractile activity, p38 MAP kinase pathway, synthetic β₂ adrenergic agonists, anabolic steroids and ski proto-oncogene.

Ski

Ski is a nuclear proto-oncogene that is essential for embryonic development. It is expressed in most post-natal tissues. Like IGF-1, it exhibits dual functions in promoting cell proliferation and muscle differentiation. In addition to transforming chicken embryo fibroblasts, Ski can induce cells derived from quail embryonic body wall to undergo myogenesis. Lines of transgenic mice carrying chicken Ski cDNAs under the control of murine sarcoma virus (MSV) long terminal repeat (LTR) preferentially express high levels of Ski mRNA and protein in skeletal muscle, even though MSV-LTR is usually active in other tissues (Sutrave et al., Reference Sutrave, Kelly and Hughes1990 and Reference Sutrave, Leferovich, Kelly and Hughes2000). This near exclusive skeletal muscle expression of Ski is associated with hypertrophy of fast glycolytic 2b fibres, without increase in fibre number or nuclear number (Sutrave et al., Reference Sutrave, Kelly and Hughes1990). The hypertrophic effect of Ski on 2b fibres is associated with reduced protein degradation rates (Costelli et al., Reference Costelli, Carbo, Busquets, Lopez-Soriano, Baccino and Argiles2003). It could be that Ski over-expression is deleterious to cells, which would account for its restricted distribution of expression in MSV-LTR-Ski transgenic mice, and an absence of expression in skeletal-α-actin promoter-driven Ski transgenic mice (Sutrave et al., Reference Sutrave, Leferovich, Kelly and Hughes2000). Indeed, satellite cells isolated from muscles of MSV-LTR-Ski transgenic mice showed accelerated deterioration in termination differentiation with increasing age (Charge et al., Reference Charge, Brack and Hughes2002).

Unlike other oncogenes, the over-expression of the wild type c-Ski is sufficient to cause transformation (Prunier et al., Reference Prunier, Pessah, Ferrand, Seo, Howe and Atfi2003). The oncoproteins from Ski and the related SnoN (ski-related novel gene) are able to interact with a variety of transcription regulatory complexes, including histone deacetylase complexes (HDACs) and tumour suppressors (Ueki and Hayman, Reference Ueki and Hayman2003). The TGF-β signalling pathway has been identified as a key interacting site of Ski. The regulation of cell growth and differentiation by TGF-β is mediated by the Smad proteins, which are important tumour suppressors. TGF-β signalling is initiated when the bound ligand induces the formation of a heteromeric complex comprising type I and type II serine/threonine receptors. Type II receptor transphosphorylates type I receptor, which in turn phosphorylates Smad2 and Smad3. Activated Smad2 and Smad3 form heterodimers with Smad4 and translocate into the nucleus where they interact with a host of complexes to bring about transcriptional activation or repression of specific genes (Figure 4). Ski was recently found to directly interact with Smad2, Smad3 and Smad4, and to block the phosphorylation of Smad2 and Smad3 by activated TGF-β type I receptor (Prunier et al., Reference Prunier, Pessah, Ferrand, Seo, Howe and Atfi2003; Ueki and Hayman, Reference Ueki and Hayman2003). Therefore, a mechanism of the transforming ability of Ski (and SnoN) is the repression of Smad function, whose inactivation prevents TGF-β-induced cell cycle arrest. The up-regulation of Ski in proliferating satellite cells points to its possible role in mediating cell proliferation in the regeneration of damaged fibres (Soeta et al., Reference Soeta, Suzuki, Suzuki, Naito, Tachi and Tojo2001). The mechanisms behind the effects of Ski on muscle differentiation and hypertrophy are unknown. One speculation is that Ski inhibits the signalling of myostatin, a member of the TGF-β superfamily, thereby enhancing cell proliferation and differentiation (Costelli et al., Reference Costelli, Carbo, Busquets, Lopez-Soriano, Baccino and Argiles2003) (Figure 4). Ski might be developed as a candidate marker for hypertrophic growth in marker-assisted selection.

Fibre types: coordinated isoform-specific expression

The plasticity of muscle fibres is not confined to its ability to undergo changes in fibre size. Physiological and biochemical properties can show wide variations between individual fibres, and such variations are further subjected to modulations by external stimuli. Traditionally, classification of muscle fibre types is based on differences in a number of biochemical parameters between fibres (Gil et al., Reference Gil, Lopez-Albors, Vazquez, Latorre, Ramirez-Zarzosa and Moreno2001; Zierath and Hawley, Reference Zierath and Hawley2006). Succinate dehydrogenase (SDH) histochemical staining, for example, is able to differentiate fibres into two or three different types, based on the relative amount of the enzyme present in each fibre. As SDH is an integral component of the citric acid cycle, strongly positive fibres are classified as oxidative fibres. Another commonly cited histochemical staining method depends on the overall myosin adenosine triphosphatase (ATPase) activity in each fibre (Brooke and Kaiser, Reference Brooke and Kaiser1970; Bancroft and Gamble, Reference Bancroft and Gamble2002). Myosin ATPase activity originates from myosin heavy chain (MyHC), the principal sacromeric protein component of the thick myofilament. Differential myosin ATPase staining is due to differences in susceptibility to pH between different MyHC isoforms. Since myosin ATPase is highly sensitive to pH change, this staining method is intrinsically prone to variability in results. Histochemical methods, such as SDH and myosin ATPase stainings, are invaluable in describing the biochemical profile of individual fibres. However, they are less reliable in the objective determination of fibre types. Different histochemical stains often provide slightly different classification outcome of individual fibres, which in the past had made findings of association studies between fibre types and meat quality traits variable and even contradictory (Essén-Gustavsson, Reference Essén-Gustavsson, Puolanne and Demeyer1993; Klont et al., Reference Klont, Brocks and Eikelenboom1998; Lefaucheur et al., Reference Lefaucheur, Milan, Eolan and Le Callennec2004).

Definition of fibre types. A recent major advance in farm animal muscle research has been the development of an objective approach to muscle fibre typing based on the identity of the primary MyHC isoform expressed in each fibre (Chang et al., Reference Chang, Fernandes and Goldspink1993 and Reference Chang, Fernandes and Dauncey1995; Chang and Fernandes, Reference Chang and Fernandes1997). MyHCs are encoded by a highly conserved multigene family, of which eight isoforms are known in mammals (2a, 2x, 2b, embryonic, perinatal, slow/β, extraocular and α), each with its own myosin ATPase activity and each encoded by a distinct gene (Weiss and Leinwand, Reference Weiss and Leinwand1996). In pre-natal mammalian muscles, the embryonic, perinatal and slow/β/type I MyHC isoforms represent the three dominant skeletal muscle fibre types in the developing foetus. Shortly after birth, the post-natal MyHC isoforms (2a, 2x and 2b) replace the expression of embryonic and perinatal MyHC genes. Thus, in post-natal muscles of pigs, dogs and rodents, there are four major fibre types (Figure 5) characterised by the expression of the slow/β/type I, 2a, 2x and 2b MyHC gene isoforms (Schiaffino and Reggiani, Reference Schiaffino and Reggiani1996; Wu et al., Reference Wu, Crumley and Caiozzo2000b). In cattle and horses, MyHC 2b fibres are effectively absent (Chikuni et al., Reference Chikuni, Muroya and Nakajima2004; Maccatrozzo et al., Reference Maccatrozzo, Patruno, Toniolo, Reggiani and Mascarello2004). Based on the MyHC approach, post-natal muscle fibres in animals can be resolved by immunocytochemistry or in situ hybridisation into three or four major types, depending on animal species. Metabolic, biochemical and biophysical characteristics, such as oxidative and glycolytic capacities, fibre size, colour, and glycogen and lipid contents, have been found to vary between MyHC fibre types (Schiaffino and Reggiani, Reference Schiaffino and Reggiani1996; Klont et al., Reference Klont, Brocks and Eikelenboom1998; Karlsson et al., Reference Karlsson, Klont and Fernandez1999) (Table 1). The slow/β and fast 2b fibres, also known as slow oxidative (red) and fast glycolytic (white) respectively, represent two extreme metabolic profiles. Slow MyHC fibres are characterised by slow isoform contractile proteins, high levels of myoglobin, high volumes of mitochondria, high oxidative capacity, high lipid contents and high capillary density. Favourable meat traits such as colour and, in the pig in particular, tenderness have been found to closely associate with the greater abundance of red or highly oxidative fibres (Klont et al., Reference Klont, Brocks and Eikelenboom1998; Karlsson et al., Reference Karlsson, Klont and Fernandez1999; Chang et al., Reference Chang, Da Costa, Blackley, Southwood, Evans, Plastow, Wood and Richardson2003; Maltin et al., Reference Maltin, Balcerzak, Tilley and Delday2003; Wood et al., Reference Wood, Nute, Richardson, Whittington, Southwood, Plastow, Mansbridge, da Costa and Chang2004). There is a general perception that leaner meat, especially pork, contains reduced intramuscular fat, resulting in increased toughness and reduced succulence (Dunshea et al., Reference Dunshea, D'Souza, Pethick, Harper and Warner2005). Red muscles possess higher lipid concentration (intra- and inter-fibre fat) which is associated with more tender / juicy meat (Hocquette et al., Reference Hocquette, Ortigues-Marty, Pethick, Herpin and Fernandez1998;Wood et al., Reference Wood, Enser, Fisher, Nute, Richardson and Sheard1999 and Reference Wood, Richardson, Nute, Fisher, Campo, Kasapidou, Sheard and Enser2003).

Figure 5

Typing of porcine muscle fibres. Serial sections of the psoas of a 22-week-old pig to illustrate the combined use of histochemistry, immunocytochemistry and in situ hybridisation to identify the four major post-natal MyHC fibre types. Succinate dehydrogenase (SDH) staining (a), sudan black staining (b), NOQ7.5.4D (slow) monoclonal antibody binding (c), and in situ hybridisations with MyHC isoform-specific fast 2a probe (d), fast 2b (e) and fast 2x probe (f) were performed. SDH and Sudan Black positive fibres closely mirrored each other and corresponded mainly to slow and fast 2a fibres. Note the presence of inter-fibre adipose tissue on panel b.

Table 1

Metabolic profiles of MyHC fibre types

† Pale, soft exudative (PSE) meat quality, independent of ryanodine receptor mutation. The abundance of MyHC 2b fibres in a normal pig is a contributory factor to PSE. ( ) indicates possibly variable levels or data not fully established in the pig.

By contrast, fast MyHC 2b fibres are the largest of the four fibre types with fast isoform contractile proteins, low amounts of myoglobin and mitochondria, high glycolytic capacity (high glycogen store), low lipid contents and low capillary density. The fast MyHC 2a and 2x fibres are intermediate fast oxidative-glycolytic fibres. Fast 2a fibres are more closely related to slow/I fibres, and fast 2x are more similar to fast 2b fibres (Table 1). Fast glycolytic fibres, in particular 2b fibres, are major contributors of hypertrophic growth and of rapid fall in muscle pH post mortem, associated with the formation of PSE pork. Hence in the modern pig, hypertrophic growth potential (meat quantity) from an abundance of MyHC2b and 2x fibres comes at a cost to meat quality (Chang et al., Reference Chang, Da Costa, Blackley, Southwood, Evans, Plastow, Wood and Richardson2003). On the other hand, the absence of MyHC 2b fibres in cattle is a likely explanation for the lack of a PSE problem in beef. Some beef studies have linked an abundance of fast-twitch fibres (defined by histochemical detection) to improved tenderness. The interpretation of fast-twitch fibres should be made with care as it is presently clear that in the absence of MyHC 2b fibres there are strictly speaking no fast-glycolytic fibres in bovine muscle (Geay et al., Reference Geay, Bauchart, Hocquette and Culioli2001; Maltin et al., Reference Maltin, Balcerzak, Tilley and Delday2003). Consequently, in a technical sense, bovine muscle may have higher oxidative potential than its porcine counterpart.

In addition to temporal regulation, each MyHC isoform is subjected to specific spatial control, such that the number and distribution of fibres expressing each isoform often vary between anatomical muscles, e.g. soleus and longissimus thoracis et lumborum (also referred by some as longissimus dorsi). Fibre type composition varies between muscles according to their functional adaptation. Postural muscles are under continual use and comprise a high proportion of oxidative fibres. Muscles that are periodically used for intensive activities like sprinting possess large numbers of fast fibres. A further degree of fibre type heterogeneity is the presence of a small number of hybrid MyHC fibres, usually presented as a mixture of two MyHC isoforms (slow/2a, 2a/2x, or 2x/2b) (Sant'ana Pereira et al., Reference Sant'ana Pereira, Wessels, Nijtmans, Moorman and Sargeant1995; Pette and Staron, Reference Pette and Staron2000). Thus fibre population in muscle is a continuum of pure and hybrid fibres that can be altered in the fast-to-slow or slow-to-fast direction under appropriate stimulatory conditions (Schiaffino and Reggiani, Reference Schiaffino and Reggiani1994).

Distinctive biochemical and biophysical differences between fibre types point to a coordinated programme of fibre-type or isoform specific expression (Hallauer and Hastings, Reference Hallauer and Hastings2002). However, compared with our knowledge of muscle hypertrophy, much less is known about the molecular mediators of fibre type specific expression. Coordinated fibre type specific expression requires the orchestrated regulation of a large number of gene isoforms consistent with the fibre phenotype. Gene family and differential splicing are features of muscle genes (Schiaffino and Reggiani, Reference Schiaffino and Reggiani1996). Understanding the complexities of fibre type specific expression, requires insights into the signalling pathways that coordinate the temporal and spatial distribution of expression of subsets of muscle gene isoforms. The emerging picture of fibre type specific regulation is that it is governed by multiple signalling pathways and factors rather than a single pathway (Spangenburg and Booth, Reference Spangenburg and Booth2003). These pathways and factors almost inevitably have additional cellular functions, such as proliferation, differentiation and hypertrophy, in addition to modulating fibre type specific expression.

IGF-1, β₂-agonists and myostatin-null signal fast fibre phenotype

As highlighted earlier, the major phenotypic effects of IGF-1 and -2 are cell proliferation, muscle differentiation and hypertrophy. Such properties of IGF-1 have been shown to be beneficial to ageing, atrophic, and dystrophic muscles (Barton-Davis et al., Reference Barton-Davis, Shoturma, Musaro, Rosenthal and Sweeney1998; Lynch et al., Reference Lynch, Cuffe, Plant and Gregorevic2001; Musarò et al., Reference Musarò, McCullagh, Paul, Houghton, Dobrowolny, Molinaro, Barton, Sweeney and Rosenthal2001). IGF-1 has the additional effect of converting fibres to a fast glycolytic phenotype, as evident by raised expression of glycolytic enzymes in IGF-1-transfected C2C12 myotubes (Semsarian et al., Reference Semsarian, Wu, Ju, Marcinec, Yeoh, Allen, Harvey and Graham1999), by modest rise in fast 2b fibres in transgenic mice carrying muscle IGF-1 isoform driven by a rat myosin light chain (MLC)-1/3 promoter (Musarò et al., Reference Musarò, McCullagh, Paul, Houghton, Dobrowolny, Molinaro, Barton, Sweeney and Rosenthal2001) and by raised type 2a and 2b fibres at the expense of slow fibres in IGF-1-treated dystrophic mice (Lynch et al., Reference Lynch, Cuffe, Plant and Gregorevic2001). As previously indicated, clenbuterol (a β₂-agonist) treated rats (Ryall et al., Reference Ryall, Gregorevic, Plant, Sillence and Lynch2002) as well as myostatin-null mice (Steelman et al., Reference Steelman, Recknor, Nettleton and Reecy2006) not only showed muscle hypertrophy but also slow-to-fast fibre conversion. The mechanism responsible for the slow-to-fast fibre type switch, however, remains elusive. In general, in the pig and ruminants, it appears that growth hormone (indirectly IGF-1) and β-agonists may negatively affect meat quality, as shown by increased shear force or sensory perception (Dunshea et al., Reference Dunshea, D'Souza, Pethick, Harper and Warner2005). Such effects may be connected to increased fast glycolytic (MyHC 2b or 2x) fibres but because fibre typing in meat quality association studies had been almost exclusively based on histochemical methods, the changes in MyHC 2b/2x fibres in the pig or MyHC 2x fibres in cattle have not been determined.

Calcineurin signals oxidative fibre type

Calcineurin (protein phosphatase 2B/PP2B) is an enzyme complex that comprises calcineurin A (CnA) catalytic subunit, calcineurin B (CnB) regulatory subunit and calcium-binding protein calmodulin (Schulz and Yutzey, Reference Schulz and Yutzey2004). It is a calcium dependent serine-threonine phosphatase that is widely distributed throughout the body. Calcineurin has been implicated in a wide variety of biological processes, including T-lymphocyte activation, vascular, neuronal and cardiac development and growth, and, more recently, skeletal muscle development (Crabtree, Reference Crabtree2001; Bueno et al., Reference Bueno, Van Rooij, Molkentin, Doevendans and De Windt2002; Horsley and Pavlath, Reference Horsley and Pavlath2002). In cardiac muscle, calcineurin signalling is necessary for cardiomyocyte maturation, heart chamber formation and cardiac hypertrophy (Schulz and Yutzey, Reference Schulz and Yutzey2004). In skeletal muscle, calcineurin appears to be needed in a number of key developmental processes, namely enhanced muscle cell differentiation, and in the fibre type context, conversion to slow (oxidative) muscle phenotype (Musarò et al., Reference Musarò, McCullagh, Naya, Olson and Rosenthal1999; Semsarian et al., Reference Semsarian, Wu, Ju, Marcinec, Yeoh, Allen, Harvey and Graham1999; Bigard et al., Reference Bigard, Sanchez, Zoll, Mateo, Rousseau, Veksler and Ventura-Clapier2000; Delling et al., Reference Delling, Tureckova, Lim, De Windt, Rotwein and Molkentin2000). It has been reported that activated calcineurin mediates the hypertrophic effect of IGF-1 (Musarò et al., Reference Musarò, McCullagh, Naya, Olson and Rosenthal1999; Semsarian et al., Reference Semsarian, Wu, Ju, Marcinec, Yeoh, Allen, Harvey and Graham1999). However, there is compelling evidence, including transgenic and knock-out data, to show that calcineurin has no effect on muscle hypertrophy but that the hypertrophic effect of IGF-1 is mediated by the PI3K pathway as detailed earlier (Naya et al., Reference Naya, Mercer, Shelton, Richardson, Williams and Olson2000; Bodine et al., Reference Bodine, Stitt, Gonzalez, Kline, Stover, Bauerlein, Zlotchenko, Scrimgeour, Lawrence, Glass and Yancopoulos2001b; Rommel et al., Reference Rommel, Bodine, Clarke, Rossman, Nunez, Stitt, Yancopoulos and Glass2001; Pallafacchina et al., Reference Pallafacchina, Calabria, Serrano, Kalhovde and Schiaffino2002). Calcineurin is activated by raised intracellular calcium, triggered by extracellular signals like nervous impulses, or hormonal input, such as IGF-1 stimulation. To date, few substrates, namely NFATs, MEF2s and PGC-1α, of calcineurin action in skeletal muscle are known.

NFATs. The best characterised calcineurin substrates are members of the phosphorylated NFAT (nuclear factor of activated T-cells) family of transcription factors. Five NFAT genes, each with a distinct cellular role, have been identified: NFATc1 (NFAT2/NFATc), NFATc2 (NFATp/NFAT1), NFATc3 (NFAT4/NFATx), NFATc4 (NFAT3), and NFAT5 (Delling et al., Reference Delling, Tureckova, Lim, De Windt, Rotwein and Molkentin2000; Horsley and Pavlath, Reference Horsley and Pavlath2002). Several NFAT isoforms are expressed in skeletal muscle, each of which undergoes activation at specific stages of myogenesis. For instance, NFATc2 is activated only in new myotubes and plays a crucial role via IL-4 in mediating myoblast fusion (Horsley et al., Reference Horsley, Jansen, Mills and Pavlath2003). Dephosphorylation of NFATs unmasks their nuclear localisation signal, resulting in nuclear translocation, where they bind to NFAT-binding sites on their own or as co-operative complexes with other factors, such as calcineurin, AP1, MEF2 and GATA2/4, to activate gene transcription (Musarò et al., Reference Musarò, McCullagh, Naya, Olson and Rosenthal1999; Sugiura et al., Reference Sugiura, Sio, Shuntoh and Kuno2001; Schulz and Yutzey, Reference Schulz and Yutzey2004). Cultured muscle fibres showed NFAT nuclear translocation only when electrically stimulated at a slow muscle pattern, which results in high intracellular calcium (100 to 300 nM) but not at a fast muscle pattern that results in low intracellular calcium ( < 50 nM) (Liu et al., Reference Liu, Cseresnyés, Randall and Schneider2001; Fraysse et al., Reference Fraysse, Desaphy, Pierno, De Luca, Liantonio, Mitolo and Camerino2003). Nuclear NFATs are deactivated and re-located back to the cytoplasm through phosphorylation by several protein kinases, including GSK-3β (Figure 3), protein kinase A, p38 MAP kinase and casein kinase (Schulz and Yutzey, Reference Schulz and Yutzey2004). In T-cells, simultaneous activation of AP-1 (Jun/Fos) and NFAT is absolutely essential for cytokine transcriptional induction (Rao et al., Reference Rao, Luo and Hogan1997). In skeletal muscle, however, only GATA2 has been shown to be a co-operative binding partner of NFATs (Paul and Rosenthal, Reference Paul and Rosenthal2002).

MEF2s. Members of the MEF2 (myocyte enhancer-binding factor 2) family belong to a class of transcription factors that are responsible for the activation of many muscle-specific genes with a conserved A/T-rich cis-acting regulatory element (Naya and Olson, Reference Naya and Olson1999) and play a crucial role in p38 MAP kinase-mediated terminal muscle differentiation (discussed above). Like NFATs, MEF2 proteins form co-operative complexes with other factors, like members of the MyoD family, to regulate transcription. Calcineurin up-regulates the transcription of MEF2 genes (Wu et al., Reference Wu, Naya, McKinsey, Mercer, Shelton, Chin, Simard, Michel, Bassel-Duby, Olson and Williams2000a). It can also dephosphorylate MEF2 directly and enhance its transactivational activity (Dunn et al., Reference Dunn, Simard, Bassel-Duby, Williams and Michel2001; Wu et al., Reference Wu, Rothermel, Kanatous, Rosenberg, Naya, Shelton, Hutcheson, DiMaio, Olson, Bassel-Duby and Williams2001). Hence members of the MEF2 family are transcriptional targets and protein substrates of calcineurin.

PGC-1α. Peroxisome proliferator-activated receptor-γ co-activator-1 (PPARγ co-activator 1α /PGC-1α), a ubiquitous transcriptional co-factor for nuclear receptors, is a potent inducer of mitochondrial biogenesis (Lin et al., Reference Lin, Wu, Tarr, Zhang, Wu, Boss, Michael, Puigserver, Isotani, Olson, Lowell, Bassel-Duby and Spiegelman2002). It was recently shown to be able to convert fast fibres to the slow phenotype when over-expressed in transgenic mice. Part of this effect is thought to be mediated through the action of calcineurin on PGC-1α as a substrate (see PPARs below) (Lin et al., Reference Lin, Wu, Tarr, Zhang, Wu, Boss, Michael, Puigserver, Isotani, Olson, Lowell, Bassel-Duby and Spiegelman2002). As a co-factor, PGC-1α is likely to exert its effects indirectly by modulating the expression of a specific group of downstream genes. Although all three substrates (NFATs, MEF2s and PGC-1α) are important mediators of calcineurin activation, none is exclusively expressed in skeletal muscle. Additional substrates not yet identified may well be involved in the signalling pathway. It is not known which regulatory target genes activated by NFAT, MEF-2 or PGC-1α are responsible for the phenotypic effects of calcineurin. Further work is needed to identify the genes regulated by the known and, possibly, other unidentified substrates of calcineurin.

Several endogenous calcineurin-specific inhibitors have been discovered. AKAP79, cain/cabin 1, and CBHP are ubiquitous factors and were found to inhibit NFAT function or translocation to the nucleus (Crabtree, Reference Crabtree2001). More recently, two additional endogenous calcineurin-specific inhibitors (DSCR1/MCIP1 and ZAKI-4/DSCR1L1/MCIP2), highly expressed in striated muscles and brain, were found (Yang et al., Reference Yang, Rothermel, Vega, Frey, McKinsey, Olson, Bassel-Duby and Williams2000; Rothermel et al., Reference Rothermel, McKinsey, Vega, Nicol, Mammen, Yang, Antos, Shelton, Bassel-Duby, Olson and Williams2001). DSCR1 expression is induced by calcineurin and hence forms a negative feedback loop to limit calcineurin activation (Yang et al., Reference Yang, Rothermel, Vega, Frey, McKinsey, Olson, Bassel-Duby and Williams2000). Its over-expression in transgenic mice prevented cardiac hypertrophy (Rothermel et al., Reference Rothermel, McKinsey, Vega, Nicol, Mammen, Yang, Antos, Shelton, Bassel-Duby, Olson and Williams2001; Van Rooij et al., Reference Van Rooij, Doevendans, Crijns, Heeneman, Lips, Van Bilsen, Williams, Olson, Bassel-Duby, Rothermel and De Windt2004). In skeletal muscle, the role of ZAKI-4 is particularly relevant because, unlike DSCR1, it is responsive to thyroid hormone stimulation (Cao et al., Reference Cao, Kambe, Miyazaki, Sarkar, Ohmori and Seo2002). Thyroid hormone is a well known pleiotropic endocrine regulator of metabolism that modulates the transcription of a large number of genes, leading to increased metabolic rate, protein breakdown and muscle loss (Clement et al., Reference Clement, Viguerie, Diehn, Alizadeh, Barbe, Thalamas, Storey, Brown, Barsh and Langin2002). The importance of thyroid hormone, however, on glucose and lipid homeostasis has not been addressed. In hyperthyroidism, muscle loss is accompanied by increase of fast fibres at the expense of slow fibres (Caiozzo et al., Reference Caiozzo, Baker, McCue and Baldwin1997). Triiodothyronine (T₃) with clenbuterol greatly enhances the slow to fast fibre type switch (Awede et al., Reference Awede, Thissen and Lebacq2002). Such changes resemble the effects of chemical inhibitors of calcineurin, cyclosporine A or FK506, on muscle (Bueno et al., Reference Bueno, Van Rooij, Molkentin, Doevendans and De Windt2002). It is possible that the effects of thyroid hormone on muscle loss and fast phenotype conversion is mediated through the inhibition of calcinuerin by ZAKI-4.

There is little doubt that the calcineurin-NFAT pathway is critical in muscle phenotype determination, in particular its roles in muscle differentiation and slow fibre conversion. However, as in other pathways, calcineurin signalling is not an absolute effect in that it does not activate all slow genes in all muscles. Some fast muscle genes, like MyHC 2b and SERCA1, are up-regulated in activated calcineurin transfected C2C12 cells (Swoap et al., Reference Swoap, Hunter, Stevenson, Felton, Kansagra, Lang, Esser and Kandarian2000). Transgenic mice over-expressing the activated calcineurin in skeletal muscle showed substantial slow fibre switch but only in certain muscles (Naya et al., Reference Naya, Mercer, Shelton, Richardson, Williams and Olson2000).

PPARs and PGC-1α influence oxidative phenotype

Peroxisome proliferator-activated receptors (PPAR) α, γ and δ/β belong to an important family of nuclear hormone receptors (transcription factors) that regulate genes that are involved in lipid metabolism (Muoio et al., Reference Muoio, Way, Tanner, Winegar, Kliewer, Houmard, Kraus and Dohm2002). PPARα, initially identified as the mediator of a class of compounds that induces peroxisomal proliferation in rodent liver, is most abundantly expressed in striated muscle (Muoio et al., Reference Muoio, Way, Tanner, Winegar, Kliewer, Houmard, Kraus and Dohm2002). Its activation increases fatty acid β-oxidation in muscle and reduces body weight (Koh et al., Reference Koh, Kim, Park, Kim, Youn, Park, Youn and Lee2003). PPARγ is more highly expressed in adipose tissues (Koh et al., Reference Koh, Kim, Park, Kim, Youn, Park, Youn and Lee2003) and, in contrast to PPARα, stimulates adipocyte differentiation (Oberkofler et al., Reference Oberkofler, Esterbauer, Linnemayr, Strosberg, Krempler and Patsch2002; Yu et al., Reference Yu, Liu, Mersmann and Ding2006). PPARδ is highly expressed in adipose tissue, heart and skeletal muscle. Recently, it was shown that activated PPARδ increases fatty acid oxidation in adipose tissue and muscle (Wang et al., Reference Wang, Lee, Tiep, Yu, Ham, Kang and Evans2003) and that during starvation its expression is up-regulated in skeletal muscle (Holst et al., Reference Holst, Luquet, Nogueira, Kristiansen, Leverve and Grimaldi2003). PPARα, PPARγ and PPARδ bind to the same consensus sequence, suggesting that mechanisms must exist that regulate PPAR type specificity (Oberkofler et al., Reference Oberkofler, Esterbauer, Linnemayr, Strosberg, Krempler and Patsch2002).

PPARγ co-activator 1α (PGC-1α), cloned from a brown fat cDNA library for its interaction with PPARγ, is a potent transcriptional activator that interacts with several nuclear hormone receptors, which include PPARs, steroid hormone receptors (glucocorticoid, oestrogen and mineralocorticoid), retinoic X receptor, thyroid hormone receptor (Oberkofler et al., Reference Oberkofler, Esterbauer, Linnemayr, Strosberg, Krempler and Patsch2002), as well as MEF2 (Handschin et al., Reference Handschin, Rhee, Lin, Tarr and Spiegelman2003). PGC-1α stimulates mitochondrial function and number (biogenesis), and fatty acid oxidation in cardiac and skeletal muscles by the induction of mitochondrial and nuclear genes involved in energy production pathways, including PPARα and nuclear respiratory factor-1 (NRF-1) (Miura et al., Reference Miura, Kai, Ono and Ezaki2003). Therefore, the phenotypic effect of PGC-1α activation, in partnership with nuclear hormone receptors, including PPARs, in skeletal muscle is increased oxidative capacity.

Markers of atrophy and hypertrophy

Markers of atrophy and hypertrophy are relevant to farm animal production from the perspective of disease and welfare. Animals suffer from a wide range of infectious and non-infectious diseases that are often manifested as reduced weight gain or net weight loss. Markers that can readily measure muscle atrophy or hypertrophy could have veterinary applications.

Biological markers that can predict the functional state of muscle are useful indicators in the diagnosis and monitoring of muscle conditions. Clearly, muscles that have undergone gross hypertrophy or atrophy would be apparent during examination. Muscles in the transitional process of remodelling are more difficult to recognise. Thus the use predictive biological markers would be valuable in identifying the underlying changes that are taking place in muscle.

E3 ubiquitin ligases as atrophic markers. Muscle atrophy is a consequence of a variety of conditions: denervation, injury, limb immobilisation, inactivity, glucocorticoid treatment, infection, diabetes, renal failure, cancer and ageing. In muscle atrophy, muscle loss is often the result of raised protein degradation and turn-over rather than simply due to reduced protein synthesis. Thus, atrophy is as much an active degradation process as a passive one from reduced stimulation of the anabolic (hyperplastic and hypertrophic) pathways. Of the different protein degradation pathways (Costelli et al., Reference Costelli, Carbo, Busquets, Lopez-Soriano, Baccino and Argiles2003), the ATP-dependent ubiquitin-proteasome proteolytic system is thought to be of major importance in skeletal muscle (Dehoux et al., Reference Dehoux, Van Beneden, Fernandez-Celemin, Lause and Thissen2003; Glass, Reference Glass2003a). In this process, ubiquitin (Ub) is activated by an ubiquitin-activating enzyme (E1 family) and conjugated to a substrate protein by ubiquitin-conjugating enzyme (E2 family) in conjunction with ubiquitin protein ligase (E3 ligase family). E3 ligases, of which hundreds have been identified, confer substrate specificity (Glass, Reference Glass2003a). During muscle atrophy, many of the genes encoding the Ub-proteasome pathway are up-regulated (Lecker et al., Reference Lecker, Jagoe, Gilbert, Gomes, Baracos, Bailey, Price, Mitch and Goldberg2004), including E2 (such as E2_14K and UbcH2) and E3 (such as E3α / UBr1) genes (Li et al., Reference Li, Chen, Li and Reid2003; Lecker et al., Reference Lecker, Jagoe, Gilbert, Gomes, Baracos, Bailey, Price, Mitch and Goldberg2004). In particular, two E3 ubiquitin ligases, MuRF1 (muscle ring finger 1) and MAFbx (muscle atrophy F-box or atrogin-1) have been identified as mediators of muscle atrophy (Bodine et al., Reference Bodine, Latres, Baumhueter, Lai, Nunez, Clarke, Poueymirou, Panaro, Na, Dharmarajan, Pan, Valenzuela, DeChiara, Stitt, Yancopoulos and Glass2001a; Dehoux et al., Reference Dehoux, Van Beneden, Fernandez-Celemin, Lause and Thissen2003). MuRF1 and MAFbx are selectively induced in skeletal muscle and heart when subjected to a variety of atrophic conditions, such as denervation and glucocorticoid treatment. Their absence in null knock-out mice led to the preservation of muscle mass under atrophying conditions (Bodine et al., Reference Bodine, Latres, Baumhueter, Lai, Nunez, Clarke, Poueymirou, Panaro, Na, Dharmarajan, Pan, Valenzuela, DeChiara, Stitt, Yancopoulos and Glass2001a). TNF-α stimulated proteolysis may be mediated through the increased expression of MAFbx and MuRF1 (Dehoux et al., Reference Dehoux, Van Beneden, Fernandez-Celemin, Lause and Thissen2003; Glass Reference Glass2005). MuRF1 contains three domains: a RING-finger domain, required for ubiquitin ligase activity, a B-box of unclear function, and a coil-coil domain, which may be required for heterodimerisation with a related protein MuRF2. Activation, by cachectic factors such as TNFα, of the NF-κB transcription pathway induces skeletal muscle atrophy, which is mediated in part by NF-κB on the up-regulation of MuRF1 (Glass, Reference Glass2005). MuRF1 physically interacts with titin, which suggests a possible role for MuRF1 in titin turn-over (Centner et al., Reference Centner, Yano, Kimura, McElhinny, Pelin, Witt, Bang, Trombitas, Granzier and Gregorio2001). MAFbx contains an F-box domain, characteristic of SCF family of E3 ubiquitin ligases. F-box E3 ligases usually bind post-translationally modified substrates, such as phosphorylation, and may target proteins involved in cell signalling. We recently found that in porcine congenital splayleg, a condition characterised with severe muscle weakness and fibre atrophy, MAFbx is abnormally up-regulated in relation to normal littermates (Ooi et al., Reference Ooi, Da Costa, Edgar and Chang2006).

FOXO proteins, a subgroup of the Forkhead family of transcription factors, have been recently identified as mediators that link the signalling pathways of muscle hypertrophy and atrophy. FOXO transcription factors are important for the induction of cell quiescence and apoptosis. FOXO factors are targets of activated Akt (Figure 3). Direct and multiple phosphorylation by Akt on FOXO factors leads to their displacement from the nucleus to the cytoplasm and to the inhibition of their transcriptional activities (Burgering and Medema, Reference Burgering and Medema2004). Under atrophic conditions, FOXO1 (Stitt et al., Reference Stitt, Drujan, Clarke, Panaro, Timofeyva, Kline, Gonzalez, Yancopoulos and Glass2004) and FOXO3 (Sandri et al., Reference Sandri, Sandri, Gilbert, Skurk, Calabria, Picard, Walsh, Schiaffino, Lecker and Goldberg2004) have been shown to up-regulate the expression of MAFbx and MuRF1. Inhibition of FOXO activity that leads to the down-regulation of MAFbx and MuRF1 is a necessary anti-atrophy step mediated by the PI3K-Akt1 pathway (Sandri et al., Reference Sandri, Sandri, Gilbert, Skurk, Calabria, Picard, Walsh, Schiaffino, Lecker and Goldberg2004; Stitt et al., Reference Stitt, Drujan, Clarke, Panaro, Timofeyva, Kline, Gonzalez, Yancopoulos and Glass2004; Tesseraud et al., Reference Tesseraud, Metayer, Duchene, Bigot, Grizard and Dupont2007). This anti-atrophy effect can be triggered by insulin as ligand which has the dual roles of enhancing protein synthesis (Figure 3) and decreasing proteolysis via FOXO inhibition (Tesseraud et al., Reference Tesseraud, Metayer, Duchene, Bigot, Grizard and Dupont2007).

GATA-2 as a hypertrophic marker. Factors involved in signalling, muscle metabolism or sarcomeric structure that are associated with the process of muscle hypertrophy could potentially be used as hypertrophic markers. GATA-2 has been recently identified as a potentially useful candidate marker gene of muscle hypertrophy (Musarò et al., Reference Musarò, McCullagh, Naya, Olson and Rosenthal1999; Paul and Rosenthal, Reference Paul and Rosenthal2002). In mammals, the GATA zinc-finger transcription factor family comprises six gene members that can be divided into 2 sub-groups, based on structure and function (LaVoie, Reference LaVoie2003). GATA-1/-2/-3 members are often associated with haematopoiesis and neural development. GATA-4/-5/-6 members are commonly associated with organ development, including heart, gut, blood vessels and parts of the genito-urinary system. With the exception of GATA-5, gene ablation of the GATA family members results in embryonic lethality. GATA-2 is necessary for mast cell development, and maintenance and expansion of multipotential progenitors and haematopoietic stem cells (Fujiwara et al., Reference Fujiwara, Chang, Williams and Orkin2004). GATA-2 is usually absent or lowly expressed in skeletal muscle at all stages of development, but is induced in muscle cell culture and in vivo by IGF-1, during muscle regeneration after bupivacaine injection, and by exercise (Paul and Rosenthal, Reference Paul and Rosenthal2002; Sakuma et al., Reference Sakuma, Nishikawa, Nakao, Watanabe, Totsuka, Nakano, Sano and Yasuhara2003). GATA-2 co-precipitates with calcineurin and NFATc1 (see below), suggesting it mediates its effect on muscle gene expression in conjunction with components of the calcineurin signalling pathway as a protein complex (Musarò et al., Reference Musarò, McCullagh, Naya, Olson and Rosenthal1999; Sakuma et al., Reference Sakuma, Nishikawa, Nakao, Watanabe, Totsuka, Nakano, Sano and Yasuhara2003). There is no direct evidence to show that GATA-2 is a hypertrophic factor, but its expression is closely associated with factors (e.g. IGF-1) and conditions (e.g. exercise, regeneration) that are involved in the process of hypertrophy and regeneration.