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Myostatin signaling through Smad2, Smad3 and Smad4 is regulated by the inhibitory Smad7 by a negative feedback mechanism. Cytokine, 26, 262–272. Zimmers, T. A., Davies, M. V., Koniaris, L. G., Haynes, P., Esquela, A. F., Tomkinson, K. N., Mcpherron, A. C., Wolfman, N. M., Lee, S. J. (2002). Induction of cachexia in mice by sys- temically administered myostatin. Science, 296, 1486–1488. 449 G.S. Lynch (ed.), Sarcopenia – Age-Related Muscle Wasting and Weakness, DOI 10.1007/978-90-481-9713-2_19, © Springer Science+Business Media B.V. 2011 Abstract While the importance of b-adrenergic signalling in the heart has been well documented for more than half a century and continues to receive significant attention, it is only more recently that we have begun to understand the importance of this signalling pathway in skeletal muscle. There is considerable evidence regarding the stimulation of the b-adrenergic system with b-adrenoceptor agonists (b-agonists) in animals and humans. Although traditionally used for the treatment of bronchospasm, it became apparent that some b-agonists, such as clenbuterol, had the ability to increase skeletal muscle mass and decrease body fat (Ricks et al. 1984; Beerman et al. 1987). These so-called “repartitioning effects” proved desirable for those working in the livestock industry trying to improve feed efficiency and meat quality (Sillence 2004). Not surprisingly, b 2 -agonists were soon being used by those engaged in competitive bodybuilding and by other athletes, especially those engaged in strength- and power-related sports (Lynch 2002; Lynch and Ryall 2008). As a consequence of their muscle anabolic actions, the effects of b-agonist administration on skeletal muscle have been examined in a number of animal models (and in humans) with the hope of discovering therapeutic applications, particularly for muscle wasting conditions including sarcopenia (age-related mus- cle wasting and associated weakness), cancer cachexia, sepsis, and other forms of metabolic stress, denervation, disuse, inactivity, unloading or microgravity, burns, HIV-acquired immunodeficiency syndrome (AIDS), chronic kidney or heart failure, chronic obstructive pulmonary disease, muscular dystrophies, and other neuromuscular disorders. For many of these conditions, the anabolic properties of b-agonists have the potential to attenuate (or potentially reverse) the muscle Role of b-Adrenergic Signalling in Skeletal Muscle Wasting: Implications for Sarcopenia James G. Ryall and Gordon S. Lynch G.S. Lynch (*) Department of Physiology, Basic and Clinical Myology Laboratory, The University of Melbourne, Victoria, Australia e-mail: gsl@unimelb.edu.au J.G. Ryall The Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health (NIH), Bethesda, MD, USA e-mail: ryallj@mail.nih.gov 450 J.G. Ryall and G.S. Lynch wasting, muscle fibre atrophy, and associated muscle weakness. b-agonists also have clinical significance for enhancing muscle repair and restoring muscle function after injury or following reconstructive surgery. In addition to having anabolic effects on skeletal muscle, b-agonists have also been associated with some undesirable side effects, including increased heart rate (tachycardia) and muscle tremor, which have so far limited their therapeutic potential. In this chapter we describe the physiological significance of b-adrenergic signalling in skeletal muscle and discuss the therapeutic potential of b-adrenergic stimulation for age-related muscle wasting and weakness. We describe the effects of current b-agonists on skeletal muscle and identify novel research strategies to minimize the unwanted side-effects associated with systemic b-adrenergic stimulation. Keywords β-adrenoceptor agonist • β-adrenergic signalling • cardiac muscle • fibre type • G-protein couple receptor • heart • muscle hypertrophy • muscle wasting • skeletal muscle 1 Overview of b-Adrenergic Signalling Before discussing the therapeutic potential of b-adrenergic stimulation for sarcopenia, it is important to characterize the role of this important signalling pathway in normal healthy skeletal muscle. b-adrenoceptors belong to the guanine nucleotide-binding G-protein coupled receptor (GPCR) family (Fredriksson et al. 2003), and are activated endogenously via adrenaline (epinephrine) and/or noradrenaline (norepinephrine). One of the defining features of the GPCR superfamily is that all of the receptors couple to heterotrimeric guanine-nucleotide-binding regulatory proteins (G-proteins). These molecules received their name from the typical three subunit composition (designated ‘abg’). All GPCRs (including b-adrenoceptors) have a conserved seven transmembrane a-helical structure forming three extracellular loops; including an amino-terminus and three intracellular loops, including a carboxy-terminus (Johnson 2006; Morris and Malbon 1999). The third-fifth intramembranous regions are believed to be important in ligand binding, while the third intracellular loop of the GPCR has a central role in G-protein coupling (Johnson 2006). The G-proteins are located in the cytoplasmic space and act intracellularly, interacting with an intracellular loop of the GPCR (Fig. 1). The G-protein bg sub- units (Gbg) form a tightly interacting dimer which is bound to the intracellular plasma membrane via an isoprenyl moiety located on the C-terminus of the g sub- unit, whereas the G-protein a subunit (Ga), in its inactive state, remains attached to the Gbg dimer (Bockaert and Pin 1999). Activation of the GPCR causes a pro- found change in the conformation of the intracellular loops and uncovers a previ- ously masked G-protein binding site (Filipek et al. 2004; Klco et al. 2005; Meng and Bourne 2001). Specifically, the third intracellular loop of the GPCR is involved in G-protein binding (Kobilka et al. 1988). Upon binding of a ligand to 451 Role of b-Adrenergic Signalling in Skeletal Muscle Wasting: Implications for Sarcopenia the GPCR, guanosine diphosphate (GDP) is released from the Ga subunit, and subsequent guanosine triphosphate (GTP) binding occurs, which activates the Ga subunit and exposes effector-interaction sites in the Gbg dimer (Bockaert and Pin 1999; Gilman 1995; Hampoelz and Knoblich 2004; Rodbell et al. 1971). The Ga-subunits can be divided into four main families, based on their primary sequence: Ga s , Ga i/o , Ga q/11 and Ga 12 , which regulate the activity of many different second messenger systems (Lohse 1999; Wilkie et al. 1992). b-adrenoceptors couple predominantly with Ga s and Ga i isoforms to initiate downstream effector pathways including adenylyl cyclase (AC), transmembrane protein kinases, and phospholipases (Dascal 2001; Wenzel-Seifert and Seifert 2000). Three subtypes of b-adrenoceptors have been identified and cloned; b 1 -, b 2 - and b 3 -adrenoceptors (Dixon et al. 1986; Emorine et al. 1989; Frielle et al. 1987), each with a 65–70% homology in their amino acid composition (Kobilka et al. 1987). Skeletal muscle contains a significant proportion of b-adrenoceptors, mostly of the b 2 -subtype, but also include approximately 7–10% b 1 -adrenoceptors (Kim et al. 1991; Williams et al. 1984) and a smaller population of a-adrenoceptors, usually in higher P-GSK3β βγ α Non-Canonical β-AR signalling Canonical β-AR signalling Extracellular Intracellular PIP 3 PI3 PKA PIP 2 PDK1/2 cAMP b-adrenoceptor P-Akt P-TSC2 Rheb mTORC1 P-FoxO1/3 G G Fig. 1 b-adrenergic signalling in skeletal muscle. Traditionally, the stimulated b-adrenoceptor has been thought to couple with the stimulatory Ga subunit (Ga s ) of the heterotrimeric G-protein (Gabg) and adenylate cyclase (AC), resulting in conversion of ATP to cAMP and the activation of protein kinase A (PKA). Stimulation of this pathway has been linked to the inhibition of proteolytic pathways and possibly to protein synthesis. In the non-canonical signalling pathway b-adrenoceptors signal via the G-protein Gbg subunits to promote phosphorylation of phosphatidylinositol-4,5- bisphosphate (i.e. PIP 2 becomes PIP 3 ) by phosphatidylinositol 3-kinase (PI3-K), leading to Akt activation. These events trigger the downstream activators, glycogen synthase kinase 3b (GSK3b), tuberous sclerosis complex 2 (TSC2, an activator of mammalian target of rapamycin complex-1, mTORC1) and the forkhead box O (FoxO) family of transcription factors. Thus, b-adrenoceptor stimulation can influence protein synthesis and degradation by several mechanisms 452 J.G. Ryall and G.S. Lynch proportions in slow-twitch muscles (Rattigan et al. 1986). Slow-twitch muscles like the soleus have a greater density of b-adrenoceptors than fast-twitch muscles, such as the extensor digitorum longus (EDL) (Martin et al. 1989; Ryall et al. 2002, 2004). Although the functional significance of this difference in b- adrenoceptor density is not yet understood fully, the response to b-agonist administration appears to be greater in fast-, than in slow-twitch skeletal muscles (Ryall et al. 2002, 2006). The Ga s -AC-cyclic AMP (cAMP) is the most well characterized of the b 2 - adrenoceptor signalling pathways and is generally thought to be, at least partially, responsible for the b 2 -adrenoceptor mediated hypertrophy in skeletal muscle (Hinkle et al. 2002; Navegantes et al. 2000). The production of cAMP results in the activation of numerous downstream signalling pathways, including the well- described protein kinase A (PKA) signalling pathways. Following cAMP activation, PKA is thought to phosphorylate and regulate the activity of numerous proteins. In addition, PKA is capable of diffusing passively into the nucleus, where it can regulate the expression of many target genes via direct phosphorylation of the cAMP response element (CRE) binding protein (CREB), or via a modulator that acts on second generation target genes (Carlezon et al. 2005; Mayr and Montminy 2001). The CRE binding protein is a nuclear transcription factor that is expressed ubiq- uitously and has been implicated in many processes, including cell proliferation, differentiation, adaptation, and survival (Mayr and Montminy 2001). CREB forms a homodimer and binds to a conserved CRE-region on DNA. Nuclear entry of PKA, phosphorylates CREB at a single serine residue site (Ser 133 ) (Hagiwara et al. 1993). Phosphorylation of Ser 133 promotes transcription at the CRE-region through recruitment of the transcriptional co-activators CREB-binding protein (CBP) and p300, which mediate transcriptional activity through their association with RNA Polymerase II (Goodman and Smolik 2000; Mayr and Montminy 2001). CREB- phosphorylation promotes activation of genes containing a CRE-region, of which there are >4,000 in the human genome (Pourquié 2005; Zhang et al. 2005). Finally, CRE-gene activation is terminated by dephosphorylation of CREB, a process regu- lated by the serine/threonine phosphatases PP-1 and PP-2A (Hagiwara et al. 1992; Wadzinski et al. 1993). One target for b-adrenoceptor mediated CRE activation in skeletal muscle is the promoter region of the orphan nuclear receptor, NOR-1 (NR4A3) (Ohkura et al. 1998; Pearen et al. 2006). b 2 -adrenoceptor activation is associated with an increased expression of NOR-1 and the related orphan nuclear receptor nur-77 (NR4A1) (Maxwell et al. 2005; Pearen et al. 2006). Interestingly, Pearen and colleagues (2006) found that siRNA mediated inhibition of NOR-1 expression was associated with a dramatic increase (>65 fold) in the levels of myostatin mRNA in C2C12 cells. Myostatin is a member of the transforming growth factor-b superfamily and a potent negative regulator of muscle mass (McPherron et al. 1997). Thus, b-adre- noceptor activation, through increased NOR-1 expression, may inhibit myostatin expression and hence promote skeletal muscle growth. The transcriptional adapters, CBP and p300, promote skeletal muscle myogenesis via the coactivation of a number of myogenic basic helix-loop-helix (bHLH) pro- 453 Role of b-Adrenergic Signalling in Skeletal Muscle Wasting: Implications for Sarcopenia teins (Eckner et al. 1996; McKinsey et al. 2002; Sartorelli et al. 1997). The family of myogenic bHLH proteins, including MyoD, myogenin, myf5 and MRF4, activate muscle gene transcription via pairing with the ubiquitously expressed E-box consen- sus sequence in the control regions of muscle-specific genes (McKinsey et al. 2002; Molkentin and Olson 1996). Sartorelli and colleagues (1997) found that p300 and CBP may positively influence myogenesis by acting as a ‘bridge’ between the myogenic bHLH and the myocyte enhancer factor 2 (MEF2) family of proteins. In addition to transcriptional coactivation, CBP and p300 have intrinsic histone acetyltransferase (HAT) activity (Goodman and Smolik 2000; Roth et al. 2003; Thompson et al. 2004). Histone acetyltransferases are believed to play an important role in transcription, since they catalyze the transfer of acetyl groups from acetyl- coenzyme A to the e-amino group of lysine side chains of specific proteins, includ- ing several transcriptional regulatory proteins (Yang 2004). Therefore, the b-adrenoceptor mediated actions of CBP and p300 could increase the accessibility of docking sites for transcriptional proteins and regulators (Ogryzko et al. 1996; Thompson et al. 2004). Chen and colleagues (2005) identified an unexpected role for PKA/CREB sig- nalling during myogenesis, proposing that myogenic gene expression of Pax3, MyoD, and Myf5 is dependent on AC/cAMP mediated phosphorylation of PKA and subsequent activation of CREB. The authors demonstrated the importance of CREB in the developing myotome, since CREB −/− mice did not express Pax3, MyoD, or Myf5 and myotome formation was defective (Chen et al. 2005). It remains to be determined whether b-adrenoceptor mediated activation of PKA/ CREB signalling has a similar response during myogenesis. Berdeaux and colleagues (2007) demonstrated a novel role of CREB in mediat- ing the activity of MEF2. They showed that b-adrenergic stimulated CREB modu- lated the phosphorylation status of the class II histone deacetylase HDAC5 in mouse skeletal muscle, by increasing the expression of salt inducible kinase 1 (SIK1). Activated SIK1 phosphorylated HDAC5, resulting in its nuclear exclusion and subsequent activation of the MEF2 myogenic program (Berdeaux et al. 2007). These exciting results demonstrated the complexity of the downstream activators of the b-adrenergic signalling pathway and highlighted the previously unappreci- ated role of this pathway in skeletal muscle. In addition to the well-described Ga s -cAMP signalling pathways, studies have implicated the Gbg subunits in various cell signalling processes, which may also play important roles in b-adrenoceptor signalling in skeletal muscle (Crespo et al. 1994; Dascal 2001; Diversé-Pierluissi et al. 2000; Ford et al. 1998; Mirshahi et al. 2002). Specifically, in vitro cell culture experiments have revealed that the Ga i linked Gbg subunits activate the phosphoinositol 3-kinase (PI3K)-AKT signalling pathway (Lopez-Ilasaca et al. 1997; Murga et al. 1998, 2000; Schmidt et al. 2001). The PI3K-AKT signalling pathway has been implicated in protein synthesis, gene transcription, cell proliferation, and cell survival (Bodine et al. 2001b; Glass 2003, 2005; Kline et al. 2007; Pallafacchina et al. 2002; Rommel et al. 2001). Although there are three distinct isoforms of AKT, the predominant 454 J.G. Ryall and G.S. Lynch skeletal muscle isoform is AKT1 (Nader 2005). Activation of PI3K phosphorylates the membrane bound PIP 2 , creating a lipid-binding site on the cell membrane for both AKT1 and 3¢-phoshphoinositide-dependent protein kinase 1 (PDK). PDK then phosphorylates AKT1 at the membrane (Nicholson and Anderson 2002). Akt activation, in turn, results in the phosphorylation of numerous downstream activa- tors, including glycogen synthase kinase 3b (GSK3b), tuberous sclerosis complex 2 (TSC2, leading to the subsequent activation of mammalian target of rapamycin complex1, mTORC1) (Garami et al. 2003; Latres et al. 2005) and members of the forkhead box O (FOXO) family of transcription factors (Sandri et al. 2004; Stitt et al. 2004). Kline and colleagues (2007) found that stimulation of the b-adrenoceptor signal- ling pathway resulted in AKT phosphorylation and subsequent activation of mTORC1. Initiation of mTORC signalling phosphorylates and subsequently acti- vates p70 s6 kinase (p70 S6K ), while concomitantly inactivating 4EBP-1 (also termed PHAS-1). p70 S6K mediates the phosphorylation of the 40S ribosomal S6 protein, resulting in the upregulation of mRNA translation encoding for ribosomal proteins and elongation factors (Jefferies et al. 1997). Inactivation of 4EBP-1 removes its inhibitory action on the protein initiation factor eukaryotic initiation factor 4E (eIF- 4E) (Lai et al. 2004; Nave et al. 1999). These findings supported those of Sneddon and colleagues (2001) who reported an increased phosphorylation of 4E-BP1 and p70 S6K in rat plantaris muscle after 3 days of clenbuterol treatment. GSK-3b is reported to be a negative regulator of protein translation and gene expression in cardiac (Hardt and Sadoshima 2002) and skeletal muscle (Childs et al. 2003; Bossola et al. 2008). Following b-adrenoceptor stimulation, GSK3b is phosphorylated and subsequently inactivated by AKT1 (Yamamoto et al. 2007), resulting in the expression of a dominant negative form of GSK3b. Since GSK3b normally acts to inhibit the translation initiation factor eIF2B, blockade of GSK3b by AKT1 might promote protein synthesis (Bodine et al. 2001b; Rommel et al. 2001). AKT1 signalling is not only involved in the signalling pathways responsible for muscle hypertrophy, but it has been implicated in the inhibition of signalling pathways responsible for “muscle atrophy”. AKT1 inactivation of FOXO leads to nuclear exclusion and inhibition of the forkhead transcriptional program. The DNA displacement and subsequent nuclear exclusion of FOXO requires the involvement of 14-3-3 proteins, which bind to FOXO following AKT1-mediated phosphorylation (Tran et al. 2003). 14-3-3 proteins are among a family of chaperone proteins that interact with specific phosphorylated protein ligands (Tran et al. 2003). Activation of the forkhead transcriptional program is necessary for induction of both muscle RING finger 1 (muRF1) and muscle atrophy F-box (MAFbx, also called atrogin-1) (Sandri et al. 2004; Stitt et al. 2004). Both muRF1 and MAFbx encode ubiq- uitin ligases which conjugate ubiquitin to protein substrates, and are upregulated in numerous models of muscle atrophy (Bodine et al. 2001a; Tintignac et al. 2005). Thus, by phosphorylating and inactivating FOXO, AKT1 blocks the induction of FOXO- mediated atrophy signalling via muRF1 and MAFbx. b-Adrenoceptor activation 455 Role of b-Adrenergic Signalling in Skeletal Muscle Wasting: Implications for Sarcopenia reduces the expression of muRF1 and MAFbx in skeletal muscle from denervated and hindlimb-suspended rats, an effect possibly mediated via AKT1-initiated inhibition of the forkhead transcriptional program (Kline et al. 2007). It is interesting to note that while FOXO1 regulates the expression of both MAFbx and muRF1 (Stitt et al. 2004), FOXO3a appears only to activate the MAFbx promoter (Sandri et al. 2004). In addition, while measurable levels of FOXO4 have been identified in skeletal muscle (Furuyama et al. 2002), very little is known about its role in skeletal muscle atrophy. Furuyama and colleagues (2002) characterized the expression pattern of FOXO1, FOXO3a and FOXO4 with ageing and caloric restriction in rats. FOXO4 mRNA expression increased from 3 to 12 months and then decreased from 12 to 26 months. A similar pattern was observed for FOXO3a expression (Furuyama et al. 2002). Interestingly, FOXO1 mRNA expression remained unchanged. In contrast, caloric restriction resulted in an increase in the expression levels of both FOXO4 and FOXO1, but not FOXO3a (Furuyama et al. 2002). These results indicate the complexity of the forkhead transcriptional program in the regulation of skeletal muscle atrophy (Kandarian and Jackman 2006). Several studies have identified a role for FOXO1 in binding to the promoter region of 4EBP-1 which resulted in increased mRNA and protein expression (Léger et al. 2006; Wu et al. 2008). Associated with the increase in 4EBP-1 was a reduction in mTORC activation and p70 S6K . Thus, in addition to previously reported roles in atrophic signalling pathways, FOXO1 also plays an active role in inhibiting protein synthesis (Yang et al. 2008). A number of researchers have identified genes that are activated by b-adreno- ceptor stimulation, but the mechanism for their activation remains unclear. For example McDaneld and colleagues (2004) examined differential gene expression in skeletal muscle after b-agonist administration to evaluate the role of genes thought responsible for muscle growth. Decreased mRNA abundance following b-adreno- ceptor stimulation was confirmed for DD143 identified as ASB15, a bovine gene encoding an ankyrin repeat and a suppressor of cytokine signalling (SOCS) box protein, in both cattle and rats (McDaneld et al. 2004, 2006; Spangenburg 2005). The authors reported that ASB15 was a member of an emerging gene family involved in a variety of cellular processes including cellular proliferation and dif- ferentiation (McDaneld et al. 2004). Similarly, Spurlock and colleagues (Spurlock et al. 2006) examined gene expression changes in mouse skeletal muscle 24 hours and 10 days after b-adreno- ceptor stimulation and identified genes involved in processes important to skeletal muscle growth, including regulators of transcription and translation, mediators of cell-signalling pathways, and genes involved in polyamine metabolism. They reported changes in mRNA abundance of multiple genes associated with myogenic differentiation relevant to the effect of b-adrenoceptor stimulation on the prolifera- tion, differentiation, and/or recruitment of satellite cells into muscle fibres to pro- mote muscle hypertrophy. Similarly, they showed an upregulation of translational initiators responsible for increasing protein synthesis (Spurlock et al. 2006). More recently, Pearen and colleagues (2009) profiled skeletal muscle gene expression in mouse tibialis anterior muscles at 1 and 4 h after systemic administration . 450 J.G. Ryall and G.S. Lynch wasting, muscle fibre atrophy, and associated muscle weakness. b-agonists also have clinical significance for enhancing muscle repair and restoring muscle function. animal models (and in humans) with the hope of discovering therapeutic applications, particularly for muscle wasting conditions including sarcopenia (age-related mus- cle wasting and associated. cardiac muscle • fibre type • G-protein couple receptor • heart • muscle hypertrophy • muscle wasting • skeletal muscle 1 Overview of b-Adrenergic Signalling Before discussing the therapeutic potential