456 J.G. Ryall and G.S. Lynch of formoterol and revealed significant expression changes in genes associated with skeletal muscle hypertrophy, myoblast differentiation, metabolism, circadian rhythm, transcription, histones, and oxidative stress. With respect to formoterol’s anabolic effects, differentially expressed genes relevant to the regulation of muscle mass and metabolism were validated by quantitative RT-PCR to examine gene expression after acute (1–24 h) and chronic administration (1–28 days) of formoterol. Following acute and chronic formoterol administration there was an attenuation of myostatin signalling (differential expression of myostatin, activin receptor IIB, and phospho-Smad3) which was a previously unreported effect of b-adrenoceptor signalling in skeletal muscle. Acute (but not chronic) formoterol administration induced expression of genes involved in oxidative metabolism, including hexoki- nase 2, sorbin and SH3 domain containing 1, and uncoupling protein 3. Interestingly, formoterol administration also appeared to influence some genes associated with the peripheral regulation of circadian rhythm (including nuclear factor interleukin 3 regulated, D site albumin promoter binding protein, and cryptochrome 2) indicat- ing crosstalk between b-adrenoceptor signalling and circadian cycling in skeletal muscle. This was the first study showing regulation of the peripheral circadian regulators in skeletal muscle by b-adrenoceptor signalling, possibly implicating b-adrenoceptor (sympathetic) signalling as a pathway coordinating communication between central and peripheral circadian clocks in skeletal muscle (Pearen et al. 2009). 2 Changes in Skeletal Muscle b-Adrenergic Signalling with Aging While there has been much conjecture as to the exact changes in catecholamine levels as a consequence of ageing, it is now accepted that there is an increase in the plasma level of noradrenaline and a decrease in adrenaline, in rats and humans (Esler et al. 1995; Kaye and Esler 2005; Larkin et al. 1996). In addition, work from our laboratory has demonstrated an age-related change in b-adrenoceptor signalling in skeletal muscle (Ryall et al. 2007). Chronic administration of the b-adrenoceptor agonist, formoterol, for 4 weeks increased the mass of the slow-twitch soleus muscle in young (3 months), but not in adult (16 months) or old (27 months) rats. In contrast, formoterol increased the mass of the fast-twitch EDL muscle of rats in all three age groups tested (Ryall et al. 2007). These findings suggest that the b-adrenergic signalling pathway and especially that pathway leading to striated muscle hypertrophy, is altered by age in slow- but not in fast-twitch skeletal mus- cles, an effect independent of b-adrenoceptor density. There is currently a dearth of knowledge regarding how ageing affects this important signalling pathway with most of our current knowledge based on studies conducted on the ageing myocardium. However, due to the differences in b-adren- ergic signalling between these two tissues it is important that future studies focus on skeletal muscle. 457 Role of b-Adrenergic Signalling in Skeletal Muscle Wasting: Implications for Sarcopenia 3 Therapeutic Potential of b-Adrenoceptor Agonists for Sarcopenia There have been numerous studies on animals and several studies on humans investigating the effects of b-agonists on skeletal muscle (for review see Lynch and Ryall 2008). In relation to attenuating the loss of muscle mass and protein content or hastening the restoration of these parameters in the elderly during periods of malnutrition or extended periods of inactivity, three early studies by Carter and Lynch (1994a, b, c) provided encouraging evidence that b-agonists could find therapeutic application for these conditions. To examine the anabolic effects of low-dose salbutamol or clenbuterol administration on aged rats, Carter and Lynch (1994b) showed that in old rats, s.c. delivery by osmotic minipumps (at daily doses of 1.03 mg/kg or 600 mg/kg) for 3 weeks, increased combined hindlimb muscle mass by 19% and 25%, respectively. Gastrocnemius muscle mass and protein content were increased by 19% and 23%, respectively, in old rats. Overall, this study found that salbutamol and clenbuterol increased skeletal muscle protein content and reduced carcass fat content, suggesting that both b-agonists could potentially stimulate muscle growth in frail elders (Carter and Lynch 1994b). In a related experiment, Carter and Lynch (1994c) studied the effect of clenbuterol on recovery of muscle mass and carcass protein content after protein malnutrition in aged rats. The rats were subjected to 3-weeks of dietary protein restriction that reduced overall body mass by 21%. During the recovery period, the rats were fed a normal diet with clenbuterol (10 mg/kg) added to the feed. The addition of clen- buterol to the diet increased hindlimb muscle mass by 30% and protein content by 25%, in aged rats (Carter and Lynch 1994c). In another experiment (Carter and Lynch 1994a), aged rats were injected daily with thyroid hormone (4–6.5 mg of triiodothyronine per 100 g body mass) for 3 weeks to cause an ~20% reduction in body mass and hindlimb muscle mass. Feeding the rats a diet containing 10 mg clenbuterol per kg during a 3-week recovery period restored body mass and muscle mass to euthyroid control levels, whereas feeding the rats a control diet did not (Carter and Lynch 1994a). Taken together, these findings suggested that clen- buterol, or other b-agonists, could find application in hastening recovery of muscle mass as a consequence of malnutrition in frail, elderly humans (Carter and Lynch 1994a, c). In aged rats, clenbuterol treatment (2 mg/kg) via daily injection for 4 weeks restored the age-associated decline in the mass and specific force (i.e. normalized force or force per muscle cross-sectional area) of diaphragm muscle strips (Smith et al. 2002). A much lower dose of clenbuterol (10 mg/kg per day), attenuated the loss of specific force in the soleus muscle only slightly (i.e. by 8%) and reduced fatigue (in response to repeated stimulation) by approximately 30% in aged rats, with considerable muscle atrophy having been subjected to 21 days of hindlimb suspension (Chen and Alway 2001). However, low-dose clenbuterol treatment did not attenuate the loss of specific force in the soleus of adult rats or in the plantaris 458 J.G. Ryall and G.S. Lynch muscles of old or adult rats. The study concluded that clenbuterol could reduce muscle fatigue in slow muscles during disuse with some clinical implications for reducing fatigue in muscles of the elderly. Findings from this and a related study (Chen and Alway 2000), indicated that low-dose clenbuterol treatment did not attenuate atrophy of fast muscles and only modestly attenuated the atrophy of slow muscles, making it largely ineffective for preventing muscle wasting from disuse atrophy in aged rats. In a study from our laboratory (Ryall et al. 2004) old rats were treated daily with a relatively high dose of the b-agonist, fenoterol (1.4 mg/kg/day, i.p.), or saline for 4 weeks. At 28 months of age, untreated old F344 rats exhibited a loss of skeletal muscle mass and a decrease in force-producing capacity, in both fast and slow muscles. Interestingly, the muscle mass, fibre size, and force-producing capacity of EDL and soleus muscles from old rats treated with fenoterol was equivalent to, or greater than, untreated adult rats (Ryall et al. 2004). Fenoterol treatment caused a small increase in the fatigability of both EDL and soleus muscles due to a decrease in oxidative metabolism. The findings highlighted the clinical potential of b-agonists to increase muscle mass and function to levels that exceeded those in adult rats. Schertzer and colleagues (2005) found that treating aged rats with fenoterol (1.4 mg/kg/day, i.p.) for 4 weeks, reversed the slowing of (twitch) relaxation in slow- and fast-twitch skeletal muscle due to increased SERCA activity and SERCA pro- tein levels (Fig. 2). That study provided evidence for an age-related alteration in the environment of the nucleotide binding domain and/or a selective nitration of the SERCA2a isoform, which was associated with depressed SERCA activity. Fig. 2 Sample recordings of twitch characteristics in the predominately fast-twitch extensor digitorum longus muscles of adult (16 mo) and aged (28 mo) Fischer 344 rats that had been treated for 4 weeks with with fenoterol (Fen; dashed line) or saline vehicle only (control, Con; solid line) (see Ryall et al. 2004; Schertzer et al. 2005 for details). Reprinted with permission 459 Role of b-Adrenergic Signalling in Skeletal Muscle Wasting: Implications for Sarcopenia Fenoterol treatment ameliorated the age-related decrease in nucleotide binding affinity and reversed the age-related accumulation of nitrotyrosine residues on the SERCA2a isoform. These changes, in combination with increases in SERCA1 protein levels, appeared to be the underlying mechanisms of fenoterol treatment reversing age-related decreases in the V max of SERCA (Schertzer et al. 2005). In a later study (Ryall et al. 2006), we demonstrated that ‘newer’ generation b-agonists, formoterol and salmeterol, could exert significant anabolic actions on skeletal muscle even at micromolar doses, compared with the millimolar doses required to elicit similar responses with older generation b-agonists such as fenot- erol or clenbuterol. Using this information, we investigated the potential of formot- erol, one of these newer generation b-agonists, to increase muscle mass and force producing capacity of EDL and soleus muscles in aged rats (Ryall et al. 2007). In addition, we studied the effects of formoterol withdrawal on parameters such as muscle mass and strength. Rats were similarly treated with either formoterol (25 mg/ kg/day, i.p.), or saline vehicle for 4 weeks, and another group of rats were similarly treated with formoterol, followed by a period of withdrawal for 4 weeks. Formoterol treatment increased EDL muscle mass and the force producing capacity of both EDL and soleus muscles, without a concomitant increase in heart mass. The hypertrophy and increased force of EDL muscles persisted for 4 weeks after withdrawal of treat- ment. This study was important because it demonstrated significant improvements in muscle function in old rats after b-agonist administration, at a dose 1/50th that of other b-agonists that had been used previously (Ryall et al. 2004). These findings have important implications for clinical trials that might utilize b-agonists for mus- cle wasting conditions (Fowler et al. 2004; Kissel et al. 1998, 2001). We and others have found that exogenous administration of clenbuterol, fenot- erol and formoterol can result in a dramatic shift in the muscle fibre phenotype from slow-oxidative to fast oxidative-glycolytic fibres (Figs. 1 and 2; Ryall et al. 2002, 2007; Zeman et al. 1988). Although previous studies have identified the mechanisms underlying a shift from a fast to a slow muscle phenotype (Handschin et al. 2007; Kim et al. 2008; Oh et al. 2005), less is known about the pathways responsible for shifts from a slow to a fast muscle phenotype (Grifone et al. 2004; Ryall et al. 2008a, b). This is relevant if b-agonists are to be considered for thera- peutic application for sarcopenia since age-related losses of fast motor units have important consequences for the preservation of fast muscles fibres during advanc- ing age. Studies in rats and mice have shown that a significant shift in slow to fast fibre proportions within skeletal muscles as a consequence of chronic b-agonist administration can dramatically affect function, particularly shortening the duration of the isometric twitch response (Schertzer et al. 2005), increasing velocity of shortening (Dodd et al. 1996), and increasing muscle fatigability (Dupont- Versteegden 1996). In our hands, these effects are largely dependent on the type and dose of b-agonist employed (Harcourt et al. 2007). Whether b-adrenergic signalling is implicated in the preservation of motor units has not been determined specifically but Zeman and colleagues (2004) reported that treating motor neuron degeneration (mnd) mice with clenbuterol enhanced regen- eration of motor neuron axons and reduced the proportion of motor neurons with 460 J.G. Ryall and G.S. Lynch eccentric nuclei, a characteristic of axonal injury and subsequent compensatory axonal sprouting. These effects were consistent with improved synaptic function and an attenuated progression of motor deficits such as the decline in grip strength (Fig. 3) (Zeman et al. 2004). 4 Novel b-Adrenoceptor Therapeutic Strategies Some of the most serious consequences of chronic b-agonist administration relate to the systemic responses to b-adrenoceptor activation (Gregorevic et al. 2005; Ryall et al. 2008b). Much research is currently focused on developing new methods of drug administration that limit unwanted systemic effects, with many having potential to improve the safe delivery of b-agonists to skeletal muscles. Fig. 3 Extensor digitorum longus (EDL) muscle sections from adult and old control rats and formoterol-treated rats reacted for mATPase at a preincubation pH of 4.3. Strongly reacting (dark) fibres are slow type I, and light gray fibres are fast-type II isoforms. EDL muscles from old control rats had a greater proportion of type I fibres, and formoterol treatment resulted in a decreased proportion of type I fibres. Note also the significant fibre hypertrophy in muscles from formoterol treated rats. Reprinted with permission (Ryall et al. 2007) 461 Role of b-Adrenergic Signalling in Skeletal Muscle Wasting: Implications for Sarcopenia 4.1 Intramuscular Administration We have examined whether direct intramuscular (i.m.) injection of the b-agonist formoterol can localize its effects to skeletal muscle directly and so minimize potential deleterious systemic effects (Ryall et al. 2008a). Two days after a single i.m. injection of formoterol, the force producing capacity of regenerating rat EDL muscles was two-fold higher than that of regenerating EDL muscles that received a single i.m. injection of saline. Importantly, i.m. administration of formoterol was not associated with cardiac hypertrophy. However, it should be noted that the increase in muscle mass and force-producing capacity after i.m. administration was lost within 5 days, and was still associated with a number of changes in cardiovas- cular function, including a transient increase in heart rate and a decrease in blood pressure. Furthermore, this mode of administration would prove problematic in a condition such as sarcopenia, where the loss of muscle mass and strength is not limited to a single muscle. More likely, this approach could find application in sports medicine and rehabilitation where functional impairments might be limited to a single muscle or muscle group. 4.2 Co-administration with a b 1 -Adrenoceptor Antagonist Blocking stimulation of the b 1 -adenoceptors is possible with highly selective b 1 -adrenoceptor antagonists such as CGP 20712A (Sillence and Matthews 1994) and the importance of blocking b 1 -adrenoceptors in heart failure to abrogate cardiotoxic b 1 -adrenoceptor-mediated effects is also well known (Ahmet et al. 2008; Molenaar and Parsonage 2005). Previous clinical trials of the older gen- eration b-agonist, albuterol, for patients with neuromuscular disorders revealed some cardiovascular complications, including palpitations and tachycardia (Fowler et al. 2004). The fact that formoterol is highly selective for the b 2 - adrenoceptor compared with older generation agonists such as albuterol and clenbuterol (Anderson 1993), and that it is efficacious in eliciting skeletal muscle anabolic effects even at micromolar doses (Ryall et al. 2006), offers the considerable advantage that simultaneous b 1 -adrenoceptor blockade may pre- vent or attenuate many of these cardiovascular side effects. Molenaar and col- leagues (2006) have suggested that the use of highly selective b 2 -agonists, in conjunction with a selective b 1 -blocker, could prevent unintended b 1 -adrenocep- tor activation and thus prevent unwanted cardiovascular effects while maintain- ing the desirable effects on skeletal muscle. This is particularly important for b 1 -adrenoceptors in the cardiovascular system, where chronic activation of b 1 - adrenoceptors is contraindicated for prevalent cardiac and vascular disorders including hypertension, ischemic heart disease, arrhythmias and heart failure where b-blockers are indicated. A pathological role of the b 1 -adrenoceptor was confirmed in transgenic mice where 15-fold overexpression led to progressive 462 J.G. Ryall and G.S. Lynch deterioration of heart function, hypertrophy and heart failure (Engelhardt et al. 1999). The importance of blocking b 1 -adrenoceptors in heart failure to abolish cardiotoxic b 1 -mediated effects have been reported previously (Ahmet et al. 2008; Molenaar and Parsonage 2005). 4.3 Phosphodiesterase Inhibitors Phosphodiesterase (PDE) is the enzyme responsible for the degradation of cAMP into 5¢-AMP, and it therefore plays an important role in terminating the PKA-cAMP signaling cascade (for review see Omori and Kotera 2007). Skeletal muscle con- tains numerous isoforms of PDE, including: PDE4, PDE7, and PDE8, however, PDE4 is believed to be predominantly responsible for cAMP degradation in this tissue (Bloom 2002). Selective inhibitors of PDE have been used to treat a diverse range of pathologi- cal conditions, including chronic obstructive pulmonary disorder, erectile dysfunc- tion, and hypertension (Benedict et al. 2007; Burnett 2008; Kass et al. 2007). However, the potential of PDE inhibitors to treat skeletal muscle wasting and weak- ness has received only limited attention. Some of the earliest studies in skeletal muscle utilized the non-selective PDE inhibitor, pentoxifylline. Hudlická and Price (1990) found that 5 weeks of tri-daily administration of pentoxifylline (3mg/kg, i.p.) to rats increased the proportion of glycolytic fibres in EDL muscles. Breuillé and colleagues (1993) demonstrated that a single injection of pentoxifylline (100mg/kg, i.p.) to rats could attenuate the atrophy of the gastrocnemius muscle associated with 6 days of induced sepsis. More recently, Hinkle and colleagues (2005) administered either rolipram or Ariflo (both selective PDE4 inhibitors) or pentoxifylline via twice-daily s.c. injections to rats and mice after denervation or during disuse atrophy (limb-casting), respectively. PDE4 selective or PDE non- selective inhibition had little or no effect on muscle mass and strength in control muscles, while all three pharmacological inhibitors prevented the loss of muscle mass associated with denervation or disuse by ~20% to 40%. The results from these studies suggested a role for PDEs in proteolytic processes, and this was confirmed by Baviera et al. (2007) who found that pentoxifylline administration to diabetic rats reduced the activity of the Ca 2+ -dependent and ATP proteasome-dependent proteolytic pathways. An attractive hypothesis is that selective PDE inhibitors may be sufficient to prevent, attenuate, or reverse muscle wasting and weakness, without the complicat- ing cardiac side-effects associated with b-agonist administration. However, it must be noted that chronic administration of the non-selective PDE pentoxifylline is associated with a rightward shift of the left ventricular end-diastolic pressure-vol- ume relationship, thinning of the left ventricular wall, and infiltration of collagen in the myocardium (Anamourlis et al. 2006). 463 Role of b-Adrenergic Signalling in Skeletal Muscle Wasting: Implications for Sarcopenia 4.4 Engineered GPCRS, RASSLs, and DREADDs An exciting avenue of research that may lead to ways that can obviate unwanted side- effects involves the use of designer GPCRs that allow for tight spatiotemporal control of GPCR signalling. This involves the development of both a synthetic receptor and an activator (neither of which activates or impairs endogenous GPCR signalling) and which therefore limits signalling to the tissue/region of interest – a result that current b-adrenoceptor agonists cannot achieve (Small et al. 2001). Roth and colleagues (in particular) are creating specific designer drug-designer receptor complexes to isolate the effects of GPCR activation (Dong et al. 2010; Conklin et al. 2008; Pei et al. 2008) recognising that exogenous ligands have off-target effects and endogenous ligands constantly modulate the activity of the native receptors (Dong et al. 2010). These include ‘Receptors Activated Solely by Synthetic Ligands’ (RASSLs) and Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) (Nichols and Roth 2009) and represent tools for investigating biological function with a high degree of specificity. Although still in development, such approaches may yet lead to the successful separation of the effects of b-agonists on skeletal and cardiac muscle, thus promoting desirable effects that can improve the functional capacity of skeletal muscles without producing cardiovascular complications. 5 Conclusions This chapter has provided evidence for the importance of b-adrenergic signalling in skeletal muscle and implicated this pathway as a potential target for the treatment of age-related muscle wasting and weakness. Although we are only beginning to understand the significance of the b-adrenergic signaling pathway in skeletal muscle, especially in relation to its role in sarcopenia, a wealth of information exists regarding the stimulation of the b-adrenergic system with b-agonists. Although there is great promise that b-agonists can be used for treating sarcopenia, and other conditions where muscle wasting is indicated, their clinical application has been limited by cardiovascular side effects, especially when b-agonists are administered chronically and at high doses. Newer generation b-agonists (such as formoterol) can elicit an anabolic response in skeletal muscle even when adminis- tered at very low doses and this has renewed enthusiasm for their clinical applica- tion, especially because they exhibit reduced effects on the heart and cardiovascular system compared with older generation b-agonists (such as fenoterol and clen- buterol). However, the potentially deleterious cardiovascular side effects associated with b-agonist administration have not been obviated completely and so it is important to refine their development and investigate novel strategies to limit b-adrenoceptor 464 J.G. Ryall and G.S. Lynch activation to skeletal muscle. 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Science, 245, 1118–1121. . skeletal muscle mass and a decrease in force-producing capacity, in both fast and slow muscles. Interestingly, the muscle mass, fibre size, and force-producing capacity of EDL and soleus muscles. signalling in skeletal muscle and implicated this pathway as a potential target for the treatment of age-related muscle wasting and weakness. Although we are only beginning to understand the significance. increased EDL muscle mass and the force producing capacity of both EDL and soleus muscles, without a concomitant increase in heart mass. The hypertrophy and increased force of EDL muscles persisted