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. If successful, these beneficial effects of b-adrenoceptor
stimulation on skeletal muscle would find application for treating sarcopenia,
where muscle wasting impacts not only upon the ability to perform the tasks of
daily living, and quality of life, but ultimately on life itself, since the maintenance
of functional muscle mass is critical for survival.
Acknowledgments Supported by research grants from the National Health & Medical Research
Council (NHMRC, Australia; project grant 509313) and the Association Française contre les
Myopathies (France). JGR is supported by a Biomedical Overseas Research Fellowship from the
National Health and Medical Research Council of Australia (520034).
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