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The β2-adrenoceptor agonist (β2-agonist) fenoterol has potent anabolic effects on rat skeletal muscle. We conducted an extensive dose–response study to determine the most efficacious dose of fenoterol for increasing skeletal muscle mass in adult rats and used this dose in testing the hypothesis that fenoterol may have therapeutic potential for ameliorating age-related muscle wasting and weakness. We used adult (16-month-old) rats that had completed their growth and development, and old (28-month-old) rats that exhibited characteristic muscle wasting and weakness, and treated them daily with either fenoterol (1.4 mg kg−1, i.p), or saline vehicle, for 4 weeks. Following treatment, functional characteristics of fast-twitch extensor digitorum longus (EDL) and predominantly slow-twitch soleus muscles of the hindlimb were assessed in vitro. Untreated old rats exhibited a loss of skeletal muscle mass and a decrease in force-producing capacity, in both fast and slow muscles, compared with adult rats (P < 0.05). However, there was no age-associated decrease in skeletal muscle β-adrenoceptor density, nor was the muscle response to chronic β-agonist stimulation reduced with age. Thus, muscle mass and force-producing capacity of EDL and soleus muscles from old rats treated with fenoterol was equivalent to, or greater than, untreated adult rats. The increase in mass and strength was attributed to a non-selective increase in the cross-sectional area of all muscle fibre types, in both the EDL and soleus. Fenoterol treatment caused a small increase in fatiguability due to a decrease in oxidative metabolism in both EDL and soleus muscles, with some cardiac hypertrophy. Further studies are needed to fully separate the desirable effects on skeletal muscle and the undesirable effects on the heart. Nevertheless, our results demonstrate that fenoterol is a powerful anabolic agent that can restore muscle mass and strength in old rats, and provide preliminary evidence of therapeutic potential for age-related muscle wasting and weakness.
Ageing is associated with a progressive loss of skeletal muscle mass (sarcopenia) and a subsequent decline in muscle strength (Brooks & Faulkner, 1994; Larsson & Ramamurthy, 2000; Morley et al. 2001). Over the past two decades much research has focused on the underlying mechanisms of age-related effects on skeletal muscle, responsible for the gradual loss of functional independence amongst the elderly. Progressive muscle fibre denervation, a loss of motor units, and potential motor unit remodelling have been implicated, but since the slowing of contraction occurs before significant muscle wasting, intrinsic changes to skeletal muscle fibres, including excitation–contraction coupling, cannot be ruled out (Faulkner et al. 1995; Larsson, 1995; Larsson & Ansved, 1995; Roos et al. 1997; Plant & Lynch, 2002).
Developing therapeutic interventions to prevent or reverse the age-related decline in function is of increasing importance for two reasons. First, many elderly people require the use of all their muscle strength to complete simple tasks such as rising from a chair, and any further impairment in muscle function (such as that following extended bed rest after surgery) can result in a loss of functional independence (Larsson & Ramamurthy, 2000). Secondly, as the proportion of the elderly within the population continues to escalate worldwide, so does the associated socio-economic impact of age-related frailty and weakness, placing an increasing burden on the healthcare system (Larsson & Ramamurthy, 2000). There is a profound need for strategies that can ameliorate the effects of ageing on muscle structure and function, and thus restore functional independence (Lynch, 2002).
Associated with normal ageing is a decrease in the circulating levels of anabolic hormones, including, but not limited to: growth hormone (GH), insulin, insulin-like growth factor I (IGF-I), and testosterone (Janssens & Vanderschueren, 2000). These hormonal changes are thought to be responsible, at least in part, for the age-related loss of muscle mass and strength. Numerous clinical studies have tried increasing the circulating levels of one or more of these hormones, with the aim of increasing muscle mass and strength. However, to date, hormone replacement therapy has produced mixed success in humans (Janssens & Vanderschueren, 2000). Studies reporting on the effects of testosterone and testosterone precursor supplementation on muscle mass and strength have produced equivocal results in elderly human subjects (Morley et al. 1993; Sih et al. 1997; Snyder et al. 1999). Whereas testosterone has been shown to increase muscle strength in hypogonadal elderly men, testosterone administration to elderly men with normal testosterone levels was associated with an increased risk of polycythemia (Morley et al. 1993; Drinka et al. 1995). Furthermore, testosterone treatment for elderly women may be inappropriate due to the masculinizing effects of androgens.
Several studies have postulated that GH and (or) IGF-I administration may prevent the muscle wasting associated with ageing. To date, GH supplementation has been shown to alter body composition by decreasing body fat mass and increasing lean body mass in elderly men. However, GH did not augment muscle strength or produce muscle hypertrophy (Lange et al. 2002). The administration or up-regulation of IGF-I has proven more promising in a number of animal models of pathologies where muscle wasting is indicated (Barton-Davis et al. 1998; Gregorevic et al. 2002), but there is concern that elevated levels of IGF-I may be implicated in tumour formation (Adams, 2000). Furthermore, both GH and IGF-I are expensive therapies that must be given daily. These findings suggest that hormone replacement alone has limited therapeutic efficacy for treating sarcopenia in the frail elderly. In the absence of successful hormone replacement therapies, muscle anabolic agents have been used in an attempt to treat sarcopenia.
Although traditionally administered locally (by inhalation) at low doses for bronchodilatation in the treatment of asthma, when given at higher doses, systemically, β2-adrenoceptor agonists (β2-agonists, such as the most widely described, clenbuterol) have potent anabolic effects in healthy muscle (Emery et al. 1984). Their experimental use in the treatment for muscle wasting conditions has also yielded promising results (Maltin et al. 1993; Sneddon et al. 2000). Carter et al. (1991), administered clenbuterol at a dose of 1.5 mg kg−1 day−1 subcutaneously for 22 days to 3, 12 and 23-month-old Fischer 344 (F344) rats. The clenbuterol-induced increase in muscle mass was equivalent in 23- and 12-month-old rats (Carter et al. 1991), supporting the hypothesis that β2-agonists might be effective for ameliorating sarcopenia. However, when administered at a much lower dose (10 μg kg−1 day−1), clenbuterol caused only a modest attenuation in the loss of muscle mass and strength associated with hindlimb suspension in the slow-twitch soleus muscle, and did not affect muscle mass or force of the fast-twitch plantaris muscle, in old (38 month) F344 × Brown Norway F1 rats (Chen & Alway, 2000). Therefore, the use of a more potent β2-agonist, and (or) a higher dose, may be necessary for treating sarcopenia.
We recently reported that at an equimolar dose to clenbuterol, fenoterol has a 10–15% greater anabolic effect on rat fast- (EDL) and slow- (soleus) twitch skeletal muscle (Ryall et al. 2002). Therefore, we chose to investigate whether fenoterol may have therapeutic potential for treating sarcopenia. To this end, we conducted an extensive dose–response examination in adult rats to determine the dose of fenoterol that would produce a maximal increase in the mass of EDL and soleus muscles. We hypothesized that at this dose, fenoterol treatment would attenuate the age-related decline in skeletal muscle mass and strength in old F344 rats, specifically by causing hypertrophy of muscle fibres.
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The most important findings of this study were that skeletal muscles from old rats were as responsive to anabolic treatment as adult rats, and that 4 weeks of fenoterol treatment can counteract the atrophy and weakness associated with sarcopenia. Our dose–response investigation determined that the most efficacious dose of fenoterol for increasing fast and slow skeletal muscle mass was between 1.0 and 2.0 mg kg−1 day−1. We found that the mass and force-producing capacity of fast- and slow-twitch skeletal muscles from old rats treated for 4 weeks with fenoterol (1.4 mg kg−1 day−1) was equal or greater than that of adult rats that exhibited no signs of sarcopenia, providing support for the hypothesis that fenoterol can reverse the muscle atrophy and weakness concomitant with ageing.
Both fast- and slow-twitch skeletal muscles from old rats exhibited the characteristics of sarcopenia: a loss of muscle mass and strength. All fibre types exhibited atrophy in the EDL muscles of old rats, the greatest atrophy observed in type IIa and IId/x fibres, which is consistent with previous findings (Brooks & Faulkner, 1994; Larsson & Ramamurthy, 2000). However, the loss of mass and strength cannot be attributed solely to muscle fibre atrophy, as there was also a decrease in sPo in EDL muscles, when fibre CSA was taken into account. Thus, the loss in mass and strength is a likely result of both fibre loss and atrophy, as well as other intrinsic factors (Brooks & Faulkner, 1994; Larsson & Ansved, 1995; Plant & Lynch, 2002).
The increase in muscle mass and strength after fenoterol treatment was explained directly by the increase in fibre CSA. Although sPo values were higher in both adult and old animals than has been reported previously (Lynch et al. 2001), the deficit in sPo observed in old rats, persisted in muscles of treated aged rats, despite the significant improvement in absolute force-producing capacity. From a physiological perspective and applying these findings to a clinical situation, an improvement in absolute force has greater implications for carrying out the tasks of daily living than changes in force per muscle cross-sectional area. The reduced sPo of the EDL muscles from old rats was not associated with increased collagen localization within the muscle, and may be explained by the presence of other non-contractile material such as fat and other connective tissue.
The loss of muscle mass and strength that occurred with ageing was associated with a slowing of the time course of contraction. Slowing of movement is a serious problem in the elderly, since it increases the susceptibility to fall-related injuries (Larsson & Ramamurthy, 2000). We also demonstrated that the slowing of contraction in EDL and soleus muscles from old rats could not be attributed to an increase in the proportion of slow type I muscle fibres. Rather, as we have demonstrated previously, impaired excitation–contraction coupling and decreased function of calcium release from the sarcoplasmic reticulum, also contribute to the age-related slowing of contraction (Plant & Lynch, 2002). In old rats, fenoterol treatment was associated with a faster isometric twitch response in the EDL muscle. Such an adaptation might be advantageous for the elderly since the ability to perform more rapid movements might help in avoidance behaviours that prevent falls and subsequent injury.
The dose of fenoterol used in this study (1.4 mg kg−1 day−1) was half that used previously (Ryall et al. 2002), but the increases in muscle mass and strength were similar in magnitude. Even at this lower dose, the mass of the EDL muscles from fenoterol-treated old rats far exceeded that of adult (untreated) rats, indicating the potential for fenoterol to actually reverse the muscle wasting associated with ageing. Thus, a dose capable of restoring mass and strength to muscles in the elderly could be within the estimated safe limits for humans (Carter & Lynch, 1994). Even a modest (∼10%) increase in strength could dramatically improve the quality of life for the elderly. We have shown that even at a low dose of 0.025 mg kg−1 day−1, fenoterol increased absolute mass of the EDL and soleus muscles in adult rats (by ∼5–10%). These findings support the notion that for the elderly it would be possible to produce a desirable increase in skeletal muscle mass even at a low dose of this β-agonist. Further examination of the dose–response relationship of fenoterol and muscle mass in old rats is warranted in order to understand the therapeutic utility of fenoterol.
The EDL muscles from both adult and old rats treated with fenoterol were more prone to fatigue, and did not recover from fatigue as well as age-matched controls. In contrast, ageing had no effect on fatigue development and only a minor effect on recovery in soleus muscles. The greater susceptibility to fatigue and impaired recovery in fenoterol-treated rats could be attributed to the reduced oxidative capacity of these muscles, evidenced by the greater proportion of fibres with low SDH activity in both the EDL and soleus muscles of adult treated rats. Whilst such effects of fenoterol are not beneficial, in practice the advantages of a greatly improved force-producing capacity and increased speed of contraction in treated old rats are likely to outweigh the slight increase in fatigue following repeated maximal contractions, in terms of clinical significance. However, further studies are needed to fully separate the potentially desirable and undesirable effects of fenoterol on skeletal muscle.
Previous studies have found an age-associated decrease in β-adrenoceptor sensitivity and density in the heart (Lakatta & Sollott, 2002), which has been attributed to down-regulation and impaired coupling of β-adrenoceptors to adenyl cyclase (Xiao et al. 1998). In contrast, β−adrenoceptor density in both fast and slow skeletal muscle is not reduced with ageing (Larkin et al. 1996; Farrar et al. 1997), as confirmed in the present study. The present results also show that the responsiveness of skeletal muscle β-adrenoceptors to chronic β-agonist treatment is not reduced in old compared with adult rats. Furthermore, our findings indicate that β-adrenoceptors in EDL muscles are more susceptible to down-regulation than β-adrenoceptors in soleus muscles, during β-agonist administration. However, the greater extent of adrenoceptor down-regulation in the EDL muscle was not associated with a reduced physiological response, as evidenced by the equivalent increases in EDL and soleus muscle mass. The differential β-adrenoceptor response might be explained by a greater level of desensitization in the soleus muscle with chronic β2-agonist treatment. Agonist-dependent desensitization is initiated by phosphorylation of the receptor by receptor kinases, which become a target for arrestin proteins, inhibiting further G-protein coupling (Claing et al. 2002). We have also demonstrated that the adrenoceptor population in the EDL muscles of old rats is less susceptible to down-regulation than in EDL muscles of adult rats. The higher adrenoceptor density in the EDL muscles of old rats was not associated with a greater increase in mass or force-producing capacity, confirming previous reports that relative adrenoceptor density does not correlate with agonist-induced increases in muscle mass and function (Ryall et al. 2002). Further investigation is warranted into the differences in adrenergic signalling between fast- and slow-twitch skeletal muscles, and the effect of ageing.
Associated with high-dose administration of a β2-agonist in adult rats is cardiac hypertrophy (Duncan et al. 2000). We have shown an equivalent increase in heart mass in adult and old rats following fenoterol treatment. Whilst the increases in skeletal muscle mass and strength of old rats could be of clear benefit to an elderly person, the associated increase in heart mass remains a limiting factor for immediate clinical application. When given in high doses to animals, other β2-agonists, such as clenbuterol, have been shown to increase resting heart rate for the first few days of administration. This tachycardia is largely preventable in cattle with coadministration of a β1-adrenoceptor antagonist (CGP20712A), which would be unlikely to counteract the beneficial effects of β-agonist administration on skeletal muscle (Hoey et al. 1995). We have shown that at a low dose of 0.025 mg kg−1 day−1, fenoterol did not increase heart mass significantly in adult rats (P > 0.05) but there was still evidence of a hypertrophic effect, with cardiac size being increased by ∼5%. Our dose–response data indicate that at the lowest dose (0.025 mg kg−1 day−1), cardiac hypertrophy was only one-fifth that observed following 1.4 mg kg−1 day−1, with a greatly reduced effect on cardiac mass. Further studies that examine whether low dose treatment with such powerful β-agonists have similar effects in old rats are warranted. Whether low dose β-agonist treatment affects cardiac function deleteriously also deserves further attention, including whether any tachycardia can be prevented effectively with a β1-adrenoceptor antagonist.
Further investigation into the β-agonist-stimulated pathways leading to cardiac hypertrophy and the effects of chronic β2-agonist administration on cardiac function is essential for the continued development of this approach for tackling sarcopenia.
The limitations of this study were that following our extensive dose–response examination of the effects of fenoterol on skeletal and cardiac muscle in adult rats, we only tested the effects of a single dose of fenoterol on the skeletal muscles of old rats. Additional dose–response studies are required to fully understand the mechanisms of fenoterol's actions on skeletal and cardiac muscle, to identify potential advantages of fenoterol over other β−agonists, such as clenbuterol, and to separate the effects of these β-agonists on skeletal and cardiac muscle. Taken together, our results demonstrate that the skeletal muscle response of adult and old rats following fenoterol treatment (at a single dose of 1.4 mg kg−1 day−1) is similar, and that 4 weeks of treatment can ameliorate the age-associated loss of skeletal muscle mass and function. These exciting preliminary findings indicate that β-agonists have a definite application for treating sarcopenia, but further research is needed to separate their desirable effects on skeletal muscle from any undesirable effects on the heart, so as to optimize their therapeutic potential.