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Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, Victoria, Australia.
Address correspondence to Gordon S. Lynch, PhD, Department of Physiology, The University of Melbourne, Melbourne, Victoria, 3010 Australia. E-mail: gsl{at}unimelb.edu.au
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While ß2-adrenoceptor agonists (ß2-agonists) are used primarily for the treatment of asthma, they also have anabolic effects on skeletal muscle when administered in millimolar doses (5–11). Previous studies examining the potential of these "old" generation ß2-agonists for treating muscle wasting and weakness have found that, although they elicit beneficial skeletal muscle hypertrophy, their therapeutic use is limited by detrimental effects such as cardiac hypertrophy. The "new" generation ß2-agonists, formoterol and salmeterol, have been shown to exert significant anabolic actions on skeletal muscle at micromolar doses (11). Formoterol elicited skeletal muscle hypertrophy at a lower dose than salmeterol, and was more selective for skeletal muscle than for the heart (11,12). These studies suggested that a dose of only 25 µg/kg/day of formoterol might ameliorate muscle wasting and weakness associated with aging without detrimental effects on the heart usually observed with other (older generation) ß2-agonists (10,13).
If formoterol is to find therapeutic application for treating age-related muscle wasting and weakness, it is important to determine the anabolic effects of administration as well as the effects of formoterol withdrawal on skeletal muscle structure and function. This is important for two reasons: first, to determine the duration of the hypertrophic response after the cessation of treatment; and second, to determine whether there are any potentially deleterious effects when treatment has concluded. Previous studies in pigs and cattle have found that after 1 or 2 months of ß-agonist administration, withdrawal resulted in either a return to pretreatment body mass, or a decrease in body mass to below that of age-matched controls (14–16). Another study, by Carta
à and colleagues (17), examined the effect of 10 days of clenbuterol administration to rats (2 mg/kg in the feed), followed by 10 days of withdrawal. They found that clenbuterol increased the mass of gastrocnemius and soleus muscles, and although withdrawal of clenbuterol resulted in soleus muscle mass returning to control levels, gastrocnemius muscle mass after treatment remained higher than control (17). These results suggest that the catabolic response to ß2-agonist withdrawal differs between predominantly fast- and slow-twitch skeletal muscles.
The aims of this study were to determine whether a low (clinically relevant) dose of formoterol could reverse the age-related changes in skeletal muscle without eliciting cardiac hypertrophy. In another group of rats, we determined what effect, if any, 4 weeks of formoterol withdrawal had on skeletal muscle structure and function, and heart mass.
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MATERIALS AND METHODS |
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Young (3 months of age, n = 32), adult (16 months of age, n = 32), and old (27 months of age, n = 32) male F344 rats obtained from Harlan Sprague Dawley (Indianapolis, IN) were allocated randomly to a control (n = 16 rats per age) or formoterol-treated group (n = 16 rats per age). All rats were housed in a pathogen-free environment in standard cages, and were provided with standard laboratory diet (rat chow) and water ad libitum. At 3 months of age, young rats undergo considerable growth and development, whereas at 16 months of age adult rats are weight stable, have completed all growth and development, and (most importantly) do not exhibit muscle wasting or weakness (18). In contrast, after the age of 24 months, old F344 rats exhibit significant muscle atrophy and a lower force-producing capacity, characteristic of age-related muscle wasting and weakness (19).
Treated rats received formoterol at 25 µg/kg (Astra-Zeneca, Molndal, Sweden) administered via i.p. injections in 0.5 mL of isotonic saline every day for 4 weeks. Control rats received a daily injection of 0.5 mL of saline vehicle only. At the completion of the 4-week treatment period, half of the total number of rats were used to examine in vitro contractile properties of the extensor digitorum longus (EDL) and soleus hind-limb muscles, and the other half were monitored for a further 4 weeks without any treatment (designated as the control and formoterol withdrawal groups, respectively). Following the withdrawal period, the in vitro contractile parameters of the EDL and soleus muscles of these rats were also examined. Rats in the withdrawal groups were 4 weeks older than the 4-week treatment group at the time of the functional analyses.
In Vitro Contractile Analysis
At the completion of the 4-week treatment and withdrawal periods, rats were anesthetized with pentobarbital sodium (60 mg/kg, i.p.), with supplemental doses administered to maintain an adequate depth of anesthesia, such that there was no response to tactile stimulation. The EDL and the soleus muscles from the left hind limb were surgically excised and placed in a custom-built Plexiglas organ bath for analysis of contractile function in vitro, as described previously (9,10). Briefly, EDL and soleus muscles were stimulated by supramaximal square wave pulses (0.2 ms duration) that were amplified (Ebony, dual channel power amplifier EP500B; Audio Assemblers, Campbellfield, Victoria, Australia) to increase and sustain current intensity at a sufficient level to produce a maximum isometric tetanic contraction (Po). All stimulation parameters and contractile parameters were controlled and measured using custom-written software (D.R. Stom Software Solutions, Ann Arbor, MI) of Labview software (National Instruments, Austin, TX).
Isometric contractions were measured at stimulation frequencies of 10, 20, 30, 50, 80, 100, 120, and 150 Hz for EDL muscles and 5, 10, 15, 20, 30, 50, 80, and 100 Hz for soleus muscles to generate a frequency–force relationship. Po was determined by the plateau of the frequency–force relationship.
Following determination of Po, muscle fatigability was determined by stimulating each muscle maximally once every 5 seconds for a period of 4 minutes, with Po recorded at 60-second intervals. At the completion of the fatigue protocol, the muscle was rested and its ability to recover from fatigue was determined from a maximal tetanic contraction at 5, 10, and 15 minutes postfatigue.
At the conclusion of the contractile measurements, the muscles were trimmed of connective tissue, blotted once on filter paper, and weighed on an analytical balance. Muscles were frozen in thawing isopentane for later histological and histochemical examination. Rats were killed by rapid surgical excision of the heart while still anesthetized deeply. The heart was then trimmed of all connective tissue, blotted once on filter paper, and weighed.
Histology and Histochemistry
To examine the effect of low-dose formoterol treatment on the structural and biochemical properties of skeletal muscle, a portion of each muscle sample was cryosectioned transversely through the midbelly region on a cryostat microtome at –20°C. Serial (8 µm thick) sections of each muscle were placed onto uncoated glass microscope slides, with one EDL and one soleus muscle section on each slide. The sections were reacted for myosin ATPase (mATPase) activity for determination of muscle fiber type proportions and cross-sectional area (CSA). Briefly, sections were preincubated (in mM; sodium acetate 100, sodium barbital 30, and HCl 50) for 5 minutes at either pH 4.3 or pH 4.55, followed by a series of washes, reactions, and subsequent coloration steps as described previously (9,10,20,21). At the completion of these steps, a stable cobaltous sulfide precipitate was evident within individual fibers of the section according to mATPase activity, as regulated by the pH of the preincubation solution. The mean CSA of individual muscle fibers was calculated by interactive determination of the circumference of no less than 120 adjacent fibers from the center of every muscle section.
Statistical Analyses
Individual variables from control and formoterol-treated, and control and formoterol withdrawal groups of young, adult, and old rats were compared using a two-way analysis of variance and Fisher's least significant differences (LSD) post hoc multiple comparison procedure where significance was detected. Significance was set at p <.05. All values are expressed as mean ± standard error of the mean (SEM) unless specified otherwise.
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RESULTS |
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Formoterol treatment was associated with a significantly greater gain in body mass in adult rats than in control rats of the same age (p <.05, Figure 1B). It increased EDL muscle mass (23%, p <.05), but not soleus muscle mass. In adult rats, formoterol treatment increased the EDL mass-to-body mass ratio by 23% and the soleus mass-to-body mass ratio by 7%. Associated with the increase in EDL muscle mass in adult rats was a 44% increase in fiber CSA (p <.05). Formoterol treatment did not alter mean fiber CSA in the soleus muscles (Table 1).
Formoterol treatment prevented the loss of body mass observed in old control rats, and was associated with a 50% increase in EDL muscle mass, such that the EDL mass-to-body mass ratio was increased by 50% (p <.05, Table 1). In contrast, formoterol treatment did not alter soleus muscle mass or the soleus mass-to-body mass ratio. Formoterol treatment increased EDL and soleus mean fiber CSA by 83% and 25%, respectively (p <.05).
Absolute heart mass of adult rats was 27% greater than that of young rats (p <.05), but not different than that of old rats. Thus, when corrected for changes in body mass, the heart mass-to-body mass ratio was reduced by 12% in adult rats compared with young rats (p <.05), whereas the heart mass-to-body mass ratio in old rats was 19% greater than that observed in adult rats (p <.05). Formoterol treatment increased heart mass in young rats in absolute terms (17%) and when normalized for body mass (15%, Table 1). Importantly, formoterol treatment did not alter heart mass or heart mass-to-body mass ratio in either adult or old rats.
EDL and Soleus Muscle Fiber Type and CSA
Compared with those in adult rats, muscle fiber type proportions were not different in the EDL muscles from young rats, but old rats had an increased proportion of type I and IId/x fibers, with a concomitant decrease in type IIb fibers compared to adult rats (p <.05, Figures 2 and 3). Compared with soleus muscles from young rats, adult rats had similar fiber-type proportions, whereas old rats had an increased proportion of type I fibers (p <.05) and a concomitant decrease in type IIa and IId/x fiber proportions (Figures 4 and 5). Compared with young rats, adult rats exhibited a 14% increase in the CSA of type IIb fibers in the EDL muscles (p <.05) and a 24% decrease in the size of type IId/x fibers in the soleus muscles (p <.05). Compared with EDL muscle fibers from adult rats, old rats exhibited selective atrophy of type IId/x and IIb muscle fibers (44% and 42%, Figure 3). All soleus muscle fibers were smaller in old rats than in adult control rats (p <.05).
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Formoterol treatment resulted in a decreased proportion of type I fibers in soleus muscles from young and adult rats (p <.05) and a concomitant increase in the proportion of type IIa fibers. However, fiber proportions were unaltered in soleus muscles from old rats after formoterol treatment (Figure 5). The proportion of type IId/x fibers in soleus muscles of young, adult, and old rats was unaltered by treatment. Formoterol treatment tended to increase the CSA of type II fibers across all age groups, while an increase in type I fiber CSA in soleus muscles was observed in young and old (p <.05), but not adult rats (Figure 5).
In Vitro Contractile Parameters of EDL and Soleus Muscles
Compared with young rats, adult rats had an increased peak twitch force (Pt) in the EDL muscle (25%, p <.05, Table 2), but Pt was unchanged in soleus muscles. Compared with adult rats, old rats exhibited a 36% decrease in EDL Pt, but soleus Pt was unchanged. The time course of the isometric twitch (time to peak twitch force, TPT) and one-half relaxation time (
RT, Table 2) tended to be prolonged in both the EDL and the soleus muscles of adult and old rats compared with young rats. Po of EDL and soleus muscles was 11% and 15% higher in adult rats, compared with young rats (Figure 6A). Compared with adult rats, old rats exhibited a decrease in Po of both the EDL and soleus muscles by 37% and 36%, respectively (p <.05).
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RT were unchanged (Table 2). Formoterol treatment increased Pt in the soleus muscles from adult and old rats (12% and 28%, respectively; p <.05), but not from young rats. In both young and adult rats, formoterol treatment tended to hasten the time course of the twitch (TPT and RT), and in adult rats this was associated with a 19% increase in the rate of Pt force development (+dP/dt). Formoterol did not alter the TPT or RT in soleus muscles from old rats. Formoterol treatment was associated with a significant increase in Po in the EDL muscle from young, adult, and old rats (12%, 13%, and 31%, respectively; Figure 6A). Soleus Po was increased by formoterol treatment in young and old, but not in adult rats (12% and 39%, respectively; Figure 6A).
There was no difference in fatigue resistance of the EDL muscle between young rats and adult rats, but in old rats, muscles fatigued less and had an improved rate of recovery compared with those of adult rats (p <.05, Figure 7A). Aging had no significant effect on the development of fatigue or the rate of recovery in soleus muscles (Figure 7A).
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Morphology of EDL and Soleus Muscles Following Formoterol Withdrawal
In young rats, formoterol withdrawal resulted in a decrease in body mass to control levels (Figure 1A), such that after 28 days of withdrawal there was no difference in body mass compared to young control rats (Table 3). EDL muscle mass remained higher than control values in formoterol-treated rats following 4 weeks of withdrawal (8%, p <.05), whereas the mass of the soleus muscle returned to control levels (Table 3). Body mass-to-muscle mass ratios were not different after withdrawal of formoterol. Absolute heart mass and heart mass-to-body mass ratios were also not different between control and formoterol withdrawal groups.
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In old rats, withdrawal of formoterol was associated with a rapid decline in body mass such that after 4 weeks body mass was not different from control rats (Figure 1C). Absolute EDL muscle mass and EDL mass-to-body mass ratios remained above control levels (44% and 34%, respectively; p <.05). Absolute soleus muscle mass tended to be higher in the old formoterol withdrawal group than control (p =.07). However, when corrected for body mass there was no difference between the two groups (Table 3). Similar to those in young and adult rats, heart mass and heart mass-to-body mass ratio in old rats were not different after formoterol withdrawal.
In Vitro Contractile Parameters Following Formoterol Withdrawal
In young rats, Po of EDL muscles was 12% higher in the formoterol withdrawal group than in control rats (p <.05), whereas in the soleus muscle, no difference in Po was observed between young formoterol withdrawal and control rats (Figure 6B).
Similar to that from young rats, Po of EDL muscles from adult rats following formoterol withdrawal remained higher than that from adult control rats (8%, p <.05), whereas in the soleus muscle Po was not different from controls (Figure 6B).
As with formoterol withdrawal in both young and adult rats, Po of EDL muscles from old rats remained higher than control values (33%, p <.05), whereas in the soleus muscle there was no difference in Po (Figure 6B).
Following 4 weeks of formoterol withdrawal, there was no difference in the fatigability or recovery of EDL or soleus muscles from young, adult, or old rats (data not shown).
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DISCUSSION |
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While previous studies suggested potentially deleterious effects of the withdrawal of ß-agonist treatment on skeletal muscle mass (14–16), our findings show that the hypertrophy and increased force-producing capacity of fast-twitch muscles was still evident 4 weeks after cessation of formoterol treatment. In contrast, the hypertrophy and increased force-producing capacity of the slow-twitch skeletal muscle had reverted to control levels. In addition, the cardiac hypertrophy that was observed solely in formoterol-treated young rats was completely abolished 4 weeks after the withdrawal of treatment. These results indicate that the cardiac hypertrophy associated with formoterol administration in young rats is completely reversible, whereas the changes to fast-twitch skeletal muscles are longer lasting. Further research is required to determine how long the anabolic actions of formoterol persist in fast-twitch skeletal muscle.
We have shown previously that fenoterol treatment increases EDL and soleus muscle mass and force-producing capacity in old rats back to values for adult rats (10). In the present study, low-dose formoterol treatment increased EDL muscle mass and force-producing capacity, and to a lesser extent, soleus force-producing capacity, but did not restore these parameters to adult control levels, an effect likely associated with the lower dose used in the present study. Most importantly, formoterol treatment was not associated with cardiac hypertrophy. Thus, although the magnitude of the increase in force-producing capacity in both the fast- and slow-twitch skeletal muscles after low-dose formoterol treatment was not as great as that after (high-dose) fenoterol treatment, we have demonstrated that a clinically relevant low-dose formoterol administration protocol can confer significant functional benefits without detrimental cardiac hypertrophy (10,11,13).
It was interesting to note that, although fast-twitch skeletal muscles from old rats were equally responsive to anabolic treatment as were those from young and adult rats, the anabolic response in slow-twitch skeletal muscles was reduced in adult rats, and increased in old rats. A similar response was observed in the heart. These findings suggest that the ß-adrenergic signalling pathways leading to striated muscle hypertrophy differ quite significantly between fast- and slow-twitch skeletal muscles and the heart. As discussed previously (9–11), differences in ß-adrenergic responsiveness do not simply reflect differences in ß-adrenoceptor density, as it has been shown that ß-adrenoceptor density is greater in the soleus than in the EDL muscle (9,22). One possibility yet to receive significant attention relates to potential differences in G
-protein coupling between skeletal and cardiac muscle.
The ß-adrenoceptor population in the slow-twitch soleus muscle has been shown to couple to both the Gs- and Gi-proteins, whereas the fast-twitch EDL muscle has been found to only couple to the Gs-protein (23,24). Gosmanov and colleagues (23) showed that incubation of the soleus muscle with the selective Gi-protein inhibitor, pertussis toxin, reduced the isoproterenol-induced increase in extracellular signal-regulated kinase (ERK) phosphorylation (23). The same response was not observed when the fast-twitch plantaris muscle was incubated with pertussis toxin. This difference in G-protein coupling could result in the inhibition of adenylate cyclase activity in the soleus muscle and thus reduce the cAMP-mediated hypertrophy. In support of this mechanism is the finding that aging is associated with an increase in Gi-protein content in the heart, which is associated with a decrease in adenylate cyclase activity (25–27).
A second potential mechanism for the observed differences in ß-adrenergic responsiveness relates to the involvement of the Gß
subunits in downstream hypertrophic signaling. A recent study by Kline and colleagues (28) demonstrated that the ß2-agonist clenbuterol elicits skeletal muscle hypertrophy, in part, through the activation of Akt and mammalian target of rapamycin (mTOR) signalling pathways (28). These exciting results demonstrate the need for further research to determine the differences in ß-adrenergic-mediated skeletal muscle hypertrophy between fast- and slow-twitch muscles.
Unlike the high millimolar dose of fenoterol administered to rats in a previous study (10,29), the micromolar dose of formoterol used in the present study was sufficient to increase the proportion of type II muscle fibers in both EDL and soleus muscles at all ages studied. This shift in fiber type proportions was associated with a decrease in twitch relaxation time in EDL and soleus muscles in old rats and with a hastened rate of contraction in young, adult, and old rats. Such an effect would be beneficial for sarcopenia, as the age-related slowing of movements contributes to the increase in falls and fall-related injuries (1). Clearly, treatment with a micromolar dose of formoterol is as capable of eliciting similar (or greater) improvements in skeletal muscle contractile properties as a milligram dose of fenoterol (10).
ß2-agonists have traditionally been associated with increased muscle fatigability (9,10,30). In the present study, low-dose formoterol treatment increased the susceptibility of the EDL muscle to fatigue in young, adult, and old rats. However, unlike what has been observed with other ß2-agonists, formoterol treatment did not alter soleus muscle fatigability at any age (10). Thus, although an increase in fatigability remains a slight impairment to overall function, this impairment was limited to fast-twitch skeletal muscle.
The findings also raise important issues for patients who are currently prescribed formoterol for the prevention of asthma (31,32). Although a direct comparison between the dose used in the present study and that used for the treatment of asthma is not feasible, it is interesting to note that the absolute dose administered (25 µg/kg/day, or 5 µg/day total) is well below that administered for the treatment of asthma (up to 98 µg/day) (31). To date, no clinical study has examined whether the dose of formoterol used by asthmatics is associated with changes in muscle size or force-producing capacity. Thus, it is possible that a drug (formoterol) that is currently in clinical use for the treatment of asthma could be prescribed at a similar dose for the treatment of severe age-related muscle wasting and weakness. These exciting findings demonstrate a clear therapeutic potential of formoterol for sarcopenia. However, the cardiac hypertrophy observed in young rats remains a concern, and any possible detrimental effects of formoterol on cardiac function must be closely examined before the true therapeutic potential of formoterol can be realized.
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We thank Astra-Zeneca, Inc., Molndal, Sweden, for providing formoterol.
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Received January 28, 2007
Accepted March 22, 2007
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à J, Segues T, Yebras M, Rothwell NJ, Stock MJ. Anabolic effects of clenbuterol after long-term treatment and withdrawal in the rat. Metabolism. 1994;43:1086-1092.[Medline] expression in senescence. Am J Physiol Heart Circ Physiol. 1993;264:H805-H814.(i2) expression, resulting in enhanced coupling to G protein-coupled receptors. J Biol Chem. 2002;277:31257-31262.
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p <.05 versus preceding age control; 
p <.05, old formoterol versus adult control
