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Beta-agonists and glucocorticoids are frequently coprescribed for chronic asthma treatment. In this study the effects of 4 week treatment with beta-agonist clenbuterol (CL) and glucocorticoid dexamethasone (DEX) on respiratory (diaphragm and parasternal) and limb (soleus and tibialis) muscles of the mouse were studied. Myosin heavy chain (MHC) distribution, fibres cross sectional area (CSA), glycolytic (phosphofructokinase, PFK; lactate dehydrogenase, LDH) and oxidative enzyme (citrate synthase, CS; cytochrome oxidase, COX) activities were determined. Muscle samples were obtained from four groups of adult C57/B16 mice: (1) Control (2) Mice receiving CL (CL, 1.5 mg kg−1 day−1 in drinking water) (3) Mice receiving DEX (DEX, 5.7 mg kg−1 day−1s.c.) (4) Mice receiving both treatments (DEX + CL). As a general rule, CL and DEX showed opposite effects on CSA, MHC distribution, glycolytic and mitochondrial enzyme activities: CL alone stimulated a slow-to-fast transition of MHCs, an increase of PFK and LDH and an increase of muscle weight and fibre CSA; DEX produced an opposite (fast-to-slow transition) change of MHC distribution, a decrease of muscle weight and fibre CSA and in some case an increase of CS. The response varied from muscle to muscle with mixed muscles, as soleus and diaphragm, being more responsive than fast muscles, as tibialis and parasternal. In combined treatments (DEX + CL), the changes induced by DEX or CL alone were generally minimized: in soleus, however, the effects of CL predominated over those of DEX, whereas in diaphragm DEX prevailed over CL. Taken together the results suggest that CL might counteract the unwanted effects on skeletal muscles of chronic treatment with glucocorticoids.
The study of the interaction between beta-agonists and glucocorticoids is of great interest because these two classes of compounds are the most prescribed drugs for chronic asthma treatment and coprescribing frequently occurs (NIHLBI/WHO, 2002). The interaction between the two drugs at the cellular level has been recently reviewed (Taylor & Hancox, 2000). Exogenous and endogenous glucocorticoids enhance the effect of beta-agonists on cardiac contractility, smooth muscle relaxation and glucose metabolism (Davies & Lefkowitz, 1984). This potentiating effect might be due to the prevention of desensitization and down regulation of beta-receptors (Mak et al. 1995b). In fact, GRE (Glucocorticoid Response Element) is present in the promoter region of the gene coding for the beta-2 receptors and glucocorticoids, acting on the response element, can stimulate the receptor expression and thus opposing to down regulation (Mak et al. 1995a). Furthermore, glucocorticoids reduce the phosphorylation of the beta-receptor, which is a determinant of desensitization (Taylor & Hancox, 2000).
The information on the actions of beta-agonists on glucocorticoid-stimulated effects is less clear and in part contradictory: reciprocal inhibition of beta-2-agonists on GRE and of glucocorticoids on CRE (cAMP Response Element) has been reported (Peters et al. 1995; Stevens et al. 1995). On the other hand beta-2-agonists can enhance the anti-inflammatory action of glucocorticoids (Oddera et al. 1998; Pang & Knox, 2000). The clinical effect is generally positive and the effectiveness of asthma control is improved (Taylor & Hancox, 2000).
An aspect, which has received relatively less attention is the interaction between the effects of beta-agonists and glucocorticoids on skeletal muscles. Both classes of compounds are very effective on skeletal muscles, but they exert an opposite effect: beta-agonists promote muscle growth and hypertrophy (Sneddon et al. 2001; Awede et al. 2002) and are used as anabolic drugs to enhance the muscle mass of athletes and farm animals, whereas glucocorticoids induce muscle atrophy and loss of contractile strength (Bodine et al. 2001; Marinovic et al. 2002; Shah et al. 2002). Moreover, both classes of compounds modify the fibre type composition of skeletal muscles: beta-agonists stimulate a slow-to-fast transition (Zeman et al. 1988; Polla et al. 2001), whereas a fast-to-slow transition is determined by chronic glucocorticoid administration (Gardiner & Edgerton, 1979; Polla et al. 1994). The condition characterized by muscle weakness, atrophy and fast-to-slow transition and indicated as ‘steroid myopathy’, is common in patients with adrenocortical dysfunction (Cushing Syndrome) as well as in patients exposed to long lasting treatments with high doses of glucocorticoids (Bowyer et al. 1985; Jansen & Decramer, 1989).
To our knowledge only a few studies have analyzed the effects of combined administration of beta-agonists and glucocorticoids on skeletal muscles and the results are controversial. Agbenyega & Wareham (1992) showed that, in mice, clenbuterol (8 mg (kg body wt)−1 in the diet) counteracts the reduction of muscle mass induced by dexamethasone treatment (5 mg (kg body wt)−1 day−1s.c.). In partial contrast with these results, Jiang and coworkers (Jiang et al. 1996) showed that in rabbits the administration of clenbuterol (2 mg (kg body wt)−1 day−1s.c.) minimizes diaphragm atrophy induced by dexamethasone (3 mg (kg body wt)−1 day−1s.c.), but does not have protective effects on dexamethasone-induced dysfunction of the diaphragm. More recently (Huang et al. 2000) showed that, in rats, administration of clenbuterol (4 mg (kg body wt)−1 in the diet) was not able to fully reverse the muscle growth inhibition caused by dexamethasone (2 mg (kg body wt)−1 day−1s.c.) and dexamethasone did not attenuate the loss of beta-2 adrenoreceptors (down regulation) induced by clenbuterol treatment in skeletal muscles.
In the present study we aimed to assess whether beta-agonist administration antagonizes the effects of glucocorticoid administration in skeletal muscles. To this end we studied the effects of separate and combined administration of clenbuterol, a beta-agonist very effective on skeletal muscle, and dexamethasone (DEX) on murine skeletal muscles. Whereas several studies have described the effects of CL in mouse muscles (Zeman et al. 1994; Dupont-Versteegden et al. 1995; Lynch et al. 1996; Hayes & Williams, 1997), only one study deals with DEX-induced atrophy in mouse muscles (Agbenyega & Wareham, 1992): this study also examines the interaction between CL and DEX, but analysis is restricted to limb muscles and does not consider variations of fibre type composition. Our study was focused on the effects at cellular level, in particular the variations of fibre size (atrophy or hypertrophy) and fibre type (slow-to-fast transitions and viceversa). The characterization of the muscle phenotype was based on: (1) myosin heavy chain (MHC) isoform composition which is considered as a marker of the fibre type (Schiaffino & Reggiani, 1996) and an indicator of energy consumption of the muscle and (2) four different metabolic enzyme activities, two related with anaerobic glycolytic processes and two related with mitochondrial oxidative function. As changes in fibre type require some weeks to arise (Pette & Staron, 1997), the treatment was prolonged to four weeks. Furthermore, the analysis was extended from slow and fast limb muscles to respiratory muscles, taking into account the specific use of beta-agonists and glucocorticoids in respiratory diseases (chronic asthma). The results obtained showed that, with few exceptions, glucocorticoids and beta agonists antagonize each other. Soleus and diaphragm were more responsive to treatments than tibialis and parasternal: whereas the effects of dexamethasone prevailed in the diaphragm, clenbuterol seemed to be more effective on soleus.
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The aim of this study was to assess the long-term effects of the combined administration of the beta-agonist clenbuterol (CL) and the glucocorticoid dexamethasone (DEX) on mouse skeletal muscles. These compounds were selected as two effective and well-characterized beta-agonist and glucocorticoid widely used in coprescription for the management of asthma (NIHLBI/WHO, 2002). The effects of CL and DEX on mouse muscles had been partially characterized by previous studies (Agbenyega & Wareham, 1992) but a detailed analysis of the fibre type transformation in limb and respiratory muscles was still lacking. Thus, the effects of the separate and combined treatments were compared to each other in order to evaluate the relative contribution of the two compounds in producing fibre type transitions.
The results obtained showed that mouse muscles changed their size and their fibre type composition in response to both glucocorticoid and beta-agonist administration. Glucocorticoids and beta-agonists had opposite effects on muscle mass. As expected (Agbenyega & Wareham, 1992), CL increased body weight and the weight of soleus, tibialis and diaphragm in close proportion to body weight, so that the ratio of muscle weight:body weight was constant. Furthermore the cross sectional area of individual soleus fibres was also increased. In accordance with previous studies (Agbenyega & Wareham, 1992; Jiang et al. 1996), the effects of DEX administration were opposite: body weight decreased, muscle weight varied in proportion and cross sectional area of soleus fibres decreased. Fibre type composition was also modified by the treatments: CL stimulated expression of fast MHC-2B and -2X and repressed that of slow MHC and MHC-2A (Polla et al. 2001) whereas DEX produced a fast-to-slow transition of MHC isoforms (Polla et al. 1994). The changes in MHC expression were accompanied by changes in metabolic enzymes activities: whereas CL increased the glycolytic enzymes activities (PFK and LDH) and depressed the oxidative enzyme activities (COX and CS), DEX had opposite effects. Interestingly, the response varied among the muscles analyzed: whereas the soleus was the most responsive to both treatments and showed clear variations of MHC isoform composition and enzyme activities, the diaphragm showed intermediate responsiveness and tibialis and parasternal muscles appeared to be less responsive. The distinct responsiveness might depend on fibre type composition; both tibialis and parasternal muscles are mainly composed of fast 2B and 2X fibres, whereas soleus and diaphragm show a more complex composition, comprising slow and fast 2A fibres in addition to 2B and 2X fibres. Slow fibres express more beta-2 receptors than fast fibres (Williams et al. 1984; Martin et al. 1989) and the anabolic action of CL on muscle is known to depend on beta-2 receptors (Navegantes et al. 2001; Hinkle et al. 2002). The higher responsiveness of fast fibres to glucocorticoids might be explained by the protective action of activity from steroid induced atrophy: slow and fast 2A fibres are more active than fast 2X and 2B fibres, as discussed by Lewis and coworkers (Lewis et al. 1992).
The transitions in MHC isoform expression induced by the treatments generally followed the well-established sequence 1↔2A↔2X↔2B (Pette & Staron, 1990; Schiaffino & Reggiani, 1996). The two fast muscles, tibialis and parasternal, which under the stimulus of DEX expressed slow MHC without expression of MHC-2A, represented a notable exception to this general rule. Atypical MHC isoform transitions without the sequence 1↔2A↔2X↔2B have been observed in rat during atrophy induced by hind limb suspension (Talmadge et al. 1996; Caiozzo et al. 1998) and after cross re-innervation of soleus and EDL (Erzen et al. 1999). Unusual isoprotein combinations have also been observed in humans after prolonged bed rest (Andersen et al. 1999) and immobilization (D'Antona et al. 2000; 2003). Under these conditions ‘jump fibres’ (Erzen et al. 1999) expressing slow MHC together with MHC-2X and/or MHC-2B can be found. Although the phenomenon has not yet been described in mouse muscles, there is no reason to exclude that it may also occur during steroid induced muscle atrophy.
In all muscles studied there was a general correlation between myosin isoform transitions and changes in metabolic enzyme activities: the shift towards fast MHC isoforms induced by CL was accompanied by an increase of glycolytic activities (PFK in soleus and diaphragm) and depression of mitochondrial activities (COX and CS). The effects of chronic glucocorticoid administration on muscle metabolism are controversial (Lewis et al. 1992; van Balkom et al. 1996; Mitsui et al. 2002): in our study the overall shift towards slow MHC isoforms was associated with a depression of glycolytic enzyme activities in tibialis and an enhancement of CS activity in soleus and in diaphragm. COX was never significantly modified by DEX treatment. In some cases, the enzyme activities, however, were more responsive to both treatments than MHC expression (Pette & Staron, 1990): for example changes in enzyme activities without any change in MHC isoform composition were observed in tibialis after CL treatment. As a general interpretation the changes in enzyme activities very likely reflected the fibre type transitions induced by the treatment and revealed also by the changes in MHC expression. In particular, taking into account the known negative effects of glucocorticoids on mitochondrial function (Martens et al. 1991; Simon et al. 1998), the increase of mitochondrial enzyme activities due to DEX administration was likely only a ‘side-effect’ of the fibre transition from fast-glycolytic to slow (or fast 2 A) oxidative caused by the treatment.
The question whether CL could antagonize the alterations induced by glucocorticoids, i.e. the so called steroid myopathy, is also of interest for therapeutic reasons as the two compounds are often administered together in patients with asthma, as previously mentioned (NIHLBI/WHO, 2002). The different way of administration (oral for CL and i.p. for DEX) mimic rather well the clinical situation where CL is administered per os and DEX via intramuscular injection. The dosages administered to the mice are much higher than those employed in humans, as expected in view of the smaller body size and in general agreement with those employed in previous studies (Jiang et al. 1996; Polla et al. 2001). Taking into account the duration of the mouse life, the treatment covers a period comparable to a few years in humans. Several studies have considered the possibility to prevent or reduce the muscle atrophy induced by steroid administration with various countermeasures, including exercise (Hickson & Davis, 1981; Lieu et al. 1993), insulin-like growth factor administration (Kanda et al. 1999) and anabolic steroid administration (van Balkom et al. 1999). CL has proved to be partially effective in preventing atrophy induced by denervation (Zeman et al. 1987) and disuse (Babij & Booth, 1988). Three studies have previously analysed the effect of CL in steroid myopathy (Agbenyega & Wareham, 1992; Jiang et al. 1996; Huang et al. 2000). The results are unequivocal: whereas CL seems to prevent the reduction of muscle mass induced by DEX in mice (Agbenyega & Wareham, 1992) and in rabbits (Jiang et al. 1996), it is not able to counteract DEX-induced contractile dysfunction of the diaphragm. According to Huang et al. (2000), however, CL cannot avoid muscle growth inhibition caused by DEX in rats.
The results obtained in this study showed that, in accordance with the opposite effects on fibre type and size observed after separate administration, the combined treatment with CL and DEX tended to minimize the variations of any parameter: for example body weight growth was stimulated by CL, inhibited by DEX and remained similar to Control in mice exposed to combined treatment. In soleus muscle, however, the effect of CL predominated over that of DEX: fibre cross sectional area was increased, expression of fast MHC isoforms was stimulated and enzymatic activities modified by the combined treatment in a way very similar to that produced by CL alone. In diaphragm, tibialis and parasternal the combined treatment could only antagonize the effects of DEX administration on fibre type (for example the expression of slow MHC in tibialis and parasternal) and counteracted the decrease of muscle weight induced by DEX.
In few cases the combined treatment had a potentiating effect: the increase of cross sectional area of 2A fibres in the soleus was significantly greater after the combined treatment than after administration of CL alone. It is possible that in the soleus muscle, which is very sensitive to beta agonists, a modest prevention of beta-adrenoreceptor desensitization caused by glucocorticoid may be sufficient to enhance the response to CL during the combined treatment. An opposite effect was observed in the diaphragm: the stimulation of MHC-2 A expression, which is an index of fast to slow transition, was higher after the combined treatment than after DEX alone. In tibialis, the depression of COX reached the statistical significance only with the combined treatment and not with CL alone.
The mechanism of interaction of the two compounds and the explanation of the diverse response of the muscles analysed falls beyond the experimental approach used in this study and can be only matter of hypothesis. The interaction between the effects of CL and DEX on muscle fibre growth and type (myosin and metabolic enzymes) appeared, in most of the cases, of reciprocal and mutual inhibition. The mutual inhibition might occur at transcriptional level: there is evidence, although unequivocal (see for a discussion: Taylor & Hancox, 2000), that beta-agonists might inhibit GR binding to GRE and viceversa glucocorticoid might inhibit CREB binding to CRE. It is, however, more likely that the action of beta-agonists and that of glucocorticoids are completely independent. Glucocorticoids have been shown to induce muscle atrophy by inhibiting the translational machinery at the level of S6K1 and eIF4F (Shah et al. 2002) or by activating ubiquitinization of muscle proteins (Bodine et al. 2001; Marinovic et al. 2002), whereas CL seems to stimulate muscle protein synthesis possibly via IGF (Sneddon et al. 2001; Awede et al. 2002). Recent evidence, however, has shown that CL might also inhibit proteolysis in skeletal muscles (Navegantes et al. 2001). Although our results cannot provide an answer to these questions, they clearly identify some points which might help future research aimed at discovering the mechanism: (1) CL antagonizes the effects of DEX (2) The response is different from muscle to muscle (3) CL is more effective in muscles with higher proportion of slow fibres as soleus.
The positive interaction, i.e. the reciprocal facilitation of glucocorticoids and beta agonists, which has been shown in cardiac and smooth muscle (Davies & Lefkowitz, 1984) as well as in glucose metabolism (Davies & Lefkowitz, 1984), was found only in very few cases in skeletal muscles. As mentioned in the Introduction section, reciprocal facilitation can be explained by the action of glucocorticoids against down regulation and desensitization of beta-2 receptor (Taylor & Hancox, 2000) and by the action of intracellular cAMP against down regulation of the Glucocorticoid Receptor (GR) (Dong et al. 1989).
In conclusion this study reports the first systematic description of the changes induced by beta-agonists and glucocorticoids on MHC expression and in metabolic enzyme activities in postural (soleus), locomotor (tibialis) and respiratory (diaphragm) murine muscles. The emerging picture is characterized by the antagonism between the effects of beta agonists and glucocorticoids and by the presence of distinct responses of various muscles. The interaction between beta agonists and glucocorticoids in long-term therapeutic administration is a matter of clinical relevance as in acute severe asthma and in chronic asthma the two compounds are often prescribed together. Clinical experience demonstrates that the combination of the two drugs improves the control of asthma. The long-term administration of each of the two compounds, however, causes on skeletal muscles unwanted effects which go in opposite directions: whereas atrophy and shift to slow phenotype are caused by glucocorticoids, hypertrophy and shift to fast phenotype are caused by CL. Here we showed that the combined administration of the two compounds causes a compensation of the effects, although to different extent in different muscles. Thus, on skeletal muscles, the combination of the two compounds might produce beneficial effects, as it does in asthma control. Clinical studies on the skeletal muscles of patients, which receive beta agonists and glucocorticoids, are worth to be pursued to confirm our results.