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Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgements

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.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgements

Animal treatment

This study was carried out on male C-57 mice (average weight 35 g) purchased from Charles River (Italy). Animals were randomly divided into four groups: control group (Control; n = 6), CL treated group (CL, 1.5 mg (kg body wt)−1 day−1 for 4 weeks in drinking water; n = 6), DEX treated group (DEX, 5.7 mg (kg body wt)−1, corresponding to 0.2 mg of DEX by subcutaneous injection, every other day for 4 weeks; n = 6) and DEX + CL group (DEX + CL; n = 6) receiving both treatments.

Animals were individually housed and had free access to water and food. During the entire experimental period, food and water assumption were daily measured and were found not significantly different in the four groups: daily food intake (mean ±s.d.) was 5.4 ± 3.1 g in Control mice, 5.7 ± 2.6 g in DEX group, 5.9 ± 2.6 g in CL group, 6.0 ± 3.7 g in DEX + CL group; daily water assumption (mean ±s.d.) was 7.2 ± 2.1 ml in Control mice, 7.8 ± 3.1 ml in DEX group, 7.4 ± 3.1 in CL group, 6.2 ± 3.7 ml in DEX + CL group.

At the end of the treatment the animals were killed by cervical dislocation as approved by the local Animal Ethic Committee. The heart (ventricles and atria) was removed, the diaphragm (both costal and crural regions and central tendon) was carefully cut along the costal insertion, the parasternal, the soleus and the tibialis muscles (two for each animal) were dissected. All the dissected muscles were then blotted on filter paper, weighed and immediately frozen in liquid nitrogen except the soleus muscles used for immunohistochemical analysis which were first included in O.C.T (Tissue-Tek) embedding medium before freezing. All the muscle samples were stored at –80 °C until they were used for subsequent analysis.

MHC isoform composition

MHC isoforms were used as molecular markers to assess the fibre type composition of each muscle, and their expression was determined by gel electrophoresis as previously described (Polla et al. 2001). Briefly, fragments of the frozen muscles were dissolved in Laemmli solution (Laemmli, 1970) and small amounts (2 μl containing about 500 ng of myosin) were applied on 8% polyacrylamide gels prepared according to the method described by Talmadge and Roy (Talmadge & Roy, 1993). Electrophoresis was run for 24 h at 250 V and gels were stained with Coomassie Brilliant Blue. In the region of MHC isoforms (molecular weight ∼€200 kDa), four bands were separated corresponding, in order of migration from bottom to top, to MHC-1 or slow, MHC-2B, MHC-2X and MHC-2 A (Fig. 1). The identification of the bands by using Western blots with anti-MHC monoclonal antibodies (BA-F8 specific for slow MHC, SC-71 specific for MHC-2 A, G6 specific for MHC-2B) has been previously reported (Pellegrino et al. 2003). The relative proportions of the four MHC isoforms were determined by means of a computerized densitometer. The areas under the peaks corresponding to the MHC isoforms on the densitometric readings were measured after background subtraction, and the area of each peak was expressed as a fraction of the total area of the four peaks. No correction for molecular weight was calculated.

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Figure 1. Electrophoretic separation (SDS-PAGE) and correspondent densitometric scans of Myosin Heavy Chain (MHC) isoforms of soleus muscles A, Dex; B, Control; C, Cl; D, Dex + Cl.

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Immunohistochemistry and muscle fibre cross-sectional area

From the soleus muscles of Control and treated animals (n= 4), transversal 10 μm thick sections were cut using a cryostat. Cryosections were immunostained using two monoclonal antibodies against MHC isoforms (BA-F8 against MHC-1, SC-71 against MHC-2 A) as previously described in detail (Bottinelli et al. 1991). A secondary rabbit antimouse IgG antibody conjugated with peroxidase (DAKO P-0260) was used to reveal the binding of the primary antibodies. Images of the stained sections were captured from a light microscope (Laborlux, Leitz, Germany) and transferred to a personal computer using a video camera (Hamamatsu Photonics KK, Japan). Two fibre types, type 1 and type 2A were identified and their cross sectional areas (CSA) were measured with Scion Image analysis software (NIH, Bethesda, MD, USA) and expressed in μm2. Approximately two hundred fibres per muscle were measured.

Glycolytic and oxidative metabolic enzymes.

Fragments of frozen muscle samples were washed with ice-cold solution of sucrose-EDTA, quickly freed from connective tissue and weighed. The tissue was then finely minced and homogenized with a Polytron tissue processor (Kinematica Instruments, Westbury, HY, USA) for 5 s and subsequently homogenized in 0.25 m sucrose and 1 mm EDTA in a precooled Potter-Braun S homogenizer. The homogenate was diluted with 0.25 m sucrose-EDTA: 100 mg of tissue in 1 ml of sucrose solution. This homogenate was then centrifuged at 800 g for 15 min in a refrigerated centrifuge (Beckman J2-21; rotor JA-20). Part of the supernatant fluid obtained was used to determine enzyme activity in the crude extract, and/or for protein evaluation. The sediment was re-homogenized in 0.25 m sucrose-EDTA and centrifuged at 800 g for 15 min. The two supernatants obtained were centrifuged at 14 000 g for 20 min. The mitochondrial sediment was gently re-suspended in sucrose solution at a final dilution of 100 mg of sediment in 1 ml of sucrose. An aliquot of this preparation was used to assess the protein content, while the remaining portion was used to evaluate enzyme activities (Polla et al. 2001).

The maximum rates (Vmax) of the following enzyme activities were evaluated in the crude extract and/or in the mitochondrial fraction: phosphofructokinase (PFK) (EC 2.7.1.11) and lactate dehydrogenase (LDH) (EC 1.1.1.27) for the anaerobic glycolytic pathway, citrate synthase (CS) (EC 4.1.3.7) for Krebs cycle and cytochrome oxidase (COX) (EC 1.9.31) for the electron transfer chain. Enzyme activities were recorded graphically for at least 3 min with a double beam recorder spectrophotometer (Beckman 35, Beckman Instruments, CA, USA) and each value was calculated from two determinations on the same sample. Enzyme specific activities were expressed as nmol of substrate metabolized min−1 mg−1 of protein.

Statistical Analysis.

Data were expressed as means ±s.d. Statistical significance of the differences between means was assessed by anova followed by Student-Newman-Keuls posthoc test. A probability of less than 5% was considered significant (P < 0.05).

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgements

Effects of treatments on body and muscle growth.

The average values of initial and final body weight, the body weight gain during the experimental period and the muscle weight–body weight ratio, are shown in Table 1. All animal groups increased their body weight at the end of the treatment except DEX mice, which showed a decrease. Moreover, the increase of body weight of CL mice was significantly greater than those of Control and DEX + CL groups. No significant change of body mass gain was observed in mice exposed to the combined treatment when compared to Control. In soleus, tibialis and diaphragm muscle weight–body weight ratio did not show any difference among groups, thus indicating that the mass of skeletal muscles increased with CL and decreased with DEX in close proportion with body weight. Heart weight–body weight ratio was determined to assess whether cardiac hypertrophy or atrophy was induced by the experimental treatments: the lack of significant variations of this parameter between Control and treated groups suggested that also cardiac muscle mass increased during clenbuterol treatment and decreased during dexamethasone administration in proportion with all other muscles.

Table 1.  Body parameters and muscle mass-body mass ratio
 nControlCLDEXDEX + CL
  1. Values are means ±s.d.. n, no. of mice. Body parameters: mi initial body mass, mf final body mass, mg body mass gain, mm/mb muscle mass-body mass ratio *significantly different versus the other groups, P < 0.05. Values for parasternal muscle were excluded from the analysis of muscle mass for the high degree of imprecision of the dissection procedure.

mi (g)637.66 ± 2.635.33 ± 2.7334.66 ± 1.034.66 ± 2.16
mf (g)641.33 ± 0.6643.16 ± 2.0433.51 ± 0.4239.33 ± 1.50
mg (g)63.66 ± 0.987.83 ± 1.47*−1.16 ± 0.31*2.66 ± 2.94
Heartm/mb (%)60.48 ± 0.040.54 ± 0.120.51 ± 0.060.51 ± 0.06
Soleusm/mb (%)60.033 ± 0.010.036 ± 0.0040.028 ± 0.010.042 ± 0.01
Tibialism/mb (%)60.22 ± 0.080.29 ± 0.080.21 ± 0.040.28 ± 0.05
Diaphragmm/mb (%)60.31 ± 0.040.29 ± 0.080.32 ± 0.010.32 ± 0.05

Effects of CL and DEX on MHC isoform expression

MHC isoform composition was determined in two hind-limb muscles (soleus and tibialis) and in two respiratory muscles (diaphragm and parasternal) as an index of the fibre type composition. Examples of electrophoretic separation of MHC isoforms in the soleus muscle of the four groups of mice are shown in Fig. 1.

Average values of the MHC isoform composition of the soleus muscle in Control and treated mice are shown in Fig. 2A. CL induced a dramatic shift of fibre type distribution towards MHC-2B with a concomitant decrease of MHC-1, MHC-2 A and MHC-2X in comparison with Control. DEX had the opposite effect inducing a significant shift towards slow MHC isoform (MHC-1) with a significant reduction of MHC-2B and MHC-2X and no statistical change of MHC-2 A in comparison with Control. DEX + CL was associated with an overall shift towards MHC-2B and a significant decrease of MHC-1 and MHC-2A in comparison with Control, suggesting a predominance of the effect of CL over that of DEX. In tibialis the effect of treatments was less pronounced than in soleus muscle. Control tibialis expressed a combination of MHC-2B and MHC-2X (Fig. 2B: CL did not induce changes in MHC composition, whereas DEX produced the appearance of MHC-1 without consistent expression of MHC-2A. DEX + CL did not cause any significant change compared to Control and Cl, but prevented the expression of slow myosin induced by DEX.

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Figure 2. Effects of treatments on MHC distribution in the soleus A and tibialis B muscle *Significantly different from Control. Significantly different versus Dex. Significantly different from Cl. Values are means ±s.d.; n= 6, P < 0.05.

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The respiratory muscles showed distinct responses to the treatments. Diaphragm appeared to be more responsive than parasternal both to CL and DEX administration. Figure 3A shows the MHC isoform composition of the diaphragm in Control and treated mice. CL induced an overall shift from slow to fast MHC isoforms in comparison with Control, whereas no significant changes were induced by DEX. DEX + CL also showed a significantly lower MHC-2B and higher MHC-2A expression in comparison with CL indicating that the effect of DEX predominates over that of CL and that the combined treatment tends to mimic the action of DEX alone. Interestingly, the increase of MHC-2A was even higher than that caused by DEX alone. The response of parasternal was reminiscent of that of tibialis, possibly in accordance with their similar nature, as both muscles express predominantly fast myosin isoforms. Parasternal did not show any significant changes in MHC isoforms expression after CL administration and the combined DEX + CL administration, whereas DEX alone produced the appearance of MHC-1 without consistent presence of MHC-2A (Fig. 3B).

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Figure 3. Effects of treatments on MHC distribution in the diaphragm A and parasternal B muscle *Significantly different from Control. Significantly different versus Dex. Significantly different versus Cl. Values are means ±s.d.; n= 6, P < 0.05.

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Effects of CL and DEX on fibre cross sectional area.

Cross sectional area (CSA) of skeletal muscle fibres was analysed in detail in the soleus muscle, as this muscle was the most responsive to the treatments. Examples of consecutive sections stained with antibodies specific for MHC-1 and MHC-2 A are shown in Fig. 4. Interestingly, no sign of necrosis is detectable, thus suggesting that the dosages used are not able to induce fibre necrosis. Changes in the number of fibres stained with each antibody are clearly visible and confirm the trends already observed by the electrophoretic separation of the MHC isoforms: CL induced an overall shift towards fast fibres whereas DEX produced an opposite effect. CSA of type 1 and type 2A fibres was measured as described in the Methods section. In the soleus, CSA of type 1 and 2A fibres were significantly increased by CL treatment and a slightly decreased by DEX; DEX + CL also caused an increase in CSA of both fibre groups (Fig. 5), the increase of 2A fibres being slightly greater than that caused by CL alone. These results clearly show that CL and DEX were able to induce fibre hypertrophy and fibre atrophy, respectively, in agreement with the data reported in Table 1 concerning body weight variations. Furthermore the results confirmed that in the soleus muscle CL predominates over DEX treatment, as already suggested by the analysis of the MHC isoforms expression.

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Figure 4. Example of immunostaining of soleus muscle with antislow MHC and anti-MHC-2 A antibody A, Control; B, Cl; C, Dex; D, Dex + Cl. The bar in B corresponds to 600 μm.

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Figure 5. Effects of treatments on CSA of muscle fibers in the soleus muscle *Significantly different versus Control. Significantly different versus Dex. Significantly different versus Cl. Values are means ±s.d.; n= 4, P < 0.05.

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Effects of CL and DEX on metabolic enzyme activities

Four enzymatic activities (COX, CS, PFK and LDH) were determined in three selected muscles (soleus, tibialis and diaphragm) in order to characterize the changes induced by the treatments in the metabolic phenotype of the muscles. In the soleus, CL treatment induced an increase of PFK activity whereas it decreased the activity of COX and CS (Fig. 6A). On the contrary, DEX treatment increased CS activity and the combined treatment DEX + CL counteracted all effects of CL except the reduction of COX activity. The effects of combined treatment on enzymatic activities are in agreement with the results of MHC and CSA analysis suggesting that the action of CL is predominant in soleus muscle.

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Figure 6. Effects of treatments on soleus (A) and tibialis (B) oxidative and glycolytic enzymatic activities Cytochrome oxidase (COX), citrate synthase (CS), phosphofructokinase (PFK) and lactate dehydrogenase (LDH). *Significantly different versus Control. Significantly different versus Dex. Significantly different versus Cl. Values are means ±s.d.; n= 6, P < 0.05.

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The overall effects of the treatments in tibialis muscle (Fig. 6B) were rather similar to those observed in soleus. Cl was associated with a general increase of glycolytic enzyme activities and decrease of the mitochondrial enzyme activities, which became statistically significant only for the depression of CS. DEX decreased PFK and LDH activities and this change appeared statistically significant for PFK. No changes in COX and CS activities were observed. The combined treatment induced: depression of COX and CS, more pronounced than that caused by CL alone and depression of LDH activity similar to that caused by DEX.

In the diaphragm (Fig. 7), the activity of PFK was increased and the activity of CS was decreased in Cl mice. DEX induced a significant increase of CS activity, whereas DEX + CL did not cause any change compared to Control, thus preventing the increase of PFK and the decrease of CS caused by CL treatment. These results are in agreement with the predominance of DEX on CL in controlling MHC expression observed in diaphragm (see Fig. 3A).

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Figure 7. Effects of treatments on diaphragm oxidative and glycolytic enzymatic activities: cytochrome oxidase (COX), citrate synthase (CS), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) *Significantly different versus Control. Significantly different versus Cl. Values are means ±s.d.; n= 6, P < 0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgements

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[LEFT RIGHT ARROW]2A[LEFT RIGHT ARROW]2X[LEFT RIGHT ARROW]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[LEFT RIGHT ARROW]2A[LEFT RIGHT ARROW]2X[LEFT RIGHT ARROW]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.

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  3. Methods
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  5. Discussion
  6. References
  7. Acknowledgements
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Acknowledgements

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgements

This work was partially supported by ASI (Italian Space Agency). The authors gratefully acknowledge the expert assistance of Silvia Ricci and Lorenza Brocca.