Effects of beta 2-agonist clenbuterol on biochemical and contractile properties of unloaded soleus fibers of rat

Carole Ricart-Firinga, Laurence Stevens, Marie-Helene Canu, Tatiana L. Nemirovskaya, and Yvonne Mounier

Laboratory of Neuromuscular Plasticity, University of Sciences and Technologies of Lille, F-59655 Villeneuve d'Ascq, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of clenbuterol beta 2-agonist administration were investigated in normal and atrophied [15-day hindlimb-unloaded (HU)] rat soleus muscles. We showed that clenbuterol had a specific effect on muscle tissue, since it reduces soleus atrophy induced by HU. The study of Ca2+ activation properties of single skinned fibers revealed that clenbuterol partly prevented the decrease in maximal tension after HU, with a preferential effect on fast-twitch fibers. Clenbuterol improved the Ca2+ sensitivity in slow- and fast-twitch fibers by shifting the tension-pCa relationship toward lower Ca2+ concentrations, but this effect was more marked after HU than in normal conditions. Whole muscle electrophoresis indicated slow-to-fast transitions of the myosin heavy chain isoforms for unloaded and for clenbuterol-treated soleus. The coupling of the two latter conditions did not, however, increase these phenotypical transformations. Our findings indicated that clenbuterol had an anabolic action and a beta 2-adrenergic effect on muscle fibers and appeared to counteract some effects of unloading disuse conditions.

beta 2-adrenergic agonist; hindlimb suspension; atrophy; single fibers; myosin heavy chains; calcium and strontium activation characteristics


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CLENBUTEROL is a beta 2-adrenergic agonist known to produce muscle hypertrophy (6, 16) and to improve functional capacity by increasing muscular strength (8, 33). Dodd et al. (8) showed that this increase in strength was due to hypertrophy of slow- and fast-twitch fibers, whereas others generally demonstrated a preferential fast-twitch fiber hypertrophy (7, 16). Numerous studies have indicated qualitative changes in the phenotypical expression of protein isoforms, i.e., transitions from slow to fast myosin heavy chain (MHC) isoforms, in slow-twitch clenbuterol-treated soleus muscle (7, 18). Such transitions in myosin isoforms are well known to influence the functional characteristics of skeletal muscles (17, 24, 31). Thus, according to the effects of clenbuterol on growth and muscular strength, the study of its action at the myofibrillar protein level appeared to be of particular interest. Moreover, many works have reported the effects of clenbuterol on whole muscles, but its action at cellular and molecular levels remained unknown. Thus a fundamental question would be whether the anabolic effects of the drug themselves can explain the improvement in functional capacity and/or whether a specific effect of the beta 2-adrenergic pathway may improve the myofilament responsiveness. For instance, stimulation of beta 2-adrenoceptors is known to induce the phosphorylation of many proteins (e.g., membrane proteins and kinases) (32).

In inherited muscle dystrophy (mdx mice) (25) and after denervation (1, 33), clenbuterol has been described as an agent able to reduce muscle atrophy. Another type of atrophy related to muscle disuse is a result of hindlimb unloading (HU). The HU conditions are generally induced in rats by the hindlimb suspension model (22), in which load bearing by the lower extremities is prevented. This experimental model, generally employed to mimic the effects of weightlessness, involves a number of muscular alterations, including 1) losses in mass and force, clearly marked in muscles used in weight bearing, such as the slow-twitch soleus muscle (26, 27, 30), 2) changes in fiber type distribution and protein isoform composition from slow to fast type (30, 31), and 3) corresponding modifications in the functional properties of soleus muscle (20, 26).

Hence, the present study was undertaken with two main objectives. The primary goal was to investigate the method of clenbuterol action at an intracellular level by using single skinned fibers. The skinned fiber preparation has a great advantage over intact fiber, in that it allows free access to contractile proteins and permits study of the Ca2+ sensitivity of the myofilaments. The second objective was to examine the effect of clenbuterol on atrophied muscles with altered contractile properties to propose a possible countermeasure to unloading atrophy. The functional characteristics were studied in correlation with the analysis of MHC isoform expression. We focused on the contractile protein myosin as the marker molecule in which to assess muscle plasticity because of its abundance in striated muscles and its highly extended range of isoforms.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Animals and Samples

Adult male Wistar rats (initial body weight ~250 g) were divided randomly into four groups: 1) control (n = 4), 2) treated with clenbuterol (Sigma Chemical, St. Louis, MO) via their drinking water with an intake of 0.6 mg/day in 20 ml of water for 2 wk (CB, n = 8), 3) HU by the tail suspension model of Morey (22) for 2 wk (HU, n = 4), and 4) treated with clenbuterol during the HU period (HU-CB, n = 9). The suspension apparatus used for the HU and HU-CB groups consisted of an overhead swivel that permitted 360° rotation and allowed the rats to walk freely on their forelimbs and have free access to food and water. Clenbuterol-treated rats received 20 ml of water plus clenbuterol daily for 2 wk. All the rats drank at least this quantity, and water alone was then provided ad libitum. This protocol ensured that each rat received the same dose of clenbuterol during the treatment. This drug concentration (30 mg/l) has been previously shown to be effective in promoting maximal growth of several types of muscles (5, 19, 33). This manner of administering the drug was chosen 1) because it enabled us to avoid manipulation of the HU rats to prevent stress and 2) because water absorption has previously been described to be as effective as injections (9, 18). All the rats were individually caged on a 12:12-h light-dark cycle at 23°C room temperature. The experiments, as well as the maintenance conditions of the animals, were authorized by the Ministries of Agriculture and Education (Veterinary Service of Health and Animal Protection Authorization 03805).

The body weight of each rat was measured before and after the different treatments. On the 15th day, the rats were anesthetized with ethyl carbamate (1 mg/kg body wt ip). The soleus muscles from all rats were immediately removed and weighed. Some control extensor digitorum longus muscles were taken and used as indicators for the electrophoretic mobility of the fast myosin isoforms.

Electrophoretic Analysis of MHC Isoforms

One soleus muscle of each rat was frozen in liquid N2 and stored at -80°C until analyzed. Fifteen 20-µm-thick muscle sections were removed from three scattered parts of each biopsy, pooled, and treated as described by Carraro and Catani (4). One microgram of protein was then loaded into each electrophoretic well. The MHC isoform content was studied by SDS-PAGE according to the method of Hämäläinen and Pette (12). The stacking and separating gels consisted of 4.5 and 7.5% polyacrylamide, respectively. Electrophoresis was performed using a vertical chamber (model SE600, Hoefer) at 12°C for 24 h (180 V constant, 13 mA per slab). After the gel run, the gel slabs were silver stained, and a laser scanning densitometer (Quantiscan Microvial Systems, Biosoft) was used to determine the relative proportion of the different MHC isoforms in each muscle.

Functional Analysis on Single Skinned Fibers

Muscle skinning. The other soleus muscle of each rat was chemically skinned by exposure to an EGTA skinning solution (see Solutions) for 24 h and stored at -20°C in a 50% glycerol-50% skinning solution (storage solution), as described by Mounier et al. (23). Experiments were carried out on single-fiber segments isolated from the skinned muscles.

Experimental procedures and force measurements. Before each experiment, the isolated fiber segment was bathed twice for 7 min in a 2% Brij solution (see Solutions). This procedure irreversibly eliminated the sarcoplasmic reticulum, whereas the actomyosin system remained intact (10). The single-fiber segment was transferred to the experimental chamber containing the relaxing solution (R solution). One end of the fiber was tied by a silk thread to a fixed forceps and the other end to a hook connected to a strain gauge force transducer (model BG10, Kulite; sensitivity 0.70 V/g). The output of the gauge signal was amplified, recorded on an ink recorder (model 6120, Gould), and analyzed with a computer program. The fiber sarcomere length was measured in the R solution with use of the diffraction pattern of an He-Ne laser beam crossing the preparation perpendicularly. It was adjusted to a length of 2.60 µm, which allowed maximal isometric tension (P0) to be elicited, and was readjusted when necessary. The mounted fiber was viewed through high-magnification (×80) binoculars with a micrometer that allowed fiber diameter measurements. All the experiments were conducted at 19 ± 1°C.

At the beginning of each experiment, P0 was elicited by application of a fully activating solution with a pCa of 4.2. The tension-pCa (T-pCa) relationship was then established as described previously (27). Briefly, each amplitude of tension (P) obtained in solutions of various pCa was normalized to P0. The corresponding ratio P/P0 was related to the pCa. The same procedure was used to obtain the Sr2+ contractile activation properties (T-pSr relationship), except the fiber was maximally activated in pSr 3.4 solution. About seven fibers per muscle were analyzed in this way. Fibers on which the entire protocol could not be applied were rejected (when the fiber broke before the end of the experiment or when a decrease in P0 of >20% was recorded).

The experimental data for the T-pCa relationship were fitted to the Hill equation: P/P0 = ([Ca2+]/K)nH /{1 - ([Ca2+]/ K)nH }, where P/P0 is the normalized tension, nH is the Hill coefficient, K is the apparent dissociation constant (pK = -logK = pCa50), and [Ca2+] is Ca2+ concentration. Several parameters can be derived from the T-pCa (T-pSr) relationship, including 1) the pCa50 (pSr50) value, which represents the pCa (pSr) necessary to develop 50% of the maximum Ca2+ (Sr2+)-activated tension response (pCa50 and pSr50 are indicators of the affinity of the contractile proteins and, more especially, of troponin C for Ca2+ and Sr2+, respectively); 2) the pCathreshold value, i.e., the Ca2+ concentration necessary for the fiber to be activated and develop a detectable tension; and 3) the nH value, defined as the slope of the curve that indicates the degree of cooperativity between the proteins of the thin filament.

Physiological Fiber Type Identification: Ca2+ and Sr2+ Affinities

As mentioned above, several characteristics can be derived from the T-pCa and T-pSr relationships, especially pCa50 and pSr50. It is generally assumed that fast-twitch skeletal muscle fibers are less sensitive to Sr2+ than slow-twitch fibers (29). The difference (pCa50 - pSr50), or Delta  value, was used to reflect the relative affinity of a fiber to Ca2+ and Sr2+. The statistical test of normality applied to the histogram of Delta  distribution frequency revealed that two fiber populations could be discriminated (2 separate Henry's straight lines could be drawn from the relationship between the relative cumulated frequencies and Delta  ranges; data not shown). In our study, a fiber exhibiting little difference between Ca2+ and Sr2+ affinities was identified as slow-type fiber (see Table 2). On the contrary, a fast-type fiber could be characterized by Delta  > 1.00 (see Table 3). Fast-type fibers could be also distinguished from slow-type fibers by a higher Ca2+ threshold (lower pCa value) and a steeper T-pCa curve (higher nH value).

Solutions

The composition of all solutions was calculated as previously described (23), with a final ionic strength of 200 mM and a pH of 7.00. The EGTA skinning solution was composed of (in mM) 2.5 ATP, 20 MOPS, 170 potassium propionate, 2.5 magnesium acetate, and 5 K2-EGTA. The following solutions were used for the experimental procedure: a washing solution composed of (in mM) 2.5 ATP, 20 MOPS, 185 potassium propionate, and 2.5 magnesium acetate; an R solution similar to the skinning solution; pCa (pSr)-activating solutions consisting of washing solution plus various concentrations of free Ca2+ (Sr2+) (from CaCO3 or SrCl2, respectively) buffered with EGTA; and a Brij solution composed of R solution plus 2% Brij 58.

To determine whether clenbuterol could act directly on the contractile proteins without requiring a second messenger, we performed a series of experiments on untreated animals (control or HU). The effect of clenbuterol was tested by adding the drug directly to the pCa solutions. Clenbuterol was applied at 50 µM, since this concentration corresponded to the level of the anabolic dose given in vivo (3). For each fiber, T-pCa relationships were established in the absence or presence of clenbuterol.

Statistical Analysis

Values are means ± SE. After one-way ANOVA, Student's t-test was used as a post hoc test to establish the intergroup comparisons. P < 0.05 was chosen as level of significance.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Morphological Characteristics

After 2 wk of clenbuterol treatment, the body weight (BW) was unaltered, whereas the muscle wet weight (MWW) of the CB group tended to increase (Table 1). Thus the MWW/BW ratio increased significantly (18.5%) for the CB group compared with the control group. Two weeks of HU conditions caused decreases in BW and MWW, leading to a ~40% reduction in the MWW/BW ratio. After 15 days of clenbuterol administration, the MWW/BW ratio became greater in the HU-CB group than in the HU group. However, with regard to the control animals, the HU-CB rats exhibited persistent decreases in BW, MWW, and MWW/BW ratio.

                              
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Table 1.   Comparison of body and muscle weights among the different groups

Muscle Biochemical Analysis: SDS-PAGE

Clenbuterol administration to the control animals led to pronounced changes in the MHC isoform pattern (Figs. 1 and 2). MHCI, the predominant isoform in control muscles, decreased by 28% in the CB muscles. The fast MHC isoforms increased, with a fourfold elevation in MHCIIa, reaching a relative concentration of ~25%. MHCIId(x) and MHCIIb, normally not detected in soleus muscles, were induced, reaching relative concentrations of 4.3 and 3.5%, respectively. In HU conditions, MHCI decreased, while MHCIId(x) (6.5%) and MHCIIb (4.2%) were induced. The profile of the HU-CB rats resembled that of the HU rats, but MHCIIa was elevated threefold compared with the control group.


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Fig. 1.   Representative profiles of control (Cont), clenbuterol-treated (CB), hindlimb-unloaded (HU), and HU-CB soleus (SOL) and control extensor digitorum longus (EDL) muscles determined by electrophoretic analysis of myosin heavy chain (MHC) isoforms.



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Fig. 2.   Distribution of MHC isoforms in each experimental group. Values are means ± SE. * Significantly different from control for each type of MHC isoform.

Properties of Single Skinned Slow- and Fast-Twitch Fibers of Clenbuterol-Treated Animals

Diameters. The diameter of the fast-twitch fibers tended to increase under the influence of clenbuterol (Table 2), but this was not significant because of a large standard error resulting from the very few fast-twitch fibers found in the control soleus (3 of 29 tested fibers). In contrast, HU significantly reduced the diameters of slow- and fast-twitch fibers. Clenbuterol administration to the HU rats (HU-CB) led to increases in slow- and fast-twitch fiber diameters compared with HU fibers. However, the slow-twitch fiber diameters of the HU-CB group were smaller than those of the control group. No significant differences existed between the diameters of fast-twitch fibers in the control and HU-CB groups.

                              
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Table 2.   Effects of CB and/or HU on contractile characteristics of slow-twitch fibers from soleus muscles

P0. Absolute and normalized P0 values of slow- and fast-twitch fibers were significantly increased after clenbuterol treatment (CB group) (Tables 2 and 3). HU conditions caused a decrease in the absolute P0 values of slow- and fast-twitch fibers, whereas normalized P0 values did not differ from control. In HU rats, clenbuterol elicited significant increases in absolute and normalized tensions compared with HU values. Absolute P0 values of HU-CB fast-twitch fibers were in the range of the control values. However, absolute P0 values of HU-CB slow-twitch fibers were lower than P0 of control slow-twitch fibers. Normalized P0 values of slow- and fast-twitch fibers from the HU-CB group were not significantly different from control values.

                              
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Table 3.   Effects of CB and/or HU on contractile characteristics of fast-twitch fibers from soleus muscles

Ca2+ and Sr2+ activation characteristics. As described in MATERIALS AND METHODS, the physiological fiber type was determined according to the Delta  criterion (pCa50 - pSr50; Fig. 3). The number of slow- and fast-twitch fibers found in each experimental group, according to the Delta  value, is indicated in Tables 2 and 3. Moreover, the following differences in Ca2+ activation properties were noted between fast- and slow-twitch fibers: fast-twitch fibers exhibited higher pCathreshold (lower pCa value) and nH values than slow-twitch fibers. As previously shown (17, 27), the pCa50 parameter did not significantly differ between slow- and fast-twitch fibers. After HU, slow-twitch fibers exhibited a decrease in the pCathreshold (significant) and in pCa50 (not significant) values that indicated a rightward shift of the T-pCa curve from HU. A higher proportion of fibers (8 of 30) appeared to be fast type after HU, exhibiting Ca2+ activation characteristics similar to those of the control fast-twitch fibers (Table 3; pCathreshold, nH). The effect of clenbuterol on the Ca2+ activation characteristics pointed to a shift of the T-pCa relationships toward higher pCa values for fibers from normal (CB group) and atrophied muscles (HU-CB group; Fig. 4). Thus pCathreshold and pCa50 values were elevated after clenbuterol treatment, but the slope of the curves appeared unaffected by clenbuterol treatment.


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Fig. 3.   Distribution of Delta  (pCa50 - pSr50) values for control (A; n = 29), CB (B; n = 64), HU (C; n = 30), and HU-CB (D; n = 34) groups. Open bars, slow-twitch fibers; solid bars, fast-twitch fibers.



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Fig. 4.   Effects of clenbuterol on tension-pCa relationships of slow- and fast-twitch fibers from control (open circle ), CB (), HU (), or HU-CB () groups. P, tension; P0, maximal isometric tension. Values are means ± SE. SE were not reported when they merged with mean points.

Effect of direct clenbuterol application on functional properties of skinned fibers. Direct clenbuterol addition (50 µM) in the pCa solutions showed no effect of this drug on the contractile proteins. Control slow-twitch fibers (n = 5) developed the same absolute P0 (no significant difference) in the absence or presence of clenbuterol: 2.78 ± 0.47 and 2.70 ± 0.48 × 10-4 N, respectively. The same applied to fast-twitch HU fibers (n = 5), the absolute tensions of which were 1.01 ± 0.14 × 10-4 and 0.93 ± 0.12 × 10-4 N in the absence and presence of clenbuterol, respectively. Moreover, T-pCa curves established in the presence and absence of clenbuterol for each type of fiber were superposed, indicating the same sensitivity to Ca2+ with or without the drug (not illustrated).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study examined the effects of clenbuterol, a beta 2-adrenoceptor agonist, on the biochemical and contractile properties of slow- and fast-twitch fibers from normal and atrophied soleus muscles. By using skinned fiber preparations, we were able to investigate the changes at a subcellular level, i.e., in maximal force and Ca2+ sensitivity of the contractile proteins, and thus to distinguish the anabolic effect of clenbuterol from its beta 2-adrenoceptor effect.

Hypertrophic Effect of Clenbuterol on Whole Muscle and Single Fibers

The MWW/BW ratio increased by ~18% in normal and atrophied clenbuterol-treated soleus and could be related to the anabolic effect of the drug. This effect appeared more focused on muscle mass than on BW, although the increases in MWW after clenbuterol treatment (CB and HU-CB groups) were not large enough to be statistically significant compared with control and HU groups, respectively. However, the larger MWW/BW ratio after clenbuterol treatment confirmed that clenbuterol induced changes in muscle protein synthesis (14), probably by increasing the ratio of RNA to protein content (6, 19).

At the single-fiber level, the hypertrophic effect of clenbuterol was not obvious, the mean fiber diameters being not significantly larger than those of the control group. Nevertheless, the fast-twitch fibers appeared more responsive than the slow-twitch fibers. The hypertrophic effect became even more evident in atrophied muscles. In fast- and slow-twitch fibers in which diameters were decreased after HU (26, 30), clenbuterol produced 1) a complete diameter recovery of the fast-twitch fibers and 2) a slighter recovery of diameters from slow-twitch fibers. Such fiber-type differences in the hypertrophic effect of beta -adrenoceptor drugs have been previously described (16, 33). We can postulate that clenbuterol was able to limit atrophy of the 2-wk unloaded soleus muscles by its specific action on fast-twitch fibers. The fact that clenbuterol can prevent the loss of alpha -actin mRNA that normally occurs after 7 days of hindlimb unloading (2) suggested a possible gene regulation by the drug of the expression of slow and fast contractile proteins.

Clenbuterol-Induced Changes in the Expression Pattern of MHC Isoforms

Two weeks of clenbuterol treatment induced a slow-to-fast conversion of normal slow-twitch soleus, as described after 6 wk of clenbuterol treatment. Contrary to our results [increase of MHCIIa and expression of MHCIId(x) and MHCIIb], no difference in MHCIIa expression was shown, but a large increase was observed in MHCIId(x). This suggested that, in our conditions, after a 2-wk clenbuterol treatment, the transition from MHCI toward fast isoforms might be limited and that a larger transformation, such as the transition to MHCIId(x), might occur with longer periods of drug treatment. Hence, our findings support the theory that slow-to-fast clenbuterol-induced transitions occurred in the order MHCI right-arrow MHCIIa right-arrow MHCIId(x) right-arrow MHCIIb, previously suggested, but in the fast-to-slow order, by Pette and Staron (24) after chronic low-frequency stimulation of extensor digitorum longus muscle. Moreover, after 2 wk of clenbuterol administration, we observed a more pronounced effect on the protein isoform expression than on the anabolic response. Thus the shift from slow to fast fiber type with longer-term beta -agonist treatment would become visible when the anabolic response was attenuated (21). Finally, it seemed that clenbuterol treatment of atrophied muscles had no additional effect on the changes in MHC isoform pattern already induced by HU, i.e., an increase in fast MHC isoforms MHCIIa, MHCIId(x), and MHCIIb and a concomitant decrease in MHCI expressions.

Effect of Clenbuterol on P0

Clenbuterol induced a reinforcement of absolute and normalized P0 developed by normal and atrophied single soleus muscle fibers. The increases in normalized P0 were larger for the fast populations (+60% for the CB group and +72% for the HU-CB group) than for the slow populations (+41% for the CB group and +30% for the HU-CB group). This observation was well correlated to the preferential effect of clenbuterol on the fast-twitch fiber. However, the anabolic effect of clenbuterol estimated by the MWW/BW ratio and the single-fiber diameters was not as obvious as that observed for the tension measurements, especially in normal muscle fibers. Therefore, the stimulating effect of clenbuterol on the maximal force development should be interpreted by one or several additional mechanisms, besides the increase due to protein accretion.

A first explanation may be that each cross bridge becomes able to develop higher strength because of some changes in contractile protein properties (see Effect of Clenbuterol on the Contractile Characteristics of Single Muscle Fibers). A second possibility is that the increase in protein content, especially myofibrillar proteins, contributes to a reduction in the interfilament spacing, promoting subsequently an increase in muscular strength (11, 20).

Effect of Clenbuterol on the Contractile Characteristics of Single Muscle Fibers

Clenbuterol treatment of normal and atrophied muscles induced an increase in the Ca2+ sensitivity of the contractile proteins (leftward shift of the tension-pCa relationships), whereas no change was observed for the cooperativity parameter (unchanged nH values). The former effect was opposed to a previous result, which described a rightward shift of the T-pCa curve (18). However, these authors carried out experiments on mice treated with clenbuterol for 15 wk. The decrease in Ca2+ sensitivity observed in these conditions might be due to species-related differences, since, in contrast to our results in the rat, clenbuterol has been shown to alter the contractile properties of fast- and slow-twitch fibers of the mouse (13), or it might be due to a toxic effect caused by high dosages resulting from long-term treatment and comparable to that observed on cardiac muscle (15).

Possible Cellular Mechanisms Underlying the Positive Action of Clenbuterol

A fundamental question concerning the mechanisms involved in the anabolic effect of clenbuterol is whether its action can be related to the stimulation of the beta 2-adrenoceptor. In the skinned fiber technique, the plasma membrane of muscle fibers is permeabilized and the beta 2-receptors are mostly removed. This compromises the possible accumulation of soluble second messengers. Interestingly, it appeared that, in agreement with another work (3), clenbuterol directly added to the activated solutions in the experimental chamber did not induce any change in the maximal force or Ca2+ sensitivity. This suggested that the clenbuterol treatment effects on muscle functional properties were due to changes in regulatory mechanisms controlled by the plasma membrane or cytosolic components that were able to modify the contractile proteins. beta -Adrenoceptor agonists are able to enhance the responsiveness of the myofilaments to Ca2+ through receptor-mediated events (phosphorylation of proteins involved in the excitation-contraction coupling) (32). Among them, a better phosphorylation of the myosin light chain II might be suggested. Indeed, the stimulation of the beta 2-adrenoceptor triggers a cascade of kinase phosphorylations (e.g., myosin light chain kinase) and, thereby, sensitizes the myofibrils to Ca2+, leading to a leftward shift of the T-pCa curve (28). This hypothesis does not rule out possible phosphorylations of other contractile or regulatory proteins producing increases in Ca2+ sensitivity. The leftward shift of the T-pCa curve appeared more evident in the atrophied slow-twitch soleus fibers, which suggests that 1) atrophied muscles are more influenced by clenbuterol than normal muscles and 2) the largest shift in slow-twitch fibers might be related to the higher density of beta 2-adrenoceptors in slow-twitch muscles (16).

In conclusion, clenbuterol in normal and atrophied soleus muscles appeared to act as an anabolic drug as well as a beta -agonist agent. The stimulating effect of the beta -adrenergic pathway appeared to produce more important changes in muscle properties than the process of clenbuterol-induced protein accretion. Because clenbuterol was able to lessen some effects of HU conditions, the potential clinical benefit of treatment by this agonist, for reducing loss of mass and forces in atrophied muscles, should be underlined.


    ACKNOWLEDGEMENTS

This work was partly supported by Centre National d'Etudes Spatiales Grant 98/2711, the Nord-Pas-de-Calais Regional Council, and the Fonds Européen de Développement Régional.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: L. Stevens, Laboratory of Neuromuscular Plasticity, Bat SN4, University of Sciences and Technologies of Lille, F-59655 Villeneuve d'Ascq, France (E-mail: Structcont.cell{at}univ-lille1.fr).

Received 15 July 1999; accepted in final form 6 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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