Laboratory of Neuromuscular Plasticity, University of Sciences and Technologies of Lille, F-59655 Villeneuve d'Ascq, France
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ABSTRACT |
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The effects of clenbuterol
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
2-adrenergic effect on
muscle fibers and appeared to counteract some effects of unloading
disuse conditions.
2-adrenergic agonist; hindlimb suspension; atrophy; single fibers; myosin heavy chains; calcium and strontium activation characteristics
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INTRODUCTION |
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CLENBUTEROL is a
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
2-adrenergic pathway may
improve the myofilament responsiveness. For instance, stimulation of
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.
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MATERIALS AND METHODS |
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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 atFunctional 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 /{1Physiological 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 (pCa50Solutions
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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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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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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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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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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Ca2+ and
Sr2+ activation
characteristics.
As described in MATERIALS AND METHODS,
the physiological fiber type was determined according to the criterion (pCa50
pSr50; Fig.
3). The number of slow- and
fast-twitch fibers found in each experimental group, according to the
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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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 × 104 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).
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DISCUSSION |
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The present study examined the effects of clenbuterol, a
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
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 -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
-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 MHCIEffect 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 theIn conclusion, clenbuterol in normal and atrophied soleus muscles
appeared to act as an anabolic drug as well as a -agonist agent. The
stimulating effect of the
-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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