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2-agonist
administration and exercise on contractile activation of skeletal
muscle fibers
Muscle and Cell Physiology Laboratory, Department of Physiology, The University of Melbourne, Parkville, Victoria 3052, Australia
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Lynch, Gordon S., Alan Hayes, Siun P. Campbell, and David A. Williams. Effects of
2-agonist administration and
exercise on contractile activation of skeletal muscle fibers.
J. Appl. Physiol. 81(4):
1610-1618, 1996.
Clenbuterol, a
2-adrenoceptor agonist, has
therapeutic potential for the treatment of muscle-wasting diseases, yet
its effects, especially at the single-fiber level, have not been fully
characterized. Male C57BL/10 mice were allocated to three groups:
Control-Treated mice were administered clenbuterol (2 mg · kg
1 · day1)
via their drinking water for 15 wk; Trained-Treated mice underwent low-intensity training (unweighted swimming, 5 days/wk, 1 h/day) in
addition to receiving clenbuterol; and Control mice were sedentary and
untreated. Contractile characteristics were determined on membrane-permeabilized fibers from the extensor digitorum longus (EDL)
and soleus muscles. Fast fibers from the EDL and soleus muscles of
Treated mice exhibited decreases in
Ca2+ sensitivity. Endurance
exercise offset clenbuterol's effects, demonstrated by similar
Ca2+ sensitivities in the
Trained-Treated and Control groups. Long-term clenbuterol treatment did
not affect the normalized maximal tension of fast or slow fibers but
increased the proportion of fast fibers in the soleus muscle. Training
increased the proportion of fibers with high and intermediate succinate
dehydrogenase activity in the EDL and soleus muscles, respectively. If
clenbuterol is to be used for treating muscle-wasting disorders, some
form of low-intensity exercise might be encouraged such that
potentially deleterious slow-to-fast fiber type transformations are
minimized. Indeed, in the mouse, low-intensity exercise appears to
prevent these effects.
fiber types; mouse; muscle contraction; skinned fibers
INTRODUCTION LONG-TERM TREATMENT with
Previous studies indicate that
MATERIALS AND METHODS Animals, Clenbuterol Adminstration, and Training
2-adrenoceptor agonist drugs
such as the sympathomimetic amine clenbuterol has been shown to cause large increases in skeletal muscle mass in normal muscle and reduce muscle wasting in dystrophic (mdx)
mice, as well as offset the muscle atrophy concomitant with denervation
or hindlimb suspension in rats (16, 23, 25, 27, 33, 34, 42, 44). The mechanism of action of clenbuterol for this hypertrophy of skeletal muscle is thought to involve increased protein synthesis and/or decreased protein degradation (5, 27, 32). Given its reported effects
on skeletal muscle, it is not surprising that some authors have
proposed administering clenbuterol to patients suffering muscle-wasting
diseases (1, 4, 22, 41, 44). Interestingly, due to its muscle anabolic
effects, clenbuterol is also widely used in athletic circles,
particularly among athletes involved in strength- and power-related
sports and bodybuilding activities (6, 31).
2-adrenoceptor agonists clearly
affect fast-twitch muscle fibers, their effect on slow-twitch fibers
being less consistent (16). Zeman et al. (42) showed that clenbuterol
caused slow- to fast-twitch fiber type conversions within the rat
soleus, with accompanying increases in contractile speed. Similarly,
Hayes and Williams (12) showed that clenbuterol significantly altered
the contractile properties of intact fast- and slow-twitch muscles of
the mouse. No studies have yet investigated the long-term effects of
this 2-agonist on skeletal
muscle function at the cellular level. Earlier studies have
demonstrated direct toxic effects of high dosages of
2-agonists on cardiac muscle, including myocardial necrosis, ischemia, and arrhythmia (15). Importantly, it has not been determined whether long-term usage of
2-agonists such as clenbuterol
may have deleterious effects on skeletal muscle, especially in large
dosages. Considering its reported therapeutic potential and its use in
athletic competition (6), it is imperative that the effects of
clenbuterol on skeletal and cardiac muscles are fully characterized
(31). In this study, we investigated the effect of long-term
clenbuterol administration on the contractile function of single
skinned fast- and slow-twitch skeletal muscle fibers. Furthermore, we
examined whether exercise modified the effects of clenbuterol treatment
on skeletal muscle.
1 · day1)
was given in the drinking water every day for the first week. The
effectiveness of clenbuterol administration via the drinking water has
been well established (27). After week
1, a 2:2:3-day on-off cycle was followed, which has
been shown to reduce the attenuation of the clenbuterol response (12).
The duration of the clenbuterol treatment was 15 wk. A relatively high
dosage of clenbuterol was administered to determine the maximum effects of the 2-agonist on skeletal
muscle and also to mimic the sort of excessive dosages used/abused by
athletes in pursuit of maximum muscle development (6). The exercise
group (Trained-Treated) underwent a 15-wk program consisting of
low-intensity unweighted swimming 1 h/day, 5 days/wk (see Ref. 11 for
further details regarding the swimming training protocol). All animals
had free access to food (GX2PLUS, Barastoc Stockfeeds, Melbourne,
Australia) and water.
Single-Fiber Analysis
At 20 wk of age, the mice were killed by cervical dislocation, and the fast-twitch extensor digitorum longus (EDL) muscle and mixed slow- and fast-twitch soleus muscle were dissected tendon to tendon from one hindlimb. The muscles were tied to capillary tubes and placed in a skinning solution [with the following composition (in mM): 125 K-propionate, 5 ethylene glycol-bis(-aminoethyl ether)-N,N,N
,N-tetraacetic
acid (EGTA), 2 ATP, 2 MgCl2, 20 imidazole, and 50% (vol/vol) glycerol, adjusted to pH 7.1 with 4 M
KOH] and stored at 
20°C for up to 6 mo until required.
The contralateral muscles were frozen in isopentane cooled in liquid
nitrogen and stored at 80°C for later histochemical analysis
to determine fiber type proportions of the whole muscle.
On the day of an experiment, a muscle was cut from its ties on the capillary tubes and pinned to the bottom of a small Sylgard-based (Dow Corning, Midland, MI) petri dish filled with skinning solution. The muscle bundle was carefuly teased with fine forceps to obtain smaller bundles. From these smaller bundles, single chemically skinned fibers were randomly selected and then attached to a sensitive force transducer (AM801, SensoNor, Horten, Norway). The fibers were activated by Ca2+- and Sr2+-buffered solutions with techniques commonly employed in this laboratory (19, 20). At the end of the day of the experiment, the muscle bundles were discarded. Fiber length and diameter were directly measured by means of a calibrated reticle in the eyepiece of a dissecting microscope (Nikon). Details regarding the determination of sarcomere length, which was set at 2.7-2.8 µm for all fibers, have been described elsewhere (10, 20, 35, 36). Details regarding fiber end compliance and sarcomere uniformity have been discussed previously (20). All contractile experiments were performed at 22°C.
Composition of Solutions
The composition of the solutions for activation and relaxation of skinned muscle fibers was similar to those employed in previous studies on mammalian muscle and have been described in detail elsewhere (10, 19, 35). Four stock solutions were used: a high-relaxing solution (type A) that contained 50 mM EGTA; a Ca2+-activating solution (type B) that contained 50 mM Ca-EGTA; a low-relaxing preactivating solution (type C) that contained 50 mM hexamethylenediamine-N,N,N,N-tetraacetic
acid and a Sr2+-activating
solution (type D) that contained 40 mM Sr-EGTA and 10 mM EGTA2.
Each solution contained (in mM) 60 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 117 K+, 36 Na+, 1 Mg2+, 1 NaN3, 8 ATP, and 10 creatine
phosphate. The apparent affinity constant values of
Ca2+ and
Sr2+ to EGTA at pH 7.10 and in the
presence of 1 mM Mg2+ were 4.78 × 106
M1 and 1.91 × 104
M1, respectively, as
previously determined (26, 35). The pH of the solutions was adjusted to
7.10 ± 0.01 at room temperature (22°C). Solutions with
differing ionized Ca2+ (pCa > 5.0) and Sr2+ (pSr > 3.7) were
obtained by mixing (in various proportions) a stock solution of
type A with type
B or type A with
type D, respectively. Solutions with
pCa < 5.0 and pSr < 3.7 were prepared separately. Potentiometric
techniques were used to determine the exact pCa of each mixture, as
previously described (35).
Contractile Activation Procedures and Analysis
The muscle fiber preparation was incubated for 1 min in high-relaxing solution (type A) and then was transferred to a low-relaxing preactivating solution (type C-type A 49:1) to minimize the fiber EGTA concentration and prepare it for activation. All fibers were activated by Ca2+ in a stepwise fashion {from lowest to highest Ca2+ concentration ([Ca2+])} and relaxed (in type A solution), and the activation sequence was repeated with Sr2+. The complete Ca2+- and Sr2+-activation process was then repeated. The absolute maximal tension values were normalized for the cross-sectional area of each fiber. Muscle fiber diameter has been shown to swell by up to 30% after removal or disruption of the sarcolemma in a relaxing solution (9). Therefore, normalized maximal tension values were obtained by dividing the maximal Ca2+-activated force response by the estimated cross-sectional area of the skinned fiber (while being measured in relaxing solution) corrected by a factor of 1.69. All submaximal isometric force responses were compared with the maximal Ca2+-activated (Sr2+-activated) force response of the same fiber and plotted as a function oflog10
[Ca2+] (pCa) and
log10
Sr2+ concentration
([Sr2+]) (pSr) to
obtain relative force-pCa (-pSr) curves from which several quantitative
characteristics were derived. These included a contraction threshold
for each activating ion, as indicated by the 10% relative force level
for Ca2+ and
Sr2+
(pCa10 and
pSr10, respectively); fiber
sensitivity to Ca2+ and
Sr2+ [half-maximal tension
(pCa50 and
pSr50, respectively)
values]; the relative sensitivity of a fiber to the activating
ions, given by the difference between
pCa50 and
pSr50
(pCa50 pSr50); and the relative
steepness of the sigmoidal force-pCa (-pSr) curves given by the
associated Hill coefficients
nCa and
nSr, representing the number n in the Hill equation
Pr = K[X2+]n/(1 + K[X2+]n,
which provided the closest fit to the experimental points and which
reflects the degree of cooperativity in the activation of tension.
K is a constant associated with the
[Ca2+] or
[Sr2+] required for
pCa50 or
pSr50 by using the equation
log10
K = nCapCa50
(nSrpSr50),
and
[X2+]
represents the [Ca2+]
or [Sr2+]. A series of
theoretical curves derived from the Hill equation for
n values changing by 0.1 unit was used
to fit the data points (18, 20). The maximal force response was taken
as the first contractile response after activation in a maximal
[Ca2+]. All fibers
satisfied the criterion that the final maximal
Ca2+-activated force (of the
second Ca2+-activation sequence)
be within 10% of the initial maximal force level.
Fiber Type Classification
On the basis of their relative force-pCa and force-pSr data, individual fibers were classified as fast or slow twitch by using the criteria detailed previously (see Refs. 18, 19, 40). To verify the physiological fiber type classification, individual fiber segments that had undergone contractile activation and for which complete force-pCa (-pSr) characteristics had been determined were also stored for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis. The procedure for separation of the myosin heavy chain (MHC) isoforms within single muscle fiber segments was slightly modified from that described by Talmadge and Roy (37) for homogenized rat skeletal muscles. Briefly, fiber segments were stored individually in small centrifuge tubes with a small volume (15 µl) of sample buffer [62.5 mM tris(hydroxymethyl)aminomethane (Tris), pH 6.6, 1% SDS, 0.01% bromophenol blue, 15% glycerol, and 5%-mercaptoethanol] at 20°C for up to 4 wk. The
separating gels were composed of 35% glycerol, 10%
acrylamide-N,N-methylene-bis-acrylamide (25:1), 0.375 M Tris (pH 8.8), and 0.4% SDS. The stacking gels were
composed of 30% glycerol, 4%
acrylamide-N,N-methylene-bis-acrylamide (25:1), 0.5 M Tris (pH 6.7), 4 mM EDTA, and 0.4% SDS. The gel constituents were prepared from stock solutions, and polymerization was
initiated with 0.2%
N,N,N,N-tetramethylethylenediamine
and 1.25% ammonium persulfate. The gels were run on a Mighty Small II
mini-gel electrophoresis unit (Hoefer Scientific Instruments, San
Francisco, CA) at 4°C at a constant voltage (90 V) with a variable
current (18-24 mA) for 24 h. At the conclusion of the running
time, the gel was placed in a 10% acetic acid solution for up to 24 h
before staining was commenced. The gels were silver stained with a
commercially produced silver-staining kit (ICN Biochemicals, Irvine,
CA). The stained gels were imaged with a charge-coupled device camera
(Mintron MTV-1801CB) and digitized with a MRC-1000 frame grabber
(Bio-Rad). The images were analyzed with the image-analysis program
Comos (Version 6.03, Bio-Rad). Fibers were classified as fast or slow
twitch according to the relative position of the MHC bands within each
lane, as described by Talmadge and Roy (37). An example illustrating
the different positions of the bands correlating with the MHC within
single fibers is shown in Fig. 1.
Muscle Histochemistry
The contralateral EDL and soleus muscles were cut on a cooled tissue cryostat (AMES cryostat II), and the 8-µm sections (taken from the midmuscle belly region) were reacted for succinate dehydrogenase (SDH) activity and myosin adenosinetriphosphatase (mATPase) activity with the metachromatic dye technique described by Ogilvie and Feeback (28). Three sections were taken from each muscle, and the results were averaged. The sections were viewed with an Olympus IMT-2 inverted microscope. Images of fiber groups were captured with a charge-coupled device camera (Mintron MTV-1801CB) attached to the microscope. The images were digitized to an eight-bit (256 gray scale) resolution with a series 151 (Imaging Technology) frame grabber under computer control (Sparc-1 workstation, Sun Microsystems). Digital images were then analyzed with a custom-written image-analysis program with an interactive mouse-driven cursor to determine fiber staining intensity. Individual fiber borders were traced, and the enclosed area of the fiber was calculated automatically. Only fibers with clearly distinguishable boundaries were included in the sample. Fibers were allocated into type I, type IIA, and type IIB fiber types on the basis of their mATPase activity according to the criteria described by Ogilvie and Feeback (28). In addition, fibers were allocated into specific types on the basis of their relative oxidative status (high, intermediate, and low) according to their relative levels of SDH activity.Statistical Analysis
All data are presented as means ± SE. Differences in muscle fiber contractile characteristics among groups were first assessed by a one-way analysis of variance followed by Newman-Keuls multiple-comparison procedure where differences were detected. Relative fiber type proportions within each muscle were similarly analyzed with analysis of variance followed by Student Newman-Keuls procedure. Differences among groups were considered significant if P < 0.05.RESULTS
Effect of Clenbuterol on Muscle and Body Mass
The effect of clenbuterol treatment on skeletal muscle and body mass is presented in Table 1. After the 15 wk of treatment, body mass was not different among groups. Muscle mass (absolute and relative to body mass) was significantly greater in the soleus but not in the EDL of the Control-Treated animals compared with the other groups. Interestingly, in another fast-twitch muscle, the plantaris, muscle mass was significantly increased both in absolute and relative terms in the Control-Treated animals compared with the other groups (data not shown).
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Effect of Clenbuterol and Exercise on Single-Fiber Contractile Characteristics
EDL. All chemically skinned fibers from the EDL exhibited similar contractile characteristics and were not able to be clearly distinguished as either EDL population I (type IIA) or EDL population II (type IIB), as has been described previously for fresh mechanically skinned fibers (20) or of fibers liberated from partial enzyme digestion of whole muscles (19). Hence all EDL fibers were designated as fast twitch, a classification we have also used previously when describing mouse EDL fibers (40). Gel electrophoresis did not always fully separate the bands corresponding to the type IIA and type IIB varieties but nevertheless verified these fibers as being composed of fast MHC. Typical force-pCa (-pSr) curves for these fast-twitch fibers have been presented elsewhere (see Refs. 10, 20) and are not repeated here.The Control-Treated fibers showed an increased threshold for contraction by and a decreased sensitivity to Ca2+ when compared with fibers from Control animals (see Table 2). However, fibers from the Trained-Treated mice exhibited similar values for the threshold for contraction and Ca2+ sensitivity to those of the Control group animals. The force-pCa relationships of fibers of the Control-Treated group were significantly less steep than those of the Control group mice. The force-pSr relationships of fibers from both clenbuterol-treated groups (Control-Treated and Trained-Treated) were less steep than those from the untreated (Control) animals. The sensitivity to Sr2+ was not significantly different between the Control-Treated and Control groups. Maximal force generation and tension development (per cross-sectional area) of fibers was not different among groups (see Table 2).
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pSr50) values were between 0.50 and 1.00. Such "intermediate" fibers have previously been
described in murine skeletal muscle (18).
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Effect of Clenbuterol on Histochemical Muscle Fiber Type Proportions
The relative fiber type proportions within the EDL and soleus muscles of the three experimental groups are presented in Figs. 2 and 3. On the basis of their mATPase activity, there existed fibers from the EDL of both the Control (14%) and Trained-Treated groups (16.5%) that reacted as type I fibers. In comparison, all fibers from the Control-Treated group reacted as type II. In the soleus, mATPase histochemistry revealed that the Control-Treated group exhibited a significant reduction in the proportion of type I fibers and a concomitant increase in the proportion of type IIA fibers compared with the Control group (see Fig. 2).
To expand and clarify the histochemical classification of fiber types, muscle cross sections were also reacted for SDH activity, which separates fibers on the basis of their relative oxidative status. The results are presented in Fig. 3. In the EDL, the Control-Treated group displayed a significant reduction in the proportion of highly oxidative fibers compared with the Control and Trained-Treated groups. Similarly, this group had an increased percentage of fibers with low SDH activity (i.e., low-oxidative status) and a decreased percentage of fibers with intermediate SDH activity compared with the Control group. The Trained-Treated group also exhibited a reduction in the proportion of intermediate oxidative fibers compared with the Control group; however, the percentage of low-oxidative fibers was not different from control levels. For the soleus muscle, the Trained-Treated group displayed a significant reduction in the proportion of highly oxidative fibers and a significant increase in the percentage of intermediate fibers compared with both the Control-Treated and Control groups.
DISCUSSION
The most important findings of this study were 1) that long-term treatment with clenbuterol caused a decrease in the sensitivity to Ca2+ in fast-twitch fibers from both the EDL and soleus muscles; 2) that low-intensity endurance exercise could prevent the decrease in Ca2+-sensitivity resulting from clenbuterol administration; 3) that clenbuterol did not affect maximal tension development of fast- or slow-twitch fibers; and 4) that clenbuterol caused slow- to fast-twitch fiber conversion within the EDL and soleus muscles, which was also offset by exercise training.
In this study, a relatively high dosage of clenbuterol (2 mg · kg1 · day1)
was administered for two reasons: 1)
to determine the maximum effects of the
2-agonist on skeletal muscle
fibers and 2) to mimic the high to
excessive dosages that are commonly used/abused by strength and
bodybuilding athletes in their quest for maximal muscle development
(6). Much lower doses, on the order of 1 µg/kg, have been proposed
for the treatment of muscle-wasting diseases in humans (22). However,
there have been numerous reports in the literature concerning the
undesirable effects of long-term 2-agonist therapy applied to
asthmatic patients (15). These included deleterious cardiovascular
effects such as tachycardia and myocardial necrosis and unwanted
skeletal muscle effects such as tremor and muscle spasm (15). Thus, to
fully characterize the maximum effects of this
2-agonist, clenbuterol was
given in a relatively high dosage, and to reduce the attenuation of the
response, the drug was administered following a staggered 2:2:3-day
on-off protocol (12, 24).
Effect of Clenbuterol and Exercise on the Contractile Characteristics of Single Muscle Fibers
Long-term clenbuterol treatment caused the force-pCa relationship of fast-twitch fibers from both the EDL and soleus to be right shifted, resulting in an increased threshold for contraction by Ca2+ and decreased fiber sensitivity to Ca2+. No change was observed in the slow-twitch fibers from the soleus in response to clenbuterol treatment. The decreased sensitivity to Ca2+ exhibited by the clenbuterol-treated (Control-Treated) fibers is most likely mediated by elevation of the cytosolic adenosine 3,5-cyclic monophosphate (cAMP)
concentration and the resultant activation of protein kinases, which is
the most commonly proposed mechanism of clenbuterol on skeletal muscle
(17, 25, 41). It may be expected that chronic clenbuterol
administration would maintain high levels of cAMP within skeletal
muscle fibers, although this was not directly measured in this study.
It has previously been demonstrated in skinned cardiac muscle that such
elevations of cAMP had a direct desensitizing effect on the contractile
proteins, causing a decreased sensitivity to
Ca2+, as reflected by a rightward
shift of the force-pCa relationship (13). This decrease in
Ca2+ sensitivity was accompanied
by an increase in phosphorylation of troponin-I by cAMP-dependent
protein kinase, adding to the decreased responsiveness of the
myofilaments to Ca2+ (13). Thus
chronic administration of clenbuterol may decrease fiber sensitivity to
Ca2+ by altering contractile and
regulatory protein interactions, with a resultant alteration in the
cross-bridge cycling process. Interestingly, fast-twitch fibers sampled
from the EDL and soleus of clenbuterol-treated mice that had undergone
15 wk of low-intensity endurance swimming did not exhibit the same
reduction in Ca2+ sensitivity. The
exercise training offset the effects of clenbuterol administration, as
reflected in the similarity of the values for Ca2+-activated contractile
characteristics of the sedentary untreated (Control) and the
swim-trained clenbuterol-treated (Trained-Treated) groups. The
mechanism for this prevention may be the result of training lowering or
maintaining the intracellular cAMP concentration, thereby offsetting
the clenbuterol-induced rightward shifting of the force-pCa
relationship. Previously, it has been shown that high-intensity
(weighted) endurance swim training can modify the contractile apparatus
within rodent skeletal muscle fibers so as to increase the myofilament
sensitivity to Ca2+, causing a
leftward shift of the force-pCa relationship (19, 20). It is
interesting to note that in this study only 1 h of unweighted swimming
5 days/wk was sufficient to offset the effects of clenbuterol
administration.
The shifting of the force-Ca relationship either to the left or right has important physiological significance. A leftward shift of the force-pCa curve would indicate an increase in the sensitivity of a fiber to released Ca2+. If the Ca2+ transient was unaffected [which would imply that the rate of Ca2+ release from the sarcoplasmic reticulum (SR) was unaltered or fixed], then greater force would be generated for each release event or at any given time of the transient. This phenomenon has physiological implications for an increase in the speed of contraction. Conversely, a rightward shift of the force-pCa curve would indicate a decrease in fiber sensitivity to Ca2+ and hence may be seen to be deleterious because of less force or slower force generation at a given [Ca2+]. However, if the SR dynamics were also changed such that the basal [Ca2+] was increased or the SR released more Ca2+, then the force will remain constant if less force is generated for a given [Ca2+], i.e., the same maximal force level, but this would only occur at a higher [Ca2+]. This may have physiological implications as a protective mechanism for the muscle in situations of chronically elevated [Ca2+] (e.g., muscular dystrophy, cardiac myopathy, ischemia, and muscle damage), where reducing residual force production (at a so-called basal [Ca2+]) would reduce the metabolic cost (lower ATP turnover) and enable the muscle fiber to manage with the altered internal environment. Hence the effects of clenbuterol-induced changes in Ca2+ sensitivity could be offset by modifications to the internal Ca2+-handling mechanisms. Direct measurements of the effect of clenbuterol on resting intracellular [Ca2+], Ca2+ transients, and the release and reuptake of Ca2+ by the SR would help clarify these issues.
Further evidence of clenbuterol modifying the contractile characteristics of single muscle fibers was evident in the EDL muscle, where the steepness of the force-pCa and force-pSr relationships of single fibers was significantly reduced in the clenbuterol-treated (Control-Treated and Trained-Treated) groups compared with the Control group. Such decreases in the steepness of the force-pCa relationship indicate a reduction in the cooperative interactions between functional regulatory groups within the thin filament in the activation process (2, 30).
Previous investigations into the effect of clenbuterol on the contractility of skeletal muscle have been at the whole muscle level. These studies have shown that clenbuterol (in doses similar to that used in the present study) decreased the time course of the isometric twitch response (12, 43). Clenbuterol has also been shown to cause significant muscle hypertrophy due to an increase in protein synthesis and possibly due to a decrease in protein degradation (16, 32). Indeed, in this study, clenbuterol caused a significant increase in the mass of the soleus but not of the EDL muscle. Swim training offset this clenbuterol-induced increase in muscle mass (see Table 1).
With intact in vitro muscle preparations, addition of sympathomimetic amines to the bathing solution has been shown to increase force production by a cAMP-dependent phosphorylation of Ca2+-release channels to facilitate SR Ca2+ release during tetanic stimulation (3). However, in this study, muscle force assessed by maximal Ca2+ activation of skinned muscle fiber preparations showed that chronic clenbuterol administration did not affect force production at the single-fiber level. Although the diameters of the single muscle fibers sampled were also not different among groups (see Tables 2 and 3), the fact that fibers swell after skinning indicates that these measurements should be used with caution in the assessment of whether clenbuterol caused fiber hypertrophy. It is possible that the lack of a clenbuterol-induced increase in single-fiber diameter and force production was due to the fact that the increases in overall mass after treatment were too small to reach significance. For example, in the soleus, muscle mass was 17% higher in the Control-Treated compared with the Control group. However, the large variability in diameter of the single fibers sampled from that muscle would be likely to obscure differences in force production at the cellular level after long-term clenbuterol treatment. The increase in maximal isometric force production observed in intact muscle after long-term clenbuterol treatment (12, 43) can most likely be explained by the concomitant increases in overall muscle mass and muscle cross-sectional area.
Effect of Clenbuterol on Muscle Fiber Type Proportions
Clenbuterol has previously been shown to affect the relative fiber type distribution within skeletal muscle, causing type I to type II fiber conversions and even alterations within the type II fiber population (21, 29). Similarly, in this study, clenbuterol caused an increase in the percentage of fast-twitch type II fibers within the soleus. In the EDL, clenbuterol also appeared to modify the type II fiber population. By using the metachromatic dye-ATPase method, it was found that in the EDL of the Control, and even the Trained-Treated, group there were fibers that reacted as type I according to the criteria described by Ogilvie and Feeback (28). However, within the EDL of the Control-Treated group, no such fibers were identified, suggesting that clenbuterol-induced alterations had occurred within this fiber pool. To supplement the fiber type classification by mATPase histochemistry, muscle cross sections were also reacted for relative SDH activity. Generally, SDH activity will only give an estimate of muscle fiber proportions but is useful for detecting exercise and/or drug treatment effects on the oxidative properties of muscle. The results further supported the fiber type classifications obtained from mATPase histochemistry. In the EDL, the Control-Treated animals displayed a reduction in the number of fibers with high and intermediate levels of SDH activity and an increase in the number of fibers with low SDH activity. The clenbuterol-treated animals that were also subjected to the low-intensity swimming program exhibited a reduction in the percentage of intermediate-oxidative fibers but showed no change in the proportion of fibers of low-oxidative status. The percentage of fibers with high levels of SDH activity was similar to that found in the EDL of the Control group. This illustrates the effectiveness of the low-intensity exercise program in preventing the clenbuterol-induced shift in fiber type proportions within the EDL.On the basis of mATPase activity, the proportion of type I and type IIA fibers within the soleus was not significantly different between the Trained-Treated and Control groups, indicating that training offset or prevented the clenbuterol-induced slow-to-fast fiber transformation exhibited in the soleus muscles of the Control-Treated group. Interestingly, we found a decrease in the percentage of fibers with high SDH activity and an increase in the percentage of fibers with intermediate levels of SDH activity within the soleus muscle of the Trained-Treated group compared with both the Control-Treated and Control groups, indicating an overall decrease in oxidative capacity. In a recent study (38), after 7 wk of clenbuterol administration in Zucker rats, citrate synthase activity was decreased in the red and white gastrocnemius but increased in the soleus muscle. Although the histochemical results of the present study cannot be directly correlated with the data of Torgan et al. (38), it is clear that clenbuterol affects enzyme activity differently in fast- and slow-twitch muscles. The reduction in the number of fibers within the soleus muscle exhibiting high levels of SDH activity after clenbuterol and training is interesting, considering that muscle oxidative status is often enhanced after endurance training (38). However, in a previous study, Hayes et al. (11) have shown that the fiber proportions (based on SDH activity) within the mouse soleus were not altered after long-term (15 wk) 2 h/day, 5 days/wk, high-intensity (weighted) swimming training. Therefore, we would expect that the changes in SDH activity observed in the fibers of the soleus muscle of the Trained-Treated group are probably caused by the combined effects of clenbuterol treatment and the swimming activity and not simply an effect of training alone. Although the low-intensity training was sufficient in offsetting the decrease in Ca2+ sensitivity in the sampled fibers, the histochemical data (SDH activity) indicate that this training did not completely prevent the clenbuterol-induced shift in fiber proportions. A comparison of the relative fiber proportions within the soleus based on the number of randomly sampled fibers (and separated into fiber types by their physiological characteristics) supports this notion. In both clenbuterol-treated groups (Control-Treated and Trained-Treated), a greater percentage of fast-twitch fibers were sampled (75 and 74%, respectively) compared with 60% from the Control group.
This study has shown that long-term clenbuterol treatment in mice
caused a decrease in the sensitivity to
Ca2+ of fast-twitch fibers within
the EDL and soleus muscles. Muscle fibers from animals that received
chronic clenbuterol treatment and also underwent an endurance training
program did not exhibit this decrease in
Ca2+ sensitivity. Clenbuterol does
cause increases in muscle mass, a desirable effect for athletes
involved in sports requiring a larger muscle bulk, and its effects on
the skeletal muscle contractile properties might also appear to be
conducive to improvements in specific muscle performance, e.g., faster
rates of contraction and relaxation. Interestingly, however, the
effects on single muscle fiber contractility in isolation appear to be
less desirable, as demonstrated by the decrease in
Ca2+ sensitivity; thus higher
[Ca2+] values are
required to achieve activation. The large doses of mass-building drugs
taken by these athletes may also be life threatening, considering the
evidence for myocardial necrosis and decreased cardiac performance
after chronic high-dose
2-agonist therapy (15). Indeed,
in other related experiments, Duncan and colleagues (7, 8)
have observed a decrease in the exercise performance of chronic
clenbuterol-treated rats during 2-h swimming sessions and short-term
high-intensity treadmill-running bouts that appear to be attributed to
decreased cardiac performance. In this study, 1 h of low-intensity
unweighted swimming was comfortably managed by the clenbuterol-treated
mice, and no attempt was made to subject the clenbuterol-treated mice
to more intense training. It should also be stressed that the
therapeutic dosage of clenbuterol would be considerably smaller than
that employed in this study, on the order of 1 µg/kg, as proposed for
the treatment of muscle-wasting diseases in humans (22). If, because of
its powerful anabolic properties, clenbuterol is to be used as a
therapeutic agent in the treatment of muscle-wasting disorders, it
might also be suggested that some form of low-intensity exercise be
encouraged such that the existing muscle mass be maintained but the
potentially deleterious slow-to-fast fiber type transformations be
minimized. It has been established that in muscular dystrophy the
larger type II fibers are more susceptible to necrosis than the smaller
caliber type I fibers (14, 39). Although a clenbuterol-induced increase or maintenance of existing muscle mass would be desirable for the
dystrophic condition, the slow-to-fast fiber transformations would be
an unwanted side effect because the potential for further necrosis
might be increased. In mice, the performance of even low-intensity
endurance exercise (e.g., unweighted swimming) appears to prevent these
deleterious effects.
ACKNOWLEDGEMENTS
This work was supported by the Australian Research Council and the National Health and Medical Research Council (Australia).
FOOTNOTES
Address for reprint requests: D. A. Williams, Muscle and Cell Physiology Laboratory, Dept. of Physiology, The Univ. of Melbourne, Parkville, Victoria 3052, Australia.
Received 23 January 1995; accepted in final form 3 May 1996.
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