Effect of clenbuterol on sarcoplasmic reticulum function in
single skinned mammalian skeletal muscle fibers
Anthony J.
Bakker1,
Stewart I.
Head2,
Anthony C.
Wareham3, and
D. George
Stephenson4
1 Department of Physiology,
University of Western Australia, Nedlands 6907;
2 School of Physiology and
Pharmacology, University of New South Wales, Sydney 2052;
4 School of Zoology, La Trobe
University, Bundoora 3083, Australia; and
3 Division of Neuroscience,
School of Biological Sciences, University of Manchester, Manchester M13
9PT, United Kingdom
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ABSTRACT |
We examined the effect of the
2-agonist clenbuterol (50 µM)
on depolarization-induced force responses and sarcoplasmic reticulum (SR) function in muscle fibers of the rat (Rattus
norvegicus; killed by halothane overdose) that had been
mechanically skinned, rendering the
2-agonist pathway inoperable.
Clenbuterol decreased the peak of depolarization-induced force
responses in the extensor digitorum longus (EDL) and soleus fibers to
77.2 ± 9.0 and 55.6 ± 5.4%, respectively, of
controls. The soleus fibers did not recover. Clenbuterol significantly
and reversibly reduced SR Ca2+
loading in EDL and soleus fibers to 81.5 ± 2.8 and 78.7 ± 4.0%, respectively, of controls. Clenbuterol also produced
an ~25% increase in passive leak of
Ca2+ from the SR of the EDL and
soleus fibers. These results indicate that clenbuterol has direct
effects on fast- and slow-twitch skeletal muscle, in the absence of the
2-agonist pathway. The
increased Ca2+ leak in the triad
region may lead to excitation-contraction coupling damage in the soleus
fibers and could also contribute to the anabolic effect of clenbuterol
in vivo.
calcium uptake; calcium leak; calcium release;
-agonist; excitation-contraction coupling; anabolism
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INTRODUCTION |
RECENT STUDIES HAVE SHOWN that clenbuterol, a
2-agonist, has different
effects in fast- and slow-twitch muscle. Clenbuterol has been shown to
potentiate electrical field stimulation-induced contractions of
isolated extensor digitorum longus (EDL; fast twitch) muscles of the
rabbit (25) but seems to have detrimental effects on soleus (slow
twitch) muscle. Clenbuterol treatment was shown to increase the
fatigability of soleus muscles of mice by 30-40% (11) and to
induce single muscle fiber injuries and significantly increase serum
creatine kinase (muscle isoenzyme CK-MM) levels (a marker of muscle
damage) in the soleus muscle of clenbuterol-treated rats (34). This
difference in sensitivity to damage may reflect higher
2-receptor density in
slow-twitch compared with fast-twitch muscle (16).
However, other
2-agonists, such
as terbutaline, have been shown to potentiate force responses in fast-
and slow-twitch muscles of the rat (4) and to slow high-frequency
fatigue in rat soleus muscle fibers (5).
Clenbuterol is also a powerful anabolic agent that significantly
increases the amount of protein synthesis in skeletal muscle and
simultaneously reduces subcutaneous body fat, and it is thought to act
via
2-receptors because the
anabolic effect is inhibited by high doses of the
2-antagonist propranolol (20)
and the selective
2-antagonist
ICI-118551 (7). However, other groups have reported that high doses of
propranolol have no effect on clenbuterol-induced anabolism (21, 29)
but do reduce
2-mediated fat
deposition (29).
These reports suggest that clenbuterol may elicit other, direct effects
on cell function in skeletal muscle, which are not related to the
2-activation pathway and which
disproportionately affect slow-twitch skeletal muscle, making it more
susceptible than fast-twitch skeletal muscle to damage. Clenbuterol is
membrane permeable, due to its high lipid solubility (30) and is one of
the few
2-agonists that readily
passes the blood-brain barrier (14) and will therefore readily enter
the sarcoplasm in vivo and accumulate within muscle fibers (23). In
this study, we used the skinned fiber technique to examine the effect
of clenbuterol on fast- and slow-twitch skeletal muscle fibers of the
rat in the absence of the
2-activation pathway. In the
skinned fiber technique, the plasma membrane of skeletal muscle fibers
is mechanically removed (9, 33). This compromises the
2-activation pathway by
preventing the accumulation of soluble second messengers, as the
cytosol of the fiber is washed out with a practically infinite pool of
bath solution. Moreover, the dissection of the sarcolemma removes all
surface membrane
2-receptors.
In addition, the skinned fiber technique has a number of other
important advantages over intact preparations for the present study.
After the sarcolemma of the fibers has been mechanically peeled away,
the transverse tubular system seals and repolarizes (9, 18, 33).
Replacing the K+-based solution
bathing the fibers with a solution in which
K+ has been replaced with
Na+ depolarizes the sealed
transverse tubular system and triggers force responses via
the normal excitation-contraction (E-C) coupling pathway. Therefore,
this preparation is unique in that one has access to the cytosol but at
the same time it is possible to trigger force responses by the normal
physiological mechanism (18). Furthermore, this preparation also allows
the quantitative examination of subcellular events occurring within the
fiber, such as sarcoplasmic reticulum (SR)
Ca2+ release, the level of
Ca2+ uptake by the SR, and the
passive SR Ca2+ leak (1),
measurement of which is not presently possible with intact
preparations. In this study, we used the skinned fiber technique to
examine the effects of clenbuterol on E-C coupling, the
Ca2+ sensitivity of the
contractile apparatus, and SR Ca2+
release, Ca2+ loading, and
Ca2+ leakage in fast- and
slow-twitch skeletal muscle fibers of the rat.
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METHODS |
Muscle fibers were isolated from the EDL and soleus muscle of Wistar
rats (Rattus norvegicus) killed by
an overdose of halothane. The muscle fibers were dissected and skinned
in paraffin oil and mounted on a force transducer to monitor isometric
force. The length and diameter of each fiber were measured at slack
length, and the fiber was then stretched by 20% to bring the sarcomere length to ~2.8-3.0 µm to maximize the force responses.
The fibers were then transferred to a 2-ml Perspex bath containing a
potassium hexamethylenediamine tetraacetate
(K+-HDTA) solution composed of (in
mM) 125 K+, 36 Na+, 50 HDTA2
, 8 ATP (total), 8.6 Mg2+ (total), 10 creatine
phosphate, 0.03 EGTA (total), 90 HEPES, and 1 NaN3 at pH 7.10 ± 0.01 and pCa 7.0 (16). NaN3 was
added to prevent mitochondrial
Ca2+ fluxes. The free
Mg2+ concentration was 1 mM.
Isolation of soleus type I fibers.
In the soleus muscle of the rat, two distinct populations of fibers are
present, a predominant population of slow-twitch (type I) fibers and a
smaller but significant population of fast-twitch (type IIa) fibers. In
this study, slow- and fast-twitch soleus fibers could be
distinguished by determining the frequency of myofibrillar
oscillations measured in response to exposure to highly
Ca2+-buffered solutions containing
a submaximal Ca2+ concentration
(12). Type I soleus fibers typically exhibited low-frequency
myofibrillar oscillations (~0.33 Hz), whereas type IIa soleus fibers
exhibited higher frequency myofibrillar oscillations (~1 Hz). Only
soleus type I fibers were used in this study.
Contractile apparatus.
The effects of clenbuterol on maximal force and the sensitivity of the
contractile apparatus to Ca2+ in
the EDL and soleus fibers were determined by exposing fibers to
solutions of different free Ca2+
concentrations in the presence and absence of 50 µM clenbuterol. The
strongly Ca2+-buffered solutions
were prepared by mixing specific proportions of EGTA-containing
solution (solution
A) and CaEGTA-containing solution
(solution
B) (32).
Solution
A contained (in mM) 117 K+, 36 Na+, 8 ATP (total), 1 free
Mg2+, 10 creatine phosphate, 50 EGTA (total), 60 HEPES, and 1 NaN3 at pH 7.10. Solution
B was similar to
solution
A, with the exception that the EGTA
and CaEGTA concentrations of solution
B were 0.3 and 49.7 mM, respectively.
The free Ca2+ concentrations of
the solutions were calculated using a
Kapparent for EGTA of 4.78 × 106
M
1 (12). Maximal force was
determined by exposure to solution B, containing a free
Ca2+ concentration of 3.5 × 10
5 M. During experiments,
force was returned to baseline between force measurements by brief
exposure to solution
A. The plateaus of the force responses
elicited by exposure to solutions of increasing free
Ca2+ concentration were expressed
as a percentage of maximum
Ca2+-activated force and plotted
as a function of pCa. The data were fitted with Hill curves using the
curve-fitting software package GraphPad Prizm (GraphPad Software). The
slopes of the curves (Hill coefficients) and the pCa values
corresponding to 50% of maximum force were determined for both
clenbuterol and control data, and the values were compared.
Depolarization-induced force responses.
The skinned muscle fibers used in this study retain normal E-C coupling
due to resealing of the transverse tubular system after mechanical
skinning. The sealed transverse tubular system can be normally
polarized by exposing the preparation to the
K+-HDTA solution and normal
activation of the voltage sensor, and ensuing SR
Ca2+ release and contraction can
be induced by depolarization of the sealed transverse tubular system
through exposure to an HDTA solution in which
K+ has been replaced by
Na+ (18).
Repolarization of the transverse tubular system between each
consecutive depolarization-induced force response was achieved by a
1-min incubation of the fiber in a
K+-HDTA solution. The
K+ and
Na+ solutions used with rat fibers
were isosmotic (295 mosmol/kg) (18). The pCa in the
K+-HDTA and
Na+-HDTA solutions was weakly
buffered to about pCa 7.0
Depolarization-induced force responses measured in the presence of 50 µM clenbuterol were compared with control responses elicited both
before and after exposure to the drug. Before depolarization in the
presence of clenbuterol, the fibers were first incubated in a
K+-HDTA solution containing the
drug for 30 s to allow time for the drug to diffuse into the fiber. The
depolarization-induced force responses elicited in the fast-twitch
fibers were found to be considerably larger than those elicited in the
slow-twitch fibers when expressed as a percentage of maximum
Ca2+-activated force (31).
Caffeine-induced
Ca2+-release
experiments.
In experiments designed to investigate the effect of clenbuterol on
caffeine-induced Ca2+ release from
the SR of the EDL and soleus fibers of the rat, the fibers were first
depleted of Ca2+ by exposure for 2 min to a K+-HDTA solution
containing low Mg2+ (0.25 mM) and
30 mM caffeine, to maximally release
Ca2+ from the SR, and 0.25 mM
EGTA, to chelate all released Ca2+
and prevent SR Ca2+
reaccumulation. The fiber was then reloaded with
Ca2+ for a particular period of
time (7 s for the soleus fibers and 10 s for the EDL fibers) by
exposure to a highly Ca2+-buffered
solution (pCa 6.55) made by combining
solutions
A and B at a ratio of 1:1. Loading was
rapidly terminated at the end of each loading period by a brief
exposure (~1-2 s) to solution A, after which the fiber was washed in
a K+-HDTA solution to remove
excess EGTA. The fiber was then reexposed to the caffeine solution
(above), and the force response was measured. The time to peak and peak
of force responses elicited after reexposure to a caffeine solution
containing 50 µM clenbuterol were compared with force responses
elicited in an identical caffeine solution without clenbuterol,
measured both before and after the clenbuterol response. Before
exposure to the caffeine solution, the fibers were incubated for 30 s
in a K+-HDTA solution containing
0.25 mM EGTA to allow time for the EGTA to equilibrate within the
fiber. Before exposure to a caffeine release solution containing
clenbuterol, the fiber was exposed to a similar
K+-HDTA solution (0.25 mM EGTA)
also containing clenbuterol to allow time for clenbuterol to enter and
equilibrate within the fiber. The caffeine release solutions were made
at double volume and were split.
Experiments to measure the effect of clenbuterol on SR
Ca2+ loading in
rat EDL and soleus fibers.
In experiments to determine the effect of clenbuterol on SR
Ca2+ loading, the fibers were
depleted and reloaded with Ca2+ in
the same way described in the previous section. The fiber was then
depleted again, and the time integral of the force response elicited by
this depletion was used as an indicator of the amount of
Ca2+ loaded during the loading
period. Depletion measurements made after loading in the presence 50 µM clenbuterol were compared with control measurements made before
and after loading with the drug to minimize errors associated with any
deterioration in the size of the control responses. Before exposure to
the load solution containing clenbuterol, the fibers were exposed to a
K+-HDTA solution containing
clenbuterol for 30 s to allow time for clenbuterol to equilibrate
within the fiber.
Experiments to measure the effect of clenbuterol on SR
Ca2+ leak in rat
EDL and soleus fibers.
The experiments used to examine the effect of clenbuterol on leakage of
Ca2+ from the SR of rat EDL and
soleus fibers were again similar to the SR
Ca2+-release experiments described
in the previous section. The SR of the fibers was first depleted of
Ca2+, and then the SR was reloaded
with Ca2+ for a specific time. In
these experiments, the fibers were then exposed to a
"Ca2+ leak"
solution before being reexposed to the caffeine depletion solution. The
Ca2+ leak solution consisted of
the K+-HDTA solution with 0.75 mM
EGTA added to sequester all leaked Ca2+. The force responses elicited
after a period of exposure to the Ca2+ leak solution were compared
with control force responses (no exposure to
Ca2+ leak solution) measured
before and after the measurement of the force responses designed to
measure SR Ca2+ leak. The
resulting control leak force responses, which represent the normal leak
associated with skeletal muscle SR, were compared with the force
responses elicited after exposure to a leak solution containing 50 µM
clenbuterol.
In the clenbuterol solutions, the clenbuterol (Sigma) was dissolved in
a K+-HDTA solution similar to that
used in the experiments. In all cases, an equal volume of
K+-HDTA solution was added to the
matching control (no clenbuterol) solution. Ascorbate (0.56 mM) was
added to all clenbuterol (and control) solutions in this study to
minimize oxidation of the drug during the course of the experiments.
All experiments were conducted at room temperature (21-22°C)
except those involving depolarization-induced force responses in
soleus, which were undertaken at 25°C. Results are expressed as
means ± SE. The results were analyzed with
t-tests using the statistical software
package INSTAT.
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RESULTS |
The effect of clenbuterol on the contractile apparatus of rat EDL
and soleus fibers.
Because the main experimental parameter measured in this study was
force, we first examined the effect of clenbuterol on the contractile
apparatus of the EDL and soleus fibers to ensure that the experimental
results in the following sections were not misconstrued due to
clenbuterol-induced changes in the sensitivity of the contractile apparatus to Ca2+. The skinned EDL
and soleus fibers were exposed to highly buffered Ca2+ solutions of different, known
free Ca2+ concentrations, and the
resulting force responses were measured. The data were plotted as
percentage of maximum force vs. pCa and fitted with Hill curves (Fig.
1, A and
B). No significant
difference in either maximum force production or the sensitivity of the
contractile apparatus to Ca2+
(Table 1) compared with control
measurements was found in the presence of 50 µM clenbuterol in the
EDL and soleus fibers.

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Fig. 1.
Effect of clenbuterol (50 µM) on force measurements elicited in
extensor digitorum longus (EDL; A) and type I soleus
(B) skinned fibers exposed to highly buffered
Ca2+ solutions of different pCa,
plotted as a percentage of maximum force and fitted with Hill curves.
No significant difference was found between force measurements made in
presence of 50 µM clenbuterol and under control conditions,
indicating that clenbuterol had no significant effect on maximum force
or sensitivity of contractile apparatus to
Ca2+ in skinned fibers.
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Table 1.
Effects of clenbuterol on Ca2+ sensitivity of
contractile apparatus and caffeine-induced SR force responses in rat
EDL and soleus fibers
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The effect of clenbuterol on the E-C coupling in rat EDL and soleus
fibers.
In these experiments, we examined the effect of clenbuterol on force
response triggered via the normal E-C coupling pathway. The skinned
fibers used in this study retain an intact E-C coupling pathway. During
the mechanical skinning process used to remove the sarcolemma from the
fibers, the transverse tubular system seals and repolarizes after
exposure to a K+-HDTA solution.
The transverse tubular system can then be depolarized, and
depolarization-induced force responses can be elicited by exposing the
preparation to an HDTA solution in which
K+ has been replaced with
Na+ (18).
Clenbuterol had a marked effect on the peak of depolarization-induced
force responses in both skinned EDL and soleus fibers (Table
2). In the EDL fibers, 50 µM clenbuterol
decreased the peak of depolarization-induced force responses by 23%
compared with initial control responses (Fig.
2A, Table
2), and force responses similar to initial control measurements could
be obtained after clenbuterol was removed
(t-test,
P = 0.78, n = 10), showing that the effect of
clenbuterol on depolarization-induced force responses in rat EDL fibers
was reversible (Fig. 2A, Table 2). The
presence of 50 µM clenbuterol had no significant effect on the half
peak width of the depolarization-induced force responses evoked in the
EDL fibers (paired t-test,
P = 0.48).
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Table 2.
Effects of clenbuterol on depolarizationinduced force responses
and SR Ca2+ loading in EDL and soleus fibers
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Fig. 2.
Effect of 50 µM clenbuterol on peak of force responses elicited by
normal excitation-contraction coupling pathway in skinned fibers from
EDL (A) and soleus
(B) muscles of rat. Clenbuterol
decreased peak of depolarization-induced force responses in both EDL
and soleus fibers. In all cases, fibers were exposed to a potassium
hexamethylenediamine tetraacetate solution for 30 s between responses
to allow repolarization of transverse tubular system. In clenbuterol
responses, depolarization was preceded by a 30-s exposure to
repolarization solution containing clenbuterol. Effect of clenbuterol
on peak of depolarization-induced responses was reversible in EDL
fibers but not in soleus fibers (last responses;
A and
B,
right). Depol, depolarization;
Repol, repolarization.
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Depolarization-induced force responses elicited in skinned soleus type
I fibers were substantially more sensitive to clenbuterol than were the
EDL fibers. The peaks of the depolarization-induced force responses
elicited in the soleus type I fibers fell by ~45% in the presence of
clenbuterol (Fig. 2B, Table 2) and
failed to significantly recover after washout of the drug (Fig.
2B, Table 2). In contrast, in
fast-twitch fibers the peak of the depolarization-induced force
responses fell by only 23% in the presence of clenbuterol, and the
effect was fully reversible. These results suggest that 50 µM
clenbuterol causes marked, irreversible damage to E-C coupling in
soleus fibers. No significant difference was found between the half
peak width (paired t-test,
P = 0.95) of the
depolarization-induced force responses elicited in the soleus fibers in
the presence and absence of 50 µM clenbuterol.
The depolarization-induced force responses elicited in this study are
equivalent to K+ depolarizations
of intact skeletal muscle fibers. Therefore, it was highly unlikely
that the effect of clenbuterol on the peak of depolarization-induced
force responses shown in this study was due to clenbuterol-induced
changes in membrane excitability and more likely that it was due to
some direct effect of clenbuterol on SR function.
The effect of clenbuterol on caffeine-induced force responses in rat
EDL and soleus fibers.
To test whether clenbuterol was inhibiting the depolarization-induced
force responses in the EDL and soleus fibers by altering the
caffeine-induced Ca2+-release
properties of the SR, fibers were initially exposed to an HDTA solution
containing 30.0 mM caffeine and 0.75 mM EGTA to completely deplete the
SR of Ca2+. The fibers were then
reloaded with Ca2+ for a known
length of time by exposure to a highly
Ca2+-buffered load solution (pCa
6.55). After this loading period, the fiber was reexposed to the
caffeine-EGTA solution in either the presence or the absence of 50 µM
clenbuterol, and the time to peak and peak of the resulting force
responses were measured. Changes in the time to peak and/or
peak of the force responses in the presence of 50 µM clenbuterol
would be indicative of clenbuterol-induced changes in the rate of SR
Ca2+ release and/or rate
of force development at the cross-bridge level.
No significant difference in the peak or time to peak of
caffeine-induced force responses was found in either EDL or soleus fibers in the presence and absence of 50 µM clenbuterol (Fig. 3, Table 1). These results indicate that 50 µM clenbuterol has no significant effect on caffeine-induced
Ca2+ release or rate of force
development in EDL and soleus fibers of the rat, and, therefore, the
reduction in the peak of depolarization-induced force responses
elicited in these fibers in the presence of clenbuterol must be due to
inhibition at another stage of the E-C coupling process.

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Fig. 3.
Effect of 50 µM clenbuterol on caffeine-induced
Ca2+ responses in EDL
(A) and soleus
(B) fibers of rat. The fibers were
loaded with Ca2+ for fixed periods
of time (EDL, 10 s; soleus, 7 s) in a highly buffered
Ca2+ solution (pCa 6.55; note that
all Ca2+ loading was undertaken in
absence of clenbuterol in this experiment) before exposure to caffeine
Ca2+-release solution.
Caffeine-induced responses in presence of clenbuterol were compared
with control responses measured before and afterward. Note that in
soleus fibers (B) the baseline after
caffeine contracture did not return to original level shown before
caffeine contracture. This was due to effect of caffeine increasing the
sensitivity of contractile apparatus to
Ca2+. Effect is visible in soleus
fibers because the contractile apparatus of soleus is more sensitive to
Ca2+ than is that of EDL (see
control values of pCa corresponding to 50% of maximum force in Table
1). After removal of caffeine, baseline always quickly returned to
original level, indicating that no damage to contractile apparatus had
occurred.
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The effect of clenbuterol on SR
Ca2+ loading in
rat EDL and soleus fibers.
To test whether clenbuterol was inhibiting the depolarization-induced
force responses at the level of SR
Ca2+ loading, we examined the
effect of 50 µM clenbuterol on SR
Ca2+ loading in skinned EDL fibers
and soleus fibers. In these experiments, the SR of the fibers was
initially depleted of Ca2+ and
then reloaded with Ca2+ for a
known length of time (see previous section). After this loading period,
the fiber was reexposed to the caffeine-EGTA solution to release all
releasable Ca2+ from the SR. The
level of Ca2+ loading that had
occurred during the loading process could then be estimated by
measuring the area under the force response resulting from reexposure
to the caffeine-EGTA solution (1). The integrals of caffeine-induced
force responses elicited after loading in the presence and absence of
50 µM clenbuterol were compared.
In the skinned EDL and soleus fibers, SR
Ca2+ loading was significantly
reduced to 81.5 (n = 38) and 78.7%
(n = 19) of control levels,
respectively, in the presence of 50 µM clenbuterol (Fig. 4, A and
B). This inhibition of SR
Ca2+ loading was significantly
reversible in both the EDL and soleus fibers, although the SR loading
did not totally recover to original control levels (Table 2).

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Fig. 4.
An example of effect of 50 µM clenbuterol on sarcoplasmic reticulum
(SR) Ca2+ loading in skinned EDL
(A) and soleus
(B) fibers. Clenbuterol was present
only in Ca2+-loading solution used
to load Ca2+ for 2nd responses
shown (A and
B,
middle). Presence of 50 µM
clenbuterol in Ca2+-loading
solution reduced SR Ca2+ loading
in both EDL and soleus fibers.
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To provide additional evidence that the effects of clenbuterol on SR
function were not related to its activity as a
2-agonist, the experiments were
repeated in the presence of propranolol (1 µM). In the presence of
both clenbuterol and propranolol, SR
Ca2+ loading in skinned EDL fibers
(n = 14) was also reduced by a similar
amount, to 82.8 ± 4.0% of control levels (paired
t-test, P = 0.004), suggesting that the
decrease in SR loading in the presence of clenbuterol is due to a
direct effect of clenbuterol on SR function. In the presence of
propranolol alone, SR Ca2+ loading
in EDL fibers (n = 14) was not
significantly different from control measurements (paired
t-test,
P = 0.068).
The effect of clenbuterol on leak of
Ca2+ from the SR
of rat EDL and soleus fibers.
The simplest hypothesis to explain the decrease in SR
Ca2+ loading induced by
clenbuterol is that this drug slows the SR
Ca2+ pump. However, substances
that slow or inhibit the SR Ca2+
pump typically produce significantly wider depolarization-induced force
responses due to decreased SR Ca2+
uptake (1). In this study (see previous section), no significant difference was found between half peak width of the
depolarization-induced force responses induced in EDL and soleus fibers
in the presence and absence of 50 µM clenbuterol, which strongly
suggests that 50 µM clenbuterol does not slow the SR
Ca2+ pump in mammalian EDL and
soleus fibers.
However, mammalian skeletal muscle has recently been shown to possess a
small passive Ca2+ leak from the
SR (1). Therefore, it is possible that clenbuterol acts to decrease SR
Ca2+ loading by increasing the
passive leak of Ca2+ from the SR
of rat EDL and soleus fibers. To test this hypothesis, rat EDL and
soleus fibers, previously depleted of
Ca2+, were loaded under control
conditions and then reexposed to the caffeine release solution after
first being exposed for 3.5 min to a
Ca2+ leak solution with or without
50 µM clenbuterol. The Ca2+ leak
solution contained 0.75 mM EGTA to chelate leaked
Ca2+ and to prevent its reuptake
by the SR.
The normal passive SR Ca2+ leak
present in EDL and soleus skeletal muscle fibers reduced
Ca2+ loading (measured as the area
under the force response) to 45.3 ± 8.9 (n = 6) and 56.8 ± 4.7% (n = 6), respectively, of initial controls under the conditions of this study. This normal passive SR
Ca2+ leakage in the EDL and soleus
fibers was significantly elevated by the presence of 50 µM
clenbuterol in the Ca2+ leak
solution (Fig. 5). Force responses obtained
in the EDL and soleus fibers after a 3.5-min exposure to the
Ca2+ leak solution containing
clenbuterol were significantly reduced to 74.6 ± 8.7%
(t-test,
P = 0.033, n = 6) and 76.4 ± 6.7%
(t-test, P = 0.017, n = 6), respectively, of responses
elicited after exposure to a Ca2+
leak solution without clenbuterol. These results suggest that the
decrease in SR Ca2+ loading
observed in EDL and soleus fibers of the rat in the presence of 50 µM
clenbuterol is predominantly due to a clenbuterol-induced increase in
passive SR Ca2+ leak.

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Fig. 5.
Effect of 50 µM clenbuterol on SR
Ca2+ leak in EDL
(A) and soleus
(B) fibers of rat. The fibers were
loaded with Ca2+ by exposure to a
highly buffered Ca2+ solution (pCa
6.55) for fixed periods of time (EDL, 10 s; soleus, 7 s) and then
exposed to a leak solution that contained 0.75 mM EGTA to sequester all
leaked Ca2+. Control
Ca2+ leak force responses elicited
after 3.5 min of exposure to Ca2+
leak solution were compared with control force responses (no exposure
to Ca2+ leak solution, not shown)
measured before and after measurement of force responses designed to
measure SR Ca2+ leak. Resulting
control leak force responses demonstrate normal leak associated with
skeletal muscle fibers (left and
right). These responses were then
compared with force responses elicited after exposure to a leak
solution containing 50 µM clenbuterol
(middle).
|
|
 |
DISCUSSION |
In this study, we examined the effect of the anabolic
drug clenbuterol, a
2-agonist,
on single fast- and slow-twitch mammalian skeletal muscle fibers of the
rat. The fibers had been mechanically skinned to wash out soluble
second messengers and remove
2-adrenergic receptors from the
surface membrane and thus render the
2-agonist pathway inoperable.
This study showed that in the absence of the
2-agonist pathway 50 µM
clenbuterol still has marked detrimental effects on skeletal muscle
depolarization-induced force responses, which, in the case of the
soleus, were irreversible. The results of this study are consistent
with previous reports of the increased detrimental effects of
clenbuterol (administered orally) on slow-twitch muscle compared with
fast-twitch muscle (11, 34).
In this study, we used a clenbuterol concentration of 50 µM because
it ensured a rapid response (50 µM clenbuterol permanently damaged
depolarization-induced force responses in all soleus fibers within 30 s) and because it represented the upper level of what might be found in
vivo when the anabolic dose is given. Recent studies examining the
anabolic effects of clenbuterol report use of a clenbuterol dose of
1-2
mg · kg
1 · day
1
injected subcutaneously (8, 26, 28). At 2 mg/kg, the maximum internal
clenbuterol concentration expected, considering the volume of the rat,
would be ~6 µM. However, clenbuterol is lipophilic and has been
shown to accumulate within muscle fibers (23), and clenbuterol has a
relatively long half-life of ~35 h (35). Therefore, chronic
administration of the anabolic dose of clenbuterol could lead to
clenbuterol levels in muscle well above 6 µM and closer to the 50 µM used in this study. It has recently been shown in adult rats that
a single subcutaneous injection of only 250 µg/kg of clenbuterol
rapidly induces degenerative changes in soleus fibers while having no
damaging effect on the fast-twitch plantaris muscle (S. J. P. Damment, Glaxo, personal communication). Furthermore, considering the
abuse of this drug within the athletic and bodybuilding communities,
where it is common practice to administer anabolic substances in
concentrations far in excess of the anabolic dose, the concentrations
of clenbuterol occurring may be even higher. For example, within
bodybuilding circles, users of anabolic steroids have been reported to
commonly use up to 26 times the therapeutic dose (3). In the case of
clenbuterol, higher doses are likely to severely reduce athletic
performance rather than enhance it. It has recently been shown that
clenbuterol significantly decreases exercise performance in mice (15,
19). Duncan (10) found that chronic clenbuterol treatment significantly
reduced the ability of rats to exercise at high intensity and that
chronic clenbuterol treatment in combination with exercise predisposed
the animals to sudden death, presumably as a result of cardiac failure.
It has been reported that clenbuterol administration increases the
proportion of fast-twitch fibers in the soleus muscle (8, 19, 36). In
this study, it is shown that clenbuterol preferentially damages soleus
type I fibers. Such damage in vivo could contribute to the increase in
the number of fast-twitch fibers by selectively destroying a proportion
of the slow-twitch type I soleus fibers.
Administration of clenbuterol to mice has been reported to decrease the
sensitivity of the contractile apparatus to
Ca2+ in fast-twitch EDL fibers
(19). However, the present study shows that clenbuterol has no effect
on the Ca2+ sensitivity of the
contractile apparatus in EDL fibers of the rat when added acutely to
the experimental solutions, suggesting that chronic clenbuterol
treatment has some permanent effect on the functional state of the
contractile and regulatory proteins themselves.
Experiments examining the effect of clenbuterol on SR function in rat
EDL and soleus fibers in the present study showed that the decrease in
the size of depolarization-induced force responses in the presence of
clenbuterol was due to a clenbuterol-induced increase in the rate of
passive Ca2+ leak from the SR,
which reduced SR Ca2+ levels,
leaving less Ca2+ available for
release in response to stimulation. A continuously raised intracellular
Ca2+ concentration in the
spatially restricted region of the triad due to increased SR
Ca2+ leakage could damage E-C
coupling, as elevated intracellular Ca2+ levels have recently been
shown to damage E-C coupling in rat EDL muscle fibers (17). However, in
this study, only the soleus fibers were irreversibly damaged by
clenbuterol; the EDL fibers recovered almost fully. Therefore, it is
possible that slow-twitch muscle is more susceptible to the damage to
E-C coupling caused by raised Ca2+
levels than that observed in fast-twitch muscle in the rat, which may
be able to cope with the increased level of intracellular Ca2+ concentration caused by the
clenbuterol-induced increase in passive leak in these fibers.
Interestingly, increased SR Ca2+
release or leak is thought to be the primary cause of
malignant hyperthermia, and clenbuterol has been shown to significantly increase body temperature in rats (6) and induce hyperthermia in rats
kept at high ambient temperature (24).
If an increased passive leak is also present in vivo in response to the
anabolic dose of clenbuterol, the resulting raised Ca2+ levels may also play a role
in promoting the anabolic effect of clenbuterol via, for example, the
activation of the
Ca2+/calmodulin-dependent protein
kinase, which has been shown to regulate gene expression in many cells
(27).
-Adrenoceptor-mediated hypertrophy in neonatal rat cardiac
myocytes has recently been shown to be mediated by a
Ca2+-dependent pathway involving
the SR rather than by a pathway involving cAMP (2).
It is highly unlikely that the effect of clenbuterol on
depolarization-induced force responses shown in the present study is
due to clenbuterol-induced changes in membrane excitability, as the
depolarization-induced force responses are activated by a mechanism
equivalent to K+ depolarization of
intact skeletal muscle fibers, in which the voltage sensors of the
transverse tubular system are depolarized directly. Considering the
substantial effects of clenbuterol on SR function, it would seem likely
that alterations of SR function are ultimately responsible for the
effect of clenbuterol on depolarization-induced force responses in
mammalian skeletal muscle. However, this study does not rule out the
possibility that clenbuterol may also alter E-C coupling by affecting
the charge movement of the voltage sensors.
In conclusion, this study shows that the anabolic drug clenbuterol has
direct effects on SR function in fast- and slow-twitch skeletal muscle
that are unrelated to the
2-activation pathway. Clenbuterol causes a net decrease in SR
Ca2+ accumulation in skeletal
muscle fibers due to a clenbuterol-induced increase in the passive
Ca2+ leak from the SR. In
slow-twitch soleus fibers, exposure to clenbuterol leads to permanent
damage to the E-C coupling process. The results of this study have
important implications for the safe use of clenbuterol in clinical
applications.
 |
ACKNOWLEDGEMENTS |
This work was supported by grants from the National Health and
Medical Research Council of Australia.
 |
FOOTNOTES |
Address for reprint requests: A. J. Bakker, Dept. of Physiology,
University of Western Australia, Nedlands 6907, Australia.
Received 10 November 1997; accepted in final form 17 February
1998.
 |
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