ClenbuterolYoung rats were offered to appetite a semi-synthetic diet either alone or clenbutsrol the beta 2-selective agonist clenbuterol 4-amino-alpha[t-butylamino methyl]-3,5-dichlorobenzyl alcohol. Animals were slaughtered after 0, 4, 8, 11, 18, 21 and 25 d of treatment. At 4, 11, 21 and clenbuterol protein synthesis d muscle protein synthesis was measured by the method of Garlick et al. Although clenbuterol increased the rate tipos de corticoides inhalados protein and RNA accretion in gastrocnemius and soleus muscles, protein synthesis was not clenbuterol protein synthesis. The results suggested that the drug had a rapid, perhaps direct, inhibitory effect on protein degradation.
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However, the cellular mechanisms of the effects of chronic clenbuterol administration on skeletal muscle are not completely understood. Male Wistar rats were chronically treated with 4 mg. EDL muscles from clenbuterol-treated animals displayed hypertrophy, a shift from slow to fast fiber type profile and increased absolute force, while the relative force remained unchanged and resistance to fatigue decreased compared to control muscles from rats treated with saline vehicle.
Conversely, calpain activity was increased by clenbuterol chronic treatment. November 13, ; Accepted: May 26, ; Published: This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
This work was supported by World Anti Doping Agency http: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors have declared that no competing interests exist. Although clenbuterol has positive effects on skeletal muscle mass, some studies also reported myotoxic effects on heart and skeletal muscles  , . Clenbuterol effects on skeletal muscle seem to be fiber-type specific. Indeed, clenbuterol treatment causes hypertrophy only of fast skeletal muscle fibers . Conversely, its myotoxic effects selectively target slow fibers  ,  ,  , . EDL muscles have a fast-glycolytic phenotype and were chosen because they are less affected by myonecrosis and they are more sensitive to clenbuterol-induced hypertrophy  , .
Surgery was performed under sodium pentobarbital anesthesia and all efforts were made to minimize suffering. Every day at 9 am for 21 days, rats were weighted and received a subcutaneous injection of clenbuterol 4 mg. Rats were sacrificed with a lethal dose of pentobarbital sodium 24 h after the last injection.
The pH was set to 7. These conditions are optimal for maintaining muscle viability in vitro for the duration of the experimental protocol. EDL muscle fiber contractile properties were assessed in vitro according to the methods described in detail previously . The optimum muscle length L 0 , i. All subsequent measurements were made at L 0. The tension-frequency response was then determined B Stimulator, Aurora Scientific Inc, Ontario, Canada using stimulation trains of ms, with pulse duration of 0.
Stimulus trains were separated by one-min interval. The maximum isometric tetanic tension P 0 was then determined. Three minutes after the tension-frequency determination, the resistance to fatigue was evaluated using a low-frequency fatigue protocol of hz trains of ms delivered every 2 s for 5 min .
After all measurements, muscles were removed from the bath, trimmed of the connective tissue, blotted dry and weighed on an analytical scale. The whole-muscle CSA was determined by dividing the muscle volume obtained by dividing the muscle mass by the muscle density, assumed to be 1. L f was determined by multiplying L 0 by 0. Muscles were then mounted under stretch between 3. Fluorescence images were acquired in line-scan mode spatial [x] vs.
Image strips of the manually identified area of a single fiber were extracted. Single fibers were manually dissected under a dissecting microscope Stemi ; Zeiss, Le Peck, France , mounted in an experimental chamber containing the internal-like buffer, and permeabilized by adding 0.
Fibers were slightly stretched to the sarcomere length approximately 2. Fluorescence images were acquired with the confocal system operated in line-scan mode x vs.
Image acquisition was performed along the fiber longitudinal axis. Potential spark areas were empirically identified using an auto-detection algorithm . The mean F value of the image was calculated by summing and averaging the temporal F at each spatial location, while ignoring potential spark areas. Following a three-point smoothing routine, potential spark locations were visualized manually and their spatio-temporal properties analyzed as described previously  , .
Fields of one or two fibers were imaged at the acquisition rate of one image every ms. The mean fluorescence was calculated in regions of interest and reported as a function of time. Calpain activity was determined in whole EDL muscle homogenates i. Proteins were transferred onto nitrocellulose membranes subjected to mA of constant electric current for 2 h. Specific interactions were detected with horseradish peroxidase conjugated secondary antibodies Santa Cruz Biotechnology, Gaithersburg, U.
The calpain fractional activation index was calculated by dividing the band intensity of the autolysed form by the sum of the band intensities of both autolysed and non-autolysed isoforms.
Statistical comparison of parameters in the two groups was carried out using a non-parametric Kruskal—Wallis test. Muscle fiber types were identified based on the extent of myosin ATPase staining Fig. As seen in Fig. The images correspond to staining for ATPase activity. Skeletal muscle mass is a key determinant of the force output. We thus investigated whether the EDL muscle hypertrophy and the shift to a fast-glycolytic muscle fiber type profile induced by CBL chronic treatment affected the force output.
Conversely, the peak tetanic force decreased more quickly in CLB-treated muscles, and T lim , used as an index of resistance to fatigue, was significantly reduced compared to CTL. Left graph, P 0: This result was confirmed by immunoblotting experiments showing that autolysis of calpain 1 and calpain 2 was significantly increased in CBL-treated EDL muscle homogenates compared to CTL from 8.
C, Calculation of the calpain fractional activation index i. Clenbuterol induces muscle hypertrophy and a phenotypic shift to a faster contractile profile.
These properties have made clenbuterol a promising candidate for the treatment of various myopathies. However, several side effects have been reported following chronic treatment with clenbuterol and their mechanisms have not been clearly elucidated yet. Taken together, these alterations may contribute to the deleterious effects on muscle functionality reported following chronic treatment with clenbuterol.
Because of its positive effect on muscle mass, clenbuterol therapeutic potential in various pathological conditions affecting muscle tissue has been extensively evaluated. Our day treatment with clenbuterol led to marked EDL muscle hypertrophy as indicated by muscle mass and muscle-fiber CSA measurements and a phenotypic shift to a faster muscle profile. These effects are consistent with what is usually reported in the literature on fast skeletal muscle  , thereby asserting the efficacy of our treatment.
In humans, 4-week clenbuterol treatment in orthopedic patients did not improve muscle absolute strength . In rats, two studies have demonstrated negative effects of chronic clenbuterol treatment on exercise performance evaluated with the run-to-exhaustion treadmill  , swimming or sprinting protocol tests . Increased skeletal muscle fatigability was also reported following clenbuterol treatment in rat  , an effect associated with reduced skeletal muscle oxidative capacity  ,  , .
Moreover, muscle hypertrophy following chronic clenbuterol treatment increases the isometric force in fast-twitch skeletal muscle . Our study confirms these previous results. The absolute force P 0 of EDL muscle was increased following clenbuterol treatment, while the specific force sP 0 was not different compared to controls, suggesting that the strength improvement was only due to the increased skeletal muscle mass.
Analysis of representative recording of the decrease in EDL tension during the fatigue protocol highlighted that the initial tension was higher and appeared earlier in clenbuterol-treated EDL muscles than in untreated controls, in accordance with the increase in muscle mass and the phenotypic shift to a faster contractile profile.
Fatigability of EDL muscles from clenbuterol-treated animals was nevertheless increased, as the peak tetanic force fell more quickly in clenbuterol-treated EDL muscles than in controls, and T lim , used as an index of resistance to fatigue, was significantly reduced.
This last result confirmed that clenbuterol treatment negatively affects muscle endurance, in agreement with the phenotypic remodeling. However, we did not find any deterioration of the specific maximal isometric strength in EDL muscles following chronic treatment.
Recent studies have confirmed that chronic clenbuterol treatment in rats leads to increased calpain activities in fast-twitch skeletal muscles  , . Previously, it has been reported that clenbuterol treatment could decrease calpain 1 activity and increase calpain 2 activity . Here, we found that chronic treatment with clenbuterol increases both calpain 1 and 2 activities in EDL muscles.
While calpain activation seems to be necessary for clenbuterol-induced hypertrophy and the fiber profile shift  , its activation might also cause deleterious effects on skeletal muscle, for example by promoting fiber necrosis, as already suggested .
This effect occurs in association with increased calpain activities. Together, these alterations might contribute to the structural and functional adaptations of skeletal muscle to chronic clenbuterol treatment. As clenbuterol is considered a good candidate for the treatment of muscle disuse, our study underlines that more experiments are necessary to further elucidate the impact of chronic clenbuterol treatment on muscle metabolism and function.
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June 27, Copyright: Muscle fiber contractile function EDL muscle fiber contractile properties were assessed in vitro according to the methods described in detail previously . Calpain activity Calpain activity was determined in whole EDL muscle homogenates i.
Effects of chronic clenbuterol treatment on EDL fiber-type profile and muscle-fiber cross-sectional area. Muscle functional features Skeletal muscle mass is a key determinant of the force output.
Effects of chronic clenbuterol treatment on intact muscle contractile properties. Effects of chronic clenbuterol treatment on EDL muscle calpain activity.
Discussion Clenbuterol induces muscle hypertrophy and a phenotypic shift to a faster contractile profile. Effects of clenbuterol treatment on skeletal muscle mass and fiber-type profile Because of its positive effect on muscle mass, clenbuterol therapeutic potential in various pathological conditions affecting muscle tissue has been extensively evaluated. Clenbuterol treatment increases calpain activities: Author Contributions Conceived and designed the experiments: View Article Google Scholar 2.
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