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. 2015 Apr 15;593(8):2071-84.
doi: 10.1113/jphysiol.2014.287060. Epub 2015 Feb 27.

Chronic Clenbuterol Treatment Compromises Force Production Without Directly Altering Skeletal Muscle Contractile Machinery

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Free PMC article

Chronic Clenbuterol Treatment Compromises Force Production Without Directly Altering Skeletal Muscle Contractile Machinery

G Py et al. J Physiol. .
Free PMC article

Abstract

Clenbuterol is a β2 -adrenergic receptor agonist known to induce skeletal muscle hypertrophy and a slow-to-fast phenotypic shift. The aim of the present study was to test the effects of chronic clenbuterol treatment on contractile efficiency and explore the underlying mechanisms, i.e. the muscle contractile machinery and calcium-handling ability. Forty-three 6-week-old male Wistar rats were randomly allocated to one of six groups that were treated with either subcutaneous equimolar doses of clenbuterol (4 mg kg(-1) day(-1) ) or saline solution for 9, 14 or 21 days. In addition to the muscle hypertrophy, although an 89% increase in absolute maximal tetanic force (Po ) was noted, specific maximal tetanic force (sPo) was unchanged or even depressed in the slow twitch muscle of the clenbuterol-treated rats (P < 0.05). The fit of muscle contraction and relaxation force kinetics indicated that clenbuterol treatment significantly reduced the rate constant of force development and the slow and fast rate constants of relaxation in extensor digitorum longus muscle (P < 0.05), and only the fast rate constant of relaxation in soleus muscle (P < 0.05). Myofibrillar ATPase activity increased in both relaxed and activated conditions in soleus (P < 0.001), suggesting that the depressed specific tension was not due to the myosin head alteration itself. Moreover, action potential-elicited Ca(2+) transients in flexor digitorum brevis fibres (fast twitch fibres) from clenbuterol-treated animals demonstrated decreased amplitude after 14 days (-19%, P < 0.01) and 21 days (-25%, P < 0.01). In conclusion, we showed that chronic clenbuterol treatment reduces contractile efficiency, with altered contraction and relaxation kinetics, but without directly altering the contractile machinery. Lower Ca(2+) release during contraction could partially explain these deleterious effects.

Figures

Figure 1
Figure 1. Fitting of force activation and relaxation in EDL (A) and SOL (B) muscles
Recording shows the fitting curve (red line) obtained from the data curve (black line) recorded at 150 and 100 Hz for EDL and SOL, respectively. The kinetics of force development was described by one exponential increase: a1(1–ekACT*t), where a1 is the value of force at the maximal curve asymptote (mN); e is the exponential function; kACT (s−1) is the rate constant of the force development; and x is the time (ms). The kinetics of force relaxation was described using two exponential slow and fast decays: a2(1–e−slow kREL*(t-td2)) + c2 and a3(1–e− fast kREL*(t-td3)) +c3 where c2 and c3 are the force levels at the beginning of each decay; slow kREL and fast kREL are the rate constants of the slow and fast phases of tension decline, respectively; a2 and a3 are the basal relaxation force level c2 and c3, respectively; and td2 and td3 are the time delays at the beginning of each decay.
Figure 2
Figure 2. Effect of clenbuterol treatment on maximal isometric tetanic power (Po), specific isometric tetanic power (sPo), and force development and force relaxation kinetics in EDL (A, C) and SOL (B, D) muscle
Mean (± SEM) EDL (A) and SOL (B) maximal isometric tetanic power (Po, left vertical axis) and specific maximal isometric tetanic power (sPo, right vertical axis) recorded from CTL-G21 (white bar) and CB-G21 animals (dark bar). C and D, recording shows representative fitting curves in a CB-treated rat (red line) and a CTL-treated rat (black line) in EDL muscle (C) and SOL muscle (D). The kinetics of force development was described by one exponential increase: a1(1–ekACT*t), where a1 is the value of force at the maximal curve asymptote (mN); e is the exponential function; kACT (s−1) is the rate constant of the force development; and x is the time (ms). The kinetics of force relaxation was described using two exponential slow and fast decays: a2(1–e−slow kREL*(t-td2)) + c2 and a3(1–e− fast kREL*(t-td3)) +c3 where c2 and c3 are the force levels at the beginning of each decay; slow kREL and fast kREL are the rate constants of slow and fast phases of tension decline, respectively; a2 and a3 are the basal relaxation forces level c2 and c3, respectively; and td2 and td3 are the time delasy at the beginning of each decay. *P < 0.05 CTL-G21 vs. CB-G21.
Figure 3
Figure 3. Time courses of SOL myosin ATPase in relaxed (A) or Ca2+-activated (B) myofibrils in control group (filled circles), and after 21 days of clenbuterol treatment (open circles)
Reaction mixtures were 3 μm myofibrils (as myosin heads) plus 30 μm [γ-32P]ATP at 4°C. The reaction mixtures were quenched at the times indicated and the total [32P]Pi concentrations were determined. In the relaxed condition, the amplitude of Pi burst and ATPase activity were 0.34 ± 0.05 vs. 0.45 ± 0.07 mol mol−1, and 0.0032 ± 0.0005 vs. 0.0083 ± 0.0011 s−1 for the control and 21-day clenbuterol-treated groups, respectively. In the Ca2+-activated condition, the slope of the fast linear phase (ATPase activity in unloaded shortening), slope of the slow linear phase, duration of the unloaded shortening phase (dashed lines) and the ATP cost of shortening were 0.025 ± 0.004 vs. 0.065 ± 0.01 s−1, 0.016 ± 0.003 vs. 0.031 ± 0.006 s−1, 87 ± 9 vs. 50 ± 5 s and 2.4 ± 0.2 vs. 4.2 ± 0.4 mol mol−1 for the control (CTL-G21) and 21-day clenbuterol-treated groups (CB-G21), respectively.
Figure 4
Figure 4. Effect of clenburerol on SOL myofibrilar ATPase activity in relaxed (A–E) and Ca2+-activated (F–H) conditions at 4°C
Reaction mixture concentrations were 3 and 30 μm for myosin heads and [γ-32P]ATP concentrations in Rapid Flow Quench experiments, respectively. In Stopped-Flow experiments (B and C), myosin head concentration was 1 μm reaction mixture, and ATP concentrations ranged from 30 μm to 1 mm. CTL-G21, control group; CB-G14, 14-day clenburerol-treated group; CB-G21, 21-day clenburerol-treated group; kSS, rate constant of the steady-state phase in relaxed condition; k2/K1, ATP binding-induced myosin detachment obtained from the initial slope of the regression of ktrypophan fast as a function ATP concentration; ktryprophan slow, rate constant observed for the slow phase of tryptophan increase when mixing myofibrils with ATP; k4, rate constant of Pi release (the isomerization step preceding diffusion of Pi); kF, rate constant of the fast steady-state phase in activated condition; tB, duration of the unloaded shortening phase, an index of unloaded shortening velocity; ATPtB, ATP consumed during the unloaded shortening phase. Because no significant difference was detected between CTL-G9, CTL-G14 and CTL-G21 groups, results of the CTL-G21 group are presented. NS, not significant. **P < 0.01; ***P < 0.001.
Figure 5
Figure 5. Effect of clenburerol on EDL myofibrilar ATPase activity in Ca2+-activated conditions at 4°C
Reaction mixture concentrations were 3 and 30 μm for myosin heads and [γ-32P]ATP concentrations in Rapid Flow Quench experiments, respectively. CTL-G21, control group; CB-G9, 9-day clenbuterol-treated group; CB-G14, 14-day clenburerol-treated group; CB-G21, 21-day clenburerol-treated group; k4, rate constant of Pi release (the isomerization step preceding diffusion of Pi); kF, rate constant of the fast steady-state phase in activated condition; tB, duration of the unloaded shortening phase, an index of unloaded shortening velocity; ATPtB, ATP consumed during the unloaded shortening phase. Because no significant difference was detected between CTL-G9, CTL-G14 and CTL-G21 groups, results of the CTL-G21 group are presented. NS, not significant.
Figure 6
Figure 6. Effect of clenbuterol treatment on FDB Ca2+ transient amplitude
Bar graphs comparing amplitude for single-fibre AP-elicited Ca2+ transients in FDB fibres of control (CTL; n = 7) or clenbuterol-treated (CB; n = 8) rats after 9, 14 and 21 days of treatment. Amplitude was calculated from individual exponential fits and averaged. **P < 0.01.

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