Fast-twitch EDL Research Articles

Skeletal Muscle Chloride Channel, a Biophysical Sensor of Dystrophic Progression in Mdx Mouse, is a Potential Target of Pro-inflammatory Mediators

A decrease in resting chloride channel conductance (gCl) characterizes myofibers of dystrophic mdx mouse in relation to both spontaneous degeneration, as in diaphragm, or exercise-induced damage as in fast-twitch EDL muscle (De Luca et al., J. Pharmacol. Exp. Ther. 2003). The molecular mechanism underlying gCl impairment might involve change in ClC-1 channel expression/turnover and/or function. Considering the role of inflammation in dystrophic damage, we tested if pro-inflammatory mediators may have ClC-1 channel as a target, through phosphorylating/dephosphorylating pathways. Two microelectrodes current clamp recordings were used to measure resting gCl in EDL and diaphragm muscle fibers from adult wild-type (wt) and mdx mice. In line with previous evidences, the in vitro application of phorbol dibutyrate (50 microM) reduced gCl of wt EDL myofibers from 2610±240 microS/cm2 (n=30) to 1265±180 microS/cm2 (n=15). The application of TNF-alpha (1-30 ng/ml), a cytokine highly expressed in dystrophic muscle, to wt EDL fibers reduced gCl in a concentration-dependent manner with a maximal significant 20% decrease at 10-30 ng/ml. Angiotensin-II (10-100nM), possibly involved in muscle degeneration and oxidative stress, produced a concentration-dependent decrease of gCl in wt EDL myofibers, with a 40 % decrease at 100 nM. The PKC-inhibitor chelerythrine (1 microM) contrasted the effect of either phorbol ester, TNF-alpha or angiotensin-II. The application of 3.3 nM IGF-1 to diaphragm and EDL muscle fibers from exercised mdx mice, significantly counteracted the 40% impairment of gCl. Okadaic acid (0.25 microM) fully prevented IGF-1 effect, supporting the involvement of a serine-threonine phosphatase in IGF-1 activity (De Luca et al., Br. J. Pharmacol. 1998). Angiotensin receptor antagonists and other tools are in use to gain further insight in the mechanisms involved in inflammation-sensitive ClC-1 impairment in muscular dystrophy (Telethon-Italy GGP05130).

Immunoneutralization of TGFβ1 Improves Skeletal Muscle Regeneration: Effects on Myoblast Differentiation and Glycosaminoglycan Content

When injured by crushing, the repair of the slow-twitch soleus rat muscle, unlike the fast-twitch EDL, is associated with fibrosis. As TGFβ1, whose activity can be controlled by glycosaminoglycans (GAG), plays a major role in fibrosis, we hypothesized that levels of TGFβ1 and GAG contents could account for this differential quality of regeneration. Here we show that the regeneration of the soleus was accompanied by elevated and more sustained TGFβ1 level than in the EDL. Neutralization of TGFβ1 effects by antibodies to TGFβ1 or its receptor TGFβ-R1 improved muscle repair, especially of the soleus muscle, increased in vitro growth of myoblasts, and accelerated their differentiation. These processes were accompanied by alterations of GAG contents. These results indicate that the control of TGFβ1 activity is important to improve regeneration of injured muscle and accelerate myoblast differentiation, in part through changes in GAG composition of muscle cell environment.

Disuse of rat muscle in vivo reduces protein kinase C activity controlling the sarcolemma chloride conductance

Muscle disuse produced by hindlimb unloading (HU) induces severe atrophy and slow-to-fast fibre type transition of the slow-twitch soleus muscle (Sol). After 2 weeks HU, the resting ClC-1 chloride conductance (g(Cl)) of sarcolemma, which controls muscle excitability, increases in Sol toward a value typical of the fast-twitch EDL muscle. After 3 days of HU, the g(Cl) increases as well before initiation of fibre type transition. Since ClC-1 channels are acutely silenced by PKC-dependent phosphorylation, we studied the modulation of g(Cl) by PKC and serine-threonine phosphatase in Sol during HU, using a number of pharmacological tools. We show that a fraction of ClC-1 channels of control Sol are maintained in an inactive state by PKC basal activity, which contributes to the lower g(Cl) in control Sol compared to EDL. After 14 days of HU, PKC/phosphatase manipulation produces effects on Sol g(Cl) that corroborate the partial slow-to-fast transition. After 3 days of HU, the early increase of g(Cl) in Sol is entirely attributable to a reduction of PKC activity and/or activation of phosphatase, maintaining ClC-1 channels in a fully active state. Accordingly, we found that HU reduces expression of PKCalpha, epsilon, and isoenzymes in Sol and EDL muscles and reduces total PKC activity. Moreover, we show that the rheobase current is increased in Sol muscle fibres as soon as after 3 days of HU, most probably in relation to the increased g(Cl). In conclusion, Sol muscle disuse is characterized by a rapid reduction of PKC activity, which reduces muscle excitability and is likely to contribute to disuse-induced muscle impairment.

Effects Of Clenbuterol On MyoD, UCP3, MCT1, CD147, And Myosin-hc Isoforms Of Rat Skeletal Muscles

Clenbuterol is one of the beta-2 adrenergic receptor agonists with powerful muscle anabolic and lipolytic effects and is prohibited to use as doping drug for athletes. Since previously we have reported that clenbuterol induced the muscular hypertrophy, the possible decreased endurance capacity by the decreased expression of lactate transporter (MCT1 and MCT4) in rat skeletal muscles, and the transition of muscle fiber type from slow to fast-type (JAP 91(1), 85–90, 2001). However, the mechanism of these changes with clenbuterol was not clear yet. PURPOSE This study was performed to examine the effects of clenbuterol on the molecular adaptation system of muscle. METHODS In the present study, clenbuterol was subcutaneously administered in 8wks female Sprague-Dawley rats (CLEB: n=8) during 4 weeks. The effects of clenbuterol on slow-twitch soleus and fast-twitch EDL muscles were examined using reverse-transcription polymerase chain reaction (RT-PCR) analysis of the mRNAs of MCT1, MyoD, CD147, and UCP3. Furtermore, the effects of CLEB on proteins were examined using polyacrylamide gel (PAGE) systems of non-denatured PAGE for LDH isozymes and denatured SDS-PAGE for the both of Myosin HCs, MCT1, and CD147. RESULTS The muscle wet weights increased in both soleus and EDL muscles. The MyoD and UCP3 mRNAs increased drastically in SOL. They may explained the accelelated higher lipolytic function in soleus muscle by the accumulated fast-twitch fibers with fiber type transition from slow-to-fast. Furthermore, the increased MyoD mRNA may explain the muscle hypertrophy in soleus, but no changes of MyoD mRNA in EDL may need another explanation for the hypertrophy like as autocrine of IGF-1. The contents of MCT1 and its mRNA decreased in EDL and they tended to decrease in SOL. The contents of CD147 significantly decreased in EDL, but the CD147 mRNA showed no change in both SOL and EDL. In EDL, the decreased MCT1 might be explained by the decreased CD147 which is important to translocate MCT1 to plasma membrane. The difference of these changes between the proteins and mRNAs might be due to the posttranslational modification. These findings suggest that clenbuterol is able to induce lactate accumulation easily and to decrease the endurance capacity. CONCLUSIONS These results suggest that the clenbuterol induced many chages on skeletal muscles via positive feedback mechanism of MyoD in myogenesis as well as via cAMP of beta-2 adrenergic receptor system. It is necessary to carry out more detail experiments to explain the specific adaptation mechanism. Supported by A grant-in-aid (No. 15500437 to T.K.) from the Ministry of Education, Science, Sports, and Culture of Japan.

Effects Of Clenbuterol On MyoD, UCP3, MCT1, CD147, And Myosin-hc Isoforms Of Rat Skeletal Muscles

Clenbuterol is one of the beta-2 adrenergic receptor agonists with powerful muscle anabolic and lipolytic effects and is prohibited to use as doping drug for athletes. Since previously we have reported that clenbuterol induced the muscular hypertrophy, the possible decreased endurance capacity by the decreased expression of lactate transporter (MCT1 and MCT4) in rat skeletal muscles, and the transition of muscle fiber type from slow to fast-type (JAP 91(1), 85–90, 2001). However, the mechanism of these changes with clenbuterol was not clear yet. PURPOSE This study was performed to examine the effects of clenbuterol on the molecular adaptation system of muscle. METHODS In the present study, clenbuterol was subcutaneously administered in 8wks female Sprague-Dawley rats (CLEB: n=8) during 4 weeks. The effects of clenbuterol on slow-twitch soleus and fast-twitch EDL muscles were examined using reverse-transcription polymerase chain reaction (RT-PCR) analysis of the mRNAs of MCT1, MyoD, CD147, and UCP3. Furtermore, the effects of CLEB on proteins were examined using polyacrylamide gel (PAGE) systems of non-denatured PAGE for LDH isozymes and denatured SDS-PAGE for the both of Myosin HCs, MCT1, and CD147. RESULTS The muscle wet weights increased in both soleus and EDL muscles. The MyoD and UCP3 mRNAs increased drastically in SOL. They may explained the accelelated higher lipolytic function in soleus muscle by the accumulated fast-twitch fibers with fiber type transition from slow-to-fast. Furthermore, the increased MyoD mRNA may explain the muscle hypertrophy in soleus, but no changes of MyoD mRNA in EDL may need another explanation for the hypertrophy like as autocrine of IGF-1. The contents of MCT1 and its mRNA decreased in EDL and they tended to decrease in SOL. The contents of CD147 significantly decreased in EDL, but the CD147 mRNA showed no change in both SOL and EDL. In EDL, the decreased MCT1 might be explained by the decreased CD147 which is important to translocate MCT1 to plasma membrane. The difference of these changes between the proteins and mRNAs might be due to the posttranslational modification. These findings suggest that clenbuterol is able to induce lactate accumulation easily and to decrease the endurance capacity. CONCLUSIONS These results suggest that the clenbuterol induced many chages on skeletal muscles via positive feedback mechanism of MyoD in myogenesis as well as via cAMP of beta-2 adrenergic receptor system. It is necessary to carry out more detail experiments to explain the specific adaptation mechanism. Supported by A grant-in-aid (No. 15500437 to T.K.) from the Ministry of Education, Science, Sports, and Culture of Japan.

Evaluation of a Hill based muscle model for the energy cost and efficiency of muscular contraction

The purpose of this study was to evaluate a Hill-based mathematical model of muscle energetics and to disclose inconsistencies in existing experimental data. For this purpose, we simulated iso-velocity contractions of mouse fast twitch EDL and slow twitch SOL fibers, and we compared the outcome to experimental results. The experimental results were extracted from two studies published in the literature, which were based on the same methodology but yielded different outcomes (B96 and B93). In the model, energy cost was modeled as the sum of heat and work. Parameters used to model heat rate were entirely independent of the experimental data-sets. Parameters describing the mechanical behavior were derived from both experimental studies. The model was found to accurately predict the muscle energetics and mechanical efficiency of data-set B96. The model could not, however, replicate the energetics and efficiency of SOL and EDL that were found in data-set B93. The model overestimated the shortening heat rate of EDL but, surprisingly, also the mechanical work rate for both muscles. This was surprising since mechanical characteristics of the model were derived directly from the experimental data. It was demonstrated that the inconsistencies in data-set B93 must have been due to some unexplained confounding artifact. It was concluded that the presented model of muscle energetics is valid for iso-velocity contractions of mammalian muscle since it accurately predicts experimental results of an independent data-set (B96). In addition, the model appeared to be helpful in revealing inconsistencies in a second data-set (B93).

Inhibition of caffeine-induced Ca2+ release by adenosine in mammalian skinned slow- and fast-twitch fibres.

The present study performed on chemically skinned skeletal fibres was designed to compare the effects of adenosine on the Ca2+ sensitivity of contractile proteins and on caffeine-induced Ca2+ release in rat slow- (soleus) and fast-twitch (edl) muscles. The tension-pCa relationships were obtained by exposing triton X-100 (1% v/v) skinned fibres sequentially to solutions of decreasing pCa in the presence or in absence of adenosine. Then, changes in caffeine contracture due to adenosine were recorded on saponin (50 microg/ml) skinned fibres. The results show that the sensitivity to Ca2+ of contractile proteins in the presence of different concentrations of caffeine was not significantly modified by adenosine. However, it was proposed that adenosine (0.1-2 mM) reduced the Ca2+ released by caffeine (0.1-10 mM) from the sarcoplasmic reticulum in slow- and fast-twitch fibres and that the soleus was more sensitive to adenosine than edl muscle. The effects of specific A2a and A1 agonists and antagonists were also tested on caffeine contractures. It was found that the A1 antagonist reduced adenosine effect on caffeine response. Then it is proposed that adenosine modulates the sarcoplasmic reticulum Ca2+ release by a direct effect on the RyR1 receptors and/or by an indirect effect mediated by A1 receptors located at the sarcoplasmic level.

A weakly coupled version of the Huxley crossbridge model can simulate energetics of amphibian and mammalian skeletal muscle.

This study aimed to establish whether quantitatively accurate predictions of the rate of crossbridge-dependent energy output from shortening muscle could be made on the basis of a 2-state model of crossbridge kinetics incorporating weak coupling between mechanical cycles and ATP hydrolysis. The model was based on Huxley's (1957) model but included rapid detachment, without ATP hydrolysis, of crossbridges when their strain energy increased sufficiently that crossbridge free energy exceeded that of the unbound state (Cooke et al., 1994). An expression was derived relating force to steady-state velocity in terms of the model's rate constants. The values of the rate constants that both provided the best fit through force-velocity data and correctly predicted crossbridge-dependent rate of energy output during an isometric contraction were found and used to predict the variation in rate of energy liberation with shortening velocity. The model predictions closely matched the estimated crossbridge energetics of frog sartorius muscle, including the decline in rate of enthalpy output at high shortening velocities. Data from fast- and slow-twitch muscles of the mouse were also simulated. The velocity-dependence of rate of energy liberation from fast-twitch EDL muscle was well described by the model. The model overestimated crossbridge-dependent energy output from slow-twitch soleus at low shortening velocities but provided accurate predictions of energy output at high velocities. In terms of this model, the distinctive energetics of fast and slow muscles cannot be explained exclusively by differences in cross-bridge detachment rate; differences in the relative rates of crossbridge attachment must also be considered to explain the different relations between energy output and shortening velocity.

A Comparison of the Electrophysiological Effects of Two Organophosphates, Mipafox and Ecothiopate, on Mouse Limb Muscles

Adult male albino mice were given single subcutaneous injections of either mipafox (110 μmol/kg) or ecothiopate (0.5 μmol/kg), two organophosphorus compounds (OPs). Acetylcholinesterase activity was measured in the soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles. At 7 and 28 days after dosing,in vitroelectrophysiological measurements were carried out in the soleus and EDL. Action potentials and end-plate potentials were evoked at 30 Hz and recorded intracellularly from single muscle fibers. The amplitudes, time course, and latencies of these potentials were measured and the variability (jitter) of latencies was calculated. Recordings after mipafox were also made with 3-Hz stimulation. Acetylcholinesterase activity was inhibited by mipafox (65% in the soleus; 76% in the EDL) and ecothiopate (59% in the soleus; 42% in the EDL). Mipafox and ecothiopate both increased postjunctional (muscle action potential) jitter in the soleus and EDL at 7 days after dosing. Organophosphates caused an increase in end-plate potential amplitudes in the soleus. Mipafox caused an increase in prejunctional (end-plate potential) jitter at 28 days after dosing in both muscles. A single dose of ecothiopate also caused an increase in prejunctional jitter at 28 days in the soleus. The OP-induced increase in jitter was different at different frequencies of stimulation. The results show that there are electrophysiological changes in both muscles after administration of organophosphorus compounds. The slow-twitch soleus appears more sensitive to prejunctional changes caused by OPs than the fast-twitch EDL.

Quantitative measurement of resting skeletal muscle [Ca 2+] i following acute and long-term downhill running exercise in mice

Alteration of resting free intracellular [Ca 2+] ([Ca 2+] i) homeostasis has been implicated in the aetiology of skeletal muscle fibre injury following damaging pliometric (lengthening or 'eccentric') contractions. Quantitative measurements of resting [Ca 2+] i in skeletal muscles following acute or long-term exercise involving such injurious contractions have not been performed. We tested the hypothesis that, following an acute bout of pliometric exercise, the maximum force production (P o) of isolated skeletal muscles would be significantly reduced and that this deficit in force would be accompanied by an elevation in resting skeletal muscle [Ca 2+] i. Further, we tested whether long-term pliometric exercise training would protect skeletal muscles from contraction-induced injury. Adult male mice were randomly assigned to either: control, 24-hour, 48-hour, or trained groups. The 24-hour and 48-hour group animals were subjected to a single acute downhill treadmill running bout (decline 16°, at a rate of 13 m/min, for 60 min) and sacrificed at 24 or 48 h, respectively. Trained animals underwent a 14 week endurance training program consisting of a daily (5 days/week) downhill running session, under identical conditions to that of the acutely exercised groups. The sedentary control animals remained in their cages. For each animal, P o was determined in the fast-twitch EDL and slow-twitch soleus muscles from one hindlimb and quantitative measurements of [Ca 2+] i were made in the contralateral muscles using fluorescence digital imaging microscopy in conjunction with Fura-2. P o was lower in the EDL and soleus muscles from the 48-hour group compared with the control group animals. P o was higher in the EDL muscles of the trained group compared with the 48-hour group. No significant difference in P o was detected in either muscle from the 24-hour or trained groups compared with muscles from control mice. In EDL muscles, [Ca 2+] i was elevated in the 48-hour group compared with the control and trained group animals, but was not different between the 24-hour group compared with control mice. [Ca 2+] i was not different in the soleus muscles among the 48-hour, trained or control group mice, but was increased in muscles from the 48-hour group compared with the 24-hour group. Endurance downhill running training conferred protection to recruited skeletal muscles against the effects of an acute bout of repeated pliometric contractions, as evidenced by [Ca 2+], and P o values similar to muscles from unexercised control mice.

Changes of macromolecular organizations in nonjunctional sarcolemmas after cross-innervation— a study of fast-and slow-twitch muscle fibres in rats

The purpose of the present study was to analyse the changes of macromolecular organizations in nonjunctional sarcolemmas of different types of skeletal muscle fibres after cross-innervation. In normal rats the mean density of square arrays (6 nm particles organized in orthogonal arrays) was 9.02 / μm2 for the nonjunctional sarcolemmas of fast-twitch extensor digitorum longus (control EDL, CE) muscle fibres and 0.34 / μm2 for the nonjunctional sarcolemmas of slow-twitch soleus (control SOL, CS) muscle fibres. After cross-innervation between the fast-twitch EDL and slow-twitch SOL muscle fibres by slow and fast muscle nerves respectively for three months, the mean density was 0.45/ μm2 for the nonjunctional sarcolemmas of the operated EDL (OE) and 8.3/ μm2 for the nonjunctional sarcolemmas of the operated SOL (OS), This indicates that the cross-innervation causes a reciprocal transformation of the number and distribution of such macromolecular organizations in the electrically excitable nonjunctional sarcolemmas.

Influence of the interval between removal and freezing of muscle samples on muscle fibre size with special reference to sample size and fibre type

To study the influences of the interval between removal and freezing of a muscle sample on fibre size, rat hindlimb muscles (soleus and extensor digitorum longus, EDL) were cut into three or five pieces and frozen at different intervals, cross-sectioned, stained and analysed morphometrically. As in a previous study (Larsson & Skogsberg 1988), a pronounced interval-dependent change in soleus muscle fibre size was observed, the fibres in soleus muscle samples frozen immediately (within 15 s) being significantly (P less than 0.01-0.001) larger than those samples frozen 4 or 10 min after removal. However, the interval-dependent change was only noted in small muscle samples, i.e. those where the maximum muscle fibre length was 6.5 mm, while samples in which the fibres were approximately twice this length showed no change. In the slow-twitch soleus, type I fibres predominate, and type II fibres were often too few in one or more cross-sections in each animal to allow reliable measurements of fibre size. The fast-twitch EDL was therefore studied, and the results indicated an interval-dependent change in fibre size which was differentiated according to enzyme-histochemical type, i.e. there was a significant change in type I and IIA fibres but not in type IIB. It is concluded that the interval between removal and freezing of small muscle samples, as in percutaneous muscle biopsies in man, is of vital importance for fibre size, and that this interval should be at least 2-4 min.

Leucine oxidation and protein degradation in the extensor digitorum longus and soleus of the tumor-bearing host

A comparison was made of the effect of tumor growth on protein degradation and leucine oxidation in the EDL and soleus. Both protein degradation, as measured by net tyrosine release by incubated muscles, and 14CO 2 production from l-[1- 14C] leucine under in vitro conditions were increased in the fast-twitch pale EDL in rats bearing a Walker 256 carcinoma whereas in the slow-twitch red soleus from these animals neither protein degradation nor leucine oxidation was altered significantly from control levels.

Histochemical changes in fast and slow muscles of nutritionally dystrophic rabbits.

Rabbits were rendered dystrophic by feeding them a diet deficient in vitamin E and their fast-twitch EDL and slow-twitch SOL muscles were examined histochemically. The soleus muscle of control rabbits consisted largely of type I fibres with occasional areas of scattered type II fibres. In the nutritionally dystrophic rabbits type II fibres were consistently found homogeneously distributed throughout the entire muscle and in increased proportion. A very similar pattern was observed in the solei of rabbits following sciatic nerve section. The normal EDL contained three fibre types (I, IIoxidative and IIglycolytic). Vitamin E deficiency appeared to be associated with a shift towards an increase in the proportion of IIglycolytic fibres at the expense of IIoxidative. In denervation as well as vitamin E deficiency the type I fibres of the EDL appeared to be spared. A small number of the E-deficient rabbits exhibited degenerative changes in their sciatic and sural nerves. When animals were both denervated and E-deprived the resulting muscle changes were very much more severe than in the case of either challenge in isolation. We suggest that although some of the signs of vitamin E deficiency resemble those of a neural defect there is, in addition, a direct myopathic effect.