Effect of cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca-ATPase, on skeletal muscles from normal and mdx mice

AIM: In this study, we investigated Ca2+ loading by the sarcoplasmic reticulum in skeletal muscle from mdx mice, an animal model of human Duchenne's muscular dystrophy, at two stages of development: 4 and 11 weeks. METHOD: Experiments were conducted on fast- (extensor digitorum longus, EDL) and slow- (soleus) twitch muscles expressing different isoforms of Ca2+-ATPase, which is responsible for the uptake of Ca2+ by the sarcoplasmic reticulum. RESULTS: In sarcoplasmic reticulum vesicles, the ATP-dependent activity and sensitivity to cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase, were similar in mdx and normal EDL muscle. Furthermore, in chemically-skinned fibres from both normal and mdx muscles, the presence of CPA induced a decrease in Ca2+ uptake by the sarcoplasmic reticulum. However, the sensitivity to CPA was lower in mdx EDL muscle than in normal muscle. In addition, in EDL muscle from 4-week-old mdx mice, the expression of the slow Ca2+-pump isoform (SERCA2a) was significantly increased, without any accompanying change in slow myosin expression. In contrast, the expression and function of the Ca2+-ATPase in mdx soleus muscles at 4- and 11-weeks of development did not differ from those in age-matched controls. CONCLUSION: These findings show that in dystrophic muscle, where the Ca2+ homeostasis was perturbed, the Ca2+ handling by the sarcoplasmic reticulum was altered in fast-twitch muscle, and this was associated with the expression of the slow isoform of SERCA. In these muscles, reduced Ca2+ uptake could then contribute to an elevated concentration of Ca2+ in the cytosol, and also to Ca2+ depletion of the sarcoplasmic reticulum.     

The action potential-evoked sarcoplasmic reticulum calcium release is impaired in mdx mouse muscle fibres

The mdx mouse, a model of the human disease Duchenne muscular dystrophy, has skeletal muscle fibres which display incompletely understood impaired contractile function. We explored the possibility that action potential-evoked Ca²⁺ release is altered in mdx fibres. Action potential-evoked Ca²⁺-dependent fluorescence transients were recorded, using both low and high affinity Ca²⁺ indicators, from enzymatically isolated fibres obtained from extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of normal and mdx mice. Fibres were immobilized using either intracellular EGTA or N -benzyl- p -toluene sulphonamide, an inhibitor of the myosin II ATPase. We found that the amplitude of the action potential-evoked Ca²⁺ transients was significantly decreased in mdx mice with no measured difference in that of the surface action potential. In addition, Ca²⁺ transients recorded from mdx fibres in the absence of EGTA also displayed a marked prolongation of the slow decay phase. Model simulations of the action potential-evoked transients in the presence of high EGTA concentrations suggest that the reduction in the evoked sarcoplasmic reticulum Ca²⁺ release flux is responsible for the decrease in the peak of the Ca²⁺ transient in mdx fibres. Since the myoplasmic Ca²⁺ concentration is a critical regulator of muscle contraction, these results may help to explain the weakness observed in skeletal muscle fibres from mdx mice and, possibly, Duchenne muscular dystrophy patients.     

Increased susceptibility of EDL muscles from mdx mice to damage induced by contractions with stretch

Summary Absence of dystrophin in mdx muscles may render the muscle more susceptible to damage when submitted to high stress levels. To test this, typically slow (soleus) and fast (EDL) limb muscles of dystrophic (mdx) and normal (C57BL/10) mice were submitted (in vitro) to a series of isometric contractions, followed by a series of contractions with stretches. Muscle injury was assessed by monitoring the force signal. Membrane damage was evaluated by bathing the muscle in Procion Red, a dye that does not penetrate intact fibres, and subsequent analysis by light microscopy. After isometric contractions, only a very small force drop (<3% of maximal isometric force) was observed which indicated that no injury had occurred in soleus and EDL muscles in either mdx or C57 strains. After contractions with a stretch, a force drop of 10% was observed in soleus muscles from both strains and in EDL muscles from C57 mice. However, in mdx mice EDL muscles displayed an irreversible force drop of 40–60%. Histological analysis of the muscles indicates that force drop is associated with membrane damage. These results show that EDL muscles from mdx mice are more vulnerable than their controls, supporting the structural role hypothesis for dystrophin. Furthermore, they suggest that contractions with stretches may contribute to the muscle damage and degeneration observed in DMD-patients.     

Functional and biochemical modifications in skeletal muscles from malarial mice

Although it is well established that patients suffering from malaria experience skeletal muscle problems (contracture, aches, fatigue, weakness), detailed studies have not been performed to investigate changes in the contractile function and biochemical properties of intact and skinned skeletal muscles of mammals infected with malaria. To this end, we investigated such features in the extensor digitorium longus (EDL, fast-twitch, glyocolytic) and in the soleus (SOL, slow-twitch, oxidative) muscles from mice infected with Plasmodium berghei. We first studied maximal tetanic force (T(max)) produced by intact control and malaria-infected muscles before, during and after fatigue. Triton-skinned muscle fibres were isolated from these muscles and used to determine isometric contractile features as well as a basic biochemical profile as analysed by silver-enhanced SDS-PAGE. We found that the T(max) of intact muscles and the maximal Ca2+-activated force (F(max)) of Triton-skinned muscle fibres were reduced by approximately 50% in malarial muscles. In addition, the contractile proteins of Triton-skinned muscle fibres from malarial muscles were significantly less sensitive to Ca2+. Biochemical analysis revealed that there was a significant loss of essential contractile proteins (e.g. troponins and myosin) in Triton-skinned muscle fibres from malarial muscles as compared to controls. The biochemical alterations (i.e., reduction of essential contractile proteins) seem to explain well the functional modifications resolved in both intact muscles and Triton-skinned muscle fibres and may provide a suitable paradigm for the aetiology of muscle symptoms associated with malaria.     

Mechanical and energetic properties of dystrophic (mdx) mouse muscle

The mechanical and energetic properties of extensor digitorum longus (EDL) and soleus muscles of X chromosome-linked muscular dystrophic mutant (mdx) mice aged 4-6 weeks were studied and compared with those of the muscles of normal mice. Maximum tetanic tension, the speed of contraction of relaxation, and the heat production of mdx soleus muscles were not significantly different from those of the normal muscles. However, in mdx EDL muscles, the tension and heat production were significantly reduced, and relaxation was prolonged. To study the cause of these changes in mdx EDL muscles, tension and heat production were measured at various muscle lengths greater than optimum for tension. Both the amount of twitch heat and the heat rate for a tetanus were linearly related to the tension and had non-zero intercepts at zero tension, the activation heat. The twitch activation heat and the tension-related heat in tetani of mdx EDL muscles were not different from those in normal muscles. On the other hand, the tetanus activation heat of mdx EDL muscles was significantly smaller than that of normal muscles. Assuming that the degenerated fibers do not contribute to the active force produced, these results suggest that the amount of Ca2+ released in a contraction is not significantly different between normal and mdx muscles, but the Ca-ATPase activity of the salcoplasmic reticulum is reduced in mdx EDL, which could cause the slowing of relaxation.     

Adaptation of mouse skeletal muscle to a novel functional overload test: changes in myosin heavy chains and SERCA and physiological consequences

We have used a new approach to study the effects of overload on skeletal muscle phenotype in mice. The method used avoids any traumatising contact with muscles and the inflammatory reaction that this may provoke. Blocks of lead embedded in silicone were inserted under the skin of the lower part of the back. After 1?month, a 17% hypertrophy was found to have occurred in the tonic soleus muscle, but no change was observed in the fast-twitch extensor digitorum longus (EDL) muscle. The main effects on the contractile properties of the soleus muscle were a decrease in the tetanic relaxation rate and a reduction in the maximal velocity of shortening. Immunohistological analysis of the soleus muscles revealed an increase in the proportion of fibres that express myosin heavy chain (MHC) 1, from 54.2% to 73.9%, with a reduction in the proportion of MHC2a-positive fibres, from 45.8% to 30.2%. These changes were accompanied by an increase in the proportion of fibres that express the slow type of sarcoplasmic reticulum calcium pump (SERCA2a), from 61.8% to 84.7%. In EDL muscles, overload induced only minor changes. Thus, this method of overload affected the soleus muscle in particular. The observed changes in the control of muscle contraction were significantly larger than the changes in typical myofibrillar properties that were observed. These results indicate that there is a temporal dissociation between the relative expression of MHCs and SERCAs.     

Functional regeneration in the hindlimb skeletal muscle of the mdx mouse

Summary The pattern of spontaneous skeletal muscle degeneration and clinical recovery in hindlimb muscles of the mdx mutant mouse was examined for functional and metabolic confirmation of apparent structural regeneration. The contractile properties, histochemical staining and myosin light chain and parvalbumin contents of extensor digitorum longus (EDL) and soleus (Sol) muscles of mdx and age-matched control mice were studied at 3–4 and 32 weeks. Histochemical staining (myofibrillar ATPase and NADH-tetrazolium reductase) revealed no significant change in slow-twitch-oxidative (SO) or fast-twitch-oxidative-glycolytic (FOG) fibre type proportions in mdx Sol apart from the normal age-related increase in SO fibres. At 32 weeks mdx EDL, however, showed significantly smaller fast-twitch-glycolytic (FG) and larger FOG proportions than those in control EDL. These fibre type distributions were confirmed by differential staining with antibodies to myosin slow-twitch and fast-twitch heavy chain isozymes. Frequency distribution of cross-sectional area for each fibre type showed a wider than normal range of areas especially in FOG fibres of mdx Sol, and FG fibres of mdx EDL, supporting previous observations using autoradiography of myofibre regeneration. Isometric twitch and tetanic tensions in Sol were significantly less than in controls at 4 weeks, but by 32 weeks, values were not different from age-matched controls. In mdx EDL at 3 weeks, twitch and tetanus tensions were significantly less, and time-to-peak twitch tensions were significantly faster than in control EDL. By 32 weeks, mdx EDL twitch and tetanus tensions expressed relative to muscle weight continued to be significantly lower than in age-matched controls, despite normal absolute tensions. The maximum velocity of shortening in 32-week mdx EDL was significantly lower than in control EDL. Myosin light chain distribution in mdx Sol exhibited significantly less light chain 2-slow (LC2s) and more light chain 1b-slow(LC1bs) at 32 weeks than age-matched control Sol. Gels of EDL from 32-week-old mdx mice showed significantly less light chain 2-fast-phosphorylated (LC2f-P) and light chain 3-fast (LC3f) and significantly more light chain 1-fast (LC1f) and light chain 2-fast (LC2f), but normal parvalbumin content compared to age-matched controls. These observations suggest that mdx hindlimb muscles are differentially affected by the disease process as it occurs in murine models of dystrophy. However, the uniqueness of mdx Sol and to a lesser extent EDL is that they also undergo an important degree of functional regeneration which is able to compensate spontaneously for degenerative influences of genetic origin. The mdx mutant may therefore be an important model for the study of regeneration by skeletal muscle, and of the nerve-muscle interactions which enable or restrict that regeneration.     

Muscle fatigue with prolonged exercise: contractile and biochemical alterations

Alterations in the contractile and biochemical properties of fast and slow skeletal muscle were studied in rats following a prolonged swim to exhaustion. The exercise produced glycogen depletion (less than 1 mg/g tissue) in muscles representative of all three fiber types; the isometric contractile properties were altered in the 84% type I soleus (SOL) and the 60% type IIa extensor digitorium longus (EDL) but not in the 100% type IIb superficial region of the vastus lateralis (SVL). Peak tetanic tension (Po) and the rate of tension development and decline all decreased after prolonged exercise in both the SOL and the EDL. The maximal isotonic shortening velocity was highly correlated with the myofibrillar ATPase activity, and both were relatively resistant to fatigue. Furthermore, the Ca2+ sensitivity of the myofibrils was unaffected by exercise in both fast and slow muscle. The Ca2+ uptake capacity of the sarcoplasmic reticulum (SR) was reduced in both the SOL and the fast-twitch type IIa deep region of the vastus lateralis, whereas the SR ATPase activity was unchanged. Our findings provide evidence that prolonged exercise produces alterations in contractile and biochemical properties of type I and IIa but not type IIb fibers and that muscle fatigue as measured by a decline in Po is not necessarily correlated with glycogen depletion.     

Effect of cyclopiazonic acid,an inhibitor of the sarcoplasmic reticulum Ca‐ATPase,on skeletal muscles from normal and mdx mice

Aim: In this study, we investigated Ca²⁺ loading by the sarcoplasmic reticulum in skeletal muscle from mdx mice, an animal model of human Duchenne's muscular dystrophy, at two stages of development: 4 and 11 weeks. Method: Experiments were conducted on fast‐ (extensor digitorum longus, EDL) and slow‐ (soleus) twitch muscles expressing different isoforms of Ca²⁺‐ATPase, which is responsible for the uptake of Ca²⁺ by the sarcoplasmic reticulum. Results: In sarcoplasmic reticulum vesicles, the ATP‐dependent activity and sensitivity to cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum Ca²⁺‐ATPase, were similar in mdx and normal EDL muscle. Furthermore, in chemically‐skinned fibres from both normal and mdx muscles, the presence of CPA induced a decrease in Ca²⁺ uptake by the sarcoplasmic reticulum. However, the sensitivity to CPA was lower in mdx EDL muscle than in normal muscle. In addition, in EDL muscle from 4‐week‐old mdx mice, the expression of the slow Ca²⁺‐pump isoform (SERCA2a) was significantly increased, without any accompanying change in slow myosin expression. In contrast, the expression and function of the Ca²⁺‐ATPase in mdx soleus muscles at 4‐ and 11‐weeks of development did not differ from those in age‐matched controls. Conclusion: These findings show that in dystrophic muscle, where the Ca²⁺ homeostasis was perturbed, the Ca²⁺ handling by the sarcoplasmic reticulum was altered in fast‐twitch muscle, and this was associated with the expression of the slow isoform of SERCA. In these muscles, reduced Ca²⁺ uptake could then contribute to an elevated concentration of Ca²⁺ in the cytosol, and also to Ca²⁺ depletion of the sarcoplasmic reticulum.     

Reduced sarcoplasmic reticulum content of releasable Ca2+ in rat soleus muscle fibres after eccentric contractions

AIM: The purpose was to evaluate the effects of fatiguing eccentric contractions (EC) on calcium (Ca2+) handling properties in mammalian type I muscles. We hypothesized that EC reduces both endogenous sarcoplasmic reticulum (SR) content of releasable Ca2+ (eSRCa2+) and myofibrillar Ca2+ sensitivity. METHODS: Isolated rat soleus muscles performed 30 EC bouts. Single fibres were isolated from the muscle and after mechanical removal of sarcolemma used to measure eSRCa2+, rate of SR Ca2+ loading and myofibrillar Ca2+ sensitivity. RESULTS: Following EC maximal force in whole muscle was reduced by 30% and 16/100 Hz force ratio by 33%. The eSRCa2+ in fibres from non-stimulated muscles was 45 +/- 5% of the maximal loading capacity. After EC, eSRCa2+ per fibre CSA decreased by 38% (P = 0.05), and the maximal capacity of SR Ca2+ loading was depressed by 32%. There were no effects of EC on either myofibrillar Ca2+ sensitivity, maximal Ca2+ activated force per cross-sectional area and rate of SR Ca2+ loading, or in SR vesicle Ca2+ uptake and release. CONCLUSIONS: We conclude that EC reduces endogenous SR content of releasable Ca2+ but that myofibrillar Ca2+ sensitivity and SR vesicle Ca2+ kinetics remain unchanged. The present data suggest that the long-lasting fatigue induced by EC, which was more pronounced at low frequencies (low frequency fatigue), is caused by reduced Ca2+ release occurring secondary to reduced SR content of releasable Ca2+.     

Effect of hyperthyroidism on the contractile and histochemical properties of fast and slow twitch skeletal muscle in the rat

Male Wistar rats were made hyperthyroid by intraperitoneal injections on alternate days for 1–6 weeks, of 200 g/kg triiodothyronine (T₃). The effects of this treatment on the contractile properties of the soleus, a slow twitch and the extensor digitorum longus (EDL), a fast twitch skeletal muscle, were studied in vivo in the anaesthetized animal. Post mortem, serial frozen sections of both muscles were stained histochemically for myosin ATPase, succinic dehydrogenase and phosphorylase. Muscle fibres were classified as either slow twitch (SO), fast twitch oxidative glycolytic (FOG) or fast twitch glycolytic (FG).Elevation of plasma T₃ levels is associated with progressive alterations in the muscle fibre populations of both muscles. In the soleus there is conversion of SO to FOG fibres while in the EDL the main effect is FG to FOG conversion. There are also marked changes, mainly confined to the soleus muscle, in contractile properties; progressive increases in isometric twitch and tetanic tension and in the rates of contraction and relaxation during both twitch and tetanus.The effect of T₃ on slow muscle contractility may be related to its effect on muscle phenotype. However, changes in the former precede detectable fibre population changes. T₃ may influence properties such as the Ca²⁺ binding activity of sarcoplasmic reticulum of existing slow twitch fibres before the later changes associated with the interconversion of fibre types occur.     

Inositol 1,4,5-trisphosphate-sensitive Ca2+ release in rat fast- and slow-twitch skinned muscle fibres

Inositol 1,4,5-trisphosphate (InsP3), an intracellular messenger, induces Ca2+ release in various types of cells, particularly smooth muscle cells. Its role in skeletal muscle, however, is controversial. The present study shows that the application of InsP3 to rat slow- and fast-twitch saponin-skinned fibres induced contractile responses that were not related to an effect of InsP3 on the properties of the contractile proteins. The amplitude of the contractures was dependent upon the Ca(2+)-loading period, and was larger in slow- than in fast-twitch muscle. In both types of skeletal muscle, these responses, unlike caffeine contractures, were not inhibited by ryanodine (100 microM), but were abolished by heparin (20 micrograms.ml-1). In soleus muscle, the concentration of heparin required to inhibit the response by 50% (IC50) was 5.7 micrograms.ml-1, a similar value to that obtained previously in smooth muscle. Furthermore, the results show that in slow-twitch muscle, the InsP3 contractures have a "bell-shaped" dependency on the intracellular Ca2+ concentration. These results show that InsP3 receptors should be present in skeletal muscle. Thus, it is possible that InsP3 participates in the regulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle, particularly in slow-twitch fibres.     

Calsequestrin expression and calcium binding is increased in streptozotocin-induced diabetic rat skeletal muscle though not in cardiac muscle

Altered mechanisms of Ca2+ transport may underlie the contractile dysfunctions that have been frequently reported to occur in diabetic cardiac and skeletal muscle tissues. Calsequestrin, a high-capacity Ca2+-binding protein, is involved in the regulation of the excitation-contraction-relaxation cycle of both skeletal and cardiac muscle fibres. We have investigated the expression of calsequestrin and Ca2+ binding in cardiac and skeletal muscle from streptozotocin-induced diabetic rat. Immunoblotting of microsomal membranes from normal and streptozotocin-induced diabetic muscle revealed no significant changes in heart, but an increase in the relative abundance of calsequestrin and calsequestrin-like proteins in skeletal muscle. In analogy, the overall Ca2+-binding capacity of sarcoplasmic reticulum vesicles from diabetic skeletal muscle was drastically increased. The expression of fast muscle marker proteins was not affected, indicating that no relevant fibre transformation occurred in streptozotocin-treated rat muscles. The up-regulation of the high-capacity Ca2+-binding element calsequestrin might represent a compensatory mechanism of diabetic skeletal muscle. An increased Ca2+-buffering capacity of the sarcoplasmic reticulum lumen might counteract elevated cytosolic Ca2+ levels in diabetes thereby preventing Ca2+-dependent myo-necrosis.     

High-frequency fatigue of skeletal muscle: role of extracellular Ca2+

The present study evaluated whether Ca(2+) entry operates during fatigue of skeletal muscle. The involvement of different skeletal muscle membrane calcium channels and of the Na(+)/Ca(2+) exchanger (NCX) has been examined. The decline of force was analysed in vitro in mouse soleus and EDL muscles submitted to 60 and 110 Hz continuous stimulation, respectively. Stimulation with this high-frequency fatigue (HFF) protocol, in Ca(2+)-free conditions, caused in soleus muscle a dramatic increase of fatigue, while in the presence of high Ca(2+) fatigue was reduced. In EDL muscle, HFF was not affected by external Ca(2+) levels either way, suggesting that external Ca(2+) plays a general protective role only in soleus. Calciseptine, a specific antagonist of the cardiac isoform (alpha1C) of the dihydropyridine receptor, gadolinium, a blocker of both stretch-activated and store-operated Ca(2+) channels, as well as inhibitors of P2X receptors did not affect the development of HFF. Conversely, the Ca(2+) ionophore A23187 increased the protective action of extracellular Ca(2+). KB-R7943, a selective inhibitor of the reverse mode of NCX, produced an effect similar to that of Ca(2+)-free solution. These results indicate that a transmembrane Ca(2+) influx, mainly through NCX, may play a protective role during HFF development in soleus muscle.     

The role of branched fibres in the pathogenesis of Duchenne muscular dystrophy

Branched fibres are a well-documented phenomenon of regenerating skeletal muscle. They are found in the muscles of boys with Duchenne muscular dystrophy (DMD), a severe condition of progressive muscle wasting caused by an absence of the sarcolemmal protein dystrophin, and in the muscles of the mdx mouse, an animal model of DMD. However, only a handful of studies have investigated how the physiological properties of these morphologically deformed fibres differ from those of normal fibres. These studies have found an association between the extent of fibre branching in mdx muscles and the susceptibility of these muscles to damage from eccentric contractions. They have also found that branched mdx muscle fibres cannot sustain maximal contractions in buffered Ca(2+) solutions, that branch points are sites of increased mechanical stress and that myofibrillar structure is greatly disturbed at branch points. These findings have important implications for understanding the function of dystrophin. It is commonly thought that the role of dystrophin is mechanical stabilization of the sarcolemma, as numerous studies have shown that eccentric contractions damage mdx muscle more than normal muscle. However, the finding that branched mdx fibres are mechanically weakened raises the question, is it the lack of dystrophin or is it the fibre branching that leads to the vulnerability of mdx muscle to contractile damage? The importance of this question to our understanding of the function of dystrophin warrants further research into the physiological properties of branched fibres and how they differ from morphologically normal fibres.     

Functional TRPV4 channels are expressed in mouse skeletal muscle and can modulate resting Ca2+ influx and muscle fatigue

Skeletal muscle contraction is basically controlled by Ca(2+) release and its reuptake into the sarcoplasmic reticulum. However, the long-term maintenance of muscle function requires an additional Ca(2+) influx from extracellular. Several mechanisms seem to contribute to the latter process, such as store-operated Ca(2+) entry, stretch-activated Ca(2+) influx and resting Ca(2+) influx. Candidate channels that may control Ca(2+) influx into muscle fibers are the STIM proteins, Orai, and the members of the transient receptor potential (TRP) family of cation channels. Here we show that TRPV4, an osmo-sensitive cation channel of the vanilloid subfamily of TRP channels is functionally expressed in mouse skeletal muscle. Western blot analysis showed the presence of TRPV4-specific bands at about 85 and 100?kDa in all tested muscles. The bands were absent when muscle proteins from TRPV4 deficient mice were analyzed. Using the manganese quench technique, we studied the resting influx of divalent cations into isolated wild-type muscle fibers. The specific TRPV4-channel activator 4α-phorbol-12,13-didecanoate (4α-PDD) stimulated resting influx by about 60% only in wild-type fibers. Electrical stimulation of soleus muscles did not reveal changes in isometric twitch contractions upon application of 4α-PDD, but tetanic contractions (at 120?Hz) were slightly increased by about 15%. When soleus muscles were stimulated with a fatigue protocol, muscle fatigue was significantly attenuated in the presence of 4α-PDD. The latter effect was not observed with muscles from TRPV4(-/-) mice. We conclude that TRPV4 is functionally expressed in mouse skeletal muscle and that TRPV4 activation modulates resting Ca(2+) influx and muscle fatigue.     

Power output of fast and slow skeletal muscles of mdx (dystrophic) and control mice after clenbuterol treatment

The mdx mouse is the most commonly used animal model for Duchenne muscular dystrophy. We tested the null hypothesis that 20 weeks of clenbuterol treatment ( approximately 2 mg kg-1 day-1) of mdx and control mice would have no effect on the absolute and specific force (Po, kN m-2) and absolute and normalised power output (W kg-1) of extensor digitorum longus (EDL) and soleus muscles. For mdx and control mice, clenbuterol treatment produced modest increases in the mass of the two muscles but did not increase absolute or specific force or normalised power output. For absolute power output, only the EDL muscles of mdx mice showed a difference following treatment, with the power output of treated mice being 118 % that of the untreated mice. The modest effects of clenbuterol treatment on the dynamic properties of skeletal muscle provide little support for any improvement in muscle function for the dystrophic condition.     

Sarcoplasmic reticulum Ca(2+) content affects 4-CmC and caffeine contractures of rat skinned skeletal muscle fibers

This study investigated whether the sarcoplasmic reticulum Ca(2+) content of rat skeletal muscle fibers affected contractile responses obtained by an application of 4-chloro-m-cresol (4-CmC) and caffeine. Contractures were elicited on saponin-skinned fibers under different Ca(2+) loading conditions. The amplitude of 4-CmC and caffeine contractures of fast-twitch muscle fibers (edl, extensor digitorum longus) differed between the different loading conditions, and this is associated with a greater change in sensitivity to 4-CmC. When the sarcoplasmic reticulum was loaded with a low Ca(2+) concentration for a short period, the 4-CmC concentration providing half-maximal response was tenfold higher than with a larger sarcoplasmic reticulum Ca(2+) loading for a longer period, whereas for caffeine this concentration was only twofold higher in the same conditions. These findings indicate that 4-CmC contractile responses of edl muscle fibers are more dependent on luminal Ca(2+) activity than those of caffeine are. Thus 4-CmC would appear to be of greater interest than caffeine for the study of muscle contractile responses where variations in intracellular Ca(2+) activity exist.     

Hydroxyl radical and glutathione interactions alter calcium sensitivity and maximum force of the contractile apparatus in rat skeletal muscle fibres

Studies on intact muscle fibres indicate that reactive oxygen species (ROS) produced during muscle activity, or applied exogenously, can cause decreased force responses primarily by reducing the Ca(2+) sensitivity of the contractile apparatus. Identification of the molecular basis of this effect is complicated by the fact that studies on skinned muscle fibres in general have not observed reduced contractile Ca(2+) sensitivity when applying ROS, predominantly H(2)O(2). Here, using skinned fibres from rat extensor digitorum longus (EDL) and soleus muscle, it is shown that although H(2)O(2) (> or = 100 microm) has little effect by itself, when added in the presence of myoglobin it causes marked reduction in the Ca(2+) sensitivity of the contractile apparatus, probably due to production of hydroxyl radicals (OH(*)). Maximum force production is also reduced, but only with larger or more prolonged treatments. The effects are not prevented by tempol, a potent superoxide scavenger. Dithiotreitol (DTT) produces little reversal of the sensitivity change if applied afterwards, but it does substantially reverse all the changes if applied before the fibre undergoes an activation sequence. When glutathione (GSH, 5 mM) is present, exposure of EDL fibres to H(2)O(2) and myoglobin causes an increase in Ca(2+) sensitivity, with longer treatments causing a subsequent decrease, whereas in soleus fibres it causes only decreases in sensitivity and maximum force. The increased Ca(2+) sensitivity in EDL fibres is evidently due to the summed actions of (i) a potentiating effect of glutathionylation, which can be reversed by DTT and only occurs in fast-twitch fibres, and (ii) a less reversible reduction in sensitivity. Western blotting showed that reductions in Ca(2+) sensitivity were not due to loss of troponin-C. The present findings help provide a mechanistic basis for diverse findings on the effects of ROS in muscle fibres and implicate OH(*) radicals and glutathione as likely mediators of the effects.     

Effects of age on sarcoplasmic reticulum properties and histochemical composition of fa stand slow-twitch rat muscles

Calcium release activity of sarcoplasmic reticulum and enzyme-histochemical properties were investigated in extensor digitorum longus (e.d.l.) and soleus muscles in young (4 months) and old (24 months) male rats. With age, the caffeine threshold concentration for calcium release from the sarcoplasmic reticulum of soleus skinned muscle fibres showed only minor modifications. On the other hand, in e.d.l. skinned muscle fibres, the caffeine threshold concentration decreased significantly (P < 0.05). The histochemical fibre type composition changed with age both in soleus and in e.d.l. muscles, showing a common transformation toward a more oxidative histochemical profile. In fact, in aged soleus, a significant (P < 0.05) increase was observed of type 1 fibres to represent almost the totality of the muscle fibres (more than 98%), while types 2C and 2A were reduced in proportion. In aged e.d.l. the percentage of type 1 (P < 0.05), 2A and 2X (a recently identified fourth component of the fast-twitch muscle types) fibres increased, with a reduction of type 2B (P < 0.01) fibres. The present results suggest that the changes in contractile properties of aged muscles may be related to the changes not only in fibre composition but also in the mechanism of calcium release from sarcoplasmic reticulum.