Mouse Extensor Digitorum Longus Muscle Research Articles

Role of reactive oxygen species in contraction‐mediated glucose transport in mouse skeletal muscle

Exercise increases glucose transport into skeletal muscle via a pathway that is poorly understood. We investigated the role of endogenously produced reactive oxygen species (ROS) in contraction-mediated glucose transport. Repeated contractions increased 2-deoxyglucose (2-DG) uptake roughly threefold in isolated, mouse extensor digitorum longus (fast-twitch) muscle. N-Acetylcysteine (NAC), a non-specific antioxidant, inhibited contraction-mediated 2-DG uptake by approximately 50% (P < 0.05 versus control values), but did not significantly affect basal 2-DG uptake or the uptake induced by insulin, hypoxia or 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR, which mimics AMP-mediated activation of AMP-activated protein kinase, AMPK). Ebselen, a glutathione peroxidase mimetic, also inhibited contraction-mediated 2-DG uptake (by almost 60%, P < 0.001 versus control values). Muscles from mice overexpressing Mn2+-dependent superoxide dismutase, which catalyses H2O2 production from superoxide anions, exhibited a approximately 25% higher rate of contraction-mediated 2-DG uptake versus muscles from wild-type control mice (P < 0.05). Exogenous H2O2 induced oxidative stress, as judged by an increase in the [GSSG]/[GSH + GSSG] (reduced glutathione + oxidized glutathione) ratio to 2.5 times control values, and this increase was substantially blocked by NAC. Similarly, NAC significantly attenuated contraction-mediated oxidative stress as judged by measurements of glutathione status and the intracellular ROS level with the fluorescent indicator 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (P < 0.05). Finally, contraction increased AMPK activity and phosphorylation approximately 10-fold, and NAC blocked approximately 50% of these changes. These data indicate that endogenously produced ROS, possibly H2O2 or its derivatives, play an important role in contraction-mediated activation of glucose transport in fast-twitch muscle.

Notexin causes greater myotoxic damage and slower functional repair in mouse skeletal muscles than bupivacaine

Although the myotoxins bupivacaine and notexin are employed for studying processes that regulate muscle regeneration after injury, no studies have compared their efficacy in causing muscle damage or assessing functional regeneration in mouse skeletal muscles. Bupivacaine causes extensive injury in rat muscles but its effects on mouse muscles are variable. We compared functional and morphological properties of regenerating mouse extensor digitorum longus (EDL) muscles after notexin or bupivacaine injection and tested the hypothesis that muscle damage would be more extensive and functional repair less complete after notexin injection. Bupivacaine caused degeneration of 45% of fibers and reduced maximum force (Po) to 42% of control after 3 days. In contrast, notexin caused complete fiber breakdown and loss of functional capacity after 3 days (P < 0.05). At 7 and 10 days after bupivacaine, Po was restored to 65% and 71% of control, respectively, whereas Po of notexin-injected muscles was only 10% and 39% of control at these time-points, respectively (P < 0.05). At 7 and 10 days after bupivacaine, approximately 30% of fibers were centrally nucleated (regenerating), whereas notexin-injected muscles were comprised entirely of regenerating fibers (P < 0.05). The results demonstrate that notexin causes a more extensive and complete injury than bupivacaine, and is a useful model for studying muscle regeneration in mice.

Contractile properties of EDL and soleus muscles of myostatin-deficient mice

Myostatin is a negative regulator of muscle mass. The impact of myostatin deficiency on the contractile properties of healthy muscles has not been determined. We hypothesized that myostatin deficiency would increase the maximum tetanic force (P(o)), but decrease the specific P(o) (sP(o)) of muscles and increase the susceptibility to contraction-induced injury. The in vitro contractile properties of extensor digitorum longus (EDL) and soleus muscles from wild-type (MSTN(+/+)), heterozygous-null (MSTN(+/-)), and homozygous-null (MSTN(-/-)) adult male mice were determined. For EDL muscles, the P(o) of both MSTN(+/-) and MSTN(-/-) mice were greater than the P(o) of MSTN(+/+) mice. For soleus muscles, the P(o) of MSTN(-/-) mice was greater than that of MSTN(+/+) mice. The sP(o) of EDL muscles of MSTN(-/-) mice was less than that of MSTN(+/+) mice. For soleus muscles, however, no difference in sP(o) was observed. Following two lengthening contractions, EDL muscles from MSTN(-/-) mice had a greater force deficit than that of MSTN(+/+) or MSTN(+/-) mice, whereas no differences were observed for the force deficits of soleus muscles. Myostatin-deficient EDL muscles had less hydroxyproline, and myostatin directly increased type I collagen mRNA expression and protein content. The difference in the response of EDL and soleus muscles to myostatin may arise from differences in the levels of a myostatin receptor, activin type IIB. Compared with the soleus, the amount of activin type IIB receptor was approximately twofold greater in EDL muscles. The results support a significant role for myostatin not only in the mass of muscles but also in the contractility and the composition of the extracellular matrix of muscles.

Denervation in murine fast-twitch muscle: short-term physiological changes and temporal expression profiling

Denervation deeply affects muscle structure and function, the alterations being different in slow and fast muscles. Because the effects of denervation on fast muscles are still controversial, and high-throughput studies on gene expression in denervated muscles are lacking, we studied gene expression during atrophy progression following denervation in mouse tibialis anterior (TA). The sciatic nerve was cut close to trochanter in adult CD1 mice. One, three, seven, and fourteen days after denervation, animals were killed and TA muscles were dissected out and utilized for physiological experiments and gene expression studies. Target cDNAs from TA muscles were hybridized on a dedicated cDNA microarray of muscle genes. Seventy-one genes were found differentially expressed. Microarray results were validated, and the expression of relevant genes not probed on our array was monitored by real-time quantitative PCR (RQ-PCR). Nuclear- and mitochondrial-encoded genes implicated in energy metabolism were consistently downregulated. Among genes implicated in muscle contraction (myofibrillar and sarcoplasmic reticulum), genes typical of fast fibers were downregulated, whereas those typical of slow fibers were upregulated. Electrophoresis and Western blot showed less pronounced changes in myofibrillar protein expression, partially confirming changes in gene expression. Isometric tension of skinned fibers was little affected by denervation, whereas calcium sensitivity decreased. Functional studies in mouse extensor digitorum longus muscle showed prolongation in twitch time parameters and shift to the left in force-frequency curves after denervation. We conclude that, if studied at the mRNA level, fast muscles appear not less responsive than slow muscles to the interruption of neural stimulation.

70 μM caffeine treatment enhances in vitro force and power output during cyclic activities in mouse extensor digitorum longus muscle

Caffeine ingestion by human athletes has been found to improve endurance performance primarily acting via the central nervous system as an adenosine receptor antagonist. However, a few studies have implied that the resultant micromolar levels of caffeine in blood plasma (70 microM maximum for humans) may directly affect skeletal muscle causing enhanced force production. In the present study, the effects of 70 microM caffeine on force and power output in isolated mouse extensor digitorum longus muscle were investigated in vitro at 35 degrees C. Muscle preparations were subjected to cyclical sinusoidal length changes with electrical stimulation conditions optimised to produce maximal work. 70 microM caffeine caused a small but significant increase (2-3%) in peak force and net work produced during work loops (where net work represents the work input required to lengthen the muscle subtracted from the work produced during shortening). However, these micromolar caffeine levels did not affect the overall pattern of fatigue or the pattern of recovery from fatigue. Our results suggest that the plasma concentrations found when caffeine is used to enhance athletic performance in human athletes might directly enhance force and power during brief but not prolonged activities. These findings potentially confirm previous in vivo studies, using humans, which implied caffeine ingestion may cause acute improvements in muscle force and power output but would not enhance endurance.

Endothelial NOS is main mediator for shear stress-dependent angiogenesis in skeletal muscle after prazosin administration.

The increase of wall shear stress in capillaries by oral administration of the alpha1-adrenergic receptor antagonist prazosin induces angiogenesis in skeletal muscles. Because endothelial nitric oxide synthase (eNOS) is upregulated in response to elevated wall shear stress, we investigated the relevance of eNOS for prazosin-induced angiogenesis in skeletal muscles. Prazosin and/or the NOS inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) were given to C57BL/6 wild-type mice and eNOS-knockout mice for 14 days. The capillary-to-fiber (C/F) ratio and capillary density (CD; no. of capillaries/mm2) were determined in frozen sections from extensor digitorum longus (EDL) muscles of these mice. Immunoblotting was performed to quantify eNOS expression in endothelial cells isolated from skeletal muscles, whereas VEGF (after precipitation with heparin-agarose) and neuronal NOS (nNOS) concentrations were determined in EDL solubilizates. In EDL muscles of C57BL/6 mice treated for 14 days, the C/F ratio was 28% higher after prazosin administration and 11% higher after prazosin and L-NAME feeding, whereas the CD increased by 21 and 13%, respectively. The C/F ratio was highest after day 4 of prazosin treatment and decreased gradually to almost constant values after day 8. Prazosin administration led to elevation of eNOS expression. VEGF levels were lowest at day 4, whereas nNOS values decreased after day 8. In EDL muscles of eNOS-knockout mice, no significant changes in C/F ratio, CD, or VEGF and nNOS expression were observed in response to prazosin administration. Our data suggest that the presence of eNOS is essential for prazosin-induced angiogenesis in skeletal muscle, albeit other signaling molecules might partially compensate for or contribute to this angiogenic activity. Furthermore, subsequent remodeling of the capillary system accompanied by sequential downregulation of VEGF and nNOS in skeletal muscle fibers characterizes shear stress-dependent angiogenesis.

Contractile skeletal muscle tissue-engineered on an acellular scaffold.

For the reconstructive surgeon, tissue-engineered skeletal muscle may offer reduced donor-site morbidity and an unlimited supply of tissue. Using an acellularized mouse extensor digitorum longus muscle as a scaffold, the authors produced engineered skeletal muscle capable of generating longitudinal force. Eight extensor digitorum longus muscles from adult mice were made acellular using a protocol developed in the authors' laboratory. The acellular muscles were then placed in a bath of 20% fetal bovine serum in Dulbecco's modified Eagle's medium and 100 U/ml penicillin for 1 week at room temperature. C2C12 myoblasts were injected into the acellular muscle matrix using a 26-gauge needle and a 100-microl syringe. The resulting constructs were placed in growth medium for 1 week at 37 degrees C under 5% carbon dioxide, with media changes every 48 hours. The constructs were then placed in differentiation medium for 1 week, with media changes every 48 hours. Isometric contractile force testing of the constructs demonstrated production of longitudinal contractile force on electrical stimulation. A length-tension, or Starling, relationship was observed. Light and electron microscopy studies demonstrated recapitulation of some of the normal histologic features of developing skeletal muscle.

Effects of caffeine on mouse skeletal muscle power output during recovery from fatigue.

The effects of 10 mM (high) and 70 microM (physiologically relevant) caffeine on force, work output, and power output of isolated mouse extensor digitorum longus (EDL) and soleus muscles were investigated in vitro during recovery from fatigue at 35 degrees C. To monitor muscle performance during recovery from fatigue, we regularly subjected the muscle to a series of cyclical work loops. Force, work, and power output during shortening were significantly higher after treatment with 10 mM caffeine, probably as a result of increased Ca2+ release from the sarcoplasmic reticulum. However, the work required to relengthen the muscle also increased in the presence of 10 mM caffeine. This was due to a slowing of relaxation and an increase in muscle stiffness. The combination of increased work output during shortening and increased work input during lengthening had different effects on the two muscles. Net power output of mouse soleus muscle decreased as a result of 10 mM caffeine exposure, whereas net power output of the EDL muscle showed a transient, significant increase. Treatment with 70 microM caffeine had no significant effect on force, work, or power output of EDL or soleus muscles, suggesting that the plasma concentrations found when caffeine is used to enhance performance in human athletes might not directly affect the contractile performance of fatigued skeletal muscle.

A 2 week routine stretching programme did not prevent contraction-induced injury in mouse muscle.

Most athletes stretch as part of their training regimen and it is commonly believed that this practice prevents muscle injury. We tested this belief using an animal model, in situ mouse extensor digitorum longus (EDL) muscle. One lower hindlimb was slowly stretched for 1 min on alternate days for 12 days; the other leg served as a control. The mouse was lightly anaesthetized during the stretching protocol (isofluorane). Both legs were tested in situ by measuring maximum isometric force and maximum work before and after an eccentric contraction that was designed to cause a contraction-induced injury. The difference between a contraction before and after (i.e. the deficit) was used as a measure of damage caused by the eccentric contraction. There was a threshold for force deficit at a peak to peak eccentric excursion amplitude of 19.5 % (i.e. L(o) +/- 9.75 %, where L(o) is muscle length at peak isometric force). There was a significant increase in force deficit, work deficit, and curve shift with an increase in eccentric excursion amplitude above the threshold. There was no statistical difference in the force deficit, work deficit, or curve shift between the stretched leg and the control leg (P > 0.05). A routine stretching programme, at least at the intensities employed in this experiment, did not prevent contraction-induced injury in the in situ mouse EDL muscle.

Effects of stretch on work from fast and slow muscles of mice: damped and undamped energy release.

Stretching active muscle increases the work performed during subsequent shortening. The effects of a preceding stretch on work done by the undamped or lightly damped series compliance (SC) and by the contractile component (CC), which includes cross bridges and damped elements, were assessed using mouse soleus (slow) and extensor digitorum longus (fast) muscles with limited tendon. Increasing stretch amplitude (0-10% fibre length) increased work done by the SC up to a limit, but did not effect work done by the CC. Increasing stretch velocity (10-100% Vmax) had almost no effect on work done by either component. Increasing the delay between the end of stretch and onset of shortening (0-60 ms) caused a decrease in SC work, with no effect on CC work. Recoil of the SC was responsible for 50-70% of the total work done during shortening after stretch. Usually only 10-40% of the energy imparted during the stretch was recovered as work during subsequent shortening; large stretches and long delays between stretch and shortening further reduced this recovery by one third to one fifth. Results are interpreted in the context of a loss of energy stored in the SC owing to forcible detachment of cross bridges with large stretches and cyclic detachment with long delays.

K+-induced twitch potentiation is not due to longer action potential.

The objective of this study was to determine whether an increased duration of the action potential contributes to the K+-induced twitch potentiation at 37 degrees C. Twitch contractions were elicited by field stimulation, and action potentials were measured with conventional microelectrodes. For mouse extensor digitorum longus (EDL) muscle, twitch force was greater at 7-13 mM K+ than at 4.7 mM (control). For soleus muscle, twitch force potentiation was observed between 7 and 11 mM K+. Time to peak and half-relaxation time were not affected by the increase in extracellular K+ concentration in EDL muscle, whereas both parameters became significantly longer in soleus muscle. Decrease in overshoot and prolongation of the action potential duration observed at 9 and 11 mM K+ were mimicked when muscles were respectively exposed to 25 and 50 nM tetrodotoxin (TTX; used to partially block Na+ channels). Despite similar action potentials, twitch force was not potentiated by TTX. It is therefore suggested that the K+-induced potentiation of the twitch in EDL muscle is not due to a prolongation of the action potential and contraction time, whereas a longer contraction, especially the relaxation phase, may contribute to the potentiation in soleus muscle.

Effect of injecting primary myoblasts versus putative muscle-derived stem cells on mass and force generation in mdx mice.

It is well established that the injection of normal myoblasts or of muscle-derived stem cells (MDSCs) into the muscle of dystrophin-deficient mdx mice results in the incorporation of a number of donor myoblasts into the host muscle. However, the effect of the injected exogenous cells on mdx muscle mass and functional capacity has not been evaluated. This study evaluates the mass and functional capacity of the extensor digitorum longus (EDL) muscles of adult, male mdx mice that received intramuscular injections of primary myoblasts or of MDSCs (isolated by a preplating technique; Qu, Z., Balkir, L., van Deutekom, J.C., Robbins, P.D., Pruchnic, R., and Huard, J., J. Cell Biol. 1998;142:1257-1267) derived from normal mice. Evaluations were made 9 weeks after cell transplantation. Uninjected mdx EDL muscles have a mass 50% greater than that of age-matched C57BL/10J (normal) EDL muscles. Injections of either primary myoblasts or MDSCs have no effect on the mass of mdx EDL muscles. EDL muscles of mdx mice generate 43% more absolute twitch tension and 43% less specific tetanic tension then do EDL muscles of C57BL/10J mice. However, the absolute tetanic and specific twitch tension of mdx and C57BL/10J EDL muscles are similar. Injection of either primary myoblasts or MDSCs has no effect on the absolute or specific twitch and tetanic tensions of mdx muscle. Approximately 25% of the myofibers in mdx EDL muscles that received primary myoblasts react positively with antibody to dystrophin. There is no significant difference in the number of dystrophin-positive myofibers when MDSCs are injected. Regardless of the source of donor cells, dystrophin is limited to short distances (60-900 microm) along the length of the myofibers. This may, in part, explain the failure of cellular therapy to alter the contractile properties of murine dystrophic muscle.

Effect of age on skeletal muscle proteolysis in extensor digitorum longus muscles of B6C3F1 mice.

The purpose of this study was to determine if age-related muscle atrophy is associated with an increased rate of protein degradation in extensor digitorum longus (EDL) muscles from young (YG; 2-4 months), middle-aged (MA; 12-17 months), and aged (AG; 22-24 months) B6C3F1 mice. EDL muscles from AG mice weighed less than EDL muscles from MA mice (p =.01). EDL muscles from MA mice weighed more than EDL muscles from YG mice (p =.02). The rate of protein degradation, as assessed by tyrosine release during in vitro incubations, was higher in EDL muscles from AG mice than it was in those from MA mice (p =.03). The rate of protein degradation was higher in EDL muscles from YG mice than it was in those from MA mice (p =.04). An inverse relationship existed between muscle mass and protein degradation (r = -.67; p =.0001). We conclude that skeletal muscle protein degradation rates decrease with maturation and increase with advancing age.

Temperature dependency of force loss and Ca(2+) homeostasis in mouse EDL muscle after eccentric contractions.

The goals of this study were first to determine the effect of temperature on the force loss that results from eccentric contractions in mouse extensor digitorum longus (EDL) muscles and then to evaluate a potential role for altered Ca(2+) homeostasis explaining the greater isometric force loss observed at the higher temperatures. Isolated muscles performed five eccentric or five isometric contractions at either 15, 20, 25, 30, 33.5, or 37 degrees C. Isometric force loss, caffeine-induced force, lactate dehydrogenase (LDH) release, muscle accumulation of (45)Ca(2+) from the bathing medium, sarcoplasmic reticulum (SR) Ca(2+) uptake, and resting muscle fiber free cytosolic Ca(2+) concentration ([Ca(2+)](i)) were measured. The isometric force loss after eccentric contractions increased progressively as temperature rose; at 15 degrees C, there was no significant loss of force, but at 37 degrees C, there was a 30-39% loss of force. After eccentric contractions, caffeine-induced force was not affected by temperature nor was it different from that of control muscles at any temperature. Loss of cell membrane integrity and subsequent influx of extracellular Ca(2+) as indicated by LDH release and muscle (45)Ca(2+) accumulation, respectively, were minimal over the 15-25 degrees C range, but both increased as an exponential function of temperature between 30 and 37 degrees C. SR Ca(2+) uptake showed no impairment as temperature increased, and the eccentric contraction-induced rise in resting fiber [Ca(2+)](i) was unaffected by temperature over the 15-25 degrees C range. In conclusion, the isometric force loss after eccentric contractions is temperature dependent, but the temperature dependency does not appear to be readily explainable by alterations in Ca(2+) homeostasis.

Sarcomere length-dependence of activity-dependent twitch potentiation in mouse skeletal muscle

BackgroundIt has been reported that potentiation of a skeletal muscle twitch response is proportional to muscle length with a negative slope during staircase, and a positive slope during posttetanic potentiation. This study was done to directly compare staircase and posttetanic responses with measurement of sarcomere length to compare their length-dependence.MethodsMouse extensor digitorum longus (EDL) muscles were dissected to small bundles of fibers, which permit measurement of sarcomere length (SL), by laser diffraction. In vitro fixed-end contractions of EDL fiber bundles were elicited at 22°C and 35°C at sarcomere lengths ranging from 2.35 μm to 3.85 μm. Twitch contractions were assessed before and after 1.5 s of 75 Hz stimulation at 22°C or during 10 s of 10 Hz stimulation at 22°C or 35°C.ResultsStaircase potentiation was greater at 35°C than 22°C, and the relative magnitude of the twitch contraction (Pt*/Pt) was proportional to sarcomere length with a negative slope, over the range 2.3 μm – 3.7 μm. Linear regression yielded the following: Pt*/Pt = -0.59·SL+3.27 (r2 = 0.74); Pt*/Pt = -0.39·SL+2.34 (r2 = 0.48); and Pt*/Pt = -0.50·SL+2.45 (r2 = 0.80) for staircase at 35°C, and 22°C and posttetanic response respectively. Posttetanic depression rather than potentiation was present at long SL. This indicates that there may be two processes operating in these muscles to modulate the force: one that enhances and a second that depresses the force. Either or both of these processes may have a length-dependence of its mechanism.ConclusionThere is no evidence that posttetanic potentiation is fundamentally different from staircase in these muscles.

Influence of ageing on the fatigability of isolated mouse skeletal muscles from mature and aged mice.

We investigated the influence of ageing on the fatiguing characteristics of the mouse extensor digitorum longus (EDL) muscle as compared to those of the soleus muscle. Fatigue was produced by an intermittent stimulation protocol. We report for mature and aged animals the effects of fatigue on force produced during stimulation patterns that in non-fatigued muscle gave maximum force (T(max), high frequency stimulation) and approximately half-maximum force (1/2T(max), low frequency stimulation). In 15-month-old (mature) mice, fatiguing stimulation decreased T(max) in EDL and soleus muscle to 10.3 +/- 1.0 % and 33.4 +/- 3.0 % of control, respectively. In 30-month-old (aged) mice, the decrease in T(max) in EDL and soleus was statistically equal to that of the younger animals. Fatiguing stimulation decreased 1/2T(max) in EDL and soleus from 15-month-old animals to 22.5 +/- 2.9 % and 45.7 +/- 0.3 % of control, respectively. In 30-month-old animals, the 1/2T(max) in EDL and soleus muscle decreased to 18.2 +/- 1.3 % and 35.0 +/- 3.6 % of control, respectively. Under all conditions, the soleus fatigued significantly less. Contractile recovery from fatiguing stimulation was complete for the soleus in both age groups after 30 min, but incomplete for the EDL. The 1/2T(max)/T(max) ratio significantly increased in EDL and soleus muscle from 15-month-old animals after fatiguing stimulation. This increase was less significant in EDL, and absent in soleus muscle, from 30-month-old animals. These results indicate that fatiguing stimulation induces a leftward shift in the force-frequency relationship in the young animals; this shift is either significantly less (EDL) or absent (soleus) in the older animals. We speculate that the leftward shift of the force-frequency relationship may reflect a protective mechanism in younger animals against some of the damaging effects of fatiguing stimulation (i.e. oxidative stress).

Denervation enhances the physiological effects of the K(ATP) channel during fatigue in EDL and soleus muscle.

The objective was to determine whether denervation reduces or enhances the physiological effects of the K(ATP) channel during fatigue in mouse extensor digitorum longus (EDL) and soleus muscle. For this, we measured the effects of 100 microM of pinacidil, a channel opener, and of 10 microM of glibenclamide, a channel blocker, in denervated muscles and compared the data to those observed in innervated muscles from the study of Matar et al. (Matar W, Nosek TM, Wong D, and Renaud JM. Pinacidil suppresses contractility and preserves energy but glibenclamide has no effect during fatigue in skeletal muscle. Am J Physiol Cell Physiol 278: C404-C416, 2000). Pinacidil increased the (86)Rb(+) fractional loss during fatigue, and this effect was 2.6- to 3.4-fold greater in denervated than innervated muscle. Pinacidil also increased the rate of fatigue; for EDL the effect was 2.5-fold greater in denervated than innervated muscle, whereas for soleus the difference was 8.6-fold. A major effect of glibenclamide was an increase in resting tension during fatigue, which was for the EDL and soleus muscle 2.7- and 1.9-fold greater, respectively, in denervated than innervated muscle. A second major effect of glibenclamide was a reduced capacity to recover force after fatigue, an effect observed only in denervated muscle. We therefore suggest that the physiological effects of the K(ATP) channel are enhanced after denervation.

Depletion of Ca2+ in the sarcoplasmic reticulum stimulates Ca2+ entry into mouse skeletal muscle fibres.

To examine whether a capacitative Ca2+ entry pathway is present in skeletal muscle, thin muscle fibre bundles were isolated from extensor digitorum longus (EDL) muscle of adult mice, and isometric tension and fura-2 signals were simultaneously measured. The sarcoplasmic reticulum (SR) in the muscle fibres was successfully depleted of Ca2+ by repetitive treatments with high-K+ solutions, initially in the absence and then in the presence of a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor. Depletion of the SR of Ca2+ enabled us for the first time to show convincingly that the vast majority of the voltage-sensitive Ca2+ store overlaps the caffeine-sensitive Ca2+ store in intact fibres from mouse EDL muscle. This conclusion was based on the observation that both high-K+ solution and caffeine failed to cause a contracture in the depleted muscle fibres. The existence of a Ca2+ influx pathway active enough to refill the depleted SR within several minutes was shown in skeletal muscle fibres. Ca2+ entry was sensitive to Ni2+, but resistant to nifedipine and was suppressed by plasma membrane depolarisation. Evidence for store-operated Ca2+ entry was provided by measurements of Mn2+ entry. Significant acceleration of Mn2+ entry was observed only when the SR was severely depleted of Ca2+. The Mn2+ influx, which was blocked by Ni2+ but not by nifedipine, was inwardly rectifying, as is the case with the Ca2+ entry. These results indicate that the store-operated Ca2+ entry is similar to the Ca2+ release-activated Ca2+ channel (CRAC) current described in other preparations.

IGF-I treatment improves the functional properties of fast- and slow-twitch skeletal muscles from dystrophic mice.

Although insulin-like growth factor-I (IGF-I) has been proposed for use by patients suffering from muscle wasting conditions, few studies have investigated the functional properties of dystrophic skeletal muscle following IGF-I treatment. 129P1 ReJ-Lama2(dy) (129 ReJ dy/dy) dystrophic mice suffer from a deficiency in the structural protein, laminin, and exhibit severe muscle wasting and weakness. We tested the hypothesis that 4 weeks of IGF-I treatment ( approximately 2 mg/kg body mass, 50 g/h via mini-osmotic pump, subcutaneously) would increase the mass and force producing capacity of skeletal muscles from dystrophic mice. IGF-I treatment increased the mass of the extensor digitorum longus (EDL) and soleus muscles of dystrophic mice by 20 and 29%, respectively, compared with untreated dystrophic mice (administered saline-vehicle only). Absolute maximum force (P(o)) of the EDL and soleus muscle was increased by 40 and 32%, respectively, following IGF-I treatment. Specific P(o) (sP(o)) was increased by 23% in the EDL muscles of treated compared with untreated mice, but in the soleus muscle sP(o) was unchanged. IGF-I treatment increased the proportion of type IIB and type IIA fibres and decreased the proportion of type I fibres in the EDL muscles of dystrophic mice. In the soleus muscles of dystrophic mice, IGF-I treatment increased the proportion of type IIA fibres and decreased the proportion of type I fibres. Average fibre cross-sectional area was increased in the EDL and soleus muscles of treated compared with untreated mice. We conclude that IGF-I treatment ameliorates muscle wasting and improves the functional properties of skeletal muscles of dystrophic mice. The findings have important implications for the role of IGF-I in ameliorating muscle wasting associated with the muscular dystrophies.

Redox modulation of maximum force production of fast-and slow-twitch skeletal muscles of rats and mice

We used intact fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles from rats and mice to test the hypothesis that exogenous application of an oxidant would increase maximum isometric force production (P(o)) of slow-twitch muscles to a greater extent than fast-twitch skeletal muscles. Exposure to an oxidant, hydrogen peroxide (H(2)O(2); 100 microM to 5 mM, 30 min), affected P(o) of rat muscles in a time- and dose-dependent manner. P(o) of rat soleus muscles was increased by 8 +/- 1 (SE) and 14 +/- 1% (P < 0.01) after incubation with 1 and 5 mM H(2)O(2), respectively, whereas in mouse soleus muscles P(o) was only increased after incubation with 500 microM H(2)O(2). P(o) of rat EDL muscles was affected by H(2)O(2) biphasically; initially there was a small increase (3 +/- 1%), but then P(o) diminished significantly after 30 min of treatment. In contrast, all concentrations of H(2)O(2) tested decreased P(o) of mouse EDL muscles. A reductant, dithiothreitol (DTT; rat = 10 mM, mouse = 1 mM), was added to quench H(2)O(2), and it reversed the potentiation in P(o) in rat soleus but not in rat EDL muscles or in any H(2)O(2)-treated mouse muscles. After prolonged equilibration (30 min) with 5 mM H(2)O(2) without prior activation, P(o) was potentiated in rat soleus but not EDL muscles, demonstrating that the effect of oxidation in the soleus muscles was also dependent on the activation history of the muscle. The results of these experiments demonstrate that P(o) of both slow- and fast-twitch muscles from rats and mice is modified by redox modulation, indicating that maximum P(o) of mammalian skeletal muscles is dependent on oxidation.