Properties of regenerated mouse extensor digitorum longus muscle following notexin injury.

Muscles of mdx mice are known to be more susceptible to contraction-induced damage than wild-type muscle. However, it is not clear whether this is because of dystrophin deficiency or because of the abnormal branching morphology of dystrophic muscle fibres. This distinction has an important bearing on our traditional understanding of the function of dystrophin as a mechanical stabilizer of the sarcolemma. In this study, we address the question: 'Does dystrophin-positive, regenerated muscle containing branched fibres also show an increased susceptibility to contraction-induced damage?' We produced a model of fibre branching by injecting dystrophin-positive extensor digitorum longus muscles with notexin. The regenerated muscle was examined at 21 days postinjection. Notexin-injected muscle contained 29% branched fibres and was not more susceptible to damage from mild eccentric contractions than contralateral saline-injected control muscle. Regenerated muscles also had greater mass, greater cross-sectional area and lower specific force than control muscles. We conclude that the number of branched fibres in this regenerated muscle is below the threshold needed to increase susceptibility to damage. However, it would serve as an ideal control for muscles of young mdx mice, allowing for clearer differentiation of the effects of dystrophin deficiency from the effects of fibre regeneration and morphology.

An active learning mammalian skeletal muscle lab demonstrating contractile and kinetic properties of fast- and slow-twitch muscle.

The fact that humans possess fast- and slow-twitch muscle in the ratio of ∼50% has profound implications for designing exercise training strategies for power and endurance activities. With the growth of exercise and sport science courses, we have seen the need to develop an undergraduate student laboratory that demonstrates the basic properties of fast- and slow-twitch mammalian skeletal muscle. This laboratory illustrates the major differences in contractile properties and fatigue profiles exhibited by the two muscle types. Students compare and contrast twitch kinetics, fused tetanus characteristics, force-frequency relationships, and fatigue properties of fast- and slow-twitch muscles. Examples of results collected by students during class are used to illustrate the type of data collected and analysis performed. During the laboratory, students are encouraged to connect factual information from their skeletal muscle lectures to their laboratory findings. This enables student learning in an active fashion; in particular, the isolated muscle preparation demonstrates that much of what makes muscle fast or slow is myogenic and not the product of the nervous or circulatory systems. This has far-reaching implications for motor control and exercise behavior and therefore is a crucial element in exercise science, with its focus on power and endurance sport activities. To measure student satisfaction with this active learning technique, a questionnaire was administered after the laboratory; 96% of the comments were positive in their support of active versus passive learning strategies.

Reduced postsynaptic GABAA receptor number and enhanced gaboxadol induced change in holding currents in Purkinje cells of the dystrophin-deficient mdx mouse.

UNLABELLED: Duchenne muscular dystrophy (DMD) is caused by the absence of a functional transcript of the protein dystrophin. DMD is associated with a range of cognitive deficits that are thought to result from a lack of the protein dystrophin in brain structures involved in cognitive functions. The CNS involvement extends to an impairment of cognitive abilities, with many DMD boys having significant reduction in IQ. In the cerebellum, dystrophin is normally localized at the postsynaptic membrane of GABAergic synapses on Purkinje cells. Here, we investigate the effect of an absence of dystrophin on the number of GABA(A) channels located at the synapse in cerebellar Purkinje cells of the dystrophin-deficient mdx mouse. Whole-cell patch-clamp recordings of spontaneous miniature inhibitory postsynaptic currents (mIPSCs) were performed in cerebellar slices from mdx and littermate control mice. Our results showed that the number of receptors at GABAergic synapses in the cerebellar Purkinje cell was significantly reduced in mdx mice (38.38 ± 2.95) compared to littermate controls (53.03 ± 4.11). Furthermore, when gaboxadol was added to the bath, the change in holding current in mdx mice was significantly enhanced (65.01 ± 5.89pA) compared to littermate controls (37.36 ± 3.82pA). The single channel unitary conductance and the rise and decay time of mIPSCs were not significantly different in these two groups of mice, indicating that those GABA(A) channels located at the postsynaptic sites in the mdx mice function normally. CONCLUSION: There is a reduction in the number of functional receptors localized at GABAergic synapses in the cerebellar Purkinje cells of dystrophin-deficient mdx mice and an increase in a gaboxadol induced holding current, which is evidence for an increase in extrasynaptic GABA(A) receptors in mdx mice. We hypothesize that the absence of dystrophin, from mdx Purkinje cells, reduces the number of post-synaptic GABA(A) receptors and as a result there is an increase in extrasynaptic receptors. If similar changes occur in the CNS in boys with DMD, it will impact on the function of neural networks and may contribute to some of the motor, behavioral and cognitive impairment apparent in many boys with DMD.

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.

GABA(A) receptor expression and inhibitory post-synaptic currents in cerebellar Purkinje cells in dystrophin-deficient mdx mice.

1. Duchenne muscular dystrophy (DMD) is the second most common fatal genetic disease and arises as a consequence of an absence or disruption of the protein dystrophin. In addition to wasting of the skeletal musculature, boys with DMD have a significant degree of cognitive impairment. 2. We show here that there is no difference between littermate control and mdx mice (a murine model of DMD) in the overall expression of the GABA(A) receptor a1-subunit, supporting the suggestion that it is the clustering at the synapse that is affected and not the expression of the GABA(A) receptor protein. 3. We report a significant reduction in both the frequency and amplitude of spontaneous inhibitory post-synaptic currents in cerebellar Purkinje cells of mdx mice compared with littermate controls, consistent with the reported reduction in the number and size of GABA(A) receptor clusters immunoreactive for a1- and a2-subunits at the post-synaptic densities. 4. These results may explain some of the behavioural problems and cognitive impairment reported in DMD.

The MyoRobot: A novel automated biomechatronics system to assess voltage/Ca2+ biosensors and active/passive biomechanics in muscle and biomaterials.

We engineered an automated biomechatronics system, MyoRobot, for robust objective and versatile assessment of muscle or polymer materials (bio-)mechanics. It covers multiple levels of muscle biosensor assessment, e.g. membrane voltage or contractile apparatus Ca2+ ion responses (force resolution 1µN, 0-10mN for the given sensor; [Ca2+] range ~ 100nM-25µM). It replaces previously tedious manual protocols to obtain exhaustive information on active/passive biomechanical properties across various morphological tissue levels. Deciphering mechanisms of muscle weakness requires sophisticated force protocols, dissecting contributions from altered Ca2+ homeostasis, electro-chemical, chemico-mechanical biosensors or visco-elastic components. From whole organ to single fibre levels, experimental demands and hardware requirements increase, limiting biomechanics research potential, as reflected by only few commercial biomechatronics systems that can address resolution, experimental versatility and mostly, automation of force recordings. Our MyoRobot combines optical force transducer technology with high precision 3D actuation (e.g. voice coil, 1µm encoder resolution; stepper motors, 4µm feed motion), and customized control software, enabling modular experimentation packages and automated data pre-analysis. In small bundles and single muscle fibres, we demonstrate automated recordings of (i) caffeine-induced-, (ii) electrical field stimulation (EFS)-induced force, (iii) pCa-force, (iv) slack-tests and (v) passive length-tension curves. The system easily reproduces results from manual systems (two times larger stiffness in slow over fast muscle) and provides novel insights into unloaded shortening velocities (declining with increasing slack lengths). The MyoRobot enables automated complex biomechanics assessment in muscle research. Applications also extend to material sciences, exemplarily shown here for spider silk and collagen biopolymers.

Synaptic plasticity in the dy2J mouse model of laminin alpha2-deficient congenital muscular dystrophy.

Laminin alpha2-deficient congenital muscular dystrophy is a debilitating disease affecting both muscle and neural tissue as a result of mutations in the LAMA2 gene. It presents at or soon after birth with muscle weakness and is further characterised by clinical central nervous system involvement. Laminin alpha2 is part of the extracellular matrix, linked to the cellular cystoskeleton via dystroglycan which is an integral part of the dystrophin-glycoprotein complex (DGC). We examined both short- and long-term synaptic plasticity in the C57BL6J/dy(2J) mouse, an animal model of laminin alpha2 deficient congenital muscular dystrophy. Using a cerebellar slice preparation, we show that the pre-synaptically mediated paired-pulse facilitation (PPF) was no different between dy(2J) and littermate controls. Approximately half (7/12) the dy(2J) Purkinje cells displayed a blunted LTD compared to littermate controls, and one third (4/12) of dy(2J) Purkinje cells displayed LTP. This study demonstrates that a defective laminin alpha2 causes a disruption in long-term synaptic plasticity at the Purkinje cell-parallel fibre synapse.

Measurement of membrane potential and myoplasmic [Ca2+] in developing rat myotubes at rest and in response to stimulation.

In this study, the membrane potential and cytosolic [Ca2+] were measured in rat myotubes developing in culture from days 6-14. It was found that as the myotubes developed in culture, the resting membrane potential (RMP) became more negative during days 6-8, and then did not significantly change until after day 13, when it started to become less negative. The mean RMP measured at days 8-13 was -59 +/- 1 mV (n = 70). The amplitude of action potentials elicited in the myotubes by anode break stimulation increased in size during development (range: 47.5-119 mV) and this closely correlated with the development of a more negative RMP. Cytosolic [Ca2+] was measured in the rat myotubes using the Ca2+ indicator Fura-2, and no significant change in the resting [Ca2+] was observed during development (days 6-14). Ca2+ responses triggered by action potentials varied from small slow increases in [Ca2+] that failed to return to the baseline to rapid [Ca2+] transients. The size of the [Ca2+] transients positively correlated with both the observed increase in the RMP during development and the size of the action potential. Larger [Ca2+] transients also had more rapid rates of [Ca2+] decay, indicating a tandem increase in the ability of the sarcoplasmic reticulum to release and resequester Ca2+ during development of rat myotubes. Repetitive stimulation (10 Hz) of the myotubes exhibiting small [Ca2+] transients produced a step-like rise in [Ca2+]. Many myotubes exhibiting larger [Ca2+]transients could not be stimulated at 10 Hz by anode break stimulation due to the presence of action potentials with large hyperpolarisations. However, when these myotubes were depolarised at 10 Hz, they produced a tetanic Ca2+ response similar to that seen in adult skeletal muscle.

Time course of calcium transients derived from Fura-2 fluorescence measurements in single fast twitch fibres of adult mice and rat myotubes developing in primary culture.

In this study, we applied a method to correct for the altered binding kinetics of Fura-2 for Ca2+ in vivo, on Ca2+ fluorescence transients (Ca2+F) measured using Fura-2 in single adult fast twitch skeletal muscle fibres of the mouse, which exhibit very fast [Ca2+] responses, and rat myotubes developing in culture which exhibit slower [Ca2+] responses (rise time [20-80% of peak] of Ca2+F transients: 1.81 +/- 0.17 ms and 16.14 +/- 2.60 ms, respectively). After correction, the [Ca2+] transients (Ca2+C) measured in both the adult mouse fibres and the myotubes rose more rapidly (mean rise time of Ca2+C transients: adult mouse fibres, 0.76 +/- 0.12 ms; rat myotubes, 8.25 +/- 2.83 ms) and often exhibited a Ca2+ spike which exceeded the peak of the Ca2+F transient. In the adult mouse fibres, correction increased the mean peak [Ca2+] of the Ca2+F transients by a factor of 7 from 0.53 +/- 0.08 microM to 3.76 +/- 0.71 microM. The accuracy of the time course of the corrected Ca2+ transients was confirmed by comparison to the time course of Ca2+ transients measured with Mag-Fura-5, which had a similar mean rise time (0.94 +/- 0.10 ms, t-test, P = 0.80). The more slowly rising Ca2+ transients measured in the rat myotubes were less affected by the correction process, increasing in mean peak [Ca2+] by a factor of only 1.2 from 0.82 +/- 0.17 microM to 0.97 +/- 0.15 microM. During the decay phase of the Ca2+ transients elicited in the adult mouse fibres and the myotubes, the corrected Ca2+C signal largely followed the unmodified Ca2+F transient. The correction process was found to have little effect on Ca2+ transients with rise time values greater than 10 ms, which included most of the Ca2+ transients measured in the myotubes.

Abnormalities in structure and function of limb skeletal muscle fibres of dystrophic mdx mice.

In this study we have shown that the skeletal muscle fibres from adult (older than 26 weeks) mdx mice have gross structural deformities. We have characterized the onset and age dependence of this feature in mdx mice. The three dimensional structure of these deformities has been visualized in isolated fibres and the orientation of these deformities was determined within the muscle by confocal laser scanning microscopy. We have also shown that the occurrence of morphologically abnormal fibres is greater in muscles with longer fibres (extensor digitorum longus (EDL) and soleus, 6-7.3 mm long), than in muscles with shorter fibres (flexor digitorum brevis (FDB), 0.3-0.4 mm long). A population of post-degenerative fibres, with both central and peripheral nuclei coexistent along the length of the fibre, has also been identified in the muscles studied. We showed that a mild protocol of lengthening (eccentric) contractions (the muscle was stretched by 12% during a tetanic contraction) caused a major reduction in the maximal tetanic force subsequently produced by mdx EDL muscle. In contrast, maximal tetanic force production in normal soleus, normal EDL and mdx soleus muscles was not altered by this protocol. We suggest that the deformed fast glycolytic fibres which are found in adult mdx EDL but not in adult mdx soleus muscles are the population of fibres damaged by the lengthening protocol.

A gene for speed: contractile properties of isolated whole EDL muscle from an alpha-actinin-3 knockout mouse.

The actin-binding protein alpha-actinin-3 is one of the two isoforms of alpha-actinin that are found in the Z-discs of skeletal muscle. alpha-Actinin-3 is exclusively expressed in fast glycolytic muscle fibers. Homozygosity for a common polymorphism in the ACTN3 gene results in complete deficiency of alpha-actinin-3 in about 1 billion individuals worldwide. Recent genetic studies suggest that the absence of alpha-actinin-3 is detrimental to sprint and power performance in elite athletes and in the general population. In contrast, alpha-actinin-3 deficiency appears to be beneficial for endurance athletes. To determine the effect of alpha-actinin-3 deficiency on the contractile properties of skeletal muscle, we studied isolated extensor digitorum longus (fast-twitch) muscles from a specially developed alpha-actinin-3 knockout (KO) mouse. alpha-Actinin-3-deficient muscles showed similar levels of damage to wild-type (WT) muscles following lengthening contractions of 20% strain, suggesting that the presence or absence of alpha-actinin-3 does not significantly influence the mechanical stability of the sarcomere in the mouse. alpha-Actinin-3 deficiency does not result in any change in myosin heavy chain expression. However, compared with alpha-actinin-3-positive muscles, alpha-actinin-3-deficient muscles displayed longer twitch half-relaxation times, better recovery from fatigue, smaller cross-sectional areas, and lower twitch-to-tetanus ratios. We conclude that alpha-actinin-3 deficiency results in fast-twitch, glycolytic fibers developing slower-twitch, more oxidative properties. These changes in the contractile properties of fast-twitch skeletal muscle from alpha-actinin-3-deficient individuals would be detrimental to optimal sprint and power performance, but beneficial for endurance performance.

Branched fibers in dystrophic mdx muscle are associated with a loss of force following lengthening contractions.

We demonstrated that the susceptibility of skeletal muscle to injury from lengthening contractions in the dystrophin-deficient mdx mouse is directly linked with the extent of fiber branching within the muscles and that both parameters increase as the mdx animal ages. We subjected isolated extensor digitorum longus muscles to a lengthening contraction protocol of 15% strain and measured the resulting drop in force production (force deficit). We also examined the morphology of individual muscle fibers. In mdx mice 1-2 mo of age, 17% of muscle fibers were branched, and the force deficit of 7% was not significantly different from that of age-matched littermate controls. In mdx mice 6-7 mo of age, 89% of muscle fibers were branched, and the force deficit of 58% was significantly higher than the 25% force deficit of age-matched littermate controls. These data demonstrated an association between the extent of branching and the greater vulnerability to contraction-induced injury in the older fast-twitch dystrophic muscle. Our findings demonstrate that fiber branching may play a role in the pathogenesis of muscular dystrophy in mdx mice, and this could affect the interpretation of previous studies involving lengthening contractions in this animal.

Membrane potential, resting calcium and calcium transients in isolated muscle fibres from normal and dystrophic mice.

1. Single skeletal muscle fibres were enzymatically isolated from the flexor digitorum brevis muscles (FDB) of dystrophic mdx and control C57BL/10 mice aged 3-9 weeks. In this age range the majority (> 95%) of the mdx fibres were morphologically normal. 2. There was no significant difference between the resting membrane potential (RMP) of mdx and control mice, -71.2 +/- 1.21 (n = 26) and -70.6 +/- 1.15 mV (n = 42), respectively. 3. At RMP more negative than -60 mV the resting calcium (recorded with fura-2, free acid ionophoresed into cell) in the dystrophic mdx cells was not significantly different from the normal animals, 45.7 +/- 4.1 (n = 10) and 46.2 +/- 3.9 nM (n = 9), respectively. 4. The resting cytosolic calcium concentration was measured simultaneously with the RMP. At RMP between -60 to -17 mV there was an increase in the resting calcium concentration in both mdx and control ranging from 79.3 to 252 nM. This increase was most probably due to the activation of the slow calcium current. 5. Fura-2 calcium transients were produced via single action potential stimulation using an intracellular microelectrode both to stimulate the cell and record potential changes. There was no significant difference between the rise time (Tp) or half-decay time (T1/2) at 22 degrees C of the calcium transient in response to a single action potential in mdx compared to normal animals, 5.9 +/- 0.34 (n = 8) and 5.4 +/- 0.36 ms (n = 7); 39.5 +/- 2.9 (n = 8) and 40.75 +/- 3.7 ms (n = 7), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Reflex actions of one proprioceptor on the motoneurones of a muscle receptor and their central modulation in the shore crab.

1. Reflex efferent control of a muscle stretch receptor by a joint proprioceptor of the same limb was studied in an isolated CNS preparation from the shore crab. The influence of 'fictive locomotor' activity on this interjoint reflex was also examined. 2. The thoracic-coxal muscle receptor organ (TCMRO) and the coxo-basal chordotonal organ (CBCO), which monitor movement and position of the first and second joints of the posterior leg, were isolated together with the whole thoracic ganglion complex. The TCMRO, functionally analogous to a mammalian muscle spindle, has two receptor motoneurones. RM1 innervating the receptor muscle alone and RM2 which also supplies the 'extrafusal' promotor muscle. The CBCO is a typical arthropod elastic strand organ, with many sensory neurones but lacking an efferent supply. The TCMRO was fixed at its mid-length, and stretch-hold-release stimuli were applied to the CBCO. Efferent activity was recorded from the cut nerve roots of the four basal limb muscles and intracellularly as excitatory junction potentials (EJPs) from the receptor muscle. 3. A dynamic increase in the frequency of action potentials in RM1 occurred on both stretch and release of the CBCO. During the hold phase the RM1 activity declined from the dynamic response but remained elevated compared to the resting tonic discharge. RM2, identified by EJPs occurring 1:1 with a unit in the promoter nerve, responded in a similar way. 4. One or more promotor motoneurones were usually co-activated with the two receptor efferents in response to input from the CBCO. In a typical example the average spike frequency of RM1 rose from 0 to 27 Hz during the dynamic phases (stretch and release) of the CBCO stimulus, falling to 2.5 Hz during the hold phase, while the corresponding promotor spike frequencies were 25 and 7.5 Hz, respectively. The other three muscle nerves recorded from generally also showed reflex driving by the CBCO. 5. The totally isolated thoracic ganglion could produce a rhythmic, bursting motor output in the absence of any sensory input. During this centrally generated activity the receptor motor innervation was strongly co-activated with the promotor bursts, and the reflex input from the CBCO was overridden or modulated in a phase-dependent manner. 6. The proximally directed interjoint reflex to the receptor muscle probably functions to maintain the tension on the sensory endings of the TCMRO, and so enable them to respond effectively at all times to movements of the basal leg joint.(ABSTRACT TRUNCATED AT 400 WORDS)

Properties of enzymatically isolated skeletal fibres from mice with muscular dystrophy.

1. Single intact muscle fibres were enzymatically isolated from the skeletal muscles of the dystrophic mouse 129/ReJ dy/dy and were subjected to a range of physiological interventions. 2. Electrophysiological measurements, diffusion of injected dyes (Lucifer Yellow), microdissection and general appearance in the light microscope have shown that the majority of skeletal fibres isolated from the soleus and extensor digitorum longus (EDL) of adult dystrophic mice (10-14 weeks old) had gross morphological abnormalities. These abnormalities ranged from simple branching of the fibre to interconnections of many fibre branches which form a complex syncitium. 3. Segments from fibres of normal appearance and from fibres with morphological deformities were chemically skinned with Triton X-100 and activated in Ca2(+)- and Sr2(+)-buffered solutions. The different characteristics of the Ca2(+)- and Sr2(+)-activation curves were also used to identify the fibre type. 4. Gross morphological abnormalities were observed both in fibres which had predominantly slow-twitch and fast-twitch characteristics. 5. A new group of fibres was found to exist in the soleus muscle of dystrophic animals and represented about 18% of the entire soleus fibre population. This group of fibres had predominantly fast-twitch characteristics and some of these fibres were also grossly malformed. 6. The activation characteristics of individual branches from the same complex syncitium were similar, indicating that the contractile and regulatory proteins were of one type in one syncitium. 7. Chemically skinned segments from malformed fibres which included a major deformity between the points of attachment were generally unable to sustain near-maximal forces. 8. The proportion of malformed fibres which remained intact decreased markedly after prolonged tetanical stimulation of the intact muscle. This strongly suggests that malformed fibres are also functionally weak and prone to progressive damage when stimulated within the intact muscle. 9. The presence in large proportions of fibres with gross morphological abnormalities may explain the symptoms of severe and progressive muscle weakness and muscle loss which are apparent in the 129/ReJ dy/dy mice and possibly even in the human dystrophies such as Duchenne muscular dystrophy.

Mechanism of action of a K+ channel activator BRL 38227 on ATP-sensitive K+ channels in mouse skeletal muscle fibres.

1. Investigations were made into the effects of BRL 38227, a potassium channel activator, on ATP-sensitive potassium channels (K+ATP channels) in single fibres dissociated from the flexor digitorum brevis muscle of C57BL/6J mice. 2. In cell-attached patches BRL 38227 (100 microM) caused activation of a glibenclamide-sensitive potassium current. Linear slope conductance of the inward current, partial rectification of the outward current and glibenclamide sensitivity indicate that K+ATP channels are the site of action of BRL 38227. 3. In the absence of ATP at the cytoplasmic side of excised inside-out patches, BRL 38227 caused direct and magnesium-dependent activation of K+ATP channels. The degree of activation diminished with successive applications of BRL 38227. 4. BRL 38227 also caused activation of K+ATP channels in the presence of low (< 100 microM) but not high (1.0 mM) ATP, particularly in patches containing large numbers of channels. 5. BRL 38227 and 5 microM MgATP failed to activate channels following complete run-down. 6. Results show that BRL 38227 caused direct activation of K+ATP in skeletal muscle and that this was mediated through a magnesium-dependent binding site rather than alleviation of inhibition by competitive displacement of ATP from the inhibitory site.

The role of nitric oxide in diaphragmatic dysfunction in endotoxemic rats.

In this study we examined the role of nitric oxide (NO) from inducible nitric oxide synthase (iNOS) and adenosine triphosphate (ATP) depletion, using aminoguanidine and 3-aminobenzamide, on diaphragm contractility in a rat model of sepsis. Intraperitoneal lipopolysaccharide (LPS) injection was used to induce septicemia in rats. The LPS treatment caused a decrease in maximal absolute force produced by the diaphragm muscle stimulated at 100 HZ, and the force-frequency curves were right-shifted with a decrease in force at 2, 5 and 15 HZ. LPS administration also made the diaphragm muscle strips more fatigable than controls. The decrease in force in LPS-treated animals was not due to an induction of pathological levels of i NOS. Increased fatigability did not appear to be due to a depletion of ATP through poly-adenosine-diphosphate-ribose polymerase (PARP) activation. This study does not support the hypothesis that the decrease in diaphragm muscle force as a result of sepsis is due to an induction of pathological levels of nitric oxide or ATP depletion.

Ca2+- and Sr2+-activation properties of muscle fibres from a muscle receptor organ and the associated extrafusal muscle of the crab and crayfish.

In this study on decapod crustaceans, we examined the Ca2+- and Sr2+-activation properties of skeletal muscle fibres from an identified proprioceptor, the thoracic coxal muscle receptor organ (TCMRO) and its extrafusal promotor muscle fibres. Proprioceptors and extrafusal muscles were isolated from a walking leg from the crayfish (Cherax destructor) and the rear swimming leg of the mud crab (Scylla serrata). The crayfish and mud crab TCMROs had very low Hill coefficient (nCa) values (1.86 +/- 0.08 and 1.64 +/- 0.03, respectively). In comparison to other skeletal muscle fibre types these low Hill coefficients would enable the length of the receptor muscles to be finely controlled over a wide range of [Ca2+]. Maximum force was found to be significantly lower in the TCMROs (crayfish: 5.76 +/- 0.98; crab: 4.80 +/- 0.56 Ncm(-2)), compared to their associated extrafusal promotor muscle fibres (crayfish: 10.69 +/- 1.63; crab: 20.07 +/- 1.98 Ncm(-2)), which is consistent with their sensory role. The muscle fibres of the crayfish TCMRO had faster contractile properties than the mud crab TCMRO, we discuss how these contractile properties relate to the type of locomotion undergone by each leg. The mud crab 'red' promotor and all crayfish promotor fibres were characterised as slow with low Hill coefficients (nCa: crayfish: 3.22 +/- 0.29; crab: 3.34 +/- 0.29) and a contractile apparatus with a high sensitivity to Ca2+ (pCa50: crayfish: 6.42 +/- 0.03; crab: 6.18 +/- 0.03). In contrast the 'white' mud crab promotor fibres from the swimming leg had contractile properties that were characteristic of fast fibres with a high mean Hill coefficient (nCa: 5.27 +/- 0.76) and a lower Ca2+ sensitivity (pCa50: 6.03 +/- 0.03). The sensitivity of the contractile apparatus to Sr2+ was very low (range of mean pSr50: 4.23 +/- 0.03-3.48 +/- 0.06) and low force levels were produced in comparison to that produced with Ca2+. The results of this study show that the muscle fibres of the sensory receptor, produce less force and have been adapted to enable the length of the receptor to be finely set in relation to the length of the extrafusal muscle. We discuss how the striated fibres of the receptor have been adapted to perform a sensory role and how this is related to the type of locomotion undergone by the legs. We also discuss how the fibre types of the extrafusal muscle have adapted to the mode of locomotion.

Resting calcium concentrations in isolated skeletal muscle fibres of dystrophic mice.

1. Single, intact muscle fibres were dissociated enzymatically from skeletal muscles of phenotypically normal (+/?) and dystrophic mice (129/ReJ dy/dy: Dystrophia muscularis), and resting Ca2+ levels were measured by image analysis of intracellular Fura-2 fluorescence in distinct parts of the fibres. 2. Fura-2 was introduced into fibres by ionophoresis with glass microelectrodes to concentrations of between 50 and 200 microM. Over this concentration range there was no apparent buffering of intracellular Ca2+ by Fura-2. 3. Fibres isolated from the soleus, flexor digitorum brevis (FDB) and extensor digitorum longus (EDL) muscles of normal animals maintained resting [Ca2+] of 106 +/- 2 nM. Ca2+ distributions within individual fibres were homogeneous. 4. Fibres from dystrophic animals maintained [Ca2+] that was elevated two- to fourfold in comparison to normal fibres. 5. The population of skeletal fibres from dystrophic mice which displayed morphology similar to that of fibres of normal animals were found to have Ca2+ levels that averaged 189 +/- 2 nM. The distribution of Ca2+ within these fibres appeared uniform. 6. The population of dystrophic fibres that possessed morphological abnormalities maintained even higher Ca2+ concentrations (368 +/- 3 nM). Several fibres from this morphological group displayed obvious heterogeneity in Ca2+ distribution with distinct, localized areas of higher Ca2+. 7. These results support the contention that Ca2+ homeostasis is markedly impaired in dystrophic muscle. The elevated Ca2+ levels are near the threshold for contraction and, together with severe morphological fibre abnormalities, are probably centrally involved in fibre necrosis apparent in muscular dystrophy.

Effects of the PKA inhibitor H-89 on excitation-contraction coupling in skinned and intact skeletal muscle fibres.

This study investigated the effects of the protein kinase A (PKA) inhibitor, H-89, in mechanically-skinned muscle fibres and intact muscle fibres, in order to determine whether PKA phosphorylation is essential for normal excitation-contraction (E-C) coupling. In skinned EDL fibres of the rat, force responses to depolarization (by ion substitution) were inhibited only slightly by 10 microM H-89, a concentration more than sufficient to fully inhibit PKA. Staurosporine (1 microM), a potent non-specific kinase inhibitor, also had little if any effect on depolarization-induced responses. At 1-2 microM, H-89 significantly slowed the repriming rate in rat skinned fibres, most likely due to it deleteriously affecting the T-system potential. With 100 microM H-89, the force response to depolarization by ion substitution was completely abolished. This inhibitory effect was reversed by washout of H-89 and was not due to block of the Ca2+ release channel in the sarcoplasmic reticulum (SR). In intact single fibres of the flexor digitorum longus (FDB) muscle of the mouse, 1-3 microM H-89 had no noticeable effect on action-potential-mediated Ca2+ transients. Higher concentrations (4-10 microM) caused Ca2+ transient failure in fibres stimulated at 20 Hz in a manner indicative of action-potential failure. At 10-100 microM, H-89 also inhibited net Ca2+ uptake by the SR and affected the Ca2+-sensitivity of the contractile apparatus in rat skinned fibres. All such effects were proportionately greater in toad muscle fibres. These results do not support the hypothesis that phosphorylation is essential for the Ca2+ release channel to open in response to voltage-sensor activation in skeletal muscle fibres.