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.

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.

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.

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.

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.

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.

Brain function in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the second most commonly occurring genetically inherited disease in humans. It is an X-linked condition that affects approximately one in 3300 live male births. It is caused by the absence or disruption of the protein dystrophin, which is found in a variety of tissues, most notably skeletal muscle and neurones in particular regions of the CNS. Clinically DMD is characterized by a severe pathology of the skeletal musculature that results in the premature death of the individual. An important aspect of DMD that has received less attention is the role played by the absence or disruption of dystrophin on CNS function. In this review we concentrate on insights into this role gained from investigation of boys with DMD and the genetically most relevant animal model of DMD, the dystrophin-deficient mdx mouse. Behavioural studies have shown that DMD boys have a cognitive impairment and a lower IQ (average 85), whilst the mdx mice display an impairment in passive avoidance reflex and in short-term memory. In DMD boys, there is evidence of disordered CNS architecture, abnormalities in dendrites and loss of neurones, all associated with neurones that normally express dystrophin. In the mdx mouse, there have been reports of a 50% decrease in neurone number and neural shrinkage in regions of the cerebral cortex and brainstem. Histological evidence shows that the density of GABA(A) channel clusters is reduced in mdx Purkinje cells and hippocampal CA1 neurones. At the biochemical level, in DMD boys the bioenergetics of the CNS is abnormal and there is an increase in the levels of choline-containing compounds, indicative of CNS pathology. The mdx mice also display abnormal bioenergetics, with an increased level of inorganic phosphate and increased levels of choline-containing compounds. Functionally, DMD boys have EEG abnormalities and there is some preliminary evidence that synaptic function is affected adversely by the absence of dystrophin. Electrophysiological studies of mdx mice have shown that hippocampal neurones have an increased susceptibility to hypoxia. These recent findings on the role of dystrophin in the CNS have implications for the clinical management of boys with DMD.

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.

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.

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.

Effect of clenbuterol on sarcoplasmic reticulum function in single skinned mammalian skeletal muscle fibers.

We examined the effect of the beta2-agonist clenbuterol (50 microM) on depolarization-induced force responses and sarcoplasmic reticulum (SR) function in muscle fibers of the rat (Rattus norvegicus; killed by halothane overdose) that had been mechanically skinned, rendering the beta2-agonist pathway inoperable. Clenbuterol decreased the peak of depolarization-induced force responses in the extensor digitorum longus (EDL) and soleus fibers to 77.2 +/- 9.0 and 55.6 +/- 5.4%, respectively, of controls. The soleus fibers did not recover. Clenbuterol significantly and reversibly reduced SR Ca2+ loading in EDL and soleus fibers to 81.5 +/- 2.8 and 78.7 +/- 4.0%, respectively, of controls. Clenbuterol also produced an approximately 25% increase in passive leak of Ca2+ from the SR of the EDL and soleus fibers. These results indicate that clenbuterol has direct effects on fast- and slow-twitch skeletal muscle, in the absence of the beta2-agonist pathway. The increased Ca2+ leak in the triad region may lead to excitation-contraction coupling damage in the soleus fibers and could also contribute to the anabolic effect of clenbuterol in vivo.

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.

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.

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.

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)

Ca2+ levels in myotubes grown from the skeletal muscle of dystrophic (mdx) and normal mice.

1. Myotubes were grown in culture from normal (C57BL/ScSn) and mdx mice and the cytosolic [Ca2+] was monitored through development (5-21 days in culture) using fura-2 loaded via ionophoresis. Simultaneous measurements of the membrane potential and cytosolic [Ca2+] were made in normal and mdx myotubes before, during and after stimulation by action potentials elicited following anode break excitation. All experiments were undertaken at 22 degrees C. All data are expressed as means +/- S.E.M. 2. A new method was developed which enabled accurate determination of the fluorescence characteristics of fura-2 in murine skeletal muscle fibres. In the under in vitro conditions by 14.60 +/- 0.05, 9.40 +/- 0.15 and 6.90 +/- 0.43% respectively. 3. The resting cytosolic [Ca2+] in the mdx myotubes was consistently higher than in the normal myotubes throughout the developmental period measured. Overall, the resting cytosolic [Ca2+] in mdx myotubes (134 +/- 9 nM, n = 22) was twofold higher than in normal myotubes (66 +/- 6 nM, n = 26). After stimulation (one to three action potentials) the cytosolic [Ca2+] of both mdx and normal myotubes remained elevated. The mdx myotubes (236 +/- 55 nM, n = 5) again had approximately double the cytosolic [Ca2+] of normal myotubes (109 +/- 19 nM, n = 9). 4. The time course and amplitude of the Ca2+ responses measured in the mdx and normal myotubes after action potential stimulation were variable. Two categories of Ca2+ response were observed in mdx and normal myotubes, the first consisted of a small, slow rise in [Ca2+] that remained elevated and the second consisted of a rapid (time to peak 7.4 +/- 1.5 ms) (n = 8) rise in [Ca2+] with amplitudes in the range 61-773 nM and a [Ca2+] decay rate constant of 4.35 +/- 1.57 s-1 (n = 8) (range 0.96-15 s-1). 5. In conclusion, the elevated cytosolic [Ca2+] reported here through development of cultured mdx myotubes suggests that this genetic disorder results in a defect which compromises the ability of the myotubes to strictly regulate cytosolic [Ca2+]. The results are consistent with the presence of functionally abnormal Ca2+ channels recently reported in mdx myotubes.

Contractile properties of skinned muscle fibres from young and adult normal and dystrophic (mdx) mice.

1. Single muscle fibres were enzymatically isolated from the soleus and extensor digitorum longus (EDL) muscles of genetically dystrophic mdx and normal (C57BL/10) mice aged 3-6 or 17-23 weeks. 2. Fibres of both muscles were chemically skinned with the non-ionic detergent Triton X-100 (2% v/v). Ca(2+)- and Sr(2+)-activated contractile responses were recorded and comparisons were made between several contractile parameters of various fibre types of normal and dystrophic mice of similar age. 3. There were no significant differences in the following contractile parameters of skinned fibres of normal and mdx mice of the same age: sensitivity to activating Ca2+ (pCa50) or Sr2+ (pSr50) and differential sensitivity to the activating ions (pCa50-pSr50). However the maximum isometric tension (Po) and the frequency of myofibrillar force oscillations in EDL fast-twitch fibres of young mdx mice were significantly lower than those of soleus fast-twitch fibres of the same animals, or fast-twitch fibres (EDL or soleus) of normal mice. 4. Age-related differences were apparent in some contractile parameters of both normal and mdx mice. In particular the steepness of force-pCa and force-pSr curves increased with age in normal mice, yet decreased with age in fibres of mdx mice. 5. A fluorescent probe, ethidium bromide, which interchelates with DNA, was used with laser-scanning confocal microscopy to determine the distribution of myonuclei in fibres. Fibres isolated from either muscle type of normal animals displayed a characteristic peripheral spiral of myonuclei. Fibres from muscles of mdx mice displayed three major patterns of nuclear distribution; the normal peripheral spiral, long central strands of nuclei, and a mixture of these two patterns. 6. The contractile characteristics of mdx fibres were not markedly influenced by the nuclear distribution pattern in that there were no discernible differences in the major contractile parameters (the Hill coefficients nCa and nSr, which are associated with the steepness of the Ca2+ and Sr2+ activation curves, pCa50, pSr50, pCa50-pSr50) of skinned fibres possessing peripheral or central nuclei. However, except for nSr, these values were all lower in individual fibres which displayed similar proportions of central and peripheral nuclei. The presence of mixed nucleation and absence of fibres with embryonic contractile characteristics in mdx mice suggest that the dystrophin-negative fibres can repair locally occurring muscle damage.