Six weeks of high-load resistance and low-load blood flow restricted training increase Na/K-ATPase sub-units α2 and ß1 equally, but does not alter ClC-1 abundance in untrained human skeletal muscle.

Contractile function of skeletal muscle relies on the ability of muscle fibers to trigger and propagate action potentials (APs). These electrical signals are created by transmembrane ion transport through ion channels and membrane transporter systems. In this regard, the Cl- ion channel 1 (ClC-1) and the Na+/K--ATPase (NKA) are central for maintaining ion homeostasis across the sarcolemma during intense contractile activity. Therefore, this randomized controlled trial aimed to investigate the changes in ClC-1 and specific NKA subunit isoform expression in response to six weeks (18 training sessions) of high-load resistance exercise (HLRE) and low-load blood flow restricted resistance exercise (BFRRE), respectively. HLRE was conducted as 4 sets of 12 repetitions of knee extensions performed at 70% of 1 repetition maximum (RM), while BFRRE was conducted as 4 sets of knee extensions at 30% of 1RM performed to volitional fatigue. Furthermore, the potential associations between protein expression and contractile performance were investigated. We show that muscle ClC-1 abundance was not affected by either exercise modality, whereas NKA subunit isoforms [Formula: see text]2 and [Formula: see text]1 increased equally by appx. 80-90% with BFRRE (p < 0.05) and 70-80% with HLRE (p < 0.05). No differential impact between exercise modalities was observed. At baseline, ClC-1 protein expression correlated inversely with dynamic knee extensor strength (r=-0.365, p = 0.04), whereas no correlation was observed between NKA subunit content and contractile performance at baseline. However, training-induced changes in NKA [Formula: see text]2 subunit (r = 0.603, p < 0.01) and [Formula: see text]1 subunit (r = 0.453, p < 0.05) correlated with exercise-induced changes in maximal voluntary contraction. These results suggest that the initial adaptation to resistance-based exercise does not involve changes in ClC-1 abundance in untrained skeletal muscle, and that increased content of NKA subunits may facilitate increases in maximal force production.

Cold exposure causes cell death by depolarization-mediated Ca2+ overload in a chill-susceptible insect.

Cold tolerance of insects is arguably among the most important traits defining their geographical distribution. Even so, very little is known regarding the causes of cold injury in this species-rich group. In many insects it has been observed that cold injury coincides with a cellular depolarization caused by hypothermia and hyperkalemia that develop during chronic cold exposure. However, prior studies have been unable to determine if cold injury is caused by direct effects of hypothermia, by toxic effects of hyperkalemia, or by the depolarization that is associated with these perturbations. Here we use a fluorescent DNA-staining method to estimate cell viability of muscle and hindgut tissue from Locusta migratoria and show that the cellular injury is independent of the direct effects of hypothermia or toxic effects of hyperkalemia. Instead, we show that chill injury develops due to the associated cellular depolarization. We further hypothesized that the depolarization-induced injury was caused by opening of voltage-sensitive Ca2+ channels, causing a Ca2+ overload that triggers apoptotic/necrotic pathways. In accordance with this hypothesis, we show that hyperkalemic depolarization causes a marked increase in intracellular Ca2+ levels. Furthermore, using pharmacological manipulation of intra- and extracellular Ca2+ concentrations as well as Ca2+ channel conductance, we demonstrate that injury is prevented if transmembrane Ca2+ flux is prevented by removing extracellular Ca2+ or blocking Ca2+ influx. Together these findings demonstrate a causal relationship between cold-induced hyperkalemia, depolarization, and the development of chill injury through Ca2+-mediated necrosis/apoptosis.

Quantitative model analysis of the resting membrane potential in insect skeletal muscle: Implications for low temperature tolerance.

Abiotic stressors, such as cold exposure, can depolarize insect cells substantially causing cold coma and cell death. During cold exposure, insect skeletal muscle depolarization occurs through a 2-stage process. Firstly, short-term cold exposure reduces the activity of electrogenic ion pumps, which depolarize insect muscle markedly. Secondly, during long-term cold exposure, extracellular ion homeostasis is disrupted causing further depolarization. Consequently, many cold hardy insects improve membrane potential stability during cold exposure through adaptations that secure maintenance of ion homeostasis during cold exposure. Less is known about the adaptations permitting cold hardy insects to maintain membrane potential stability during the initial phase of cold exposure, before ion balance is disrupted. To address this problem it is critical to understand the membrane components (channels and transporters) that determine the membrane potential and to examine this question the present study constructed a mathematical "charge difference" model of the insect muscle membrane potential. This model was parameterized with known literature values for ion permeabilities, ion concentrations and membrane capacitance and the model was then further developed by comparing model predictions against empirical measurements following pharmacological inhibitors of the Na+/K+ ATPase, Cl- channels and symporters. Subsequently, we compared simulated and recorded membrane potentials at 0 and 31 °C and at 10-50 mM extracellular [K+] to examine if the model could describe membrane potentials during the perturbations occurring during cold exposure. Our results confirm the importance of both Na+/K+ ATPase activity and ion-selective Na+, K+ and Cl- channels, but the model also highlights that additional electroneutral flux of Na+ and K+ is needed to describe how membrane potentials respond to temperature and [K+] in insect muscle. While considerable further work is still needed, we argue that this "charge difference" model can be used to generate testable hypotheses of how insects can preserve membrane polarization in the face of stressful cold exposure.

Chloride Channels Take Center Stage in Acute Regulation of Excitability in Skeletal Muscle: Implications for Fatigue.

Initiation and propagation of action potentials in muscle fibers is a key element in the transmission of activating motor input from the central nervous system to their contractile apparatus, and maintenance of excitability is therefore paramount for their endurance during work. Here, we review current knowledge about the acute regulation of ClC-1 channels in active muscles and its importance for muscle excitability, function, and fatigue.

Protein kinase C-dependent regulation of ClC-1 channels in active human muscle and its effect on fast and slow gating.

KEY POINTS: Regulation of ion channel function during repeated firing of action potentials is commonly observed in excitable cells. Recently it was shown that muscle activity is associated with rapid, protein kinase C (PKC)-dependent ClC-1 Cl(-) channel inhibition in rodent muscle. While this PKC-dependent ClC-1 inhibition during muscle activity was shown to be important for the maintenance of contractile endurance in rat muscle it is unknown whether a similar regulation exists in human muscle. Also, the molecular mechanisms underlying the observed PKC-dependent ClC-1 inhibition are unclear. Here we present the first demonstration of ClC-1 inhibition in active human muscle fibres, and we determine the changes in ClC-1 gating that underlie the PKC-dependent ClC-1 inhibition in active muscle using human ClC-1 expressed in Xenopus oocytes. This activity-induced ClC-1 inhibition is suggested to represent a mechanism by which human muscle fibres maintain their excitability during sustained activity. ABSTRACT: Repeated firing of action potentials (APs) is known to trigger rapid, protein kinase C (PKC)-dependent inhibition of ClC-1 Cl(-) ion channels in rodent muscle and this inhibition is important for contractile endurance. It is currently unknown whether similar regulation exists in human muscle, and the molecular mechanisms underlying PKC-dependent ClC-1 inhibition are unclear. This study first determined whether PKC-dependent ClC-1 inhibition exists in active human muscle, and second, it clarified how PKC alters the gating of human ClC-1 expressed in Xenopus oocytes. In human abdominal and intercostal muscles, repeated AP firing was associated with 30-60% reduction of ClC-1 function, which could be completely prevented by PKC inhibition (1 µm GF109203X). The role of the PKC-dependent ClC-1 inhibition was evaluated from rheobase currents before and after firing 1000 APs: while rheobase current was well maintained after activity under control conditions it rose dramatically if PKC-dependent ClC-1 inhibition had been prevented with the inhibitor. This demonstrates that the ClC-1 inhibition is important for maintenance of excitability in active human muscle fibres. Oocyte experiments showed that PKC activation lowered the overall open probability of ClC-1 in the voltage range relevant for AP initiation in muscle fibres. More detailed analysis of this reduction showed that PKC mostly affected the slow gate of ClC-1. Indeed, there was no effect of PKC activation in C277S mutated ClC-1 in which the slow gate is effectively locked open. It is concluded that regulation of excitability of active human muscle fibres relies on PKC-dependent ClC-1 inhibition via a gating mechanism.

Skeletal muscle dysfunction in the db/db mouse model of type 2 diabetes.

INTRODUCTION: In this study we examined the mechanisms of motor dysfunction in type 2 diabetes. METHODS: Contractile force was measured in isolated nerve-muscle preparations of db/db mice using various protocols for electrical stimulation. Sarcoplasmic reticulum Ca(2+) adenosine triphosphatase protein (SERCA) was quantified by comparing Ca(2+) -dependent and non-specific phosphorylation. RESULTS: Compared with controls, the muscle-nerve preparations of db/db mice displayed muscle atrophy, reduced axonal excitability, and force deficit when stimulated via the nerve. Muscle relaxation after contraction was slowed, and SERCA content was reduced. In contrast, the sensitivity of the neuromuscular junction to tubocurarine and muscle fiber excitability were not affected. CONCLUSIONS: The force deficit in db/db muscles was caused by atrophy and failure of neuromuscular signal transmission related to motor nerve axonal dysfunction. The slowed relaxation rate generally observed in diabetic muscles can, to a large extent, be explained by decreased SERCA pump content. Muscle Nerve 54: 460-468, 2016.

Reduced L-type Ca2+ current and compromised excitability induce loss of skeletal muscle function during acute cooling in locust.

Low temperature causes most insects to enter a state of neuromuscular paralysis, termed chill coma. The susceptibility of insect species to chill coma is tightly correlated to their distribution limits and for this reason it is important to understand the cellular processes that underlie chill coma. It is known that muscle function is markedly depressed at low temperature and this suggests that chill coma is partly caused by impairment in the muscle per se. To find the cellular mechanism(s) underlying muscle dysfunction at low temperature, we examined the effect of low temperature (5°C) on several events in excitation-contraction coupling in the migratory locust (Locusta migratoria). Intracellular membrane potential recordings during single nerve stimulations showed that 70% of fibers at 20°C produced an action potential (AP), while only 55% of fibers were able to fire an AP at 5°C. Reduced excitability at low temperature was caused by an ∼80% drop in L-type Ca(2+) current and a depolarizing shift in its activation of around 20 mV, which means that a larger endplate potential would be needed to activate the muscle AP at low temperature. In accordance, we showed that intracellular Ca(2+) transients were largely absent at low temperature following nerve stimulation. In contrast, maximum contractile force was unaffected by low temperature in chemically skinned muscle bundles, which demonstrates that the function of the contractile filaments is preserved at low temperature. These findings demonstrate that reduced L-type Ca(2+) current is likely to be the most important factor contributing to loss of muscle function at low temperature in locust.

Extracellular magnesium and calcium reduce myotonia in isolated ClC-1 chloride channel-inhibited human muscle.

INTRODUCTION: Experimental myotonia induced in rat muscle by ClC-1 chloride channel-inhibited has been shown to be related inversely to extracellular concentrations of Mg(2+) and Ca(2+) ([Mg(2+) ]o and [Ca(2+) ]o) within physiological ranges. Because this implicates a role for [Mg(2+)]o and [Ca(2+)]o in the variability of symptoms among myotonia congenita patients, we searched for similar effects of [Mg(2+)]o and [Ca(2+)]o on myotonia in human muscle. METHODS: Bundles of muscle fibers were isolated from abdominal rectus in patients undergoing abdominal surgery. Myotonia was induced by ClC-1 inhibition using 9-anthracene carboxylic acid (9-AC) and was assessed from integrals of force induced by 5-Hz stimulation for 2 seconds. RESULTS: Myotonia disappeared gradually when [Mg(2+)]o or [Ca(2+)]o were elevated throughout their physiological ranges. These effects of [Mg(2+)]o and [Ca(2+)]o were additive and interchangeable. CONCLUSIONS: These findings suggest that variations in symptoms in myotonia congenita patients may arise from physiological variations in serum Mg(2+) and Ca(2+).

Determination of cable parameters in skeletal muscle fibres during repetitive firing of action potentials.

Recent studies in rat muscle fibres show that repetitive firing of action potentials causes changes in fibre resting membrane conductance (Gm) that reflect regulation of ClC-1 Cl(-) and KATP K(+) ion channels. Methodologically, these findings were obtained by inserting two microelectrodes at close proximity in the same fibres enabling measurements of fibre input resistance (Rin) in between action potential trains. Since the fibre length constant (λ) could not be determined, however, the calculation of Gm relied on the assumptions that the specific cytosolic resistivity (Ri) and muscle fibre volume remained constant during the repeated action potential firing. Here we present a three-microelectrode technique that enables determinations of multiple cable parameters in action potential-firing fibres including Rin and λ as well as waveform and conduction velocities of fully propagating action potentials. It is shown that in both rat and mouse extensor digitorum longus (EDL) fibres, action potential firing leads to substantial changes in both muscle fibre volume and Ri. The analysis also showed, however, that regardless of these changes, rat and mouse EDL fibres both exhibited initial decreases in Gm that were eventually followed by a ∼3-fold, fully reversible increase in Gm after the firing of 1450-1800 action potentials. Using this three-electrode method we further show that the latter rise in Gm was closely associated with excitation failures and loss of action potential signal above -20 mV.

Cold-induced depolarization of insect muscle: differing roles of extracellular K+ during acute and chronic chilling.

Insects enter chill coma, a reversible state of paralysis, at temperatures below their critical thermal minimum (CTmin), and the time required for an insect to recover after a cold exposure is termed chill coma recovery time (CCRT). The CTmin and CCRT are both important metrics of insect cold tolerance that are used interchangeably, although chill coma recovery is not necessarily permitted by a direct reversal of the mechanism causing chill coma onset. Nevertheless, onset and recovery of coma have been attributed to loss of neuromuscular function due to depolarization of muscle fibre membrane potential (Vm). Here we test the hypothesis that muscle depolarization at chill coma onset and repolarization during chill coma recovery are caused by changes in extracellular [K(+)] and/or other effects of low temperature. Using Locusta migratoria, we measured in vivo muscle resting potentials of the extensor tibialis during cooling, following prolonged exposure to -2°C and during chill coma recovery, and related changes in Vm to transmembrane [K(+)] balance and temperature. Although Vm was rapidly depolarized by cooling, hemolymph [K(+)] did not rise until locusts had spent considerable time in the cold. Nonetheless, a rise in hemolymph [K(+)] during prolonged cold exposure further depressed muscle resting potential and slowed recovery from chill coma upon rewarming. Muscle resting potentials had a bimodal distribution, and with elevation of extracellular [K(+)] (but not temperature) muscle resting potentials become unimodal. Thus, a disruption of extracellular [K(+)] does depolarize muscle resting potential and slow CCRT following prolonged cold exposure. However, onset of chill coma at the CTmin relates to an as-yet-unknown effect of temperature on neuromuscular function.

Why do insects enter and recover from chill coma? Low temperature and high extracellular potassium compromise muscle function in Locusta migratoria.

When exposed to low temperatures, many insect species enter a reversible comatose state (chill coma), which is driven by a failure of neuromuscular function. Chill coma and chill coma recovery have been associated with a loss and recovery of ion homeostasis (particularly extracellular [K(+)], [K(+)]o) and accordingly onset of chill coma has been hypothesized to result from depolarization of membrane potential caused by loss of ion homeostasis. Here, we examined whether onset of chill coma is associated with a disturbance in ion balance by examining the correlation between disruption of ion homeostasis and onset of chill coma in locusts exposed to cold at varying rates of cooling. Chill coma onset temperature changed maximally 1°C under different cooling rates and marked disturbances of ion homeostasis were not observed at any of the cooling rates. In a second set of experiments, we used isolated tibial muscle to determine how temperature and [K(+)]o, independently and together, affect tetanic force production. Tetanic force decreased by 80% when temperature was reduced from 23°C to 0.5°C, while an increase in [K(+)]o from 10 mmol l(-1) to 30 mmol l(-1) at 23°C caused a 40% reduction in force. Combining these two stressors almost abolished force production. Thus, low temperature alone may be responsible for chill coma entry, rather than a disruption of extracellular K(+) homeostasis. As [K(+)] also has a large effect on tetanic force production, it is hypothesized that recovery of [K(+)]o following chill coma could be important for the time to recovery of normal neuromuscular function.

Role of recovery of acetylcholine release in compromised neuromuscular junction function.

Everyday physical activities, such as walking, are enabled by repeated skeletal muscle contractions and require a well-functioning neuromuscular transmission. In myasthenic disorders, activities of daily living are debilitated by a compromised neuromuscular transmission leading to muscle weakness and fatiguability in patients. To enable physical activity, acetylcholine (ACh) is released repeatedly from the motor nerve, however, the role of the nerve terminals' capacity to sustain ACh release to support repetitive contractions under compromised neuromuscular transmission remains unclear. To explore this, we studied synaptic and contractile function during repeated contractions in healthy rat skeletal muscles under conditions of pharmacological induced compromised neuromuscular transmission. Using recordings of endplate potentials, compound muscle action potential (CMAP) and force production in isolated skeletal muscles and living, anesthetized animals, we found that force and CMAP were markedly reduced by even very light activity performed up to 5 s prior to contraction showing that recovery of ACh release was insufficient to maintain synaptic transmission strength. Our results suggest that the timing of depletion and restoration of ACh release may impact clinical signs of weakness and fatigability in patients with impaired neuromuscular transmission and affect the sensitivity of electromyographic recordings in the clinic.

Relationship between membrane Cl- conductance and contractile endurance in isolated rat muscles.

Resting skeletal muscle fibres have a large membrane Cl(-) conductance (G(Cl)) that dampens their excitability. Recently, however, muscle activity was shown to induce PKC-mediated reduction in G(Cl) in rat muscles of 40-90%. To examine the physiological significance of this PKC-mediated G(Cl) reduction for the function of muscles, this study explored effects of G(Cl) reductions on contractile endurance in isolated rat muscles. Contractile endurance was assessed from the ability of muscle to maintain force during prolonged stimulation under conditions when G(Cl) was manipulated by: (i) inhibition of PKC, (ii) reduction of solution Cl(-) or (iii) inhibition of ClC-1 Cl(-) channels using 9-anthracene-carboxylic acid (9-AC). Experiments showed that contractile endurance was optimally preserved by reductions in G(Cl) similar to what occurs in active muscle. Contrastingly, further G(Cl) reductions compromised the endurance. The experiments thus show a biphasic relationship between G(Cl) and contractile endurance in which partial G(Cl) reduction improves endurance while further G(Cl) reduction compromises endurance. Intracellular recordings of trains of action potentials suggest that this biphasic dependency of contractile endurance on G(Cl) reflects that lowering G(Cl) enhances muscle excitability but low G(Cl) also increases the depolarisation of muscle fibres during excitation and reduces their ability to re-accumulate K(+) lost during excitation. If G(Cl) becomes very low, the latter actions dominate causing reduced endurance. It is concluded that the PKC-mediated ClC-1 channel inhibition in active muscle reduces G(Cl) to a level that optimises contractile endurance during intense exercise.

Chloride channel inhibition improves neuromuscular function under conditions mimicking neuromuscular disorders.

AIM: The skeletal muscle Cl- channels, the ClC-1 channels, stabilize the resting membrane potential and dampen muscle fibre excitability. This study explored whether ClC-1 inhibition can recover nerve-stimulated force in isolated muscle under conditions of compromised neuromuscular transmission akin to disorders of myasthenia gravis and Lambert-Eaton syndrome. METHODS: Nerve-muscle preparations were isolated from rats. Preparations were exposed to pre-or post-synaptic inhibitors (ω-agatoxin, elevated extracellular Mg2+ , α-bungarotoxin or tubocurarine). The potential of ClC-1 inhibition (9-AC or reduced extracellular Cl- ) to recover nerve-stimulated force under these conditions was assessed. RESULTS: ClC-1 inhibition recovered force in both slow-twitch soleus and fast-twitch EDL muscles exposed to 0.2 µmol/L tubocurarine or 3.5 mmol/L Mg2+ . Similarly, ClC-1 inhibition recovered force in soleus muscles exposed to α-bungarotoxin or ω-agatoxin. Moreover, the concentrations of tubocurarine and Mg2+ required for reducing force to 50% rose from 0.14 ± 0.02 µmol/L and 4.2 ± 0.2 mmol/L in control muscles to 0.45 ± 0.03 µmol/L and 4.7 ± 0.3 mmol/L in muscles with 9-AC respectively (P < .05, paired T test). Inhibition of acetylcholinesterase (neostigmine) and inhibition of voltage-gated K+ channels (4-AP) relieve symptoms in myasthenia gravis and Lambert-Eaton syndrome, respectively. Neostigmine and 9-AC additively increased the tubocurarine concentration required to reduce nerve-stimulated force to 50% (0.56 ± 0.05 µmol/L with 9-AC and neostigmine) and, similarly, 4-AP and 9-AC additively increased the Mg2+ concentration required to reduce nerve-stimulated force to 50% (6.5 ± 0.2 mmol/L with 9-AC and 4-AP). CONCLUSION: This study shows that ClC-1 inhibition can improve neuromuscular function in pharmacological models of compromised neuromuscular transmission.

Effect of purinergic receptor activation on Na+-K+ pump activity, excitability, and function in depolarized skeletal muscle.

Activity-induced elevation of extracellular purines and pyrimidines has been associated with autocrine and paracrine signaling in many tissues. Here we investigate the effect of purinergic signaling for the excitability and contractility of depolarized skeletal muscle. Muscle excitability was experimentally depressed by elevating the extracellular K(+) from 4 to 10 mM, which reduced the tetanic force to 24 +/- 2% of the force at 4 mM K(+). Upon addition of 1 mM ATP, however, the force recovered to 65 +/- 8% of the control force (P < 0.001, n = 5). A similar recovery was seen with ADP, but not with UTP or adenosine. The ATP-induced force recovery could be inhibited by P2Y(1) receptor antagonists (3 muM SCH-202676 or 1 muM MRS-2500). A fourfold increase in M-wave area demonstrated that the ATP-induced force recovery was associated with restoration of muscle excitability (P < 0.05, n = 4). Experiments using (86)Rb(+) as a tracer for K(+) showed that ATP also induced a twofold increase in the activity of muscle Na(+)-K(+) pumps. The force recovery and the stimulation of the Na(+)-K(+) pump activity by ATP were inhibited by 50 muM of the phospholipase C inhibitor U-73122. It is concluded that purinergic signaling can increase the Na(+)-K(+) pump activity and improve force and excitability of depolarized skeletal muscles. This novel purinergic regulation may be important for the maintenance of muscle excitability during intense exercise, where the extracellular K(+) can increase substantially.

Lactate per se improves the excitability of depolarized rat skeletal muscle by reducing the Cl- conductance.

Studies on rats have shown that lactic acid can improve excitability and function of depolarized muscles. The effect has been related to the ensuing reduction in intracellular pH causing inhibition of muscle fibre Cl(-) channels. However, since several carboxylic acids with structural similarities to lactate can inhibit muscle Cl(-) channels it is possible that lactate per se can increase muscle excitability by exerting a direct effect on these channels. We therefore examined the effects of lactate on the function of intact muscles and skinned fibres together with effects on pH and Cl(-) conductance (G(cl)). In muscles where extracellular compound action potentials (M-waves) and tetanic force response to excitation were reduced by (mean ± s.e.m.) 82 ± 4% and 83 ± 2%, respectively, by depolarization with 11 mm extracellular K(+), both M-waves and force exhibited an up to 4-fold increase when 20 mm lactate was added. This effect was present already at 5 mm and saturated at 15 mm lactate, and was associated with a 31% reduction in G(Cl). The effects of lactate were completely blocked by Cl(-) channel inhibition or use of Cl(-)-free solutions. Finally, both experiments where effects of lactate on intracellular pH in intact muscles were mimicked by increased CO2 tension and experiments with skinned fibres showed that the effects of lactate could not be related to reduced intracellular pH. It is concluded that addition of lactate can inhibit ClC-1 Cl(-) channels and increase the excitability and contractile function of depolarized rat muscles via mechanisms not related to a reduction in intracellular pH.

Depletion of ATP Limits Membrane Excitability of Skeletal Muscle by Increasing Both ClC1-Open Probability and Membrane Conductance.

Activation of skeletal muscle contractions require that action potentials can be excited and propagated along the muscle fibers. Recent studies have revealed that muscle fiber excitability is regulated during repeated firing of action potentials by cellular signaling systems that control the function of ion channel that determine the resting membrane conductance (G m ). In fast-twitch muscle, prolonged firing of action potentials triggers a marked increase in G m , reducing muscle fiber excitability and causing action potential failure. Both ClC-1 and KATP ion channels contribute to this G m rise, but the exact molecular regulation underlying their activation remains unclear. Studies in expression systems have revealed that ClC-1 is able to bind adenosine nucleotides, and that low adenosine nucleotide levels result in ClC-1 activation. In three series of experiments, this study aimed to explore whether ClC-1 is also regulated by adenosine nucleotides in native skeletal muscle fibers, and whether the adenosine nucleotide sensitivity of ClC-1 could explain the rise in G m muscle fibers during prolonged action potential firing. First, whole cell patch clamping of mouse muscle fibers demonstrated that ClC-1 activation shifted in the hyperpolarized direction when clamping pipette solution contained 0 mM ATP compared with 5 mM ATP. Second, three-electrode G m measurement during muscle fiber stimulation showed that glycolysis inhibition, with 2-deoxy-glucose or iodoacetate, resulted in an accelerated and rapid >400% G m rise during short periods of repeated action potential firing in both fast-twitch and slow-twitch rat, and in human muscle fibers. Moreover, ClC-1 inhibition with 9-anthracenecarboxylic acid resulted in either an absence or blunted G m rise during action potential firing in human muscle fibers. Third, G m measurement during repeated action potential firing in muscle fibers from a murine McArdle disease model suggest that the rise in G m was accelerated in a subset of fibers. Together, these results are compatible with ClC-1 function being regulated by the level of adenosine nucleotides in native tissue, and that the channel operates as a sensor of skeletal muscle metabolic state, limiting muscle excitability when energy status is low.

Cold acclimation modulates voltage gated Ca2+ channel currents and fiber excitability in skeletal muscles of Locusta migratoria.

Cold exposure is known to induce stressful imbalances in chill susceptible insects, including loss of hemolymph water, hyperkalemia and cell depolarization. Cold induced depolarization induces uncontrolled Ca2+ influx and accumulation of injury through necrosis/apoptosis. Conversely cold induced Ca2+ influx has been shown to induce rapid cold hardening and therefore also play a role to reduce cold injury. Cold acclimation is known to reduce cold injury in insects and due to the involvement of depolarization and Ca2+ in the pathophysiology of hypothermia, we hypothesized that cold acclimation modulates voltage gated Ca2+ channels and fiber excitability. Using intracellular electrodes or force transducers, we measured the Ca2+ currents, fiber excitability and muscle contractility in warm (31 °C) and cold (11 °C) acclimated locusts. Experiments were performed under conditions ranging from mild conditions where the membrane potential is well regulated to stressful conditions, where the membrane potential is very depolarized and the tissue is at risk of accumulating injury. These experiments found that cold acclimation modulates Ca2+ currents and fiber excitability in a manner that depends on the cold exposure. Thus, under mild conditions, Ca2+ currents and fiber excitability was increased whilst muscle contractility was unaffected by cold acclimation. Conversely, fiber excitability and muscle contractility was decreased under stressful conditions. Further work is required to fully understand the adaptive effects of these modulations. However, we propose a model which reconciles the dualistic role of the Ca2+ ion in cold exposure and cold acclimation. Thus, increased Ca2+ currents at mild temperatures could help to enhance cold sensing capacity whereas reduced fiber excitability under stressful conditions could help to reduce catastrophic Ca2+ influx during periods of severe cold exposure.

Role of physiological ClC-1 Cl- ion channel regulation for the excitability and function of working skeletal muscle.

Electrical membrane properties of skeletal muscle fibers have been thoroughly studied over the last five to six decades. This has shown that muscle fibers from a wide range of species, including fish, amphibians, reptiles, birds, and mammals, are all characterized by high resting membrane permeability for Cl(-) ions. Thus, in resting human muscle, ClC-1 Cl(-) ion channels account for ∼80% of the membrane conductance, and because active Cl(-) transport is limited in muscle fibers, the equilibrium potential for Cl(-) lies close to the resting membrane potential. These conditions-high membrane conductance and passive distribution-enable ClC-1 to conduct membrane current that inhibits muscle excitability. This depressing effect of ClC-1 current on muscle excitability has mostly been associated with skeletal muscle hyperexcitability in myotonia congenita, which arises from loss-of-function mutations in the CLCN1 gene. However, given that ClC-1 must be drastically inhibited (∼80%) before myotonia develops, more recent studies have explored whether acute and more subtle ClC-1 regulation contributes to controlling the excitability of working muscle. Methods were developed to measure ClC-1 function with subsecond temporal resolution in action potential firing muscle fibers. These and other techniques have revealed that ClC-1 function is controlled by multiple cellular signals during muscle activity. Thus, onset of muscle activity triggers ClC-1 inhibition via protein kinase C, intracellular acidosis, and lactate ions. This inhibition is important for preserving excitability of working muscle in the face of activity-induced elevation of extracellular K(+) and accumulating inactivation of voltage-gated sodium channels. Furthermore, during prolonged activity, a marked ClC-1 activation can develop that compromises muscle excitability. Data from ClC-1 expression systems suggest that this ClC-1 activation may arise from loss of regulation by adenosine nucleotides and/or oxidation. The present review summarizes the current knowledge of the physiological factors that control ClC-1 function in active muscle.

Neuro-muscular function in the wobbler murine model of primary motor neuronopathy.

The wobbler mouse represents a model for neurodegenerative disease affecting motor neurons. This study explored the importance of fiber type specific changes for the contractile dysfunction of soleus and extensor digitorum longus (EDL) muscles from wobbler mice using a specific inhibitor of force generation by the type II myosin protein. Generally, wobbler condition was associated with ~50% reductions in muscle mass and contractile capacity in both muscles. In soleus, an increase in the relative abundance of type I myosin protein was observed. Since, however, only ~40% of the fibers containing type I myosin had functional innervation whereas almost all fibers containing type II myosin were innervated, the shift toward type I myosin was without significance for the in vivo contractile phenotype. Soleus muscles from wobbler mice were further characterized by a 2-fold increase in the width of the twitches, which was associated with a reduction in the excitation frequency necessary to elicit tetanic contractions. Since the SR Ca(2+) ATPase in wobbler soleus was reduced from 22 ± 5 to 10 ± 2 nmol/g muscle tissue (P=0.0006), the increase in twitch width was most likely caused by delayed recovery of cytosolic Ca(2+). Such changes were not observed in EDL. It is concluded that the shift in myosin protein from type II to type I previously reported in both innervated and denervated wobbler muscles primarily takes place in the population of denervated muscle fibers. Since these muscles do not contribute to force generation, the transition is, therefore, of limited relevance for the contractile phenotype of the muscles. Instead, the slow contractile phenotype of wobbler soleus muscles seemed to be a consequence of reduced SR content of Ca(2+) ATPase.