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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 + 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.

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.

During intense exercise, efflux of K+ from working muscles increases extracellular K+ ([K+]o) to levels that can compromise muscle excitability and hence cause fatigue. In this context, the reduction in the exercise-induced elevation of [K+]o observed after training in humans is suggested to contribute to the increased performance after training. Although a similar effect could be obtained by an increase in the tolerance of muscle to elevated [K+]o, this possibility has not been investigated. To examine this, isolated soleus muscles from sedentary (sedentary) rats and from rats that had voluntarily covered 13.1 ± 0.7 km/day in an unloaded running wheel for 8 wk (active) were compared. In muscles from active rats, the loss of force induced by exposure to an elevated [K+]o of 9 mM was 42% lower than in muscles from sedentary rats ( P < 0.001). This apparent increase in K+ tolerance in active rats was associated with an increased excitability as evident from a 33% reduction in the electrical current needed to excite individual muscle fibers ( P < 0.0009). Moreover, muscles from active rats had lower Cl− conductance, higher maximal rate of rise of single-fiber action potentials (AP), and higher Na+/K+ pump content. When stimulated intermittently at 6.5 mM K+, muscles from active rats displayed better endurance than muscles from sedentary rats, whereas no difference was found when the muscles were stimulated continuously at 30 or 120 Hz. We conclude that voluntary running increases muscle excitability, leading to improved tolerance to elevated [K+]o.

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 P2Y1 receptor antagonists (3 μM SCH-202676 or 1 μM 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 86Rb+ 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 μM 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.

A commentary on Viewpoint: Maximal Na+-K+-ATPase activity is upregulated in association with muscle activity by C. Juel, published in Journal of Applied Physiology, 112(12), pp. 2121-3.

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.

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.

Brain edema is suggested to be the principal mechanism underlying the symptoms in acute hyponatremia. Identification of the mechanisms responsible for global and regional cerebral water homeostasis during hyponatremia is, therefore, of utmost importance. To examine the osmotic behavior of different brain regions and muscles, in vivo-determined water content (WC) was related to plasma sodium concentration ([Na+]) and brain/muscle electrolyte content. Acute hyponatremia was induced with desmopressin acetate and infusion of a 2.5% glucose solution in anesthetized pigs. WC in different brain regions and skeletal muscle was estimated in vivo from T1 maps determined by magnetic resonance imaging (MRI). WC, expressed in gram water per 100 g dry weight, increased significantly in slices of the whole brain [342(SD = 14) to 363(SD = 21)] (6%), thalamus [277(SD = 13) to 311(SD = 24)] (12%) and white matter [219(SD = 7) to 225(SD = 5)] (3%). However, the WC increase in the whole brain and white mater WC was less than expected from perfect osmotic behavior, whereas in the thalamus, the water increase was as expected. Brain sodium content was significantly reduced. Muscle WC changed passively with plasma [Na+]. WC determined with deuterium dilution and tissue lyophilzation correlated well with MRI-determined WC. In conclusion, acute hyponatremia induces brain and muscle edema. In the brain as a whole and in the thalamus, regulatory volume decrease (RVD) is unlikely to occur. However, RVD may, in part, explain the observed lower WC in white matter. This may play a potential role in osmotic demyelination.

Elevated plasma HCO3−can improve exercise endurance in humans. This effect has been related to attenuation of the work-induced reduction in muscle pH, which is suggested to improve performance via at least two mechanisms: 1) less inhibition of muscle enzymes and 2) reduced opening of muscle KATPchannels with less ensuing reduction in excitability. Aiming at determining whether the ergogenic effect of HCO3−is related to effects on muscles, we examined the effect of elevating extracellular HCO3−from 25 to 40 mM (pH from 7.4 to 7.6) on fatigue, intracellular pH (pHi), and K+efflux in isolated rat skeletal muscles contracting isometrically. Fatigue induced by 30-Hz stimulation at 30 and 37°C was similar between soleus muscles incubated in high and normal HCO3−concentrations. In extensor digitorum longus muscles stimulated at 60 Hz, elevated HCO3−did not affect fatigue at 30°C. In soleus muscles, 30-Hz stimulation induced a ∼0.2 unit reduction in pHi, as determined by using the pH-sensitive probe 2′,7′-bis(2-carboxyethyl)-5( 6 )-carboxyfluorescein. This reduction in pHiwas not affected by elevated HCO3−. Estimation of K+efflux using86Rb+showed that elevated HCO3−did not affect K+efflux at rest or during contractions. Similarly, other modifications of the intra- and extracellular pH had little effect on K+efflux during contraction. In conclusion, elevated extracellular HCO3−had no significant effect on muscle fatigue, pHi, and K+efflux. These findings indicate that alternative mechanisms must be considered for the ergogenic effect of HCO3−observed in integral exercise studies.