Ole Bækgaard Nielsen

Relations between excitability and contractility in rat soleus muscle: role of the Na+-K+ pump and Na+/K+ gradients

1 The effects of reduced Na+/K+ gradients and Na+‐K+ pump stimulation on compound action potentials (M waves) and contractile force were examined in isolated rat soleus muscles stimulated through the nerve. 2 Exposure of muscles to buffer containing 85 mM Na+ and 9 mM K+ (85 Na+/9 K+ buffer) produced a 54 % decrease in M wave area and a 50 % decrease in tetanic force compared with control levels in standard buffer containing 147 mM Na+ and 4 mM K+. Subsequent stimulation of active Na+‐K+ transport, using the β2‐adrenoceptor agonist salbutamol, induced a marked recovery of M wave area and tetanic force (to 98 and 87 % of the control level, respectively). Similarly, stimulation of active Na+‐K+ transport with insulin induced a significant recovery of M wave area and tetanic force. 3 During equilibration with 85 Na+/9 K+ buffer and after addition of salbutamol there was a close linear correlation between M wave area and tetanic force (r= 0·92, P< 0·001). Similar correlations were found in muscles where tetrodotoxin was used to reduce excitability and in muscles fatigued by 120 s of continuous stimulation at a frequency of 30 Hz. 4 These results show a close correlation between excitability and tetanic force. Furthermore, in muscles depressed by a reduction in the Na+/K+ gradients, β‐adrenergic stimulation of the Na+‐K+ pump induces a recovery of excitability which can fully explain the previously demonstrated recovery of tetanic force following Na+‐K+ pump stimulation. Moreover, the data indicate that loss of excitability is an important factor in fatigue induced by high‐frequency (30 Hz) stimulation.

Additive protective effects of the addition of lactic acid and adrenaline on excitability and force in isolated rat skeletal muscle depressed by elevated extracellular K

During strenuous exercise, extracellular K+ ([K+]o) is increased, which potentially can reduce muscle excitability and force production. In addition, exercise leads to accumulation of lactate and H+ and increased levels of circulating catecholamines. Individually, reduced pH and increased catecholamines have been shown to counteract the depressing effect of elevated K+. This study examines (i) whether the effects of addition of lactic acid and adrenaline on the excitability of isolated muscles are caused by separate mechanisms and are additive and (ii) whether the effect of adding lactic acid or increasing CO2 is related to a reduction of intra‐ or extracellular pH. Rat soleus muscles were incubated at a [K+]o of 15 mm, which reduced tetanic force by 85%. Subsequent addition of 20 mm lactic acid or 10−5m adrenaline led to a small recovery of force, but when added together induced an almost complete force recovery. Compound action potentials showed that the force recovery was associated with recovery of muscle excitability. The improved excitability after addition of adrenaline was associated with increased Na+–K+ pump activity resulting in hyperpolarization and an increase in the chemical Na+ gradient. In contrast, addition of lactic acid had no effect on the membrane potential or the Na+–K+ pump activity, but most likely increased excitability via a reduction in intracellular pH. It is concluded that the protective effects of acidosis and adrenaline on muscle excitability and force took place via different mechanisms and were additive. The results suggest that circulating catecholamines and development of acidosis during exercise may improve the tolerance of muscles to elevated [K+]o.

Loss of force induced by high extracellular [K+] in rat muscle: effect of temperature, lactic acid and β2-agonist

Loss of K+ from active muscles, leading to increased [K+]o, has been proposed to cause muscle fatigue by reducing excitability. Since exercise increases muscle temperature, we investigated the influence of temperature on muscle [K+]o sensitivity. Intact rat soleus or extensor digitorum longus (EDL) muscles were mounted on force transducers and stimulated electrically to evoke short isometric tetani at regular intervals. In each experiment, control force at 4 mM K+ was initially determined at every temperature used. In soleus muscles at 20 °C, 9 mM K+ reduced force to 33 ± 5 % of control force. Increasing the temperature to 30 °C restored force to 89 ± 5 % of control force. Likewise, at 30 °C 11 mM K+ reduced force to 16 ± 4 % and increasing the temperature to 35 °C restored force to 35 ± 5 %. Similar results were obtained using EDL. The force recovery induced by elevating temperature, reflecting reduced [K+]o sensitivity, was associated with improved excitability assessed from compound action potentials. Force recovery induced by a temperature elevation from 20 to 30 °C was associated with hyperpolarization (5 mV), reduced [Na+]i and a 93 % increase in Na+‐K+ pump activity. The force recovery was blocked by ouabain. Since intensive exercise leads to lactic acidosis and increased plasma catecholamines, the effect of these two factors was also investigated. At 11 mM K+, force was completely restored by combining temperature elevation (30 to 35 °C), L‐lactic acid (10 mM) and the β2‐agonist salbutamol (10−5 M). We suggest an exercise scenario where the depressing action of exercise‐induced hyperkalaemia is counteracted by elevated muscle temperature, lactic acidosis and catecholamines.

Excitation- and β2-agonist-induced activation of the Na+−K+ pump in rat soleus muscle

In rat skeletal muscle, Na+‐K+ pump activity increases dramatically in response to excitation (up to 20‐fold) or β2‐agonists (2‐fold), leading to a reduction in intracellular Na+. This study examines the time course of these effects and whether they are due to an increased affinity of the Na+‐K+ pump for intracellular Na+. Isolated rat soleus muscles were incubated at 30 oC in Krebs‐Ringer bicarbonate buffer. The effects of direct electrical stimulation on 86Rb+ uptake rate and intracellular Na+ concentration ([Na+]i) were characterized in the subsequent recovery phase. [Na+]i was varied using monensin or buffers with low Na+. In the [Na+]i range 21–69 mm, both the β2‐agonist salbutamol and electrical stimulation produced a left shift of the curves relating 86Rb+ uptake rate to [Na+]i. In the first 10 s after 1 or 10 s pulse trains of 60 Hz, [Na+]i showed no increase, but 86Rb+ uptake rate increased by 22 and 86 %, respectively. Muscles excited in Na+‐free Li+‐substituted buffer and subsequently allowed to rest in standard buffer also showed a significant increase in 86Rb+ uptake rate and decrease in [Na+]i. Na+ loading induced by monensin or electroporation also stimulated 86Rb+ uptake rate but, contrary to excitation, increased [Na+]i. The increase in the rate of 86Rb+ uptake elicited by electrical stimulation was abolished by ouabain, but not by bumetanide. The results indicate that excitation (like salbutamol) induces a rapid increase in the affinity of the Na+‐K+ pump for intracellular Na+. This leads to a Na+‐K+ pump activation that does not require Na+ influx, but possibly the generation of action potentials. This improves restoration of the Na+‐K+ homeostasis during work and optimizes excitability and contractile performance of the working muscle.

Potassium, Na+,K+‐pumps and fatigue in rat muscle

During contractile activity, skeletal muscles undergo a net loss of cytoplasmic K+ to the interstitial space. During intense exercise, plasma K+ in human arterial blood may reach 8 mm, and interstitial K+ 10–12 mm. This leads to depolarization, loss of excitability and contractile force. However, little is known about the effects of these physiological increases in extracellular K+ ([K+]o) on contractile endurance. Soleus muscles from 4‐week‐old rats were mounted on transducers for isometric contractions in Krebs–Ringer bicarbonate buffer containing 4–10 mm K+, and endurance assessed by recording the rate of force decline during continuous stimulation at 60 Hz. Increasing [K+]o from 4 to 8 or 10 mm and equilibrating the muscles for 40 or 20 min augmented the rate of force decline 2.4‐fold and 7.2‐fold, respectively (P < 0.001). The marked loss of endurance elicited by exposure to 8 or 10 mm K+ was alleviated or significantly reduced by stimulating the Na+,K+‐pumps by intracellular Na+ loading, the β2‐agonist salbutamol, adrenaline, calcitonin gene related peptide, insulin or repeated excitation. In conclusion, excitation‐induced increase in [K+]o is an important cause of high‐frequency fatigue, and the Na+,K+‐pumps are essential for the maintenance of contractile force in the physiological range of [K+]o. Recordings of contractile force during continuous stimulation at 8–10 mm K+ may be used to analyse the effects of agents or conditions influencing the excitability of working isolated muscles.

Regulation of ClC-1 and KATP channels in action potential–firing fast-twitch muscle fibers

Action potential (AP) excitation requires a transient dominance of depolarizing membrane currents over the repolarizing membrane currents that stabilize the resting membrane potential. Such stabilizing currents, in turn, depend on passive membrane conductance (Gm), which in skeletal muscle fibers covers membrane conductances for K+ (GK) and Cl− (GCl). Myotonic disorders and studies with metabolically poisoned muscle have revealed capacities of GK and GCl to inversely interfere with muscle excitability. However, whether regulation of GK and GCl occur in AP-firing muscle under normal physiological conditions is unknown. This study establishes a technique that allows the determination of GCl and GK with a temporal resolution of seconds in AP-firing muscle fibers. With this approach, we have identified and quantified a biphasic regulation of Gm in active fast-twitch extensor digitorum longus fibers of the rat. Thus, at the onset of AP firing, a reduction in GCl of ∼70% caused Gm to decline by ∼55% in a manner that is well described by a single exponential function characterized by a time constant of ∼200 APs (phase 1). When stimulation was continued beyond ∼1,800 APs, synchronized elevations in GK (∼14-fold) and GCl (∼3-fold) caused Gm to rise sigmoidally to ∼400% of its level before AP firing (phase 2). Phase 2 was often associated with a failure to excite APs. When AP firing was ceased during phase 2, Gm recovered to its level before AP firing in ∼1 min. Experiments with glibenclamide (KATP channel inhibitor) and 9-anthracene carboxylic acid (ClC-1 Cl− channel inhibitor) revealed that the decreased Gm during phase 1 reflected ClC-1 channel inhibition, whereas the massively elevated Gm during phase 2 reflected synchronized openings of ClC-1 and KATP channels. In conclusion, GCl and GK are acutely regulated in AP-firing fast-twitch muscle fibers. Such regulation may contribute to the physiological control of excitability in active muscle.

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.

Comparison of regulated passive membrane conductance in action potential-firing fast- and slow-twitch muscle

In several pathological and experimental conditions, the passive membrane conductance of muscle fibers (Gm) and their excitability are inversely related. Despite this capacity of Gm to determine muscle excitability, its regulation in active muscle fibers is largely unexplored. In this issue, our previous study (Pedersen et al. 2009. J. Gen. Physiol. doi:10.1085/jgp.200910291) established a technique with which biphasic regulation of Gm in action potential (AP)-firing fast-twitch fibers of rat extensor digitorum longus muscles was identified and characterized with temporal resolution of seconds. This showed that AP firing initially reduced Gm via ClC-1 channel inhibition but after ∼1,800 APs, Gm rose substantially, causing AP excitation failure. This late increase of Gm reflected activation of ClC-1 and KATP channels. The present study has explored regulation of Gm in AP-firing slow-twitch fibers of soleus muscle and compared it to Gm dynamics in fast-twitch fibers. It further explored aspects of the cellular signaling that conveyed regulation of Gm in AP-firing fibers. Thus, in both fiber types, AP firing first triggered protein kinase C (PKC)-dependent ClC-1 channel inhibition that reduced Gm by ∼50%. Experiments with dantrolene showed that AP-triggered SR Ca2+ release activated this PKC-mediated ClC-1 channel inhibition that was associated with reduced rheobase current and improved function of depolarized muscles, indicating that the reduced Gm enhanced muscle fiber excitability. In fast-twitch fibers, the late rise in Gm was accelerated by glucose-free conditions, whereas it was postponed when intermittent resting periods were introduced during AP firing. Remarkably, elevation of Gm was never encountered in AP-firing slow-twitch fibers, even after 15,000 APs. These observations implicate metabolic depression in the elevation of Gm in AP-firing fast-twitch fibers. It is concluded that regulation of Gm is a general phenomenon in AP-firing muscle, and that differences in Gm regulation may contribute to the different phenotypes of fast- and slow-twitch muscle.

Effects of reduced electrochemical Na+ gradient on contractility in skeletal muscle: role of the Na+-K+ pump

Abstract Continued excitation of skeletal muscle may induce a combination of a low extracellular Na+ concentration ([Na+]o) and a high extracellular K+ concentration ([K+]o) in the T-tubular lumen, which may contribute to fatigue. Here, we examine the role of the Na+-K+ pump in the maintenance of contractility in isolated rat soleus muscles when the Na+, K+ gradients have been altered. When [Na+]o is lowered to 25 mM by substituting Na+ with choline, tetanic force is decreased to 30% of the control level after 60 min. Subsequent stimulation of the Na+-K+ pump with insulin or catecholamines induces a decrease in [Na+]i and hyperpolarization. This is associated with a force recovery to 80–90% of the control level which can be abolished by ouabain. This force recovery depends on hyperpolarization and is correlated to the decrease in [Na+]i (r = 0.93; P<0.001). The inhibitory effect of a low [Na+]o on force development is considerably potentiated by increasing [K+]o. Again, stimulation of the Na+-K+ pump leads to rapid force recovery. The Na+-K+ pump has a large potential for rapid compensation of the excitation-induced rundown of Na+, K+ gradients and contributes, via its electrogenic effect, to the membrane potential. We conclude that these actions of the Na+-K+ pump are essential for the maintenance of excitability and contractile force.

N-Benzyl-p-toluene sulphonamide allows the recording of trains of intracellular action potentials from nerve- stimulated intact fast-twitch skeletal muscle of the rat

In skeletal muscle, the intracellular recording of trains of action potentials is difficult owing to the movement of the muscle upon stimulation. A potential tool for the removal of muscle movement is the cross‐bridge cycle blocker, N‐benzyl‐p‐toluene sulphonamide (BTS), although the effects of BTS on the passive and active membrane properties of intact muscle fibres are not known. Rat extensor digitorum longus (EDL) muscle was used to show that 50 μm BTS reduced tetanic force to ∼10% of control force, without markedly altering muscle excitability. Incubation with BTS did not alter intracellular K+ content or Na+–K+ pump activity, but produced minor decreases in intracellular Na+ content (7%), resting 22Na+ influx (14%) and excitation‐induced 22Na+ influx (29%). Despite these alterations to Na+ fluxes, BTS did not impair muscle excitability, since membrane conductance, resting membrane potential (RMP), rheobase current and the amplitude, overshoot and maximum rate of depolarization of the action potential were all unaltered. However, BTS did induce a small (8%) decrease in the maximum rate of repolarization of the action potential and an increase in the refractory period. The minor effects of BTS on muscle membrane properties did not compromise the ability of the muscle to propagate action potentials, even during tetanic stimulation. In conclusion, BTS can be used successfully to reduce contractility, allowing the intracellular recording of action potentials during both twitch and tetanic contraction of nerve‐stimulated muscle, thus making it an excellent tool for the study of electrophysiology in fast‐twitch skeletal muscle.