Thomas Holm Pedersen

Increased excitability of acidified skeletal muscle : Role of chloride conductance

Generation of the action potentials (AP) necessary to activate skeletal muscle fibers requires that inward membrane currents exceed outward currents and thereby depolarize the fibers to the voltage threshold for AP generation. Excitability therefore depends on both excitatory Na+ currents and inhibitory K+ and Cl− currents. During intensive exercise, active muscle loses K+ and extracellular K+ ([K+]o) increases. Since high [K+]o leads to depolarization and ensuing inactivation of voltage-gated Na+ channels and loss of excitability in isolated muscles, exercise-induced loss of K+ is likely to reduce muscle excitability and thereby contribute to muscle fatigue in vivo. Intensive exercise, however, also leads to muscle acidification, which recently was shown to recover excitability in isolated K+-depressed muscles of the rat. Here we show that in rat soleus muscles at 11 mM K+, the almost complete recovery of compound action potentials and force with muscle acidification (CO2 changed from 5 to 24%) was associated with reduced chloride conductance (1731 ± 151 to 938 ± 64 μS/cm2, P < 0.01) but not with changes in potassium conductance (405 ± 20 to 455 ± 30 μS/cm2, P < 0.16). Furthermore, acidification reduced the rheobase current by 26% at 4 mM K+ and increased the number of excitable fibers at elevated [K+]o. At 11 mM K+ and normal pH, a recovery of excitability and force similar to the observations with muscle acidification could be induced by reducing extracellular Cl− or by blocking the major muscle Cl− channel, ClC-1, with 30 μM 9-AC. It is concluded that recovery of excitability in K+-depressed muscles induced by muscle acidification is related to reduction in the inhibitory Cl− currents, possibly through inhibition of ClC-1 channels, and acidosis thereby reduces the Na+ current needed to generate and propagate an AP. Thus short term regulation of Cl− channels is important for maintenance of excitability in working muscle.

Cooperative hegemony: power, ideas and institutions in regional integration

For realists regionalism remains a difficult phenomenon to explicate. A particular puzzle for realists is why major states should want to pursue regional institutionalisation. Nor are pluralist accounts satisfactory given the empirical evidence of state actor prominence in processes of regional institutionalisation. This article sets out to account for the formative phase of regionalist endeavours, proposing an ideational–institutional realism as the basis for understanding regionalism. On this basis a specific theory of co-operative hegemony is developed. Stressing the importance of the grand strategies of major regional powers and their responses to the balance-of-threat in a region, the author argues that major states may advance their interests through non-coercive means by applying a strategy of co-operative hegemony which implies an active role in regional institutionalisation and the use of, for instance, side payments, power-sharing and differentiation. The article outlines a number of preconditions for regional institutionalisation, stressing what is called the capacity for power-sharing; the power aggregation capacity and the commitment capacity of the biggest power in a region. While regionalising state elites are constrained, they possess a much greater freedom of choice than neo-realism claims.

Scn3b knockout mice exhibit abnormal ventricular electrophysiological properties

We report for the first time abnormalities in cardiac ventricular electrophysiology in a genetically modified murine model lacking the Scn3b gene (Scn3b−/−). Scn3b−/− mice were created by homologous recombination in embryonic stem (ES) cells. RT-PCR analysis confirmed that Scn3b mRNA was expressed in the ventricles of wild-type (WT) hearts but was absent in the Scn3b−/− hearts. These hearts also showed increased expression levels of Scn1b mRNA in both ventricles and Scn5a mRNA in the right ventricles compared to findings in WT hearts. Scn1b and Scn5a mRNA was expressed at higher levels in the left than in the right ventricles of both Scn3b−/− and WT hearts. Bipolar electrogram and monophasic action potential recordings from the ventricles of Langendorff-perfused Scn3b−/− hearts demonstrated significantly shorter ventricular effective refractory periods (VERPs), larger ratios of electrogram duration obtained at the shortest and longest S1–S2 intervals, and ventricular tachycardias (VTs) induced by programmed electrical stimulation. Such arrhythmogenesis took the form of either monomorphic or polymorphic VT. Despite shorter action potential durations (APDs) in both the endocardium and epicardium, Scn3b−/− hearts showed ΔAPD90 values that remained similar to those shown in WT hearts. The whole-cell patch-clamp technique applied to ventricular myocytes isolated from Scn3b−/− hearts demonstrated reduced peak Na+ current densities and inactivation curves that were shifted in the negative direction, relative to those shown in WT myocytes. Together, these findings associate the lack of the Scn3b gene with arrhythmic tendencies in intact perfused hearts and electrophysiological features similar to those in Scn5a+/− hearts.

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.

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.

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.

Relationships between resting conductances, excitability, and t-system ionic homeostasis in skeletal muscle

Activation of skeletal muscle fibers requires rapid sarcolemmal action potential (AP) conduction to ensure uniform excitation along the fiber length, as well as successful tubular excitation to initiate excitation–contraction coupling. In our companion paper in this issue, Pedersen et al. (2011. J. Gen. Physiol. doi:10.1085/jgp.201010510) quantify, for subthreshold stimuli, the influence upon both surface conduction velocity and tubular (t)-system excitation of the large changes in resting membrane conductance (GM) that occur during repetitive AP firing. The present work extends the analysis by developing a multi-compartment modification of the charge–difference model of Fraser and Huang to provide a quantitative description of the conduction velocity of actively propagated APs; the influence of voltage-gated ion channels within the t-system; the influence of t-system APs on ionic homeostasis within the t-system; the influence of t-system ion concentration changes on membrane potentials; and the influence of Phase I and Phase II GM changes on these relationships. Passive conduction properties of the novel model agreed with established linear circuit analysis and previous experimental results, while key simulations of AP firing were tested against focused experimental microelectrode measurements of membrane potential. This study thereby first quantified the effects of the t-system luminal resistance and voltage-gated Na+ channel density on surface AP propagation and the resultant electrical response of the t-system. Second, it demonstrated the influence of GM changes during repetitive AP firing upon surface and t-system excitability. Third, it showed that significant K+ accumulation occurs within the t-system during repetitive AP firing and produces a baseline depolarization of the surface membrane potential. Finally, it indicated that GM changes during repetitive AP firing significantly influence both t-system K+ accumulation and its influence on the resting membrane potential. Thus, the present study emerges with a quantitative description of the changes in membrane potential, excitability, and t-system ionic homeostasis that occur during repetitive AP firing in skeletal muscle.