Sodium Channel Availability Research Articles

Effect of HIV‐1 Tat on enteric neuropathogenesis (653.6)

The gastrointestinal (GI) tract is a major target and reservoir of the HIV virus. Acute and chronic HIV infection induces a higher level of CD4 T cell depletion in the GI tract compared to peripheral blood and affects motility and digestion. Since the enteric nervous system regulates these processes, it may be a potential target of HIV infection. The majority of HIV effects are not achieved by lytic propagation of the virus but by viral proteins. HIV‐1 Tat, a regulatory protein between 86 to 101 amino acids is released by infected cells and together with other HIV proteins has been shown to modulate neuronal function in the central nervous system. However little is known about its effects on the GI tract. In this study, we examined the effects of Tat on single isolated enteric neurons and in isolated ileum tissue. At the cellular level, in whole cell patch clamp experiments, Tat increased enteric neuronal excitability both in Tat treated (n=20) and in ileum neurons isolated from mice expressing the tat gene (n=9) by a significant increase in number of action potentials evoked, decrease in rheobase (from 20pA to 10pA) and threshold potential (from ‐13.2mV to ‐16.1 mV). This increase was mediated by a leftward shift of the sodium channel activation curve (‐ 10mV decrease in V0.5 of activation) thereby increasing the steady state availability of sodium channels. Tat selectively increased the mRNA and protein expression of Nav1.7 and Nav1.8 isoforms of the sodium channel. In isolated ileum tissues, Tat elicited an inflammatory response which was observed by an up regulation of pro‐inflammatory cytokines and increased GI motility thereby contributing to the HIV mediated GI dysmotilities observed in HIV patientsGrant Funding Source: Supported by the National Institute of Health grants DK046367 and DA024009 and the VCU‐IMSD 541332

Effects of HIV‐1 Tat and morphine on the biophysical properties of sodium channels (653.5)

HIV‐infection continues to be a major clinical manifestation in the combination antiretroviral therapy era (cART), that is enhanced in opioid‐drug abusers. The gastrointestinal (GI) mucosa plays a major role in HIV‐infection as HIV is mostly transmitted across mucosal surfaces. The enteric nervous system (ENS) controls several GI processes including motility and secretion which are specifically affected in HIV‐infected patients. Opioids directly affect the ENS, causing severe constipation. Because the virus does not infect neurons, it is suggested that viral toxins, such as Tat, modulate neuronal function in HIV‐1‐infected patients. Tat has been shown to increase neuronal excitability but not much is known about effects of Tat on enteric neurons in combination with drug abuse. Recent experimental studies in our lab have shown that Tat increases enteric neuronal excitability by shifting the voltage‐dependence of activation of sodium channels to more negative potentials. In contrast, morphine alone decreases the availability of sodium channels in enteric neurons reducing excitability. Voltage gated sodium channels (VGSCs) are responsible for the generation and propagation of action potentials in neurons. In order to determine the effects of Tat (100 nM) in combination with morphine (0.3µM, 3µM) on the voltage dependence of steady‐state activation/ inactivation of sodium channels, we used a double‐pulse protocol in which a variable conditioning pulse was applied from −100mV to +50mV in 10mV steps for 50 ms followed by a test pulse. In Tat‐treated neurons, the sensitivity to morphine was significantly enhanced. The V0.5 for steady‐state inactivation was shifted in Tat‐treated neurons at 0.3 uM morphine with 50% decrease in maximal availability at ‐100 mV. Morphine did not alter the steady‐state activation. Taken together, data suggest that Tat increases the sensitivity to morphine in enteric neurons that may exacerbate the deleterious effects of morphine on GI motility.Grant Funding Source: Supported by NIDA R01 DA018633, K02 DA027374, K99 DA033878, DK046367.

Kv2 channels regulate firing rate in pyramidal neurons from rat sensorimotor cortex

The largest outward potassium current in the soma of neocortical pyramidal neurons is due to channels containing Kv2.1 α subunits. These channels have been implicated in cellular responses to seizures and ischaemia, mechanisms for intrinsic plasticity and cell death, and responsiveness to anaesthetic agents. Despite their abundance, knowledge of the function of these delayed rectifier channels has been limited by the lack of specific pharmacological agents. To test for functional roles of Kv2 channels in pyramidal cells from somatosensory or motor cortex of rats (layers 2/3 or 5), we transfected cortical neurons with DNA for a Kv2.1 pore mutant (Kv2.1W365C/Y380T: Kv2.1 DN) in an organotypic culture model to manipulate channel expression. Slices were obtained from rats at postnatal days (P7-P14) and maintained in organotypic culture. We used biolistic methods to transfect neurons with gold 'bullets' coated with DNA for the Kv2.1 DN and green fluorescent protein (GFP), GFP alone, or wild type (WT) Kv2.1 plus GFP. Cells that fluoresced green, contained a bullet and responded to positive or negative pressure from the recording pipette were considered to be transfected cells. In each slice, we recorded from a transfected cell and a control non-transfected cell from the same layer and area. Whole-cell voltage-clamp recordings obtained after 3-7 days in culture showed that cells transfected with the Kv2.1 DN had a significant reduction in outward current (∼45% decrease in the total current density measured 200 ms after onset of a voltage step from -78 to -2 mV). Transfection with GFP alone did not affect current amplitude and overexpression of the Kv2.1 WT resulted in greatly increased currents. Current-clamp experiments were used to assess the functional consequences of manipulation of Kv2.1 expression. The results suggest roles for Kv2 channels in controlling membrane potential during the interspike interval (ISI), firing rate, spike frequency adaptation (SFA) and the steady-state gain of firing. Specifically, firing rate and gain were reduced in the Kv2.1 DN cells. The most parsimonious explanation for the effects on firing is that in the absence of Kv2 channels, the membrane remains depolarized during the ISIs, preventing recovery of Na(+) channels from inactivation. Depolarization and the number of inactivated Na(+) channels would build with successive spikes, resulting in slower firing and enhanced spike frequency adaptation in the Kv2.1 DN cells.

Network‐specific mechanisms may explain the paradoxical effects of carbamazepine and phenytoin

A common notion of the mechanism by which the antiepileptic drugs (AEDs) carbamazepine and phenytoin act is that they block sodium channels by binding preferentially to the inactivated state, thereby allowing normal neuronal firing while blocking ictal activity. However, these drugs have unpredictable efficacy and, in some cases, may exacerbate seizures. Previous studies have suggested that reducing sodium channel availability in the dentate gyrus (DG) paradoxically increases excitability. We used a biophysically detailed computer model of the DG to test the hypothesis that AEDs increase excitability by disproportionately reducing negative feedback mechanisms. We built a Markov model of sodium channel gating that reproduces responses to voltage clamp experiments in the presence of carbamazepine and phenytoin. We incorporated this validated Markov model into a biophysically realistic computer model of DG neurons and networks. Simulated drug concentrations were similar to those measured in cerebral spinal fluid in medicated patients. Single neuron models were stimulated with current injections, and networks were stimulated with perforant path synaptic input. In the network model, environmental effects were studied by introducing mossy fiber sprouting. As expected, drugs reduced sodium channel availability, which in turn reduced action potential amplitude. This had only a small effect on action potential (AP) firing rate during brief (100 msec) current injections. Paradoxically, long current injections (2,500 msec) increased AP firing rates. This was caused by reduced calcium entry and consequently reduced activation of calcium activated potassium channels. It is important to note that the main determinant of drug effect was resting membrane potential (RMP) and not action potential firing rate. Binding of phenytoin and carbamazepine is slow and, thus drug effects are largely determined by the long term state of the RMP. This paradoxical AP firing increase was dependent on the unusually large calcium-activated potassium conductances expressed by DG granule cells. This predicts that drug efficacy in a given network will depend on the precise makeup of conductances in the network. RMP is expected to vary with the level of activity in the network. We simulated the effects of drugs on single shot stimulus responses in networks with mossy fiber sprouting and varied the RMP in all neurons as a model for network activity. For an RMP of -50 mV, representing an active network, drugs had no effect, or in some cases, increased excitability. Drugs had an increasingly larger inhibitory effect on network responses as RMP decreased. An important prediction is that drugs will be unable to block ictal activity invading an active network. Our key findings are that drug effects depend on both intrinsic properties of the network and its behavioral state. This may explain the paradoxical and unpredictable effects of some AEDs on seizure control in some patients.

Mutagenesis Studies Investigating Slow Inactivation of Sodium Channel Nav1.6

Slow inactivation (SI) determines the availability of voltage-gated sodium channels and effects action potential firing patterns. Neuronally-expressed Nav1.6 was suggested to play an important role in maintaining high firing frequencies, possibly due to a reduced SI compared to other sodium channel subtypes. With this study we characterized SI of WT Nav1.6R and introduced mutations that severely altered the channel's propensity to slow inactivate. Conserved residues in the S6 segment of DII and DIII were identified as SI-sensitive hot spots: V966C in DII and N1455A in DIII. WT and mutant channels were transiently expressed in ND7/23 cells and patch-clamped. The N1455A mutation greatly enhanced SI whereas the V966C mutation shifted its voltage-dependence in the depolarizing direction by ∼25 mV. Fast inactivation was shifted slightly to more hyperpolarized potentials (∼3 mV for V966C and ∼8.5 mV for N1455A). We applied trains of depolarizing stimuli to imitate high-frequency firing. At a stimulation frequency of 50 and 100 Hz, only the N1455A mutation displayed enhanced run down, whereas V966C behaved similar to WT, probably due to the slight enhancement of fast inactivation of V966C. Our findings show that Nav1.6 SI can be severely altered by mutations in DII and DIII S6 segments, thereby confirming the importance of these regions to SI. N1455A induces loss of Nav1.6 during prolonged high frequency stimulation, which will allow us to test for the specific role of Nav1.6 vs. Nav1.7 in action potential (AP) generation: when transfected into DRGs, N1455A may attenuate prolonged high-frequency firing, whereas the threshold (thought to be set by Nav1.7) may remain unaffected. With this mutations we produced a useful molecular tool and approach for elucidating the role and influence of Nav1.6 SI on firing patterns in sensory neurons.

Dynamic reciprocity of sodium and potassium channel expression in a macromolecular complex controls cardiac excitability and arrhythmia

The cardiac electrical impulse depends on an orchestrated interplay of transmembrane ionic currents in myocardial cells. Two critical ionic current mechanisms are the inwardly rectifying potassium current (I(K1)), which is important for maintenance of the cell resting membrane potential, and the sodium current (I(Na)), which provides a rapid depolarizing current during the upstroke of the action potential. By controlling the resting membrane potential, I(K1) modifies sodium channel availability and therefore, cell excitability, action potential duration, and velocity of impulse propagation. Additionally, I(K1)-I(Na) interactions are key determinants of electrical rotor frequency responsible for abnormal, often lethal, cardiac reentrant activity. Here, we have used a multidisciplinary approach based on molecular and biochemical techniques, acute gene transfer or silencing, and electrophysiology to show that I(K1)-I(Na) interactions involve a reciprocal modulation of expression of their respective channel proteins (Kir2.1 and Na(V)1.5) within a macromolecular complex. Thus, an increase in functional expression of one channel reciprocally modulates the other to enhance cardiac excitability. The modulation is model-independent; it is demonstrable in myocytes isolated from mouse and rat hearts and with transgenic and adenoviral-mediated overexpression/silencing. We also show that the post synaptic density, discs large, and zonula occludens-1 (PDZ) domain protein SAP97 is a component of this macromolecular complex. We show that the interplay between Na(v)1.5 and Kir2.1 has electrophysiological consequences on the myocardium and that SAP97 may affect the integrity of this complex or the nature of Na(v)1.5-Kir2.1 interactions. The reciprocal modulation between Na(v)1.5 and Kir2.1 and the respective ionic currents should be important in the ability of the heart to undergo self-sustaining cardiac rhythm disturbances.

Pharmacological IKs Activation Slows Cardiac Conduction and Exacerbates the Effect of INa Blockade

Background: The Kv7.1 channel underlies the IKs current and plays a key role in cardiac repolarization. Loss of IKs function can lead to the type 1 long QT syndrome (LQT1). The Kv7.1 activator R-L3 has been suggested as a potential antiarrhythmic therapy in LQT1. Having previously demonstrated that other K+ currents can modulate cardiac conduction, we tested here the effects of pharmacological IKs activation on cardiac conduction and its dependence on the sodium current (INa).Methods and Results: Conduction velocity (CV) was quantified by optical mapping during LV or RV pacing. Under control conditions transverse CV (CVT) was significantly greater (15±1 %, p<0.05) in RV than LV with no difference in longitudinal CV (CVL) between ventricles. During partial blockade of INa (flecainide, 1 µM), RV CVT decreased by 36±5 % while LV CVT decreased by 24±5 %. These data demonstrate greater RV conduction dependence on sodium channel availability. Partial IKs blockade by R-L3 (10 µM) significantly decreased both CVT (by 15±4 % in RV & 8±2 % in LV) and CVL (by 11±2% in RV & 6±1 % in LV) relative to control. Further, R-L3 exacerbated the effects of INa blockade: 1 µM flecainide now decreased CVT by 45±3% and 28±4% in RV and LV respectively.Conclusion: Pharmacological IKs activation by R-L3 slows cardiac conduction and shifts the conduction velocity - INa relationship downward. These findings may have significant implications for the use of Kv7.1 activator as antiarrhythmic drugs.

A Sodium Channel Blocker, Pilsicainide, Produces Atrial Post-Repolarization Refractoriness through the Reduction of Sodium Channel Availability

Atrial fibrillation (AF) is the most common tachyarrhythmia. Shortening of atrial action potential duration (APD) and effective refractory period (ERP) is one of the crucial factors in the occurrence and maintenance of AF. ERP is usually shorter than APD, but ERP can be prolonged beyond action potential repolarization in some situations. It is termed as post-repolarization refractoriness (PRR) that is thought to be one of main anti-arrhythmic mechanisms of class I sodium channel blockers (SCBs). Most of anti-arrhythmic agents, including SCBs, have multi-channel blocking effects. It is unknown whether atrial PRR with SCBs is associated with the reduction of sodium channel availability. We therefore explored the relationship between the reduction of sodium channel availability with a pure SCB (pilsicainide or tetrodotoxin) and atrial PRR using the left atrial appendage from male guinea pigs (each group, n = 3~10). Employing a standard microelectrode technique, we evaluated APD measured at 90% repolarization (APD(90)) and the sodium channel availability, judged from the maximal rate of rise of action potential (Vmax). At a 500-msec basic cycle length (BCL), pilsicainide prolonged atrial ERP assessed by a single extra-stimulus in response to the decrement of the Vmax in a dose-dependent manner without affecting APD(90). Furthermore, pilsicainide increased the ERP and decreased the Vmax in a rate-dependent manner without APD(90) prolongation at a shorter BCL (200 msec). Importantly, tetrodotoxin reproduced the effects of pilsicainide on atrial ERP, APD(90), and Vmax. These results indicate that SCBs produce atrial PRR through the reduction of sodium channel availability.

Incomplete Inactivation and Rapid Recovery of Voltage-Dependent Sodium Channels During High-Frequency Firing in Cerebellar Purkinje Neurons

Purkinje neurons can spike very rapidly for sustained periods. We examined the cycle of sodium channel gating during high-frequency firing of Purkinje neurons, focusing on the kinetics of sodium channel inactivation and recovery during and after spikes. To analyze sodium channel availability during spiking, we recorded the firing patterns of acutely dissociated Purkinje neurons in current clamp and used these records as command voltages in voltage-clamp experiments in the same cell, adding step depolarizations at various points to assay availability. Sodium channel availability decreased abruptly during the spike, as expected, but never reached zero. During spontaneous firing (∼ 40 Hz at 37°C), availability decreased from nearly 90% before the spike to about 10-20% after the spike. With fast steady firing stimulated by current injection (∼ 300 Hz at 37°C), the availability decreased from about 60% between spikes to roughly 15-20% after the spike. Thus even at the fastest firing rates, sodium channel inactivation is incomplete after a spike, leaving a substantial fraction of sodium channels immediately available for activation. Also, inactivation recovered quickly during the early interspike interval (time constant ∼ 1 ms at 37°C), but developed slowly during the depolarization of the late interspike interval, ensuring high availability until spike threshold. These features of sodium channel gating, especially the availability remaining after the spike, reduce the refractory period and facilitate rapid repetitive firing.

Muscle membrane dysfunction in critical illness myopathy assessed by velocity recovery cycles

Objective To test the hypothesis that muscle fibers are depolarized in patients with critical illness myopathy by measuring velocity recovery cycles (VRCs) of muscle action potentials. Methods VRCs were recorded from brachioradialis muscle by direct muscle stimulation in 10 patients in intensive care with evidence of critical illness myopathy (CIM). Two sets of recordings were made, mean 3.9 d apart, and compared with those from 10 age-matched controls. Results Muscle supernormality was reduced in the patients by 50% compared with controls ( P < 0.002) and relative refractory period was increased by 59% ( P < 0.01). Supernormality was correlated with plasma potassium levels ( R = −0.753, P < 0.001), and the slope of this relationship was much steeper than previously reported for non-critically ill patients with renal failure ( P < 0.01). Conclusions The abnormal excitability properties indicate that the muscle fibers in CIM were depolarized, and/or that sodium channel inactivation was increased. The heightened sensitivity to potassium is consistent with the hypothesis that an endotoxin reduces sodium channel availability in depolarized muscle fibers. Significance VRCs provide a practicable means to monitor muscle membrane changes in intensive care and to investigate the pathogenesis of CIM.

Pharmacological Activation of IKr Impairs Conduction in Guinea Pig Hearts

The hERG (Kv11.1) potassium channel underlies cardiac I(Kr) and is important for cardiac repolarization. Recently, hERG agonists have emerged as potential antiarrhythmic drugs. As modulation of outward potassium currents has been suggested to modulate cardiac conduction, we tested the hypothesis that pharmacological activation of I(Kr) results in impaired cardiac conduction. Cardiac conduction was assessed in Langendorff-perfused guinea pig hearts. Application of the hERG agonist NS3623 (10 microM) prolonged the QRS rate dependently. A significant prolongation (16 +/- 6%) was observed at short basic cycle length (BCL 90 ms) but not at longer cycle lengths (BCL 250 ms). The effect could be reversed by the I(Kr) blocker E4031 (1 microM). While partial I(Na) inhibition with flecainide (1 microM) alone prolonged the QRS (34 +/- 3%, BCL 250 ms), the QRS was further prolonged by 19 +/- 2% when NS3623 was added in the presence of flecainide. These data suggest that the effect of NS3623 was dependent on sodium channel availability. Surprisingly, in the presence of the voltage sensitive dye di-4-ANEPPS a similar potentiation of the effect of NS3623 was observed. With di-4-ANEPPS, NS3623 prolonged the QRS significantly (26 +/- 4%, BCL 250 ms) compared to control with a corresponding decrease in conduction velocity. Pharmacological activation of I(Kr) by the hERG agonist NS3623 impairs cardiac conduction. The effect is dependent on sodium channel availability. These findings suggest a role for I(Kr) in modulating cardiac conduction and may have implications for the use of hERG agonists as antiarrhythmic drugs.

Mechanisms of Sustained High Firing Rates in Two Classes of Vestibular Nucleus Neurons: Differential Contributions of Resurgent Na, Kv3, and BK Currents

To fire at high rates, neurons express ionic currents that work together to minimize refractory periods by ensuring that sodium channels are available for activation shortly after each action potential. Vestibular nucleus neurons operate around high baseline firing rates and encode information with bidirectional modulation of firing rates up to several hundred Hz. To determine the mechanisms that enable these neurons to sustain firing at high rates, ionic currents were measured during firing by using the action potential clamp technique in vestibular nucleus neurons acutely dissociated from transgenic mice. Although neurons from the YFP-16 line fire at rates higher than those from the GIN line, both classes of neurons express Kv3 and BK currents as well as both transient and resurgent Na currents. In the fastest firing neurons, Kv3 currents dominated repolarization at all firing rates and minimized Na channel inactivation by rapidly transitioning Na channels from the open to the closed state. In slower firing neurons, BK currents dominated repolarization at the highest firing rates and sodium channel availability was protected by a resurgent blocking mechanism. Quantitative differences in Kv3 current density across neurons and qualitative differences in immunohistochemically detected expression of Kv3 subunits could account for the difference in firing range within and across cell classes. These results demonstrate how divergent firing properties of two neuronal populations arise through the interplay of at least three ionic currents.

Augmented Sodium Currents Contribute to the Enhanced Excitability of Small Diameter Capsaicin-Sensitive Sensory Neurons Isolated From Nf1+/− Mice

Neurofibromin, the product of the Nf1 gene, is a guanosine triphosphatase activating protein (GAP) for p21ras (Ras) that accelerates conversion of active Ras-GTP to inactive Ras-GDP. Sensory neurons with reduced levels of neurofibromin likely have augmented Ras-GTP activity. We reported previously that sensory neurons isolated from a mouse model with a heterozygous mutation of the Nf1 gene (Nf1+/⁻) exhibited greater excitability compared with wild-type mice. To determine the mechanism giving rise to the augmented excitability, differences in specific membrane currents were examined. Consistent with the enhanced excitability of Nf1+/⁻ neurons, peak current densities of both tetrodotoxin-resistant sodium current (TTX-R I(Na)) and TTX-sensitive (TTX-S) I(Na) were significantly larger in Nf1+/⁻ than in wild-type neurons. Although the voltages for half-maximal activation (V(0.5)) were not different, there was a significant depolarizing shift in the V(0.5) for steady-state inactivation of both TTX-R and TTX-S I(Na) in Nf1+/⁻ neurons. In addition, levels of persistent I(Na) were significantly larger in Nf1+/⁻ neurons. Neither delayed rectifier nor A-type potassium currents were altered in Nf1+/⁻ neurons. These results demonstrate that enhanced production of action potentials in Nf1+/⁻ neurons results, in part, from larger current densities and a depolarized voltage dependence of steady-state inactivation for I(Na) that potentially leads to a greater availability of sodium channels at voltages near the firing threshold for the action potential.

Mossy fiber sprouting interacts with sodium channel mutations to increase dentate gyrus excitability

Idiopathic epilepsy is caused by the complex interaction of genetic and environmental factors. The purpose of this study was to use computational approaches to explore the interaction between changes in sodium channel availability caused by mutations and mossy fiber sprouting. We used a previously published biophysically realistic computer model of dentate gyrus neurons and networks. A sensitivity analysis probed the effects of typical mutation-like changes in either single- or multiple-gating parameters. Isolated neuron models were stimulated with current injections, and networks were stimulated with perforant path synaptic input. The gene-environment interaction was studied by including mossy fiber sprouting into these networks. Single neuron responses to current injections were complex, with increased sodium channel availability paradoxically reducing firing rates. In the absence of mossy fiber sprouting, control networks showed strong accommodation supporting the role of the dentate gyrus as a gate. Availability changes alone were not able to drive the networks into ictal states, even though they reduced the effectiveness of the dentate gyrus gate. Interestingly, the presence of electrophysiologic changes substantially increased the ability of mossy fiber sprouting to induce ictal activity. (1) Increased sodium channel availability does not necessarily lead to increased firing rates, (2) network excitability is most sensitive to changes in steady state activation of sodium channels, (3) mutation-induced changes in availability reduce the effectiveness of the dentate gyrus gate, and (4) mutations interact strongly with structural network changes to allow ictal-like activity in the dentate gyrus.

Selective γ-ketoaldehyde scavengers protect Na V1.5 from oxidant-induced inactivation

The cardiac sodium channel (SCN5A, Na V1.5) is a key determinant of electrical impulse conduction in cardiac tissue. Acute myocardial infarction leads to diminished sodium channel availability, both because of decreased channel expression and because of greater inactivation of channels already present. Myocardial infarction leads to significant increases in reactive oxygen species and their downstream effectors including lipoxidation products. The effects of reactive oxygen species on Na V1.5 function in whole hearts can be modeled in cultured myocytes, where oxidants shift the availability curve of I Na to hyperpolarized potentials, decreasing cardiac sodium current at the normal activation threshold. We recently examined potential mediators of the oxidant-induced inactivation and found that one specific lipoxidation product, the isoketals, recapitulated the effects of oxidant on sodium currents. Isoketals are highly reactive γ-ketoaldehydes formed by the peroxidation of arachidonic acid that covalently modify the lysine residues of proteins. We now confirm that exposure to oxidants induces lipoxidative modification of Na V1.5 and that the selective isoketal scavengers block voltage-dependent changes in sodium current by the oxidant tert-butylhydroperoxide, both in cells heterologously expressing Na V1.5 and in a mouse cardiac myocyte cell line (HL-1). Thus, inhibition of this lipoxidative modification pathway is sufficient to protect the sodium channel from oxidant induced inactivation and suggests the potential use of isoketal scavengers as novel therapeutics to prevent arrhythmogenesis during myocardial infarction.

The role of endotoxin in critical illness myopathy and polyneuropathy

Sirs: We read the article by Dr. Mohammadi and colleagues dealing with parameters that correlate with the severity of early stage critical illness polyneuropathy (CIP) with great interest [5]. The authors performed nerve conduction studies and electromyography in 20 consecutive patients with severe sepsis or septic shock from day 1 to day 14 after onset of sepsis. The essential finding was a reduction in the amplitude in the compound action potential of the median and/or peroneal nerve which was reduced in 75% of patients already at day 1, and in 88% of patients at day 7. Remarkably, the reduction in the amplitude of the compound motor action potential correlated with the serum concentrations of endotoxin and interleukin-2-receptors. We have recently shown a direct interaction of endotoxin with heterologously expressed voltage-gated skeletal muscle sodium channels in vitro [4]. Endotoxin reduced sodium channel availability at depolarized membrane potentials during acute application of high concentrations (C50 ng/ml) and after prolonged exposure (1 h) to a clinically relevant concentration (300 pg/ml). After 24 h of in vitro incubation with 300 pg/ml endotoxin, the reduction of sodium channel availability was irreversible and independent from the membrane potential. In septic patients, CIP and critical illness myopathy (CIM) often coexist [2, 3], and the incidence in patients with sepsis or septic shock varies between 70% and 80% [1]. Taken together, the results of the clinical study performed by Dr. Mohammadi and colleagues, and our in vitro study on the interaction of endotoxin with voltage-gated sodium channels suggest that endotoxin might be a crucial factor in the development of neuromuscular sequelae of sepsis already at a very early stage after onset of sepsis. This highlights the need for development of anti-endotoxin strategies to prevent CIM and CIP.

The cardiac sodium channel displays differential distribution in the conduction system and transmural heterogeneity in the murine ventricular myocardium

Cardiac sodium channels are responsible for conduction in the normal and diseased heart. We aimed to investigate regional and transmural distribution of sodium channel expression and function in the myocardium. Sodium channel Scn5a mRNA and Nav1.5 protein distribution was investigated in adult and embryonic mouse heart through immunohistochemistry and in situ hybridization. Functional sodium channel availability in subepicardial and subendocardial myocytes was assessed using patch-clamp technique. Adult and embryonic (ED14.5) mouse heart sections showed low expression of Nav1.5 in the HCN4-positive sinoatrial and atrioventricular nodes. In contrast, high expression levels of Nav1.5 were observed in the HCN4-positive and Cx43-negative AV or His bundle, bundle branches and Purkinje fibers. In both ventricles, a transmural gradient was observed, with a low Nav1.5 labeling intensity in the subepicardium as compared to the subendocardium. Similar Scn5a mRNA expression patterns were observed on in situ hybridization of embryonic and adult tissue. Maximal action potential upstroke velocity was significantly lower in subepicardial myocytes (mean ± SEM 309 ± 32 V/s; n = 14) compared to subendocardial myocytes (394 ± 32 V/s; n = 11; P < 0.05), indicating decreased sodium channel availability in subepicardium compared to subendocardium. Scn5a and Nav1.5 show heterogeneous distribution patterns within the cardiac conduction system and across the ventricular wall. This differential distribution of the cardiac sodium channel may have profound consequences for conduction disease phenotypes and arrhythmogenesis in the setting of sodium channel disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-009-0012-8) contains supplementary material, which is available to authorized users.

Endotoxin reduces availability of voltage-gated human skeletal muscle sodium channels at depolarized membrane potentials*

Critical illness myopathy is a common cause for difficulties in weaning from the respirator and prolonged rehabilitation of patients recovering from sepsis. Several studies have shown that the primary cause of acute generalized muscle weakness is loss of muscle membrane excitability. This study was designed to investigate a potential direct interaction of lipopolysaccharides from Escherichia coli with voltage-gated human skeletal muscle sodium channels (NaV1.4) in vitro. In vitro laboratory investigation. University laboratory. NaV1.4 sodium channel alpha-subunits stably expressed in human embryonic kidney (HEK293) cells. We investigated the effect of lipopolysaccharide on voltage-dependent sodium channel gating by using two distinct modes of application: 1) acute perfusion (pharmacologic lipopolysaccharide concentrations between 5 ng/mL and 50 microg/mL) in order to establish a concentration-effect relationship; and 2) incubation with a clinically relevant concentration of lipopolysaccharide (300 pg/mL). Lipopolysaccharide did not alter the kinetics of sodium current activation or inactivation when depolarizations were started from hyperpolarized holding potentials. However, when either fast or slow inactivation was induced by membrane depolarization before the test pulse, lipopolysaccharide reversibly reduced channel availability during the test pulse at concentrations of > or = 50 ng/mL revealed by a maximum hyperpolarizing shift of -25 mV in the voltage dependence of fast and slow inactivation, respectively. Incubation with a lipopolysaccharide concentration of 300 pg/mL for 1 hr reproduced the effects on slow but not on fast inactivation. After 20 hrs of low-dose lipopolysaccharide, the peak sodium current was significantly reduced. Our results show that lipopolysaccharide interacts with voltage-gated sodium channels, reducing channel availability at depolarized membrane potentials during acute application, independent of the membrane potential after chronic exposure. These effects may contribute to reduced muscle membrane excitability in sepsis.

Conduction velocity is regulated by sodium channel inactivation in unmyelinated axons innervating the rat cranial meninges

Axonal conduction velocity varies according to the level of preceding impulse activity. In unmyelinated axons this typically results in a slowing of conduction velocity and a parallel increase in threshold. It is currently held that Na(+)-K(+)-ATPase-dependent axonal hyperpolarization is responsible for this slowing but this has long been equivocal. We therefore examined conduction velocity changes during repetitive activation of single unmyelinated axons innervating the rat cranial meninges. In direct contradiction to the currently accepted postulate, Na(+)-K(+)-ATPase blockade actually enhanced activity-induced conduction velocity slowing, while the degree of velocity slowing was curtailed in the presence of lidocaine (10-300 microm) and carbamazepine (30-500 microm) but not tetrodotoxin (TTX, 10-80 nm). This suggests that a change in the number of available sodium channels is the most prominent factor responsible for activity-induced changes in conduction velocity in unmyelinated axons. At moderate stimulus frequencies, axonal conduction velocity is determined by an interaction between residual sodium channel inactivation following each impulse and the retrieval of channels from inactivation by a concomitant Na(+)-K(+)-ATPase-mediated hyperpolarization. Since the process is primarily dependent upon sodium channel availability, tracking conduction velocity provides a means of accessing relative changes in the excitability of nociceptive neurons.

Mechanisms underlying increased right ventricular conduction sensitivity to flecainide challenge

The cardiac sodium current (I(Na)) is a major determinant of conduction. Mechanisms underlying regionally heterogeneous conduction slowing secondary to reduced I(Na) in diseases such as the Brugada syndrome and heart failure remain incompletely understood. Right precordial electrophysiological manifestations during flecainide challenge suggest a decreased right ventricular depolarization reserve. We hypothesized that heterogeneous cardiac sodium channel (Na(v)1.5) distribution between ventricles causes interventricular depolarization heterogeneities. Western blotting analysis revealed Na(v)1.5, and Kir2.1 protein expressions were 18.2 and 12.0% lower, respectively, in the guinea pig right ventricle (RV) compared with the left ventricle (LV). Conduction velocity (theta) heterogeneities were quantified by optical mapping during LV or RV pacing. Although RV transverse theta((thetaT)) was significantly greater than LV (thetaT) by 33.09 +/- 1.38% under control conditions, there were no differences in longitudinal theta. During partial sodium channel blockade (flecainide, 0.5 microM), RV theta decreased by 35.3 +/- 1.3%, whereas LV theta decreased by 29.2 +/- 1.0%. These data demonstrate that the RV has an increased conduction dependence on sodium channel availability. Partial blockade of the inward rectifier potassium current (I(K1)) by BaCl(2) (10 microm) significantly increased theta in both ventricles under control conditions. However, BaCl(2) only increased conduction dependence on sodium channel availability in the LV. This suggests that the LV may have an increased depolarization reserve compared with the RV, but the larger I(K1) depresses control LV theta. Interventricular I(K1) heterogeneities may underlie conduction heterogeneities observed under control conditions. However, under conditions where I(Na) is functionally reduced in disease or during pharmacological sodium channel blockade, the heterogeneity in Na(v)1.5 expression may become a significant determinant of conduction heterogeneities.