Toll-like Receptor 4 Activation Promotes Cardiac Arrhythmias By Decreasing The Transient Outward Potassium Current (ito) Through An Irf3-dependent And Myd88-independent Pathway.

Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll-like receptors (TLRs) seem to be involved in cardiac complications. In the present study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias, and the signaling pathway involved in these effects. Membrane potential was recorded in Wistar rat ventricle. Ca(2+) transients, as well as the L-type Ca(2+) current (ICaL) and the transient outward K(+) current (Ito), were recorded in isolated myocytes after 24 h exposure to the TLR4 agonist, lipopolysaccharide (LPS, 1 μg/ml). TLR4 stimulation in vitro promoted a cardiac electrical remodeling that leads to action potential prolongation associated with arrhythmic events, such as delayed afterdepolarization and triggered activity. After 24 h LPS incubation, Ito amplitude, as well as Kv4.3 and KChIP2 mRNA levels were reduced. The Ito decrease by LPS was prevented by inhibition of interferon regulatory factor 3 (IRF3), but not by inhibition of interleukin-1 receptor-associated kinase 4 (IRAK4) or nuclear factor kappa B (NF-κB). Extrasystolic activity was present in 25% of the cells, but apart from that, Ca(2+) transients and ICaL were not affected by LPS; however, Na(+)/Ca(2+) exchanger (NCX) activity was apparently increased. We conclude that TLR4 activation decreased Ito, which increased AP duration via a MyD88-independent, IRF3-dependent pathway. The longer action potential, associated with enhanced Ca(2+) efflux via NCX, could explain the presence of arrhythmias in the LPS group.

Effect of trimetazidine treatment on the transient outward potassium current of the left ventricular myocytes of rats with streptozotocin-induced type 1 diabetes mellitus

Cardiovascular complications are a leading cause of mortality in patients with diabetes mellitus (DM). The present study was designed to investigate the effects of trimetazidine (TMZ), an anti-angina drug, on transient outward potassium current (Ito) remodeling in ventricular myocytes and the plasma contents of free fatty acid (FFA) and glucose in DM. Sprague-Dawley rats, 8 weeks old and weighing 200-250 g, were randomly divided into three groups of 20 animals each. The control group was injected with vehicle (1 mM citrate buffer), the DM group was injected with 65 mg/kg streptozotocin (STZ) for induction of type 1 DM, and the DM + TMZ group was injected with the same dose of STZ followed by a 4-week treatment with TMZ (60 mg·kg-1·day-1). All animals were then euthanized and their hearts excised and subjected to electrophysiological measurements or gene expression analyses. TMZ exposure significantly reversed the increased plasma FFA level in diabetic rats, but failed to change the plasma glucose level. The amplitude of Ito was significantly decreased in left ventricular myocytes from diabetic rats relative to control animals (6.25 ± 1.45 vs 20.72 ± 2.93 pA/pF at +40 mV). The DM-associated Ito reduction was attenuated by TMZ. Moreover, TMZ treatment reversed the increased expression of the channel-forming alpha subunit Kv1.4 and the decreased expression of Kv4.2 and Kv4.3 in diabetic rat hearts. These data demonstrate that TMZ can normalize, or partially normalize, the increased plasma FFA content, the reduced Ito of ventricular myocytes, and the altered expression Kv1.4, Kv4.2, and Kv4.3 in type 1 DM.

Transient outward potassium current and Ca2+ homeostasis in the heart: beyond the action potential

Normal central nervous system development relies on accurate intrinsic cellular programs as well as on extrinsic informative cues provided by extracellular molecules. Migration of neuronal progenitors from defined proliferative zones to their final location is a key event during embryonic and postnatal development. Extracellular matrix components play important roles in these processes, and interactions between neurons and extracellular matrix are fundamental for the normal development of the central nervous system. Guidance cues are provided by extracellular factors that orient neuronal migration. During cerebellar development, the extracellular matrix molecules laminin and fibronectin give support to neuronal precursor migration, while other molecules such as reelin, tenascin, and netrin orient their migration. Reelin and tenascin are extracellular matrix components that attract or repel neuronal precursors and axons during development through interaction with membrane receptors, and netrin associates with laminin and heparan sulfate proteoglycans, and binds to the extracellular matrix receptor integrins present on the neuronal surface. Altogether, the dynamic changes in the composition and distribution of extracellular matrix components provide external cues that direct neurons leaving their birthplaces to reach their correct final location. Understanding the molecular mechanisms that orient neurons to reach precisely their final location during development is fundamental to understand how neuronal misplacement leads to neurological diseases and eventually to find ways to treat them.

A transient outward-rectifying K+ channel current down-regulated by cytosolic Ca2+ in Arabidopsis thaliana guard cells

Sustained (noninactivating) outward-rectifying K+ channel currents have been identified in a variety of plant cell types and species. Here, in Arabidopsis thaliana guard cells, in addition to these sustained K+ currents, an inactivating outward-rectifying K+ current was characterized (plant A-type current: IAP). IAP activated rapidly with a time constant of 165 ms and inactivated slowly with a time constant of 7.2 sec at +40 mV. IAP was enhanced by increasing the duration (from 0 to 20 sec) and degree (from +20 to −100 mV) of prepulse hyperpolarization. Ionic substitution and relaxation (tail) current recordings showed that outward IAP was mainly carried by K+ ions. In contrast to the sustained outward-rectifying K+ currents, cytosolic alkaline pH was found to inhibit IAP and extracellular K+ was required for IAP activity. Furthermore, increasing cytosolic free Ca2+ in the physiological range strongly inhibited IAP activity with a half inhibitory concentration of ≈ 94 nM. We present a detailed characterization of an inactivating K+ current in a higher plant cell. Regulation of IAP by diverse factors including membrane potential, cytosolic Ca2+ and pH, and extracellular K+ and Ca2+ implies that the inactivating IAP described here may have important functions during transient depolarizations found in guard cells, and in integrated signal transduction processes during stomatal movements.

Outward potassium current oscillations in macrophage polykaryons: extracellular calcium entry and calcium-induced calcium release

Outward current oscillations associated with transient membrane hyperpolarizations were induced in murine macrophage polykaryons by membrane depolarization in the absence of external Na+. Oscillations corresponded to a cyclic activation of Ca2+-dependent K+ currents (IKCa) probably correlated with variations in intracellular Ca2+ concentration. Addition of external Na+ (8 mM) immediately abolished the outward current oscillations, suggesting that the absence of the cation is necessary not only for their induction but also for their maintenance. Oscillations were completely blocked by nisoldipine. Ruthenium red and ryanodine reduced the number of outward current cycles in each episode, whereas quercetin prolonged the hyperpolarization 2- to 15-fold. Neither low molecular weight heparin nor the absence of a Na+ gradient across the membrane had any influence on oscillations. The evidence suggests that Ca2+ entry through a pathway sensitive to Ca2+ channel blockers is elicited by membrane depolarization in Na+-free medium and is essential to initiate oscillations, which are also dependent on the cyclic release of Ca2+ from intracellular Ca2+-sensitive stores; Ca2+ ATPase acts by reducing intracellular Ca2+, thus allowing slow deactivation of IKCa. Evidence is presented that neither a Na+/Ca2+ antiporter nor Ca2+ release from IP3-sensitive Ca2+ stores participate directly in the mechanism of oscillation

A novel rare variant in SCN1Bb linked to Brugada syndrome and SIDS by combined modulation of Na(v)1.5 and K(v)4.3 channel currents.

BACKGROUND Cardiac sodium channel β-subunit mutations have been associated with several inherited cardiac arrhythmia syndromes. OBJECTIVE To identify and characterize variations in SCN1Bb associated with Brugada syndrome (BrS) and sudden infant death syndrome (SIDS). METHODS All known exons and intron borders of the BrS-susceptibility genes were amplified and sequenced in both directions. Wild type (WT) and mutant genes were expressed in TSA201 cells and studied using co-immunoprecipitation and whole-cell patch-clamp techniques. RESULTS Patient 1 was a 44-year-old man with an ajmaline-induced type 1 ST-segment elevation in V1 and V2 supporting the diagnosis of BrS. Patient 2 was a 62-year-old woman displaying a coved-type BrS electrocardiogram who developed cardiac arrest during fever. Patient 3 was a 4-month-old female SIDS case. A R214Q variant was detected in exon 3A of SCN1Bb (Na(v)1B) in all three probands, but not in any other gene previously associated with BrS or SIDS. R214Q was identified in 4 of 807 ethnically-matched healthy controls (0.50%). Co-expression of SCN5A/WT + SCN1Bb/R214Q resulted in peak sodium channel current (I(Na)) 56.5% smaller compared to SCN5A/WT + SCN1Bb/WT (n = 11-12, P<0.05). Co-expression of KCND3/WT + SCN1Bb/R214Q induced a Kv4.3 current (transient outward potassium current, I(to)) 70.6% greater compared with KCND3/WT + SCN1Bb/WT (n = 10-11, P<0.01). Co-immunoprecipitation indicated structural association between Na(v)β1B and Na(v)1.5 and K(v)4.3. CONCLUSION Our results suggest that R214Q variation in SCN1Bb is a functional polymorphism that may serve as a modifier of the substrate responsible for BrS or SIDS phenotypes via a combined loss of function of sodium channel current and gain of function of transient outward potassium current.

cAMP-dependent, long-lasting inhibition of a K+ current in mammalian neurons.

We report the long-term modulation of K+ channels by cAMP in cultured murine colliculi neurons. A short (1-2 s) application of 8-Br-cAMP induced a long-lasting broadening of the action potential, a loss of after-hyperpolarization, and a reduction in spike accommodation. In agreement with these changes, 8-Br-cAMP produced a long-lasting (2 hr) inhibition of a K+ current. These effects were also observed after a short activation of the pituitary adenylyl cyclase-activating polypeptide, beta-adrenergic, and 5-hydroxytryptamine type 4 (5-HT4) receptors, all known to increase cAMP. A transient activation of the cAMP-dependent protein kinase and a long-lasting inhibition of phosphatases (up to 2 hr) were detected. The blockade of the K+ current resulting from a brief application of 8-Br-cAMP or 5-hydroxytryptamine was prolonged from 2 to 4 hr when protein-serine/threonine phosphatases 1 and 2A were inhibited with 10 nM okadaic acid. The critical steps following the cAMP-dependent protein kinase activation and resulting in a long-term blockade of phosphatases are discussed in this report.

Cardiomyocyte dysfunction during the chronic phase of Chagas disease

Chagas disease, which is caused by the parasite Trypanosoma cruzi, is an important cause of heart failure. We investigated modifications in the cellular electrophysiological and calcium-handling characteristics of an infected mouse heart during the chronic phase of the disease. The patch-clamp technique was used to record action potentials (APs) and L-type Ca2+ and transient outward K+ currents. [Ca2+]i changes were determined using confocal microscopy. Infected ventricular cells showed prolonged APs, reduced transient outward K+ and L-type Ca2+ currents and reduced Ca2+ release from the sarcoplasmic reticulum. Thus, the chronic phase of Chagas disease is characterised by cardiomyocyte dysfunction, which could lead to heart failure.

Suppression of a pro-apoptotic K+ channel as a mechanism for hepatitis C virus persistence

An estimated 3% of the global population are infected with hepatitis C virus (HCV), and the majority of these individuals will develop chronic liver disease. As with other chronic viruses, establishment of persistent infection requires that HCV-infected cells must be refractory to a range of pro-apoptotic stimuli. In response to oxidative stress, amplification of an outward K(+) current mediated by the Kv2.1 channel, precedes the onset of apoptosis. We show here that in human hepatoma cells either infected with HCV or harboring an HCV subgenomic replicon, oxidative stress failed to initiate apoptosis via Kv2.1. The HCV NS5A protein mediated this effect by inhibiting oxidative stress-induced p38 MAPK phosphorylation of Kv2.1. The inhibition of a host cell K(+) channel by a viral protein is a hitherto undescribed viral anti-apoptotic mechanism and represents a potential target for antiviral therapy.

Anesthetic Activation of Central Respiratory Chemoreceptor Neurons Involves Inhibition of a THIK-1-Like Background K(+) Current

At surgical depths of anesthesia, inhalational anesthetics cause a loss of motor response to painful stimuli (i.e., immobilization) that is characterized by profound inhibition of spinal motor circuits. Yet, although clearly depressed, the respiratory motor system continues to provide adequate ventilation under these same conditions. Here, we show that isoflurane causes robust activation of CO(2)/pH-sensitive, Phox2b-expressing neurons located in the retrotrapezoid nucleus (RTN) of the rodent brainstem, in vitro and in vivo. In brainstem slices from Phox2b-eGFP mice, the firing of pH-sensitive RTN neurons was strongly increased by isoflurane, independent of prevailing pH conditions. At least two ionic mechanisms contributed to anesthetic activation of RTN neurons: activation of an Na(+)-dependent cationic current and inhibition of a background K(+) current. Single-cell reverse transcription-PCR analysis of dissociated green fluorescent protein-labeled RTN neurons revealed expression of THIK-1 (TWIK-related halothane-inhibited K(+) channel, K(2P)13.1), a channel that shares key properties with the native RTN current (i.e., suppression by inhalational anesthetics, weak rectification, inhibition by extracellular Na(+), and pH-insensitivity). Isoflurane also increased firing rate of RTN chemosensitive neurons in urethane-anesthetized rats, again independent of CO(2) levels. In these animals, isoflurane transiently enhanced activity of the respiratory system, an effect that was most prominent at low levels of respiratory drive and mediated primarily by an increase in respiratory frequency. These data indicate that inhalational anesthetics cause activation of RTN neurons, which serve an important integrative role in respiratory control; the increased drive provided by enhanced RTN neuronal activity may contribute, in part, to maintaining respiratory motor activity under immobilizing anesthetic conditions.

NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines

Pulmonary neuroepithelial bodies (NEB) are widely distributed throughout the airway mucosa of human and animal lungs. Based on the observation that NEB cells have a candidate oxygen sensor enzyme complex (NADPH oxidase) and an oxygen-sensitive K+ current, it has been suggested that NEB may function as airway chemoreceptors. Here we report that mRNAs for both the hydrogen peroxide sensitive voltage gated potassium channel subunit (KH2O2) KV3.3a and membrane components of NADPH oxidase (gp91phox and p22phox) are coexpressed in the NEB cells of fetal rabbit and neonatal human lungs. Using a microfluorometry and dihydrorhodamine 123 as a probe to assess H2O2 generation, NEB cells exhibited oxidase activity under basal conditions. The oxidase in NEB cells was significantly stimulated by exposure to phorbol esther (0.1 μM) and inhibited by diphenyliodonium (5 μM). Studies using whole-cell voltage clamp showed that the K+ current of cultured fetal rabbit NEB cells exhibited inactivating properties similar to KV3.3a transcripts expressed in Xenopus oocyte model. Exposure of NEB cells to hydrogen peroxide (H2O2, the dismuted by-product of the oxidase) under normoxia resulted in an increase of the outward K+ current indicating that H2O2 could be the transmitter modulating the O2-sensitive K+ channel. Expressed mRNAs or orresponding protein products for the NADPH oxidase membrane cytochrome b as well as mRNA encoding KV3.3a were identified in small cell lung carcinoma cell lines. The studies presented here provide strong evidence for an oxidase-O2 sensitive potassium channel molecular complex operating as an O2 sensor in NEB cells, which function as chemoreceptors in airways and in NEB related tumors. Such a complex may represent an evolutionary conserved biochemical link for a membrane bound O2-signaling mechanism proposed for other cells and life forms.

The weaver mutation of GIRK2 results in a loss of inwardly rectifying K+ current in cerebellar granule cells.

The weaver mutation in mice results in a severe ataxia that is attributable to the degeneration of cerebellar granule cells and dopaminergic neurons in the substantia nigra. Recent genetic studies indicate that the GIRK2 gene is altered in weaver. This gene codes for a G-protein-activated, inwardly rectifying K+ channel protein (8). The mutation results in a single amino acid substitution (glycine-->serine) in the pore-forming H5 region of the channel. The functional consequences of this mutation appear to depend upon the co-expression of other GIRK subunits--leading to either a gain or loss of function. Here, we show that G-protein-activated inwardly rectifying K+ currents are significantly reduced in cerebellar granule cells from animals carrying the mutant allele. The reduction is most pronounced in homozygous neurons. These findings suggest that the death of neurons in weaver is attributable to the loss of GIRK2-mediated currents, not to the expression of a nonspecific cation current.

The Neuromuscular Activity Of Bothriopsis Bilineata Smaragdina (forest Viper) Venom And Its Toxin Bbil-tx (asp49 Phospholipase A2) On Isolated Mouse Nerve-muscle Preparations.

The presynaptic action of Bothriopsis bilineata smaragdina (forest viper) venom and Bbil-TX, an Asp49 PLA2 from this venom, was examined in detail in mouse phrenic nerve-muscle (PND) preparations in vitro and in a neuroblastoma cell line (SK-N-SH) in order to gain a better insight into the mechanism of action of the venom and associated Asp49 PLA2. In low Ca(2+) solution, venom (3μg/ml) caused a quadriphasic response in PND twitch height whilst at 10μg/ml the venom additionally induced an abrupt and marked initial contracture followed by neuromuscular facilitation, rhythmic oscillations of nerve-evoked twitches, alterations in baseline and progressive blockade. The venom slowed the relaxation phase of muscle twitches. In low Ca(2+), Bbil-TX [210nM (3μg/ml)] caused a progressive increase in PND twitch amplitude but no change in the decay time constant. Venom (10μg/ml) and Bbil-TX (210nM) caused minor changes in the compound action potential (CAP) amplitude recorded from sciatic nerve preparations, with no significant effect on rise time and latency; tetrodotoxin (3.1nM) blocked the CAP at the end of the experiments. In mouse triangularis sterni nerve-muscle (TSn-m) preparations, venom (10μg/ml) and Bbil-TX (210nM) significantly reduced the perineural waveform associated with the outward K(+) current while the amplitude of the inward Na(+) current was not significantly affected. Bbil-TX (210nM) caused a progressive increase in the quantal content of TSn-m preparations maintained in low Ca(2+) solution. Venom (3μg/ml) and toxin (210nM) increased the calcium fluorescence in SK-N-SH neuroblastoma cells loaded with Fluo3 AM and maintained in low or normal Ca(2+) solution. In normal Ca(2+), the increase in fluorescence amplitude was accompanied by irregular and frequent calcium transients. In TSn-m preparations loaded with Fluo4 AM, venom (10μg/ml) caused an immediate increase in intracellular Ca(2+) followed by oscillations in fluorescence and muscle contracture; Bbil-TX did not change the calcium fluorescence in TSn-m preparations. Immunohistochemical analysis of toxin-treated PND preparations revealed labeling of junctional ACh receptors but a loss of the presynaptic proteins synaptophysin and SNAP25. Together, these data confirm the presynaptic action of Bbil-TX and show that it involves modulation of K(+) channel activity and presynaptic protein expression.

Electrical Properties of Isolated Cardiomyocytes in a Rat Model of Thiamine Deficiency

In modern society, thiamine deficiency (TD) remains an important medical condition linked to altered cardiac function. There have been contradictory reports about the impact of TD on heart physiology, especially in the context of cardiac excitability. In order to address this particular question, we used a TD rat model and patch-clamp technique to investigate the electrical properties of isolated cardiomyocytes from epicardium and endocardium. Neither cell type showed substantial differences on the action potential waveform and transient outward potassium current. Based on our results we can conclude that TD does not induce major electrical remodeling in isolated cardiac myocytes in either endocardium or epicardium cells.

Localization and function of the Kv3.1b subunit in the rat medulla oblongata: focus on the nucleus tractus solitarii

The voltage-gated potassium channel subunit Kv3.1 confers fast firing characteristics to neurones. Kv3.1b subunit immunoreactivity (Kv3.1b-IR) was widespread throughout the medulla oblongata, with labelled neurones in the gracile, cuneate and spinal trigeminal nuclei. In the nucleus of the solitary tract (NTS), Kv3.1b-IR neurones were predominantly located close to the tractus solitarius (TS) and could be GABAergic or glutamatergic. Ultrastructurally, Kv3.1b-IR was detected in NTS terminals, some of which were vagal afferents. Whole-cell current-clamp recordings from neurones near the TS revealed electrophysiological characteristics consistent with the presence of Kv3.1b subunits: short duration action potentials (4.2 +/- 1.4 ms) and high firing frequencies (68.9 +/- 5.3 Hz), both sensitive to application of TEA (0.5 mm) and 4-aminopyridine (4-AP; 30 mum). Intracellular dialysis of an anti-Kv3.1b antibody mimicked and occluded the effects of TEA and 4-AP in NTS and dorsal column nuclei neurones, but not in dorsal vagal nucleus or cerebellar Purkinje cells (which express other Kv3 subunits, but not Kv3.1b). Voltage-clamp recordings from outside-out patches from NTS neurones revealed an outward K(+) current with the basic characteristics of that carried by Kv3 channels. In NTS neurones, electrical stimulation of the TS evoked EPSPs and IPSPs, and TEA and 4-AP increased the average amplitude and decreased the paired pulse ratio, consistent with a presynaptic site of action. Synaptic inputs evoked by stimulation of a region lacking Kv3.1b-IR neurones were not affected, correlating the presence of Kv3.1b in the TS with the pharmacological effects.