A mutation in the cardiac KV7.1 channel possibly disrupts interaction with Yotiao protein.

Here, we characterized the p.Arg583His (R583H) Kv7.1 mutation, identified in two unrelated families suffered from LQT syndrome. This mutation is located in the HС-HD linker of the cytoplasmic portion of the Kv7.1 channel. This linker, together with HD helix are responsible for binding the A-kinase anchoring protein 9 (AKAP9), Yotiao. We studied the electrophysiological characteristics of the mutated channel expressed in CHO-K1 along with KCNE1 subunit and Yotiao protein, using the whole-cell patch-clamp technique. We found that R583H mutation, even at the heterozygous state, impedes IKs activation. Molecular modeling showed that HС and HD helixes of the C-terminal part of Kv7.1 channel are swapped along the C-terminus length of the channel and that R583 position is exposed to the outer surface of HC-HD tandem coiled-coil. Interestingly, the adenylate cyclase activator, forskolin had a smaller effect on the mutant channel comparing with the WT protein, suggesting that R583H mutation may disrupt the interaction of the channel with the adaptor protein Yotiao and, therefore, may impair phosphorylation of the KCNQ1 channel.

The metamorphosis of amphibian myocardium: moving to the heart of the matter.

Amphibians are a classical object for physiological studies, and they are of great value for developmental studies owing to their transition from an aquatic larval form to an adult form with a terrestrial lifestyle. Axolotls (Ambystoma mexicanum) are of special interest for such studies because of their neoteny and facultative pedomorphosis, as in these animals, metamorphosis can be induced and fully controlled in laboratory conditions. It has been suggested that their metamorphosis, associated with gross anatomical changes in the heart, also involves physiological and electrical remodeling of the myocardium. We used whole-cell patch clamp to investigate possible changes caused by metamorphosis in electrical activity and major ionic currents in cardiomyocytes isolated from paedomorphic and metamorphic axolotls. T4-induced metamorphosis caused shortening of atrial and ventricular action potentials (APs), with no changes in resting membrane potential or maximum velocity of AP upstroke, favoring higher heart rate possible in metamorphic animals. Potential-dependent potassium currents in axolotl myocardium were represented by delayed rectifier currents IKr and IKs, and upregulation of IKs caused by metamorphosis probably underlies AP shortening. Metamorphosis was associated with downregulation of inward rectifier current IK1, probably serving to increase the excitability of myocardium in metamorphic animals. Metamorphosis also led to a slight increase in fast sodium current INa with no changes in its steady-state kinetics and to a significant upregulation of ICa in both atrial and ventricular cells, indicating stronger Ca2+ influx for higher cardiac contractility in metamorphic salamanders. Taken together, these changes serve to increase cardiac reserve in metamorphic animals.

Sodium current preserves electrical excitability in the heart of hibernating ground squirrel (Citellus undulatus).

Hibernating mammals are capable of maintaining normal cardiac function at low temperatures. Excitability of cardiac myocytes crucially depends on the fast sodium current (INa), which is decreased in hypothermia due to both depolarization of resting membrane potential and direct negative effect of low temperature. Therefore, INa in hibernating mammals should have specific features allowing to maintain excitability of myocardium at low temperatures. The current-voltage dependence of INa, its steady-state inactivation and activation and recovery from inactivation were studied in winter hibernating (WH) and summer active (SA) ground squirrels and in rats using whole-cell patch clamp at 10 °C and 20 °C. INa peak amplitude and the parameters of steady-state activation and inactivation curves did not differ between SA and WH ground squirrels at both temperatures. However, at both temperatures strong positive shift of activation and inactivation curves by 5-12 mV was observed in both WH and SA ground squirrels if compared to rats. This peculiarity of cardiac INa in ground squirrels helps to maintain excitability in conditions of depolarized resting membrane potential. The time course of INa recovery from inactivation at 10 °C was faster in WH than in SA ground squirrels, which could ensure normal activation of myocardium during hibernation.

Ionic currents underlying different patterns of electrical activity in working cardiac myocytes of mammals and non-mammalian vertebrates.

The orderly contraction of the vertebrate heart is determined by generation and propagation of cardiac action potentials (APs). APs are generated by the integrated activity of time- and voltage-dependent ionic channels which carry inward Na+ and Ca2+ currents, and outward K+ currents. This review compares atrial and ventricular APs and underlying ion currents between different taxa of vertebrates. We have collected literature data and attempted to find common electrophysiological features for two or more vertebrate groups, show differences between taxa and cardiac chambers, and indicate gaps in the existing data. Although electrical excitability of the heart in all vertebrates is based on the same superfamily of channels, there is a vast variability of AP waveforms between atrial and ventricular myocytes, between different species of the same vertebrate class and between endothermic and ectothermic animals. The wide variability of AP shapes is related to species-specific differences in animal size, heart rate, stage of ontogenetic development, excitation-contraction coupling, temperature and oxygen availability. Some of the differences between taxa are related to evolutionary development of genomes, which appear e.g. in the expression of different Na+ and K+ channel orthologues in cardiomyocytes of vertebrates. There is a wonderful variability of AP shapes and underlying ion currents with which electrical excitability of vertebrate heart can be generated depending on the intrinsic and extrinsic conditions of animal body. This multitude of ionic mechanisms provides excellent material for studying how the function of the vertebrate heart can adapt or acclimate to prevailing physiological and environmental conditions.

Adrenergic prolongation of action potential duration in rainbow trout myocardium via inhibition of the delayed rectifier potassium current, IKr.

Catecholamines mediate the 'fight or flight' response in a wide variety of vertebrates. The endogenous catecholamine adrenaline increases heart rate and contractile strength to raise cardiac output. The increase in contractile force is driven in large part by an increase in myocyte Ca2+ influx on the L-type Ca current (ICaL) during the cardiac action potential (AP). Here, we report a K+- based mechanism that prolongs AP duration (APD) in fish hearts following adrenergic stimulation. We show that adrenergic stimulation inhibits the delayed rectifier K+ current (IKr) in rainbow trout (Oncorhynchus mykiss) cardiomyocytes. This slows repolarization and prolongs APD which may contribute to positive inotropy following adrenergic stimulation in fish hearts. The endogenous ligand, adrenaline (1 µM), which activates both α- and ß-ARs reduced maximal IKr tail current to 61.4 ± 3.9% of control in atrial and ventricular myocytes resulting in an APD prolongation of ~20% at both 50 and 90% repolarization. This effect was reproduced by the α-specific adrenergic agonist, phenylephrine (1 µM), but not the ß-specific adrenergic agonist isoproterenol (1 µM). Adrenaline (1 µM) in the presence of ß1 and ß2-blockers (1 µM atenolol and 1 µM ICI-118551, respectively) also inhibited IKr. Thus, IKr suppression following α-adrenergic stimulation leads to APD prolongation in the rainbow trout heart. This is the first time this mechanism has been identified in fish and may act in unison with the well-known enhancement of ICaL following adrenergic stimulation to prolong APD and increase cardiac inotropy.

Disruption of a Conservative Motif in the C-Terminal Loop of the KCNQ1 Channel Causes LQT Syndrome.

We identified a single nucleotide variation (SNV) (c.1264A > G) in the KCNQ1 gene in a 5-year-old boy who presented with a prolonged QT interval. His elder brother and mother, but not sister and father, also had this mutation. This missense mutation leads to a p.Lys422Glu (K422E) substitution in the Kv7.1 protein that has never been mentioned before. We inserted this substitution in an expression plasmid containing Kv7.1 cDNA and studied the electrophysiological characteristics of the mutated channel expressed in CHO-K1, using the whole-cell configuration of the patch-clamp technique. Expression of the mutant Kv7.1 channel in both homo- and heterozygous conditions in the presence of auxiliary subunit KCNE1 results in a significant decrease in tail current densities compared to the expression of wild-type (WT) Kv7.1 and KCNE1. This study also indicates that K422E point mutation causes a dominant negative effect. The mutation was not associated with a trafficking defect; the mutant channel protein was confirmed to localize at the cell membrane. This mutation disrupts the poly-Lys strip in the proximal part of the highly conserved cytoplasmic A−B linker of Kv7.1 that was not shown before to be crucial for channel functioning.

Effects of Na+ channel isoforms and cellular environment on temperature tolerance of cardiac Na+ current in zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss).

Heat tolerance of heart rate in fish is suggested to be limited by impaired electrical excitation of the ventricle due to the antagonistic effects of high temperature on Na+ (INa) and K+ (IK1) ion currents (INa is depressed at high temperatures while IK1 is resistant to them). To examine the role of Na+ channel proteins in heat tolerance of INa, we compared temperature dependencies of zebrafish (Danio rerio, warm-dwelling subtropical species) and rainbow trout (Oncorhynchus mykiss, cold-active temperate species) ventricular INa, and INa generated by the cloned zebrafish and rainbow trout NaV1.4 and NaV1.5 Na+ channels in human embryonic kidney (HEK) cells. Whole-cell patch-clamp recordings showed that zebrafish ventricular INa has better heat tolerance and slower inactivation kinetics than rainbow trout ventricular INa. In contrast, heat tolerance and inactivation kinetics of zebrafish and rainbow trout NaV1.4 channels are similar when expressed in the identical cellular environment of HEK cells. The same applies to NaV1.5 channels. These findings indicate that thermal adaptation of ventricular INa is largely achieved by differential expression of Na+ channel alpha subunits: zebrafish that tolerate higher temperatures mainly express the slower NaV1.5 isoform, while rainbow trout that prefer cold waters mainly express the faster NaV1.4 isoform. Differences in elasticity (stiffness) of the lipid bilayer and/or accessory protein subunits of the channel assembly may also be involved in thermal adaptation of INa. The results are consistent with the hypothesis that slow Na+ channel kinetics are associated with increased heat tolerance of cardiac excitation.

The snake heart pacemaker is localized near the sinoatrial valve.

To provide the first description of the exact location of primary pacemaker of the squamate heart, we used sharp microelectrode impalements and optical mapping of isolated sinus venosus preparations from Burmese pythons. We located the dominant pacemaker site at the base of the right leaflet of the sinoatrial valve (SAV), but latent pacemakers were also identified in a circular region around the SAV. Acetylcholine (10-5 mol l-1) or noradrenaline (10-6 mol l-1) induced shifts of the leading pacemaker site to other points near the SAV. The ionic currents of most of the cardiomyocytes isolated enzymatically from the SAV region resembled those of typical working myocytes from the sinus venosus. However, seven cells lacked the background inward rectifier current (IK1) and had a time-dependent hyperpolarization-induced inward current identified as the 'funny' pacemaker current (If). Therefore, the region proximal to SAV demonstrates pacemaking activity and contains cells that resemble the electrophysiological properties of mammalian pacemaker myocytes.

Repolarizing potassium currents in working myocardium of Japanese quail: a novel translational model for cardiac electrophysiology.

Birds developed endothermy and four-chambered high-performance heart independently from mammals. Though avian embryos are extensively studied and widely used as various models for heart research, little is known about cardiac physiology of adult birds. Meanwhile, cardiac electrophysiology is in search for easily accessible and relevant model objects which resemble human myocardium in the pattern of repolarizing currents (IKr, IKs, IKur and Ito). This study focuses on the configuration of electrical activity and electrophysiological phenotype of working myocardium in adult Japanese quails (Coturnix japonica). The resting membrane potential and action potential (AP) waveform in quail atrial myocardium were similar to that in working myocardium of rodents. Using whole-cell patch clamp and sharp glass microelectrodes, we demonstrated that the repolarization of quail atrial and ventricular myocardium is determined by voltage-dependent potassium currents IKr, IKs and Ito - the latter was previously considered as an exclusive evolutionary feature of mammals. The specific blockers of these currents, dofetilide (3 µmol l-1), HMR 1556 (30 µmol l-1) and 4-aminopyridine (3 mmol l-1), prolonged AP in both ventricular and atrial myocardial preparations. The expression of the corresponding channels responsible for these currents in quail myocardium was investigated with quantitative RT-PCR and western blotting. In conclusion, the described pattern of repolarizing ionic currents and channels in quail myocardium makes this species a novel and suitable experimental model for translational cardiac research and reveals new information related to the evolution of cardiac electrophysiology in vertebrates.

Warmer, faster, stronger: Ca2+ cycling in avian myocardium.

Birds occupy a unique position in the evolution of cardiac design. Their hearts are capable of cardiac performance on par with, or exceeding that of mammals, and yet the structure of their cardiomyocytes resembles those of reptiles. It has been suggested that birds use intracellular Ca2+ stored within the sarcoplasmic reticulum (SR) to power contractile function, but neither SR Ca2+ content nor the cross-talk between channels underlying Ca2+-induced Ca2+ release (CICR) have been studied in adult birds. Here we used voltage clamp to investigate the Ca2+ storage and refilling capacities of the SR and the degree of trans-sarcolemmal and intracellular Ca2+ channel interplay in freshly isolated atrial and ventricular myocytes from the heart of the Japanese quail (Coturnix japonica). A trans-sarcolemmal Ca2+ current (ICa) was detectable in both quail atrial and ventricular myocytes, and was mediated only by L-type Ca2+ channels. The peak density of ICa was larger in ventricular cells than in atrial cells, and exceeded that reported for mammalian myocardium recorded under similar conditions. Steady-state SR Ca2+ content of quail myocardium was also larger than that reported for mammals, and reached 750.6±128.2 µmol l-1 in atrial cells and 423.3±47.2 µmol l-1 in ventricular cells at 24°C. We observed SR Ca2+-dependent inactivation of ICa in ventricular myocytes, indicating cross-talk between sarcolemmal Ca2+ channels and ryanodine receptors in the SR. However, this phenomenon was not observed in atrial myocytes. Taken together, these findings help to explain the high-efficiency avian myocyte excitation-contraction coupling with regard to their reptilian-like cellular ultrastructure.

Cardiophysiological responses of the air-breathing Alaska blackfish to cold acclimation and chronic hypoxic submergence at 5°C.

The Alaska blackfish (Dallia pectoralis) remains active at cold temperatures when experiencing aquatic hypoxia without air access. To discern the cardiophysiological adjustments that permit this behaviour, we quantified the effect of acclimation from 15°C to 5°C in normoxia (15N and 5N fish), as well as chronic hypoxic submergence (6-8 weeks; ∼6.3-8.4 kPa; no air access) at 5°C (5H fish), on in vivo and spontaneous heart rate (fH), electrocardiogram, ventricular action potential (AP) shape and duration (APD), the background inward rectifier (IK1) and rapid delayed rectifier (IKr) K+ currents and ventricular gene expression of proteins involved in excitation-contraction coupling. In vivo fH was ∼50% slower in 5N than in 15N fish, but 5H fish did not display hypoxic bradycardia. Atypically, cold acclimation in normoxia did not induce shortening of APD or alter resting membrane potential. Rather, QT interval and APD were ∼2.6-fold longer in 5N than in 15N fish because outward IK1 and IKr were not upregulated in 5N fish. By contrast, chronic hypoxic submergence elicited a shortening of QT interval and APD, driven by an upregulation of IKr The altered electrophysiology of 5H fish was accompanied by increased gene expression of kcnh6 (3.5-fold; Kv11.2 of IKr), kcnj12 (7.4-fold; Kir2.2 of IK1) and kcnj14 (2.9-fold; Kir2.4 of IK1). 5H fish also exhibited a unique gene expression pattern that suggests modification of ventricular Ca2+ cycling. Overall, the findings reveal that Alaska blackfish exposed to chronic hypoxic submergence prioritize the continuation of cardiac performance to support an active lifestyle over reducing cardiac ATP demand.

Temperature and external K+ dependence of electrical excitation in ventricular myocytes of cod-like fishes.

Electrical excitability (EE) is vital for cardiac function and strongly modulated by temperature and external K+ concentration ([K+]o), as formulated in the hypothesis of temperature-dependent deterioration of electrical excitability (TDEE). As little is known about EE of arctic stenothermic fishes, we tested the TDEE hypothesis on ventricular myocytes of polar cod (Boreogadus saida) and navaga (Eleginus nawaga) of the Arctic Ocean and those of temperate freshwater burbot (Lota lota). Ventricular action potentials (APs) were elicited in current-clamp experiments at 3, 9 and 15°C, and AP characteristics and the current needed to elicit APs were examined. At 3°C, ventricular APs of polar cod and navaga were similar but differed from those of burbot in having a lower rate of AP upstroke and a higher rate of repolarization. EE of ventricular myocytes - defined as the ease with which all-or-none APs are triggered - was little affected by acute temperature changes between 3 and 15°C in any species. However, AP duration (APD50) was drastically reduced at higher temperatures. Elevation of [K+]o from 3 to 5.4 mmol l-1 and further to 8 mmol l-1 at 3, 9 and 15°C strongly affected EE and AP characteristics in polar cod and navaga, but had a lesser effect in burbot. In all species, ventricular excitation was resistant to acute temperature elevations, while small increases in [K+]o severely compromised EE, in particular in the marine stenotherms. This suggests that EE of the heart in these Gadiformes species is resistant against acute warming, but less so against the simultaneous temperature and exercise stresses.

Thermal acclimation and seasonal acclimatization: a comparative study of cardiac response to prolonged temperature change in shorthorn sculpin.

Seasonal thermal remodelling (acclimatization) and laboratory thermal remodelling (acclimation) can induce different physiological changes in ectothermic animals. As global temperatures are changing at an increasing rate, there is urgency to understand the compensatory abilities of key organs such as the heart to adjust under natural conditions. Thus, the aim of the present study was to directly compare the acclimatization and acclimatory response within a single eurythermal fish species, the European shorthorn sculpin (Myoxocephalus scorpio). We used current- and voltage-clamp to measure ionic current densities in both isolated atrial and ventricular myocytes from three groups of fish: (1) summer-caught fish kept at 12°C ('summer-acclimated'); (2) summer-caught fish kept at 3°C ('cold acclimated'); and (3) fish caught in March ('winter-acclimatized'). At a common test temperature of 7.5°C, action potential (AP) was shortened by both winter acclimatization and cold acclimation compared with summer acclimation; however, winter acclimatization caused a greater shortening than did cold acclimation. Shortening of AP was achieved mostly by a significant increase in repolarizing current density (IKr and IK1) following winter acclimatization, with cold acclimation having only minor effects. Compared with summer acclimation, the depolarizing L-type calcium current (ICa) was larger following winter acclimatization, but again, there was no effect of cold acclimation on ICa Interestingly, the other depolarizing current, INa, was downregulated at low temperatures. Our further analysis shows that ionic current remodelling is primarily due to changes in ion channel density rather than current kinetics. In summary, acclimatization profoundly modified the electrical activity of the sculpin heart while acclimation to the same temperature for >1.5 months produced very limited remodelling effects.

Extracellular ATP and ß-NAD alter electrical properties and cholinergic effects in the rat heart in age-specific manner.

Extracellular ATP and nicotinamide adenine dinucleotide (ß-NAD) demonstrate properties of neurotransmitters and neuromodulators in peripheral and central nervous system. It has been shown previously that ATP and ß-NAD affect cardiac functioning in adult mammals. Nevertheless, the modulation of cardiac activity by purine compounds in the early postnatal development is still not elucidated. Also, the potential influence of ATP and ß-NAD on cholinergic neurotransmission in the heart has not been investigated previously. Age-dependence of electrophysiological effects produced by extracellular ATP and ß-NAD was studied in the rat myocardium using sharp microelectrode technique. ATP and ß-NAD could affect ventricular and supraventricular myocardium independent from autonomic influences. Both purines induced reduction of action potentials (APs) duration in tissue preparations of atrial, ventricular myocardium, and myocardial sleeves of pulmonary veins from early postnatal rats similarly to myocardium of adult animals. Both purine compounds demonstrated weak age-dependence of the effect. We have estimated the ability of ATP and ß-NAD to alter cholinergic effects in the heart. Both purines suppressed inhibitory effects produced by stimulation of intracardiac parasympathetic nerve in right atria from adult animals, but not in preparations from neonates. Also, ATP and ß-NAD suppressed rest and evoked release of acetylcholine (ACh) in adult animals. ß-NAD suppressed effects of parasympathetic stimulation and ACh release stronger than ATP. In conclusion, ATP and ß-NAD control the heart at the postsynaptic and presynaptic levels via affecting the cardiac myocytes APs and ACh release. Postsynaptic and presynaptic effects of purines may be antagonistic and the latter demonstrates age-dependence.

Transcripts of Kv7.1 and MinK channels and slow delayed rectifier K+ current (IKs) are expressed in zebrafish (Danio rerio) heart.

Zebrafish are increasingly used as a model for human cardiac electrophysiology, arrhythmias, and drug screening. However, K+ ion channels of the zebrafish heart, which determine the rate of repolarization and duration of cardiac action potential (AP) are still incompletely known and characterized. Here, we provide the first evidence for the presence of the slow component of the delayed rectifier K+channels in the zebrafish heart and characterize electrophysiological properties of the slow component of the delayed rectifier K+current, IKs. Zebrafish atrium and ventricle showed strong transcript expression of the kcnq1 gene, which encodes the Kv7.1 α-subunit of the slow delayed rectifier K+ channel. In contrast, the kcne1 gene, encoding the MinK ß-subunit of the delayed rectifier, was expressed at 21 and 17 times lower level in ventricle and atrium, respectively, in comparison to the kcnq1. IKs was observed in 62% of ventricular myocytes with mean (± SEM) density of 1.23 ± 0.37 pA/pF at + 30 mV. Activation rate of IKs was 38% faster (τ50 = 1248 ± 215 ms) than kcnq1:kcne1 channels (1725 ± 792 ms) expressed in 3:1 ratio in Chinese hamster ovary cells. Microelectrode experiments demonstrated the functional relevance of IKs in the zebrafish heart, since 100 µM chromanol 293B produced a significant prolongation of AP in zebrafish ventricle. We conclude that AP repolarization in zebrafish ventricle is contributed by IKs, which is mainly generated by homotetrameric Kv7.1 channels not coupled to MinK ancillary ß-subunits. This is a clear difference to the human heart, where MinK is an essential component of the slow delayed rectifier K+channel.

Maximum heart rate in brown trout (Salmo trutta fario) is not limited by firing rate of pacemaker cells.

Temperature-induced changes in cardiac output (Q̇) in fish are largely dependent on thermal modulation of heart rate (fH), and at high temperatures Q̇ collapses due to heat-dependent depression of fH This study tests the hypothesis that firing rate of sinoatrial pacemaker cells sets the upper thermal limit of fH in vivo. To this end, temperature dependence of action potential (AP) frequency of enzymatically isolated pacemaker cells (pacemaker rate, fPM), spontaneous beating rate of isolated sinoatrial preparations (fSA), and in vivo fH of the cold-acclimated (4°C) brown trout (Salmo trutta fario) were compared under acute thermal challenges. With rising temperature, fPM steadily increased because of the acceleration of diastolic depolarization and shortening of AP duration up to the break point temperature (TBP) of 24.0 ± 0.37°C, at which point the electrical activity abruptly ceased. The maximum fPM at TBP was much higher [193 ± 21.0 beats per minute (bpm)] than the peak fSA (94.3 ± 6.0 bpm at 24.1°C) or peak fH (76.7 ± 2.4 at 15.7 ± 0.82°C) (P < 0.05). These findings strongly suggest that the frequency generator of the sinoatrial pacemaker cells does not limit fH at high temperatures in the brown trout in vivo.

Effects of new antiarrhythmic agent SS-68 on excitation conduction, electrical activity in Purkinje fibers and pulmonary veins: Assessment of safety and side effects risk.

The compound SS-68 has been selected among numerous new derivatives of indole and demonstrated antiarrhythmic effects in animal models. The present study concerns several aspects of SS-68 safety and efficacy as a potential antiarrhythmic drug. The first estimation of atrioventricular conduction in mammalian heart under SS-68 has been carried out; effects of SS-68 in Purkinje fibers and myocardium of pulmonary veins have been investigated. The drug weakly affects cardiac atrioventricular conduction: only high concentrations of SS-68 (≥10 µmol/L) significantly decrease this parameter. Also, the drug weakly affects Purkinje fibers automaticity, but effectively alters action potential waveform in Purkinje fibers in a concentration-dependent manner. SS-68 (0.1-100 µmol/L) failed to induce any early or delayed afterdepolarizations in Purkinje fibers both in basal conditions and under provocation of proarrhythmic activity by norepinephrine (NE). Moreover, 10 µmol/L SS-68 suppressed NE-induced extra-beats and rapid firing in Purkinje fibers. In pulmonary veins only high concentrations of SS-68 significantly increased action potential duration, while lower concentrations (0.1-1 µmol/L) were ineffective. Also, 0.1-100 µmol/L SS-68 was unable to elicit arrhythmogenic alternations of action potential waveform in pulmonary veins. In conclusion, SS-68 has no proarrhythmic effects, such as afterdepolarizations or abnormal automaticity in used experimental models.

A New Class III Antiarrhythmic Drug Niferidil Prolongs Action Potentials in Guinea Pig Atrial Myocardium via Inhibition of Rapid Delayed Rectifier.

PURPOSE: A new class III antiarrhythmic drug niferidil (RG-2) has been introduced as a highly effective therapy for cases of persistent atrial fibrillation, but ionic mechanisms of its action are poorly understood. In the present study, the effects of niferidil on action potential (AP) waveform and potassium currents responsible for AP repolarization were investigated in guinea pig atrial myocardium. METHODS: APs were recorded with sharp glass microelectrodes in multicellular atrial preparations. Whole-cell patch-clamp technique was used to measure K+ currents in isolated myocytes. RESULTS: In multicellular atrial preparations, 10-8 M niferidil effectively prolonged APs by 15.2 ± 2.8% at 90% repolarization level. However, even the highest tested concentrations, 10-6 M and 10-5 M failed to prolong APs more than 32.5% of control duration. The estimated concentration of niferedil for half-maximal AP prolongation was 1.13 × 10-8 M. Among the potassium currents responsible for AP repolarization phase, I K1 was found to be almost insensitive to niferidil. However, another inward rectifier, I KACh, was effectively suppressed by micromolar concentrations of niferidil with IC50 = 9.2 × 10-6 M. I KATP was much less sensitive to the drug with IC50 = 2.26 × 10-4 M. The slow component of delayed rectifier, I Ks, also demonstrated low sensitivity to niferidil-the highest used concentration, 10-4 M, decreased peak I Ks density to 46.2 ± 5.5% of control. Unlike I Ks, the rapid component of delayed rectifier, I Kr, appeared to be extremely sensitive to niferidil. The IC50 was 1.26 × 10-9 M. I Kr measured in ventricular myocytes was found to be less sensitive to niferidil with IC50 = 3.82 × 10-8 M. CONCLUSIONS: Niferidil prolongs APs in guinea pig atrial myocardium via inhibition of I Kr.

Effects of exogenous nicotinamide adenine dinucleotide (NAD+) in the rat heart are mediated by P2 purine receptors.

BACKGROUND: Recently, NAD+ has been considered as an essential factor, participating in nerve control of physiological functions and intercellular communication. NAD+ also has been supposed as endogenous activator of P1 and P2 purinoreceptors. Effects of extracellular NAD+ remain poorly investigated in cardiac tissue. This study aims to investigate the effects of extracellular NAD+ in different types of supraventricular and ventricular working myocardium from rat and their potential mechanisms. METHODS: The standard technique of sharp microelectrode action potential recording in cardiac multicellular preparations was used to study the effects of NAD+. RESULTS: Extracellular NAD+ induced significant changes in bioelectrical activity of left auricle (LA), right auricle (RA), pulmonary veins (PV) and right ventricular wall (RV) myocardial preparations. 10-100 µM NAD+ produced two opposite effects in LA and RA - quickly developing and transient prolongation of action potentials (AP) and delayed sustained AP shortening, which follows the initial positive effect. In PV and RV only AP shortening was observed in response to NAD+ application. In PV preparations AP shortening induced by NAD+ may be considered as a potential proarrhythmic effect. Revealed cardiotropic effects of NAD+ are likely to be mediated by P2 purine receptors, since P1 blocker DPCPX failed to affect them and P2 antagonist suramin abolished NAD + -induced alterations of electrical activity. P2X receptors may be responsible for NAD + -induced short-lasting AP prolongation, while P2Y receptors mediate persistent AP shortening. The latter effect is partially removed by PLC inhibitor U73122 showing the potential involvement of phosphoinositide signaling pathway in mediation of NAD+ cardiotropic effects. CONCLUSIONS: Extracellular NAD+ is supposed to be a novel regulator of cardiac electrical activity. P2 receptors represent the main target of NAD+ at least in the rat heart.

Effects of a new antiarrhythmic drug SS-68 on electrical activity in working atrial and ventricular myocardium of mouse and their ionic mechanisms.

SS-68 is a derivative of indole, which demonstrated strong antiarrhythmic effects not associated with significant QT prolongation in dog models of atrial fibrillation. Therefore, SS-68 was proposed as a new antiarrhythmic drug and the present study is the first describing its effects on action potentials (APs) configuration and elucidating the ionic mechanisms of these effects. Sharp microelectrodes were used to record APs in isolated preparations of mouse atrial and ventricular myocardium. In both types of myocardium 10(-6) M SS-68 produced reduction of AP duration, 3 × 10(-6) M failed to alter AP waveform and 10(-5) - 3 × 10(-5) M prolonged APs. Sensitivity of main ionic currents to SS-68 was determined using whole-cell patch clamp. Transient potassium current Ito was slightly inhibited by SS-68 with IC50 = 1.43 × 10(-4) M. IKur was more sensitive with IC50 = 1.84 × 10(-5) M. Background inward rectifier showed very low sensitivity to SS-68 - only 10(-4) M SS-68 caused significant reduction of IK1. ICaL was significantly inhibited by 10(-6)M - 3 × 10(-5) M SS-68. The IC50 value for the ICaL was 1.84 × 10(-6) M. Thus, main ionic currents of mouse cardiomyocytes are inhibited by SS-68 in the following order of potency: ICaL > IKur > Ito > IK1. While lower concentration of SS-68 shorten APs via suppression of ICaL, higher concentrations inhibit K(+)-currents leading to APs prolongation.