Cardiac electrophysiological remodeling associated with enhanced arrhythmia susceptibility in a canine model of elite exercise.

The health benefits of regular physical exercise are well known. Even so, there is increasing evidence that the exercise regimes of elite athletes can evoke cardiac arrhythmias including ventricular fibrillation and even sudden cardiac death (SCD). The mechanism of exercise-induced arrhythmia and SCD is poorly understood. Here, we show that chronic training in a canine model (12 sedentary and 12 trained dogs) that mimics the regime of elite athletes induces electrophysiological remodeling (measured by ECG, patch-clamp, and immunocytochemical techniques) resulting in increases of both the trigger and the substrate for ventricular arrhythmias. Thus, 4 months sustained training lengthened ventricular repolarization (QTc: 237.1±3.4 ms vs. 213.6±2.8 ms, n=12; APD90: 472.8±29.6 ms vs. 370.1±32.7 ms, n=29 vs. 25), decreased transient outward potassium current (6.4±0.5 pA/pF vs. 8.8±0.9 pA/pF at 50 mV, n=54 vs. 42), and increased the short-term variability of repolarization (29.5±3.8 ms vs. 17.5±4.0 ms, n=27 vs. 18). Left ventricular fibrosis and HCN4 protein expression were also enhanced. These changes were associated with enhanced ectopic activity (number of escape beats from 0/hr to 29.7±20.3/hr) in vivo and arrhythmia susceptibility (elicited ventricular fibrillation: 3 of 10 sedentary dogs vs. 6 of 10 trained dogs). Our findings provide in vivo, cellular electrophysiological and molecular biological evidence for the enhanced susceptibility to ventricular arrhythmia in an experimental large animal model of endurance training.

The reverse mode of the Na+/Ca2+ exchanger contributes to the pacemaker mechanism in rabbit sinus node cells.

Sinus node (SN) pacemaking is based on a coupling between surface membrane ion-channels and intracellular Ca2+-handling. The fundamental role of the inward Na+/Ca2+ exchanger (NCX) is firmly established. However, little is known about the reverse mode exchange. A simulation study attributed important role to reverse NCX activity, however experimental evidence is still missing. Whole-cell and perforated patch-clamp experiments were performed on rabbit SN cells supplemented with fluorescent Ca2+-tracking. We established 2 and 8 mM pipette NaCl groups to suppress and enable reverse NCX. NCX was assessed by specific block with 1 µM ORM-10962. Mechanistic simulations were performed by Maltsev-Lakatta minimal computational SN model. Active reverse NCX resulted in larger Ca2+-transient amplitude with larger SR Ca2+-content. Spontaneous action potential (AP) frequency increased with 8 mM NaCl. When reverse NCX was facilitated by 1 µM strophantin the Ca2+i and spontaneous rate increased. ORM-10962 applied prior to strophantin prevented Ca2+i and AP cycle change. Computational simulations indicated gradually increasing reverse NCX current, Ca2+i and heart rate with increasing Na+i. Our results provide further evidence for the role of reverse NCX in SN pacemaking. The reverse NCX activity may provide additional Ca2+-influx that could increase SR Ca2+-content, which consequently leads to enhanced pacemaking activity.

The Inhibition of the Small-Conductance Ca2+-Activated Potassium Channels Decreases the Sinus Node Pacemaking during Beta-Adrenergic Activation.

Sinus pacemaking is based on tight cooperation of intracellular Ca2+ handling and surface membrane ion channels. An important player of this synergistic crosstalk could be the small-conductance Ca2+-activated K+-channel (ISK) that could contribute to the sinoatrial node (SAN) pacemaking driven by the intracellular Ca2+ changes under normal conditions and beta-adrenergic activation, however, the exact role is not fully clarified. SK2 channel expression was verified by immunoblot technique in rabbit SAN cells. Ionic currents and action potentials were measured by patch-clamp technique. The ECG R-R intervals were obtained by Langendorff-perfusion method on a rabbit heart. Apamin, a selective inhibitor of SK channels, was used during the experiments. Patch-clamp experiments revealed an apamin-sensitive current. When 100 nM apamin was applied, we found no change in the action potential nor in the ECG R-R interval. In experiments where isoproterenol was employed, apamin increased the cycle length of the SAN action potentials and enhanced the ECG R-R interval. Apamin did not amplify the cycle length variability or ECG R-R interval variability. Our data indicate that ISK has no role under normal condition, however, it moderately contributes to the SAN automaticity under beta-adrenergic activation.

In vivo and cellular antiarrhythmic and cardiac electrophysiological effects of desethylamiodarone in dog cardiac preparations.

BACKGROUND AND PURPOSE: The aim of the present study was to study the antiarrhythmic effects and cellular mechanisms of desethylamiodarone (DEA), the main metabolite of amiodarone (AMIO), following acute and chronic 4-week oral treatments (25-50 mg·kg-1 ·day-1 ). EXPERIMENTAL APPROACH: The antiarrhythmic effects of acute iv. (10 mg·kg-1 ) and chronic oral (4 weeks, 25 mg·kg-1 ·day-1 ) administration of DEA were assessed in carbachol and tachypacing-induced dog atrial fibrillation models. Action potentials were recorded from atrial and right ventricular tissue following acute (10 µM) and chronic (p.o. 4 weeks, 50 mg·kg-1 ·day-1 ) DEA application using the conventional microelectrode technique. Ionic currents were measured by the whole cell configuration of the patch clamp technique in isolated left ventricular myocytes. Pharmacokinetic studies were performed following a single intravenous dose (25 mg·kg-1 ) of AMIO and DEA intravenously and orally. In chronic (91-day) toxicological investigations, DEA and AMIO were administered in the oral dose of 25 mg·kg-1 ·day-1 ). KEY RESULTS: DEA exerted marked antiarrhythmic effects in both canine atrial fibrillation models. Both acute and chronic DEA administration prolonged action potential duration in atrial and ventricular muscle without any changes detected in Purkinje fibres. DEA decreased the amplitude of several outward potassium currents such as IKr , IKs , IK1 , Ito , and IKACh , while the ICaL and late INa inward currents were also significantly depressed. Better drug bioavailability and higher volume of distribution for DEA were observed compared to AMIO. No neutropenia and less severe pulmonary fibrosis was found following DEA compared to that of AMIO administration. CONCLUSION AND IMPLICATIONS: Chronic DEA treatment in animal experiments has marked antiarrhythmic and electrophysiological effects with better pharmacokinetics and lower toxicity than its parent compound. These results suggest that the active metabolite, DEA, should be considered for clinical trials as a possible new, more favourable option for the treatment of cardiac arrhythmias including atrial fibrillation.

The development of L-type Ca2+ current mediated alternans does not depend on the restitution slope in canine ventricular myocardium.

Cardiac alternans have crucial importance in the onset of ventricular fibrillation. The early explanation for alternans development was the voltage-driven mechanism, where the action potential (AP) restitution steepness was considered as crucial determining factor. Recent results suggest that restitution slope is an inadequate predictor for alternans development, but several studies still claim the role of membrane potential as underlying mechanism of alternans. These controversial data indicate that the relationship of restitution and alternans development is not completely understood. APs were measured by conventional microelectrode technique from canine right ventricular papillary muscles. Ionic currents combined with fluorescent measurements were recorded by patch-clamp technique. APs combined with fluorescent measurements were monitored by sharp microelectrodes. Rapid pacing evoked restitution-independent AP duration (APD) alternans. When non-alternating AP voltage command was used, Ca2+i-transient (CaT) alternans were not observed. When alternating rectangular voltage pulses were applied, CaT alternans were proportional to ICaL amplitude alternans. Selective ICaL inhibition did not influence the fast phase of APD restitution. In this study we found that ICaL has minor contribution in shaping the fast phase of restitution curve suggesting that ICaL-if it plays important role in the alternans mechanism-could be an additional factor that attenuates the reliability of APD restitution slope to predict alternans.

Antiarrhythmic and cardiac electrophysiological effects of SZV-270, a novel compound with combined Class I/B and Class III effects, in rabbits and dogs.

Cardiovascular diseases are the leading causes of mortality. Sudden cardiac death is most commonly caused by ventricular fibrillation (VF). Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and a major cause of stroke and heart failure. Pharmacological management of VF and AF remains suboptimal due to limited efficacy of antiarrhythmic drugs and their ventricular proarrhythmic adverse effects. In this study, the antiarrhythmic and cardiac cellular electrophysiological effects of SZV-270, a novel compound, were investigated in rabbit and canine models. SZV-270 significantly reduced the incidence of VF in rabbits subjected to coronary artery occlusion/reperfusion and reduced the incidence of burst-induced AF in a tachypaced conscious canine model of AF. SZV-270 prolonged the frequency-corrected QT interval, lengthened action potential duration and effective refractory period in ventricular and atrial preparations, blocked I Kr in isolated cardiomyocytes (Class III effects), and reduced the maximum rate of depolarization (V max) at cycle lengths smaller than 1000 ms in ventricular preparations (Class I/B effect). Importantly, SZV-270 did not provoke Torsades de Pointes arrhythmia in an anesthetized rabbit proarrhythmia model characterized by impaired repolarization reserve. In conclusion, SZV-270 with its combined Class I/B and III effects can prevent reentry arrhythmias with reduced risk of provoking drug-induced Torsades de Pointes.

Muscarinic agonists inhibit the ATP-dependent potassium current and suppress the ventricle-Purkinje action potential dispersion.

Activation of the parasympathetic nervous system has been reported to have an antiarrhythmic role during ischemia-reperfusion injury by decreasing the arrhythmia triggers. Furthermore, it was reported that the parasympathetic neurotransmitter acetylcholine is able to modulate the ATP-dependent potassium current (I K-ATP), a crucial current activated during hypoxia. However, the possible significance of this current modulation in the antiarrhythmic mechanism is not fully clarified. Action potentials were measured using the conventional microelectrode technique from canine left ventricular papillary muscle and free-running Purkinje fibers, under normal and hypoxic conditions. Ionic currents were measured using the whole-cell configuration of the patch-clamp method. Acetylcholine at 5 µmol/L did not influence the action potential duration (APD) either in Purkinje fibers or in papillary muscle preparations. In contrast, it significantly lengthened the APD and suppressed the Purkinje-ventricle APD dispersion when it was administered after 5 µmol/L pinacidil application. Carbachol at 3 µmol/L reduced the pinacidil-activated I K-ATP under voltage-clamp conditions. Acetylcholine lengthened the ventricular action potential under simulated ischemia condition. In this study, we found that acetylcholine inhibits the I K-ATP and thus suppresses the ventricle-Purkinje APD dispersion. We conclude that parasympathetic tone may reduce the arrhythmogenic substrate exerting a complex antiarrhythmic mechanism during hypoxic conditions.

Increased Ca2+ content of the sarcoplasmic reticulum provides arrhythmogenic trigger source in swimming-induced rat athlete's heart model.

Sudden cardiac death among top athletes is very rare, however, it is 2-4 times more frequent than in the age-matched control population. In the present study, the electrophysiological consequences of long-term exercise training were investigated on Ca2+ homeostasis and ventricular repolarization, together with the underlying alterations of ion channel expression, in a rat athlete's heart model. 12-week swimming exercise-trained and control Wistar rats were used. Electrophysiological data were obtained by using ECG, patch clamp and fluorescent optical measurements. Protein and mRNA levels were determined by the Western immunoblot and qRT-PCR techniques. Animals in the trained group exhibited significantly lower resting heart rate, higher incidence of extrasystoles and spontaneous Ca2+ release events. The Ca2+ content of the sarcoplasmic reticulum (SR) and the Ca2+ transient amplitude were significantly larger in the trained group. Intensive physical training is associated with elevated SR Ca2+ content, which could be an important part of physiological cardiac adaptation mechanism to training. However, it may also sensitize the heart for the development of spontaneous Ca2+ release and extrasystoles. Training-associated remodeling may promote elevated incidence of life threatening arrhythmias in top athletes.

Discovery and characterization of ORM-11372, a novel inhibitor of the sodium-calcium exchanger with positive inotropic activity.

BACKGROUND AND PURPOSE: The lack of selective sodium-calcium exchanger (NCX) inhibitors has hampered the exploration of physiological and pathophysiological roles of cardiac NCX 1.1. We aimed to discover more potent and selective drug like NCX 1.1 inhibitor. EXPERIMENTAL APPROACH: A flavan series-based pharmacophore model was constructed. Virtual screening helped us identify a novel scaffold for NCX inhibition. A distinctively different NCX 1.1 inhibitor, ORM-11372, was discovered after lead optimization. Its potency against human and rat NCX 1.1 and selectivity against other ion channels was assessed. The cardiovascular effects of ORM-11372 were studied in normal and infarcted rats and rabbits. Human cardiac safety was studied ex vivo using human ventricular trabeculae. KEY RESULTS: ORM-11372 inhibited human NCX 1.1 reverse and forward currents; IC50 values were 5 and 6 nM respectively. ORM-11372 inhibited human cardiac sodium 1.5 (INa ) and hERG KV 11.1 currents (IhERG ) in a concentration-dependent manner; IC50 values were 23.2 and 10.0 µM. ORM-11372 caused no changes in action potential duration; short-term variability and triangulation were observed for concentrations of up to 10 µM. ORM-11372 induced positive inotropic effects of 18 ± 6% and 35 ± 8% in anaesthetized rats with myocardial infarctions and in healthy rabbits respectively; no other haemodynamic effects were observed, except improved relaxation at the lowest dose. CONCLUSION AND IMPLICATIONS: ORM-11372, a unique, novel, and potent inhibitor of human and rat NCX 1.1, is a positive inotropic compound. NCX inhibition can induce clinically relevant improvements in left ventricular contractions without affecting relaxation, heart rate, or BP, without pro-arrhythmic risk.

The Cardiac Pacemaker Story-Fundamental Role of the Na+/Ca2+ Exchanger in Spontaneous Automaticity.

The electrophysiological mechanism of the sinus node automaticity was previously considered exclusively regulated by the so-called "funny current". However, parallel investigations increasingly emphasized the importance of the Ca2+-homeostasis and Na+/Ca2+ exchanger (NCX). Recently, increasing experimental evidence, as well as insight through mechanistic in silico modeling demonstrates the crucial role of the exchanger in sinus node pacemaking. NCX had a key role in the exciting story of discovery of sinus node pacemaking mechanisms, which recently settled with a consensus on the coupled-clock mechanism after decades of debate. This review focuses on the role of the Na+/Ca2+ exchanger from the early results and concepts to recent advances and attempts to give a balanced summary of the characteristics of the local, spontaneous, and rhythmic Ca2+ releases, the molecular control of the NCX and its role in the fight-or-flight response. Transgenic animal models and pharmacological manipulation of intracellular Ca2+ concentration and/or NCX demonstrate the pivotal function of the exchanger in sinus node automaticity. We also highlight where specific hypotheses regarding NCX function have been derived from computational modeling and require experimental validation. Nonselectivity of NCX inhibitors and the complex interplay of processes involved in Ca2+ handling render the design and interpretation of these experiments challenging.

Evaluation of Possible Proarrhythmic Potency: Comparison of the Effect of Dofetilide, Cisapride, Sotalol, Terfenadine, and Verapamil on hERG and Native IKr Currents and on Cardiac Action Potential.

The proarrhythmic potency of drugs is usually attributed to the IKr current block. During safety pharmacology testing analysis of IKr in cardiomyocytes was replaced by human ether-a-go-go-related gene (hERG) test using automated patch-clamp systems in stable transfected cell lines. Aim of this study was to compare the effect of proarrhythmic compounds on hERG and IKr currents and on cardiac action potential. The hERG current was measured by using both automated and manual patch-clamp methods on HEK293 cells. The native ion currents (IKr, INaL, ICaL) were recorded from rabbit ventricular myocytes by manual patch-clamp technique. Action potentials in rabbit ventricular muscle and undiseased human donor hearts were studied by conventional microelectrode technique. Dofetilide, cisapride, sotalol, terfenadine, and verapamil blocked hERG channels at 37°C with an IC50 of 7 nM, 18 nM, 343 µM, 165 nM, and 214 nM, respectively. Using manual patch-clamp, the IC50 values of sotalol and terfenadine were 78 µM and 31 nM, respectively. The IC50 values calculated from IKr measurements at 37°C were 13 nM, 26 nM, 52 µM, 54 nM, and 268 nM, respectively. Cisapride, dofetilide, and sotalol excessively lengthened, terfenadine, and verapamil did not influence the action potential duration. Terfenadine significantly inhibited INaL and moderately ICaL, verapamil blocked only ICaL. Automated hERG assays may over/underestimate proarrhythmic risk. Manual patch-clamp has substantially higher sensitivity to certain drugs. Action potential studies are also required to analyze complex multichannel effects. Therefore, manual patch-clamp and action potential experiments should be a part of preclinical safety tests.

Novel Na+/Ca2+ Exchanger Inhibitor ORM-10962 Supports Coupled Function of Funny-Current and Na+/Ca2+ Exchanger in Pacemaking of Rabbit Sinus Node Tissue.

BACKGROUND AND PURPOSE: The exact mechanism of spontaneous pacemaking is not fully understood. Recent results suggest tight cooperation between intracellular Ca2+ handling and sarcolemmal ion channels. An important player of this crosstalk is the Na+/Ca2+ exchanger (NCX), however, direct pharmacological evidence was unavailable so far because of the lack of a selective inhibitor. We investigated the role of the NCX current in pacemaking and analyzed the functional consequences of the If-NCX coupling by applying the novel selective NCX inhibitor ORM-10962 on the sinus node (SAN). EXPERIMENTAL APPROACH: Currents were measured by patch-clamp, Ca2+-transients were monitored by fluorescent optical method in rabbit SAN cells. Action potentials (AP) were recorded from rabbit SAN tissue preparations. Mechanistic computational data were obtained using the Yaniv et al. SAN model. KEY RESULTS: ORM-10962 (ORM) marginally reduced the SAN pacemaking cycle length with a marked increase in the diastolic Ca2+ level as well as the transient amplitude. The bradycardic effect of NCX inhibition was augmented when the funny-current (If) was previously inhibited and vice versa, the effect of If was augmented when the Ca2+ handling was suppressed. CONCLUSION AND IMPLICATIONS: We confirmed the contribution of the NCX current to cardiac pacemaking using a novel NCX inhibitor. Our experimental and modeling data support a close cooperation between If and NCX providing an important functional consequence: these currents together establish a strong depolarization capacity providing important safety factor for stable pacemaking. Thus, after individual inhibition of If or NCX, excessive bradycardia or instability cannot be expected because each of these currents may compensate for the reduction of the other providing safe and rhythmic SAN pacemaking.

Inotropic effect of NCX inhibition depends on the relative activity of the reverse NCX assessed by a novel inhibitor ORM-10962 on canine ventricular myocytes.

Na+/Ca2+ exchanger (NCX) is the main Ca2+ transporter in cardiac myocytes. Its inhibition could be expected to exert positive inotropic action by accumulation of cytosolic Ca2+ ([Ca2+]i). However, we have observed only a marginal positive inotropic effect upon selective inhibition of NCX, which was enhanced when forward activity was facilitated. Here we attempted to clarify the underlying mechanism of the limited inotropic action of selective NCX inhibition by a novel inhibitor ORM-10962 on canine ventricular myocytes. 1µM ORM-10962 reduced the Ca2+ content of sarcoplasmic reticulum (SR) when the reverse NCX was favoured, while SR Ca2+ content was increased by ORM-10962 under conditions favouring the forward activity, like elevation of [Ca2+]i. L-type Ca2+ current (ICa) was not affected by 1µM ORM-10962 in the absence of SR Ca2+ release, while ICa was suppressed by ORM-10962 during normal Ca2+ cycling. The apparent degree of forward NCX inhibition was dependent on the elevation of [Ca2+]i, suggesting that an increased driving force of forward NCX can also limit the accumulation of [Ca2+i]. We concluded that in healthy myocardium the possible positive inotropic potential of NCX inhibition is considerably weaker than it was expected earlier by theoretical assumptions. The underlying mechanism may involve the autoregulation of Ca2+ handling and/or the preserved inducibility of forward NCX by high [Ca2+]i. This limitation of selective NCX inhibition seen in undiseased myocardium requires further studies in failing heart, which may allow correct evaluation of the potential therapeutic value of selective NCX inhibitors in the treatment of heart failure.

The Effect of a Novel Highly Selective Inhibitor of the Sodium/Calcium Exchanger (NCX) on Cardiac Arrhythmias in In Vitro and In Vivo Experiments.

BACKGROUND: In this study the effects of a new, highly selective sodium-calcium exchanger (NCX) inhibitor, ORM-10962 were investigated on cardiac NCX current, Ca2+ transients, cell shortening and in experimental arrhythmias. The level of selectivity of the novel inhibitor on several major transmembrane ion currents (L-type Ca2+ current, major repolarizing K+ currents, late Na+ current, Na+/K+ pump current) was also determined. METHODS: Ion currents in single dog ventricular cells (cardiac myocytes; CM), and action potentials in dog cardiac multicellular preparations were recorded utilizing the whole-cell patch clamp and standard microelectrode techniques, respectively. Ca2+ transients and cell shortening were measured in fluorescent dye loaded isolated dog myocytes. Antiarrhythmic effects of ORM-10962 were studied in anesthetized ouabain (10 µg/kg/min i.v.) pretreated guinea pigs and in ischemia-reperfusion models (I/R) of anesthetized coronary artery occluded rats and Langendorff perfused guinea pigs hearts. RESULTS: ORM-10962 significantly reduced the inward/outward NCX currents with estimated EC50 values of 55/67 nM, respectively. The compound, even at a high concentration of 1 µM, did not modify significantly the magnitude of ICaL in CMs, neither had any apparent influence on the inward rectifier, transient outward, the rapid and slow components of the delayed rectifier potassium currents, the late and peak sodium and Na+/K+ pump currents. NCX inhibition exerted moderate positive inotropic effect under normal condition, negative inotropy when reverse, and further positive inotropic effect when forward mode was facilitated. In dog Purkinje fibres 1 µM ORM-10962 decreased the amplitude of digoxin induced delayed afterdepolarizations (DADs). Pre-treatment with 0.3 mg/kg ORM-10962 (i.v.) 10 min before starting ouabain infusion significantly delayed the development and recurrence of ventricular extrasystoles (by about 50%) or ventricular tachycardia (by about 30%) in anesthetized guinea pigs. On the contrary, ORM-10962 pre-treatment had no apparent influence on the time of onset or the severity of I/R induced arrhythmias in anesthetized rats and in Langendorff perfused guinea-pig hearts. CONCLUSIONS: The present study provides strong evidence for a high efficacy and selectivity of the NCX-inhibitory effect of ORM-10962. Selective NCX inhibition can exert positive as well as negative inotropic effect depending on the actual operation mode of NCX. Selective NCX blockade may contribute to the prevention of DAD based arrhythmogenesis, in vivo, however, its effect on I/R induced arrhythmias is still uncertain.

The investigation of the cellular electrophysiological and antiarrhythmic effects of a novel selective sodium-calcium exchanger inhibitor, GYKB-6635, in canine and guinea-pig hearts.

The sodium-calcium exchanger (NCX) is considered as the major transmembrane transport mechanism that controls Ca2+ homeostasis. Its contribution to the cardiac repolarization has not yet been directly studied due to lack of specific inhibitors, so that an urgent need for more selective compounds. In this study, the electrophysiological effects of GYKB-6635, a novel NCX inhibitor, on the NCX, L-type calcium, and main repolarizing potassium currents as well as action potential (AP) parameters were investigated. Ion currents and AP recordings were investigated by applying the whole-cell patch clamp and standard microelectrode techniques in canine heart at 37 °C. Effects of GYKB-6635 were studied in ouabain-induced arrhythmias in isolated guinea-pig hearts. At a concentration of 1 µmol/L, GYKB significantly reduced both the inward and outward NCX currents (57% and 58%, respectively). Even at a high concentration (10 µmol/L), GYKB-6635 did not change the ICaL, the maximum rate of depolarization (dV/dtmax), the main repolarizing K+ currents, and the main AP parameters. GYKB-6635 pre-treatment significantly delayed the time to the development of ventricular fibrillation (by about 18%). It is concluded that GYKB-6635 is a potent and highly selective inhibitor of the cardiac NCX and, in addition, it is suggested to also contribute to the prevention of DAD-based arrhythmias.

A novel transgenic rabbit model with reduced repolarization reserve: long QT syndrome caused by a dominant-negative mutation of the KCNE1 gene.

BACKGROUND AND PURPOSE: The reliable assessment of proarrhythmic risk of compounds under development remains an elusive goal. Current safety guidelines focus on the effects of blocking the KCNH2/HERG ion channel-in tissues and animals with intact repolarization. Novel models with better predictive value are needed that more closely reflect the conditions in patients with cardiac remodelling and reduced repolarization reserve. EXPERIMENTAL APPROACH: We have developed a model for the long QT syndrome type-5 in rabbits (LQT5 ) with cardiac-specific overexpression of a mutant (G52R) KCNE1 ß-subunit of the channel that carries the slow delayed-rectifier K(+) -current (IKs ). ECG parameters, including short-term variability of the QT interval (STVQT ), a biomarker for proarrhythmic risk, and arrhythmia development were recorded. In vivo, arrhythmia susceptibility was evaluated by i.v. administration of the IKr blocker dofetilide. K(+) currents were measured with the patch-clamp technique. KEY RESULTS: Patch-clamp studies in ventricular myocytes isolated from LQT5 rabbits revealed accelerated IKs and IKr deactivation kinetics. At baseline, LQT5 animals exhibited slightly but significantly prolonged heart-rate corrected QT index (QTi) and increased STVQT . Dofetilide provoked Torsade-de-Pointes arrhythmia in a greater proportion of LQT5 rabbits, paralleled by a further increase in STVQT . CONCLUSION AND IMPLICATIONS: We have created a novel transgenic LQT5 rabbit model with increased susceptibility to drug-induced arrhythmias that may represent a useful model for testing proarrhythmic potential and for investigations of the mechanisms underlying arrhythmias and sudden cardiac death due to repolarization disturbances.

Identification and functional characterisation of a novel KCNJ2 mutation, Val302del, causing Andersen-Tawil syndrome.

Loss-of-function mutations of the KCNJ2 gene encoding for the inward rectifier potassium channel subunit Kir2.1 cause Andersen-Tawil Syndrome (ATS), a rare genetic disorder characterised by periodic paralysis, ventricular arrhythmias, and dysmorphic features. Clinical manifestations of the disease appear to vary greatly with the nature of mutation, therefore, functional characterisation of ATS-causing mutations is of clinical importance. In this study, we describe the identification and functional analysis of a novel KCNJ2 mutation, Val302del, identified in a patient with ATS. Heterologously expressed wild type (WT) and Val302del mutant alleles showed similar subcellular distribution of the Kir2.1 protein with high intensity labelling from the membrane region, demonstrating normal membrane trafficking of the Val302del Kir2.1 variant. Cells transfected with the WT allele displayed a robust current with strong inward rectification, while no current above background was detected in cells expressing the Val302del Kir2.1 subunit. Co-transfection of CHO cells with the WT and the Val302del Kir2.1 revealed a dose-dependent inhibitory effect of the Val302del Kir2.1 mutant subunit on WT Kir2.1 currents. These observations indicate that the WT and the Val302del mutant subunits co-assemble in the cell membrane and that the mutation affects potassium conductivity and (or) gating of the WT/Val302del heteromeric Kir2.1 channels.

Selective Na(+) /Ca(2+) exchanger inhibition prevents Ca(2+) overload-induced triggered arrhythmias.

BACKGROUND AND PURPOSE: Augmented Na(+) /Ca(2+) exchanger (NCX) activity may play a crucial role in cardiac arrhythmogenesis; however, data regarding the anti-arrhythmic efficacy of NCX inhibition are debatable. Feasible explanations could be the unsatisfactory selectivity of NCX inhibitors and/or the dependence of the experimental model on the degree of Ca(2+) i overload. Hence, we used NCX inhibitors SEA0400 and the more selective ORM10103 to evaluate the efficacy of NCX inhibition against arrhythmogenic Ca(2+) i rise in conditions when [Ca(2+) ]i was augmented via activation of the late sodium current (INaL ) or inhibition of the Na(+) /K(+) pump. EXPERIMENTAL APPROACH: Action potentials (APs) were recorded from canine papillary muscles and Purkinje fibres by microelectrodes. NCX current (INCX ) was determined in ventricular cardiomyocytes utilizing the whole-cell patch clamp technique. Ca(2+) i transients (CaTs) were monitored with a Ca(2+) -sensitive fluorescent dye, Fluo-4. KEY RESULTS: Enhanced INaL increased the Ca(2+) load and AP duration (APD). SEA0400 and ORM10103 suppressed INCX and prevented/reversed the anemone toxin II (ATX-II)-induced [Ca(2+) ]i rise without influencing APD, CaT or cell shortening, or affecting the ATX-II-induced increased APD. ORM10103 significantly decreased the number of strophanthidin-induced spontaneous diastolic Ca(2+) release events; however, SEA0400 failed to restrict the veratridine-induced augmentation in Purkinje-ventricle APD dispersion. CONCLUSIONS AND IMPLICATIONS: Selective NCX inhibition - presumably by blocking rev INCX (reverse mode NCX current) - is effective against arrhythmogenesis caused by [Na(+) ]i -induced [Ca(2+) ]i elevation, without influencing the AP waveform. Therefore, selective INCX inhibition, by significantly reducing the arrhythmogenic trigger activity caused by the perturbed Ca(2+) i handling, should be considered as a promising anti-arrhythmic therapeutic strategy.

Ionic mechanisms limiting cardiac repolarization reserve in humans compared to dogs.

The species-specific determinants of repolarization are poorly understood. This study compared the contribution of various currents to cardiac repolarization in canine and human ventricle. Conventional microelectrode, whole-cell patch-clamp, molecular biological and mathematical modelling techniques were used. Selective IKr block (50-100 nmol l(-1) dofetilide) lengthened AP duration at 90% of repolarization (APD90) >3-fold more in human than dog, suggesting smaller repolarization reserve in humans. Selective IK1 block (10 µmol l(-1) BaCl2) and IKs block (1 µmol l(-1) HMR-1556) increased APD90 more in canine than human right ventricular papillary muscle. Ion current measurements in isolated cardiomyocytes showed that IK1 and IKs densities were 3- and 4.5-fold larger in dogs than humans, respectively. IKr density and kinetics were similar in human versus dog. ICa and Ito were respectively ~30% larger and ~29% smaller in human, and Na(+)-Ca(2+) exchange current was comparable. Cardiac mRNA levels for the main IK1 ion channel subunit Kir2.1 and the IKs accessory subunit minK were significantly lower, but mRNA expression of ERG and KvLQT1 (IKr and IKs α-subunits) were not significantly different, in human versus dog. Immunostaining suggested lower Kir2.1 and minK, and higher KvLQT1 protein expression in human versus canine cardiomyocytes. IK1 and IKs inhibition increased the APD-prolonging effect of IKr block more in dog (by 56% and 49%, respectively) than human (34 and 16%), indicating that both currents contribute to increased repolarization reserve in the dog. A mathematical model incorporating observed human-canine ion current differences confirmed the role of IK1 and IKs in repolarization reserve differences. Thus, humans show greater repolarization-delaying effects of IKr block than dogs, because of lower repolarization reserve contributions from IK1 and IKs, emphasizing species-specific determinants of repolarization and the limitations of animal models for human disease.