Mechano-electrical interactions and heterogeneities in wild-type and drug-induced long QT syndrome rabbits.

Electromechanical reciprocity - comprising electro-mechanical (EMC) and mechano-electric coupling (MEC) - provides cardiac adaptation to changing physiological demands. Understanding electromechanical reciprocity and its impact on function and heterogeneity in pathological conditions - such as (drug-induced) acquired long QT syndrome (aLQTS) - might lead to novel insights in arrhythmogenesis. Our aim is to investigate how electrical changes impact on mechanical function (EMC) and vice versa (MEC) under physiological conditions and in aLQTS. To measure regional differences in EMC and MEC in vivo, we used tissue phase mapping cardiac MRI and a 24-lead ECG vest in healthy (control) and IKr -blocker E-4031-induced aLQTS rabbit hearts. MEC was studied in vivo by acutely increasing cardiac preload, and ex vivo by using voltage optical mapping (OM) in beating hearts at different preloads. In aLQTS, electrical repolarization (heart rate corrected RT-interval, RTn370) was prolonged compared to control (P < 0.0001) with increased spatial and temporal RT heterogeneity (P < 0.01). Changing electrical function (in aLQTS) resulted in significantly reduced diastolic mechanical function and prolonged contraction duration (EMC), causing increased apico-basal mechanical heterogeneity. Increased preload acutely prolonged RTn370 in both control and aLQTS hearts (MEC). This effect was more pronounced in aLQTS (P < 0.0001). Additionally, regional RT-dispersion increased in aLQTS. Motion-correction allowed us to determine APD-prolongation in beating aLQTS hearts, but limited motion correction accuracy upon preload-changes prevented a clear analysis of MEC ex vivo. Mechano-induced RT-prolongation and increased heterogeneity were more pronounced in aLQTS than in healthy hearts. Acute MEC effects may play an additional role in LQT-related arrhythmogenesis, warranting further mechanistic investigations. KEY POINTS: Electromechanical reciprocity comprising excitation-contraction coupling (EMC) and mechano-electric feedback loops (MEC) is essential for physiological cardiac function. Alterations in electrical and/or mechanical heterogeneity are known to have potentially pro-arrhythmic effects. In this study, we aimed to investigate how electrical changes impact on the mechanical function (EMC) and vice versa (MEC) both under physiological conditions (control) and in acquired long QT syndrome (aLQTS). We show that changing the electrical function (in aLQTS) results in significantly altered mechanical heterogeneity via EMC and, vice versa, that increasing the preload acutely prolongs repolarization duration and increases electrical heterogeneity, particularly in aLQTS as compared to control. Our results substantiate the hypothesis that LQTS is an ?electro-mechanical', rather than a 'purely electrical', disease and suggest that acute MEC effects may play an additional role in LQT-related arrhythmogenesis.

A computational model of rabbit geometry and ECG: Optimizing ventricular activation sequence and APD distribution.

Computational modeling of electrophysiological properties of the rabbit heart is a commonly used way to enhance and/or complement findings from classic lab work on single cell or tissue levels. Yet, thus far, there was no possibility to extend the scope to include the resulting body surface potentials as a way of validation or to investigate the effect of certain pathologies. Based on CT imaging, we developed the first openly available computational geometrical model not only of the whole heart but also the complete torso of the rabbit. Additionally, we fabricated a 32-lead ECG-vest to record body surface potential signals of the aforementioned rabbit. Based on the developed geometrical model and the measured signals, we then optimized the activation sequence of the ventricles, recreating the functionality of the Purkinje network, and we investigated different apico-basal and transmural gradients in action potential duration. Optimization of the activation sequence resulted in an average root mean square error between measured and simulated signal of 0.074 mV/ms for all leads. The best-fit T-Wave, compared to measured data (0.038 mV/ms), resulted from incorporating an action potential duration gradient from base to apex with a respective shortening of 20 ms and a transmural gradient with a shortening of 15 ms from endocardium to epicardium. By making our model and measured data openly available, we hope to give other researchers the opportunity to verify their research, as well as to create the possibility to investigate the impact of electrophysiological alterations on body surface signals for translational research.

Oxytocin exerts harmful cardiac repolarization prolonging effects in drug-induced LQTS.

Background: Oxytocin is used therapeutically in psychiatric patients. Many of these also receive anti-depressant or anti-psychotic drugs causing acquired long-QT-syndrome (LQTS) by blocking HERG/IKr. We previously identified an oxytocin-induced QT-prolongation in LQT2 rabbits, indicating potential harmful effects of combined therapy. We thus aimed to analyze the effects of dual therapy with oxytocin and fluoxetine/risperidone on cardiac repolarization. Methods: Effects of risperidone, fluoxetine and oxytocin on QT/QTc, short-term variability (STV) of QT, and APD were assessed in rabbits using in vivo ECG and ex vivo monophasic AP recordings in Langendorff-perfused hearts. Underlying mechanisms were assessed using patch clamp in isolated cardiomyocytes. Results: Oxytocin, fluoxetine and risperidone prolonged QTc and APD in whole hearts. The combination of fluoxetine + oxytocin resulted in further QTc- and APD-prolongation, risperidone + oxytocin tended to increase QTc and APD compared to monotherapy. Temporal QT instability, STVQTc was increased by oxytocin, fluoxetine / fluoxetine + oxytocin and risperidone / risperidone + oxytocin. Similar APD-prolonging effects were confirmed in isolated cardiomyocytes due to differential effects of the compounds on repolarizing ion currents: Oxytocin reduced IKs, fluoxetine and risperidone reduced IKr, resulting in additive effects on IKtotal-tail. In addition, oxytocin reduced IK1, further reducing the repolarization reserve. Conclusion: Oxytocin, risperidone and fluoxetine prolong QTc / APD. Combined treatment further prolongs QTc/APD due to differential effects on IKs and IK1 (block by oxytocin) and IKr (block by risperidone and fluoxetine), leading to pronounced impairment of repolarization reserve. Oxytocin should be used with caution in patients in the context of acquired LQTS.

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.

Docosahexaenoic acid normalizes QT interval in long QT type 2 transgenic rabbit models in a genotype-specific fashion.

AIM: Long QT syndrome (LQTS) is a cardiac channelopathy predisposing to ventricular arrhythmias and sudden cardiac death. Since current therapies often fail to prevent arrhythmic events in certain LQTS subtypes, new therapeutic strategies are needed. Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid, which enhances the repolarizing IKs current. METHODS AND RESULTS: We investigated the effects of DHA in wild type (WT) and transgenic long QT Type 1 (LQT1; loss of IKs), LQT2 (loss of IKr), LQT5 (reduction of IKs), and LQT2-5 (loss of IKr and reduction of IKs) rabbits. In vivo ECGs were recorded at baseline and after 10 µM/kg DHA to assess changes in heart-rate corrected QT (QTc) and short-term variability of QT (STVQT). Ex vivo monophasic action potentials were recorded in Langendorff-perfused rabbit hearts, and action potential duration (APD75) and triangulation were assessed. Docosahexaenoic acid significantly shortened QTc in vivo only in WT and LQT2 rabbits, in which both α- and ß-subunits of IKs-conducting channels are functionally intact. In LQT2, this led to a normalization of QTc and of its short-term variability. Docosahexaenoic acid had no effect on QTc in LQT1, LQT5, and LQT2-5. Similarly, ex vivo, DHA shortened APD75 in WT and normalized it in LQT2, and additionally decreased AP triangulation in LQT2. CONCLUSIONS: Docosahexaenoic acid exerts a genotype-specific beneficial shortening/normalizing effect on QTc and APD75 and reduces pro-arrhythmia markers STVQT and AP triangulation through activation of IKs in LQT2 rabbits but has no effects if either α- or ß-subunits to IKs are functionally impaired. Docosahexaenoic acid could represent a new genotype-specific therapy in LQT2.

Transgenic rabbit models for cardiac disease research.

To study the pathophysiology of human cardiac diseases and to develop novel treatment strategies, complex interactions of cardiac cells on cellular, tissue and on level of the whole heart need to be considered. As in vitro cell-based models do not depict the complexity of the human heart, animal models are used to obtain insights that can be translated to human diseases. Mice are the most commonly used animals in cardiac research. However, differences in electrophysiological and mechanical cardiac function and a different composition of electrical and contractile proteins limit the transferability of the knowledge gained. Moreover, the small heart size and fast heart rate are major disadvantages. In contrast to rodents, electrophysiological, mechanical and structural cardiac characteristics of rabbits resemble the human heart more closely, making them particularly suitable as an animal model for cardiac disease research. In this review, various methodological approaches for the generation of transgenic rabbits for cardiac disease research, such as pronuclear microinjection, the sleeping beauty transposon system and novel genome-editing methods (ZFN and CRISPR/Cas9)will be discussed. In the second section, we will introduce the different currently available transgenic rabbit models for monogenic cardiac diseases (such as long QT syndrome, short-QT syndrome and hypertrophic cardiomyopathy) in detail, especially in regard to their utility to increase the understanding of pathophysiological disease mechanisms and novel treatment options. LINKED ARTICLES: This article is part of a themed issue on Preclinical Models for Cardiovascular disease research (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.5/issuetoc.

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.

Transgenic Rabbit Models in Proarrhythmia Research.

Drug-induced proarrhythmia constitutes a potentially lethal side effect of various drugs. Most often, this proarrhythmia is mechanistically linked to the drug's potential to interact with repolarizing cardiac ion channels causing a prolongation of the QT interval in the ECG. Despite sophisticated screening approaches during drug development, reliable prediction of proarrhythmia remains very challenging. Although drug-induced long-QT-related proarrhythmia is often favored by conditions or diseases that impair the individual's repolarization reserve, most cellular, tissue, and whole animal model systems used for drug safety screening are based on normal, healthy models. In recent years, several transgenic rabbit models for different types of long QT syndromes (LQTS) with differences in the extent of impairment in repolarization reserve have been generated. These might be useful for screening/prediction of a drug's potential for long-QT-related proarrhythmia, particularly as different repolarizing cardiac ion channels are impaired in the different models. In this review, we summarize the electrophysiological characteristics of the available transgenic LQTS rabbit models, and the pharmacological proof-of-principle studies that have been performed with these models-highlighting the advantages and disadvantages of LQTS models for proarrhythmia research. In the end, we give an outlook on potential future directions and novel models.

Transgenic LQT2, LQT5, and LQT2-5 rabbit models with decreased repolarisation reserve for prediction of drug-induced ventricular arrhythmias.

BACKGROUND AND PURPOSE: Reliable prediction of pro-arrhythmic side effects of novel drug candidates is still a major challenge. Although drug-induced pro-arrhythmia occurs primarily in patients with pre-existing repolarisation disturbances, healthy animals are employed for pro-arrhythmia testing. To improve current safety screening, transgenic long QT (LQTS) rabbit models with impaired repolarisation reserve were generated by overexpressing loss-of-function mutations of human HERG (HERG-G628S, loss of IKr ; LQT2), KCNE1 (KCNE1-G52R, decreased IKs ; LQT5), or both transgenes (LQT2-5) in the heart. EXPERIMENTAL APPROACH: Effects of K+ channel blockers on cardiac repolarisation and arrhythmia susceptibility were assessed in healthy wild-type (WT) and LQTS rabbits using in vivo ECG and ex vivo monophasic action potential and ECG recordings in Langendorff-perfused hearts. KEY RESULTS: LQTS models reflect patients with clinically "silent" (LQT5) or "manifest" (LQT2 and LQT2-5) impairment in cardiac repolarisation reserve: they were more sensitive in detecting IKr -blocking (LQT5) or IK1 /IKs -blocking (LQT2 and LQT2-5) properties of drugs compared to healthy WT animals. Impaired QT-shortening capacity at fast heart rates was observed due to disturbed IKs function in LQT5 and LQT2-5. Importantly, LQTS models exhibited higher incidence, longer duration, and more malignant types of ex vivo arrhythmias than WT. CONCLUSION AND IMPLICATIONS: LQTS models represent patients with reduced repolarisation reserve due to different pathomechanisms. As they demonstrate increased sensitivity to different specific ion channel blockers (IKr blockade in LQT5 and IK1 and IKs blockade in LQT2 and LQT2-5), their combined use could provide more reliable and more thorough prediction of (multichannel-based) pro-arrhythmic potential of novel drug candidates.

Comparison of the effects of IK,ACh, IKr, and INa block in conscious dogs with atrial fibrillation and on action potentials in remodeled atrial trabeculae.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and a major cause of morbidity and mortality. Traditional antiarrhythmic agents used for restoration of sinus rhythm have limited efficacy in long-term AF and they may possess ventricular proarrhythmic adverse effects, especially in patients with structural heart disease. The acetylcholine receptor-activated potassium channel (IK,ACh) represents an atrial selective target for future AF management. We investigated the effects of the IK,ACh blocker tertiapin-Q (TQ), a derivative of the honeybee toxin tertiapin, on chronic atrial tachypacing-induced AF in conscious dogs, without the influence of anesthetics that modulate a number of cardiac ion channels. Action potentials (APs) were recorded from right atrial trabeculae isolated from dogs with AF. TQ significantly and dose-dependently reduced AF incidence and AF episode duration, prolonged atrial effective refractory period, and prolonged AP duration. The reference drugs propafenone and dofetilide, both used in the clinical management of AF, exerted similar effects against AF in vivo. Dofetilide prolonged atrial AP duration, whereas propafenone increased atrial conduction time. TQ and propafenone did not affect the QT interval, whereas dofetilide prolonged the QT interval. Our results show that inhibition of IK,ACh may represent a novel, atrial-specific target for the management of AF in chronic AF.

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.

Combined inhibition of key potassium currents has different effects on cardiac repolarization reserve and arrhythmia susceptibility in dogs and rabbits.

A reliable assessment of the pro-arrhythmic potential for drugs in the development phase remains elusive. Rabbits and dogs are commonly used to create models of pro-arrhythmia, but the differences between them with respect to repolarizing potassium currents are poorly understood. We investigated the incidence of drug-induced torsades de pointes (TdP) and measured conventional ECG parameters and the short-term variability of the QT interval (STVQT) following combined pharmacological inhibition of IK1+IKs and IK1+IKr in conscious dogs and anesthetized rabbits. A high incidence of TdP was observed following the combined inhibition of IK1+IKs in dogs (67% vs. 14% in rabbits). Rabbits exhibited higher TdP incidence after inhibition of IK1+IKr (72% vs. 14% in dogs). Increased TdP incidence was associated with significantly larger STVQT in both models. The relatively different roles of IK1 and IKs in dog and rabbit repolarization reserve should be taken into account when extrapolating the results from animal models of pro-arrhythmia to humans. A stronger repolarization reserve in dogs (likely due to stronger IK1 and IKs), and the more human-like susceptibility to arrhythmia of rabbits argues for the preferred use of rabbits in the evaluation of adverse pro-arrhythmic effects.

Mechanisms of ventricular rate adaptation as a predictor of arrhythmic risk.

Protracted QT interval (QTI) adaptation to abrupt heart rate (HR) changes has been identified as a clinical arrhythmic risk marker. This study investigates the ionic mechanisms of QTI rate adaptation and its relationship to arrhythmic risk. Computer simulations and experimental recordings in human and canine ventricular tissue were used to investigate the ionic basis of QTI and action potential duration (APD) to abrupt changes in HR with a protocol commonly used in clinical studies. The time for 90% QTI adaptation is 3.5 min in simulations, in agreement with experimental and clinical data in humans. APD adaptation follows similar dynamics, being faster in mid-myocardial cells (2.5 min) than in endocardial and epicardial cells (3.5 min). Both QTI and APD adapt in two phases following an abrupt HR change: a fast initial phase with time constant < 30 s, mainly related to L-type calcium and slow-delayed rectifier potassium current, and a second slow phase of >2 min driven by intracellular sodium concentration ([Na(+)](i)) dynamics. Alterations in [Na(+)](i) dynamics due to Na(+)/K(+) pump current inhibition result in protracted rate adaptation and are associated with increased proarrhythmic risk, as indicated by action potential triangulation and faster L-type calcium current recovery from inactivation, leading to the formation of early afterdepolarizations. In conclusion, this study suggests that protracted QTI adaptation could be an indicator of altered [Na(+)](i) dynamics following Na(+)/K(+) pump inhibition as it occurs in patients with ischemia or heart failure. An increased risk of cardiac arrhythmias in patients with protracted rate adaptation may be due to an increased risk of early after-depolarization formation.