Generation Of Early Afterdepolarizations Research Articles

Dynamical mechanism for generation of arrhythmogenic early afterdepolarizations in cardiac myocytes: Insights from in silico electrophysiological models.

We analyze the dynamical mechanisms underlying the formation of arrhythmogenic early afterdepolarizations (EADs) in two mathematical models of cardiac cellular electrophysiology: the Sato etal. biophysically detailed model of a rabbit ventricular myocyte of dimension 27 and a reduced version of the Luo-Rudy mammalian myocyte model of dimension 3. Based on a comparison of the two models, with detailed bifurcation analysis using spike-counting techniques and continuation methods in the simple model and numerical explorations in the complex model, we locate the point where the first EAD originates in an unstable branch of periodic orbits. These results serve as a basis to propose a conjectured scheme involving a hysteresis mechanism with the creation of alternans and EADs in the unstable branch. This theoretical scheme fits well with electrophysiological experimental data on EAD generation and hysteresis phenomena. Our findings open the door to the development of novel methods for pro-arrhythmia risk prediction related to EAD generation without actual induction of EADs.

Bifurcation Analysis on a Generation of Early Afterdepolarization in a Mathematical Cardiac Model

Electrical activity occurs in the cell membrane of cardiomyocytes. This electrical activity forms the action potential that generates pumping of the heart. An abnormality in the action potential turns into arrhythmia, which may cause sudden death. Studies of arrhythmias using mathematical models are important to reduce the risk of sudden death. In this study, we investigate bifurcations related to the generation of early afterdepolarizations (EADs) in a mathematical model. We clarify the transition process from a normal state to a persistent EAD through a transient EAD while changing only one parameter (multiple of conductance of L-type calcium channel current) value. The dependence of the transient EAD generation on parameters is shown through bifurcation analysis in a [Na]i-parameterized system.

KEY FACTORS TO THE RATE DEPENDENCE OF EARLY AFTERDEPOLARIZATIONS: A SIMULATION STUDY

Early afterdepolarizations (EADs) in cardiac myocytes have been reported to be associated with a series of cardiac arrhythmias. The generation of EADs has a high correlation with the excitation period, leading to repolarization dispersion over the cardiac tissue. However, the mechanism of EAD rate dependence has not been thoroughly revealed. In this study, the simulation approach was used to investigate the mechanism underlying EAD rate dependence. The results indicated that the gating variable of the delayed rectifier potassium current ([Formula: see text]-gate) and the intracellular sodium ion concentration ([Na[Formula: see text]][Formula: see text] were key factors contributing to EAD rate dependence. Also, different mathematical models showed different types of EAD rate dependence, which needs to be considered in the future simulation research related to EADs.

Inhibition of cardiac potassium currents by oxidation-activated protein kinase A contributes to early afterdepolarizations in the heart.

Reactive oxygen species (ROS) have been shown to prolong cardiac action potential duration resulting in afterdepolarizations, the cellular basis of triggered arrhythmias. As previously shown, protein kinase A type I (PKA I) is readily activated by oxidation of its regulatory subunits. However, the relevance of this mechanism of activation for cardiac pathophysiology is still elusive. In this study, we investigated the effects of oxidation-activated PKA I on cardiac electrophysiology. Ventricular cardiomyocytes were isolated from redox-dead PKA-RI Cys17Ser knock-in (KI) and wild-type (WT) mice and exposed to H2O2 (200 µmol/L) or vehicle (Veh) solution. In WT myocytes, exposure to H2O2 significantly increased oxidation of the regulatory subunit I (RI) and thus its dimerization (threefold increase in PKA RI dimer). Whole cell current clamp and voltage clamp were used to measure cardiac action potentials (APs), transient outward potassium current (Ito) and inward rectifying potassium current (IK1), respectively. In WT myocytes, H2O2 exposure significantly prolonged AP duration due to significantly decreased Ito and IK1 resulting in frequent early afterdepolarizations (EADs). Preincubation with the PKA-specific inhibitor Rp-8-Br-cAMPS (10 µmol/L) completely abolished the H2O2-dependent decrease in Ito and IK1 in WT myocytes. Intriguingly, H2O2 exposure did not prolong AP duration, nor did it decrease Ito, and only slightly enhanced EAD frequency in KI myocytes. Treatment of WT and KI cardiomyocytes with the late INa inhibitor TTX (1 µmol/L) completely abolished EAD formation. Our results suggest that redox-activated PKA may be important for H2O2-dependent arrhythmias and could be important for the development of specific antiarrhythmic drugs.NEW & NOTEWORTHY Oxidation-activated PKA type I inhibits transient outward potassium current (Ito) and inward rectifying potassium current (IK1) and contributes to ROS-induced APD prolongation as well as generation of early afterdepolarizations in murine ventricular cardiomyocytes.

Mathematical Analysis of the Role of Heterogeneous Distribution of Excitable and Non-excitable Cells on Early Afterdepolarizations

Early afterdepolarizations (EADs) are abnormal oscillations during the plateau phase of the cardiac action potential and have been linked to cardiac arrhythmias. At the cellular level, EADs can be caused by reactivation of the L-type calcium (Ca2+) channels, spontaneous Ca2+ releases from the sarcoplasmic reticulum, or both. In tissue, these EADs can trigger action potentials in neighboring cells, which may propagate as a nonlinear wave. In this scenario, EADs are attributed to cellular/subcellular/channel properties. In this study, we show a novel mechanism of EADs due to heterogeneous distribution of excitable and non-excitable cells in tissue, using a physiologically detailed computational model and mathematical analysis. In tissue, excitability of cells depends on the cell type and physiological and pathological conditions. Non-excitable cells create a non-excitable gap in tissue, which has been thought to be a cause of slow waves and reflected waves. Here, we show that the non-excitable gap also can be responsible for EAD generation. However, EADs occur only when the non-excitable gap size is optimal. If the gap size is too small, no EADs occur. If the gap size is too large, the action potential wave cannot propagate through the gap region. We also demonstrate that EADs caused by the non-excitable gap can initiate reentry in tissue, which has been linked to ventricular tachycardia and fibrillation. Thus, the non-excitable gap can lead to both focal and reentrant arrhythmias. EADs shown in this study are spatial phenomena and require tissue heterogeneity. Our study sheds light on the role of tissue heterogeneity on focal and reentrant arrhythmias.

Distinct Occurrence of Proarrhythmic Afterdepolarizations in Atrial Versus Ventricular Cardiomyocytes: Implications for Translational Research on Atrial Arrhythmia.

Background: Principal mechanisms of arrhythmia have been derived from ventricular but not atrial cardiomyocytes of animal models despite higher prevalence of atrial arrhythmia (e.g., atrial fibrillation). Due to significant ultrastructural and functional differences, a simple transfer of ventricular proneness toward arrhythmia to atrial arrhythmia is critical. The use of murine models in arrhythmia research is widespread, despite known translational limitations. We here directly compare atrial and ventricular mechanisms of arrhythmia to identify critical differences that should be considered in murine models for development of antiarrhythmic strategies for atrial arrhythmia.Methods and Results: Isolated murine atrial and ventricular myocytes were analyzed by wide field microscopy and subjected to a proarrhythmic protocol during patch-clamp experiments. As expected, the spindle shaped atrial myocytes showed decreased cell area and membrane capacitance compared to the rectangular shaped ventricular myocytes. Though delayed afterdepolarizations (DADs) could be evoked in a similar fraction of both cell types (80% of cells each), these led significantly more often to the occurrence of spontaneous action potentials (sAPs) in ventricular myocytes. Interestingly, numerous early afterdepolarizations (EADs) were observed in the majority of ventricular myocytes, but there was no EAD in any atrial myocyte (EADs per cell; atrial myocytes: 0 ± 0; n = 25/12 animals; ventricular myocytes: 1.5 [0–43]; n = 20/12 animals; p < 0.05). At the same time, the action potential duration to 90% decay (APD90) was unaltered and the APD50 even increased in atrial versus ventricular myocytes. However, the depolarizing L-type Ca2+ current (ICa) and Na+/Ca2+-exchanger inward current (INCX) were significantly smaller in atrial versus ventricular myocytes.Conclusion: In mice, atrial myocytes exhibit a substantially distinct occurrence of proarrhythmic afterdepolarizations compared to ventricular myocytes, since they are in a similar manner susceptible to DADs but interestingly seem to be protected against EADs and show less sAPs. Key factors in the generation of EADs like ICa and INCX were significantly reduced in atrial versus ventricular myocytes, which may offer a mechanistic explanation for the observed protection against EADs. These findings may be of relevance for current studies on atrial level in murine models to develop targeted strategies for the treatment of atrial arrhythmia.

Intracellular angiotensin-(1-12) changes the electrical properties of intact cardiac muscle.

In the present work, the influence of intracellular injection of angiotensin-(1-12) [Ang-(1-12)] on the electrical properties of the intact left ventricle of Wistar Kyoto rats was investigated with electrophysiological methods. Particular attention was given to the role of chymostatin on the effect of the peptide. The results indicated that intracellular administration of the peptide elicited a depolarization of the surface cell membrane and an increase of duration of the action potential followed by the generation of early afterdepolarizations. The increment of action potential duration caused by Ang-(1-12) (100nM) was due to a decrease of total potassium current recorded from single cardiomyocytes using the whole cell configuration of pCAMP. The decrease of potassium current was related to the activation of protein kinase C (PKC) because the specific inhibitor of kinase C, Bis-1 (10-9M), abolished Ang-(1-12) effects on the potassium current. The question of whether the effect of Ang-(1-12) was related to the formation of Ang II by chymase was investigated.The results revealed that the intracellular administration of chymostatin, a chymase inhibitor (10-9M) abolished the effect of intracellular Ang-(1-12) on the potassium current. Moreover, intracellular Ang II (100nM), by itself, reduced the potassium current, an effect decreased by intracellular valsartan (100nM). Valsartan (10-9M) dialyzed into the cell abolished the effect of Ang-(1-12) (100nM). These observations demonstrate that the effect of Ang-(1-12) on potassium current was related to the formation of Ang II and that the peptide has arrhythmogenic properties.

Electrophysiological properties of iPS cell-derived cardiomyocytes from a patient with long QT syndrome type 1 harboring the novel mutation M437V of KCNQ1.

IntroductionLong QT syndrome type 1 (LQT1) is caused by mutations in KCNQ1 coding slowly-activating delayed-rectifier K+ channels. We identified the novel missense mutation M437V of KCNQ1 in a LQT1 patient. Here, we employed iPS cell (iPSC)-derived cardiomyocytes to investigate electrophysiological properties of the mutant channel and LQT1 cardiomyocytes.MethodsTo generate iPSCs from the patient and a healthy subject, peripheral blood T cells were reprogrammed by Sendai virus vector encoding human OCT3/4, SOX2, KLF4, and c-MYC. Cardiomyocytes were prepared from iPSCs and human embryonic stem cells using a cytokine-based two-step differentiation method and were subjected to patch clamp experiments.ResultsLQT1 iPSC-derived cardiomyocytes exhibited prolongation of action potential duration (APD), which was due to a reduction of the KCNQ1-mediated current IKs; Na+, Ca2+ and other K+ channel currents were comparable. When expressed in HEK293 and COS7 cells, the mutant KCNQ1 was normally expressed in the plasma membrane but generated smaller currents than the wild type. Isoproterenol significantly prolonged APDs of LQT1 cardiomyocytes, while shortening those of healthy ones. A mathematical model for IKs-reduced human ventricular myocytes reproduced APD prolongation and generation of early afterdepolarizations (EADs) under β-adrenergic stimulation.ConclusionsQT prolongation of the LQT1 patient with the mutation M437V of KCNQ1 was caused by IKs reduction, which may render the patient vulnerable to generation of EADs and arrhythmias.

Molecular Basis of Hypokalemia-Induced Ventricular Fibrillation.

Hypokalemia is known to promote ventricular arrhythmias, especially in combination with class III antiarrhythmic drugs like dofetilide. Here, we evaluated the underlying molecular mechanisms. Arrhythmias were recorded in isolated rabbit and rat hearts or patch-clamped ventricular myocytes exposed to hypokalemia (1.0-3.5 mmol/L) in the absence or presence of dofetilide (1 μmol/L). Spontaneous early afterdepolarizations (EADs) and ventricular tachycardia/fibrillation occurred in 50% of hearts at 2.7 mmol/L [K] in the absence of dofetilide and 3.3 mmol/L [K] in its presence. Pretreatment with the Ca-calmodulin kinase II (CaMKII) inhibitor KN-93, but not its inactive analogue KN-92, abolished EADs and hypokalemia-induced ventricular tachycardia/fibrillation, as did the selective late Na current (INa) blocker GS-967. In intact hearts, moderate hypokalemia (2.7 mmol/L) significantly increased tissue CaMKII activity. Computer modeling revealed that EAD generation by hypokalemia (with or without dofetilide) required Na-K pump inhibition to induce intracellular Na and Ca overload with consequent CaMKII activation enhancing late INa and the L-type Ca current. K current suppression by hypokalemia and dofetilide alone in the absence of CaMKII activation were ineffective at causing EADs. We conclude that Na-K pump inhibition by even moderate hypokalemia plays a critical role in promoting EAD-mediated arrhythmias by inducing a positive feedback cycle activating CaMKII and enhancing late INa. Class III antiarrhythmic drugs like dofetilide sensitize the heart to this positive feedback loop.

Mechanistic Investigation of the Arrhythmogenic Role of Oxidized CaMKII in the Heart

Oxidative stress and calcium (Ca2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) both play important roles in the pathogenesis of cardiac disease. Although the pathophysiological relevance of reactive oxygen species (ROS) and CaMKII has been appreciated for some time, recent work has shown that ROS can directly oxidize CaMKII, leading to its persistent activity and an increase of the likelihood of cellular arrhythmias such as early afterdepolarizations (EADs). Because CaMKII modulates the function of many proteins involved in excitation-contraction coupling, elucidation of its role in cardiac function, in both healthy and oxidative stress conditions, is challenging. To investigate this role, we have developed a model of CaMKII activation that includes both the phosphorylation-dependent and the newly identified oxidation-dependent activation pathways. This model is incorporated into our previous local-control model of the cardiac myocyte that describes excitation-contraction coupling via stochastic simulation of individual Ca2+ release units and CaMKII-mediated phosphorylation of L-type Ca2+ channels (LCCs), ryanodine receptors and sodium (Na+) channels. The model predicts the experimentally measured slow-rate dependence of H2O2-induced EADs. Upon increased H2O2, simulations suggest that selective activation of late Na+ current (INaL), although it prolongs action potential duration, is not by itself sufficient to produce EADs. Similar results are obtained if CaMKII effects on LCCs and ryanodine receptors are considered separately. However, EADs emerge upon simultaneous activation of both LCCs and Na+ channels. Further modeling results implicate activation of the Na+-Ca2+ exchanger (NCX) as an important player in the generation of EADs. During bradycardia, the emergence of H2O2-induced EADs was correlated with a shift in the timing of NCX current reversal toward the plateau phase earlier in the action potential. Using the timing of NCX current reversal as an indicator event for EADs, the model identified counterintuitive ionic changes—difficult to experimentally dissect—that have the greatest influence on ROS-related arrhythmia propensity.

Cytosolic calcium changes affect the incidence of early afterdepolarizations in canine ventricular myocytes.

This study was designed to investigate the influence of cytosolic Ca(2+) levels ([Ca(2+)]i) on action potential duration (APD) and on the incidence of early afterdepolarizations (EADs) in canine ventricular cardiomyocytes. Action potentials (AP) of isolated cells were recorded using conventional sharp microelectrodes, and the concomitant [Ca(2+)]i was monitored with the fluorescent dye Fura-2. EADs were evoked at a 0.2 Hz pacing rate by inhibiting the rapid delayed rectifier K(+) current with dofetilide, by activating the late sodium current with veratridine, or by activating the L-type calcium current with BAY K8644. These interventions progressively prolonged the AP and resulted in initiation of EADs. Reducing [Ca(2+)]i by application of the cell-permeant Ca(2+) chelator BAPTA-AM lengthened the AP at 1.0 Hz if it was applied alone, in the presence of veratridine, or in the presence of BAY K8644. However, BAPTA-AM shortened the AP if the cells were pretreated with dofetilide. The incidence of the evoked EADs was strongly reduced by BAPTA-AM in dofetilide, moderately reduced in veratridine, whereas EAD incidence was increased by BAPTA-AM in the presence of BAY K8644. Based on these experimental data, changes in [Ca(2+)]i have marked effects on APD as well as on the incidence of EADs; however, the underlying mechanisms may be different, depending on the mechanism of EAD generation. As a consequence, reduction of [Ca(2+)]i may eliminate EADs under some, but not all, experimental conditions.

Abstract 18560: Defective Ca 2 + Homeostasis Contributes to Arrhythmia in Transgenic Rabbit Model of LQT2 Syndrome

Introduction: Loss-of function mutations in HERG potassium channels in Long QT2 syndrome are associated with sudden cardiac death triggered by sympathetic surge. In various models of cardiac disease changes in calcium (Ca) homeostasis, associated with upregulated plasmalemmal Na/Ca exchanger (NCX) and/or hyperactive ryanodine receptors (RyRs), have been linked to increased risk of arrhythmia. Methods and Results: The aims of this study were to investigate possible changes in Ca homeostasis in cardiomyocytes derived from from the hearts of LQT2 rabbits and to determine whether similar changes contribute to the triggering of early afterdepolarizations (EADs) characteristic of LQT2 myocytes under β-adrenergic stimulation. Parameters of Ca handling were measured in ventricular myocytes enzymatically isolated from LQT2 and littermate control hearts using confocal Ca imaging and whole cell patch clamp. Ca imaging revealed no changes in amplitude of Ca transients in intact myocytes and sarcoplasmic reticulum (SR) Ca content assessed by measuring amplitude of transients evoked by rapid application of caffeine. A two-fold increase in rate of decay of caffeine-induced Ca transients in LQT2 myocytes vs. controls indicated increased NCX activity. Experiments in saponin-permeabilized cells using a SR-entrapped low-affinity Ca indicator revealed ~2-fold increase in RyR-mediated SR Ca leak in LQT2 myocytes in comparison with controls. In contrast to controls, current-clamped LQT2 myocytes exposed to β-adrenergic agonist isoproterenol exhibited prolonged plateau of action potentials (AP) at -10 - 0 mV accompanied by aberrant Ca releases and phase 3 EADs. Importantly, complete exhaustion of SR [Ca] by incubation of LQT2 cells with SERCA inhibitor thapsigargin or 10 mM caffeine, shortened APD and completely abolished EADs. Conclusion: Functional changes in RyR and NCX underlie preserved systolic Ca transients in LQT2 myocytes under basal conditions and may contribute to stress-induced arrhythmia by maintaining the plateau of AP at voltages ideal for reactivation of plasmalemmal Ca channels and generation of EADs.

TTX Converts Polymorphic VT to Monomorphic VT in Transgenic Rabbit Model of LQT1

Long QT syndrome Type 1 (LQT1) is a congenital disease defined by loss of function mutations in KCNQ1, and associated with syncope and sudden death preceded by polymorphic ventricular tachycardia (pVT). We hypothesized that inhibition of sodium channel function suppresses pVT formation. using a transgenic rabbit model of LQT1 (n=4), we simultaneously mapped activation patterns from a voltage sensitive dye and microelectrode. The AV node was ablated to slow intrinsic heart rate, isoproterenol (140nM) was injected to mimic sympathetic stimulation, and pVTs were subsequently observed (avg = 3-10 s). The associated activation patterns revealed multifocal activities and complex waveform propagation varying in spatial origin and direction. Subsequent perfusion of a sodium channel blocker, TTX (2 μM), notably converted pVTs toward monomorphic activation patterns. We also report evidence of fully rotating spiral waves, and their role in maintaining monomorphic VTs. Taken together, these results strongly suggest that 1) early afterdepolarizations (EADs) play an important role in maintaining pVTs complexity, 2) sodium currents significantly influence EAD regeneration by their continuous firing during pVTs, 3) sodium channel inhibition by TTX reduces EAD generation and stabilizes reentry into monomorphic VTs.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Transient receptor potential melastatin 4 inhibitor 9‐phenanthrol abolishes arrhythmias induced by hypoxia and re‐oxygenation in mouse ventricle

Hypoxia and subsequent re-oxygenation are associated with cardiac arrhythmias such as early afterdepolarizations (EADs), which may be partly explained by perturbations in cytosolic calcium concentration. Transient receptor potential melastatin 4 (TRPM4), a calcium-activated non-selective cation channel, is functionally expressed in the heart. Based on its biophysical properties, it is likely to participate in EADs. Hence, modulators of TRPM4 activity may influence arrhythmias. The aim of this study was to investigate the possible anti-arrhythmic effect of 9-phenanthrol, a TRPM4 inhibitor in a murine heart model of hypoxia and re-oxygenation-induced EADs. Mouse heart was removed, and the right ventricle was pinned in a superfusion chamber. After a period of normoxia, the preparation was superfused for 2 h with a hypoxic solution and then re-oxygenated. Spontaneous electrical activity was investigated by intracellular microelectrode recordings. In normoxic conditions, the ventricle exhibited spontaneous action potentials. Application of the hypoxia and re-oxygenation protocol unmasked hypoxia-induced EADs, the occurrence of which increased under re-oxygenation. The frequency of these EADs was reduced by superfusion with either flufenamic acid, a blocker of Ca(2+) -dependent cation channels or with 9-phenanthrol. Superfusion with 9-phenanthrol (10(-5) or 10(-4) mol·L(-1) ) caused a dramatic dose-dependent abolition of EADs. Hypoxia and re-oxygenation-induced EADs can be generated in the mouse heart model. 9-Phenanthrol abolished EADs, which strongly suggests the involvement of TRPM4 in the generation of EAD. This identifies non-selective cation channels inhibitors as new pharmacological candidates in the treatment of arrhythmias.

Cardiac Myofibroblast-Myocyte Gap Junction Coupling Promotes After Depolarizations

Tissue fibrosis, as seen in diseased and aged hearts, promotes ventricular arrhythmias. We hypothesize that de novo gap junction coupling between myofibroblasts, which proliferate following cardiac stress injury, with neighboring ventricular myocytes may facilitate arrhythmia triggers such as early afterdepolarizations (EADs). A novel hybrid computational and biological approach was employed: virtual fibroblasts with programmable properties embedded in the dynamic clamp were coupled to a real patch-clamped rabbit ventricular myocyte exposed to oxidative (0.1 mM H2O2) or ionic (2.7 mM K+o) stressors. A virtual gap junction current of programmable conductance was added in real time to myocyte currents and their combined effects on myocyte action potentials and EAD genesis were evaluated. Exposure of myocytes to basal conditions failed to induce EADs, whereas exposure to stress led to ‘bradycardia-dependent’ EADs that occurred only during slow, but not during fast, pacing. However, when stressed myocytes were then coupled to a virtual fibroblast, EADs emerged independently of pacing rates. Fibroblast coupling alone to unstressed myocytes failed to induce EADs at any pacing rate. The virtual gap junction current has 2 components, but the earlier transient outward component was most critical for EAD generation; EADs disappeared when the virtual fibroblast was uncoupled from the myocyte during the initial 100 ms of the AP, but not when uncoupled for all but the first 100 ms of the AP. Our findings demonstrate that gap junction coupling of ventricular myocytes to myofibroblasts may directly induce EADs and the probability of EAD induction correlates with the myofibroblast-myocyte gap junction coupling strength. Elucidating the mechanism of myofibroblast-induced arrhythmogenesis may suggest new therapeutic strategies for preventing ventricular arrhythmias based on inhibiting fibroblast proliferation and/or uncoupling fibroblasts from myocytes.

Torsades: adjacent and triggering electrocardiographic events

Torsades de pointes (TdP) is a particular variant of ventricular arrhythmia associated with the long QT syndrome. The background of the latter is essentially 2-fold: patients under treatment with QT-prolonging drugs and subjects with congenital ionopathies. A third category is composed of subjects with both of these backgrounds. The fundamental feature of TdP is its provocation by pause-related augmentation of the repolarizing TU wave. The substrate electrocardiogram (ECG) shows prominent U waves in regular rhythm. The exaggeration of the U wave voltage following a pause is more marked the longer the pause and, for a given pause, more marked the faster the prepause rate. The pause-related sequences figure frequently adjacent to that which actually triggers the attack of TdP and continues to be seen after the event, serving to advise the physician as to the diagnosis, even following cardiac resuscitation, so that preventive measures can be taken (pacing, intravenous magnesium sulfate, or infusion with isoproterenol). The U wave of the regular rhythm ECG may show amplitude instability: an especially tall U triggering a premature ventricular complexe (PVC) that then in turn generates a pause-related sequence. TU alternans is common. Because these patients may not be in a monitored bed, recognition of pause-related phenomena in a patient with a long QT requires the ECG reader of the day to alert the floor as to the running danger. The mechanism of the pause-related TU augmentation is the generation of early afterdepolarizations.

Calcium oscillations and T-wave lability precede ventricular arrhythmias in acquired long QT type 2

Alternans of intracellular Ca(2+) (Ca(i)) underlies T-wave alternans, a predictor of cardiac arrhythmias. A related phenomenon, T-wave lability (TWL), precedes torsades de pointes (TdP) in patients and animal models with impaired repolarization. However, the role of Ca(i) in TWL remains unexplored. This study investigated the role of Ca(i) dynamics on TWL in a noncryoablated rabbit model of long QT syndrome type 2 (LQT2) using simultaneous measurements of Ca(i) transient (CaT), action potentials (APs), and electrocardiogram (ECG) during paced rhythms and focused on events that precede ventricular ectopy. APs and CaTs were mapped optically from paced Langendorff female rabbit hearts (n = 8) at 1.2-s cycle length, after atrioventricular node ablation. Hearts were perfused with normal Tyrode solution, then with dofetilide (0.5 μM), and reduced [K(+)] (2 mM) and [Mg(2+)] (0.5 mM) to elicit LQT2. Lability of ECG, voltage, and Ca(i) signals were evaluated during regular paced rhythm, before and after dofetilide perfusion. In LQT2, lability of Ca(i), voltage, and ECG signals increased during paced rhythm, before the appearance of early afterdepolarizations (EADs). LQT2 resulted in AP prolongation and multiple (1 to 3) additional Ca(i) upstrokes, whereas APs remained monophasic. When EADs appeared, Ca(i) rose before voltage upstrokes at the origins of propagating EADs. Interventions (i.e., ryanodine and thapsigargin, n = 3 or low [Ca](o) and nifedipine, n = 4) that suppressed Ca(i) oscillations also abolished EADs. In LQT2, Ca(i) oscillations (Ca(i)O) precede EADs by minutes, indicating that they result from spontaneous sarcoplasmic reticulum Ca(2+) release rather than spontaneous I(Ca,L) reactivation. Ca(i)O likely produce oscillations of Na/Ca exchange current, I(NCX). Depolarizing I(NCX) during the AP plateau contributes to the generation of EADs by reactivating Ca(2+) channels that have recovered from inactivation. TWL reflects CaTs and APs lability that occur before EADs and TdP.

Abstract 3503: Hydrogen Peroxide-Induced Afterdepolarizations are Dependent on Calmodulin Kinase II Signaling

Background: In the heart, hydrogen peroxide (H 2 O 2 ) has been shown to cause early afterdepolarizations (EADs) and triggered activity by impairing Na current (I Na ) inactivation. Since H 2 O 2 has been recently shown to activate Ca 2+ /calmodulin kinase II (CaMKII), and since CaMKII activation has also been reported to impair I Na inactivation and predispose to EADs, we hypothesized that CaMKII activation by H 2 O 2 may be an important factor in the genesis of EADs induced by oxidative stress. Methods and Results: Patch-clamped Fluo-4 AM-loaded rabbit ventricular myocytes were exposed to H 2 O 2 (0.1–1mM), which induced spontaneous EADs after 5–15 min. Both the I Na blocker tetrodoxtin (TTX, 10 μM) and the I Ca,L blocker nifedipine shortened AP duration (APD) and suppressed EADs. H 2 O 2 increased both peak and steady-state I Ca,L under square-pulse voltage clamp, and enhanced I Ca,L to a greater extent during the AP plateau than during the AP upstroke under AP clamp conditions. In addition, by prolonging the AP plateau and increasing Ca influx via maintained I Ca,L , H 2 O 2 -induced EADs frequently caused DADs delayed afterdepolarizations (DADs) due to spontaneous SR Ca release waves after repolarization. KN-93(1 μM), a CaMKII inhibitor, prevented H 2 O 2 -induced EADs (n=4), whereas the inactive analogue KN-92 did not (n=5). Conclusion: These findings indicate that H 2 O 2 -induced EADs depend on both impaired I Na inactivation to reduce repolarization reserve and enhanced I Ca,L to reverse repolarization. Intact CaMKII signaling is necessary for EAD generation in this setting, presumably via its actions on I Na and I Ca,L , although direct redox effects on other ion channels/transporters may also be important. Our observations support a link between increased oxidative stress, CaMKII activation and afterdepolarizations as triggers of lethal ventricular arrhythmias in diseased heart. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Stochastic Aspects of Cardiac Arrhythmias

Abnormal cardiac rhythms (cardiac arrhythmias) often display complex changes over time that can have a random or haphazard appearance. Mathematically, these changes can on occasion be identified with bifurcations in difference or differential equation models of the arrhythmias. One source for the variability of these rhythms is the fluctuating environment. However, in the neighborhood of bifurcation points, the fluctuations induced by the stochastic opening and closing of individual ion channels in the cell membrane, which results in membrane noise, may lead to randomness in the observed dynamics. To illustrate this, we consider the effects of stochastic properties of ion channels on the resetting of pacemaker oscillations and on the generation of early afterdepolarizations. The comparison of the statistical properties of long records showing arrhythmias with the predictions from theoretical models should help in the identification of different mechanisms underlying cardiac arrhythmias.

Calmodulin Antagonist W‐7 Prevents Sparfloxacin‐Induced Early Afterdepolarizations (EADs) in Isolated Rabbit Purkinje Fibers: Importance of Beat‐to‐Beat Instability of the Repolarization

The occurrence of early afterdepolarizations (EADs) has been related to the incidence of torsades de pointes in drug-induced long QT (LQT). The generation of EADs may be facilitated by Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase). In the present study, we investigated a possible involvement of Ca(2+)/Calmodulin dependent protein kinase in the generation of sparfloxacin-induced EADs in isolated rabbit Purkinje fibers by means of a calmodulin antagonist W-7. EADs were evident in 8 of the 10 preparations perfused with sparfloxacin at 1 x 10(-4) M and stimulated at 0.2 Hz. The induction of EADs by sparfloxacin was associated with a large prolongation of the duration of the action potential (APD), an increase in the triangulation, and the short-term instability of the repolarization. CaM kinase blockade with the calmodulin antagonist W-7 inhibited sparfloxacin-induced EADs in a concentration-dependent manner (EADs were induced in 3 of 10, 1 of 10, and 0 of 8 preparations in the presence of W-7 at 5 x 10(-7) M, 5 x 10(-6) M, and 5 x 10(-5) M, respectively; P < 0.01 at 5 x 10(-6) M and 5 x 10(-5) M). The inhibition of sparfloxacin-induced EADs by W-7 at 5 x 10(-7) M and 5 x 10(-6) M was associated with a significant decrease in the beat-to-beat instability but not associated with a significant shortening of the APD and reduction of V(max). The present findings support the hypothesis that CaM kinase may be a proarrhythmic signaling molecule and demonstrate that CaM kinase may be involved in the generation of EADs in drug-induced LQT and enhanced beat-to-beat instability of repolarization is essential for the genesis of EADs in rabbit in vitro.