Early Afterdepolarizations Formation Research Articles

Mechanisms of fever-induced QT prolongation and torsades de pointes in patients with KCNH2 mutation.

Patients with particular mutations of type-2 long QT syndrome (LQT2) are at an increased risk for malignant arrhythmia during fever. This study aimed to determine the mechanism by which KCNH2 mutations cause fever-induced QT prolongation and torsades de pointes (TdP). We evaluated three KCNH2 mutations, G584S, D609G, and T613M, in the Kv11.1 S5-pore region, identified in patients with marked QT prolongation and TdP during fever. We also evaluated KCNH2 M124T and R269W, which are not associated with fever-induced QT prolongation. We characterized the temperature-dependent changes in the electrophysiological properties of the mutant Kv11.1 channels by patch-clamp recording and computer simulation. The average tail current densities (TCDs) at 35°C for G584S, WT+D609G, and WT+T613M were significantly smaller and less increased with rising temperature from 35°C to 40°C than those for WT, M124T, and R269W. The ratios of the TCDs at 40°C to 35°C for G584S, WT+D609G, and WT+T613M were significantly smaller than for WT, M124T, and R269W. The voltage dependence of the steady-state inactivation curve for WT, M124T, and R269W showed a significant positive shift with increasing temperature; however, that for G584S, WT+D609G, and WT+T613M showed no significant change. Computer simulation demonstrated that G584S, WT+D609G, and WT+T613M caused prolonged action potential durations and early afterdepolarization formation at 40°C. These findings indicate that KCNH2 G584S, D609G, and T613M in the S5-pore region reduce the temperature-dependent increase in TCDs through an enhanced inactivation, resulting in QT prolongation and TdP at a febrile state in patients with LQT2.

Initation of early afterdepolarizations by complex spatial dynamics in cardiac tissue.

Early afterdepolarizations (EADs) are thought to be complex depolarizing phenomena arising in the plateau phase of a cardiac action potential that can be lethal in certain conditions as they can cause arrhythmias. One possible mechanism of EADs is the reactivation of the L-type calcium channel (LTCC) in the repolarizing phase of the action potential. This reactivation of LTCC has been attributed to the cellular level properties such as increases of inward currents, decreases of outward currents, or uncharacteristically long pacing cycle lengths that promote longer action potential. In this study, we will show that geometry at the tissue level is also critical for the formation of EADs. We investigated how spatial action potential dynamics in tissue can give rise to EADs. We used a physiologically detailed model of a rabbit action potential to simulate a variety of different tissue geometries. We created 2-dimensional tissue (600×100 cells) featuring a horizontal channel of diffusive cells and a vertical source-sink gap of diffusive cells in the channel (both 2-20 cells wide). When an action potential was generated at one end of the channel, it propagates normally until colliding with the gap. At the source-sink gap, we found that the action potential exhibited three phenomena. When the size of the channel exceeded the size of the gap, the action potential continued to propagate normally. When the size of the channel was exceeded by the size of the gap, we observed conduction failure. When the size of the channel and the size of the gap were nearly the same, EADs formed in the cells right in front of the gap. Our findings underscore that tissue geometry is critical not only for the action potential wave propagation but also for the formation of EADs.

Mechanisms underlying age-associated manifestation of cardiac sodium channel gain-of-function

Cardiac action potentials are initiated by sodium ion (Na+) influx through voltage-gated Na+ channels. Na+ channel gain-of-function (GOF) can arise in inherited conditions due to mutations in the gene encoding the cardiac Na+ channel, such as Long QT syndrome type 3 (LQT3). LQT3 can be a “concealed” disease, as patients with LQT3-associated mutations can remain asymptomatic until later in life; however, arrhythmias can also arise early in life in LQT3 patients, demonstrating a complex age-associated manifestation. We and others recently demonstrated that cardiac Na+ channels preferentially localize at the intercalated disc (ID) in adult cardiac tissue, which facilitates ephaptic coupling and formation of intercellular Na+ nanodomains that regulate pro-arrhythmic early afterdepolarization (EAD) formation in tissue with Na+ channel GOF. Several properties related to ephaptic coupling vary with age, such as cell size and Na+ channel and gap junction (GJ) expression and distribution: neonatal cells have immature IDs, with Na+ channels and GJs primarily diffusively distributed, while adult myocytes have mature IDs with preferentially localized Na+ channels and GJs. Here, we perform an in silico study varying critical age-dependent parameters to investigate mechanisms underlying age-associated manifestation of Na+ channel GOF in a model of guinea pig cardiac tissue. Simulations predict that total Na+ current conductance is a critical factor in action potential duration (APD) prolongation. We find a complex cell size/ Na+ channel expression relationship: increases in cell size (without concurrent increases in Na+ channel expression) suppress EAD formation, while increases in Na+ channel expression (without concurrent increases in cell size) promotes EAD formation. Finally, simulations with neonatal and early age-associated parameters predict normal APD with minimal dependence on intercellular cleft width; however, variability in cellular properties can lead to EADs presenting in early developmental stages. In contrast, for adult-associated parameters, EAD formation is highly dependent on cleft width, consistent with a mechanism underlying the age-associated manifestation of the Na+ channel GOF.

Circadian Rhythms of Early Afterdepolarizations and Ventricular Arrhythmias in a Cardiomyocyte Model

Sudden cardiac arrest is a malfunction of the heart’s electrical system, typically caused by ventricular arrhythmias, that can lead to sudden cardiac death (SCD) within minutes. Epidemiological studies have shown that SCD and ventricular arrhythmias are more likely to occur in the morning than in the evening, and laboratory studies indicate that these daily rhythms in adverse cardiovascular events are at least partially under the control of the endogenous circadian timekeeping system. However, the biophysical mechanisms linking molecular circadian clocks to cardiac arrhythmogenesis are not fully understood. Recent experiments have shown that L-type calcium channels exhibit circadian rhythms in both expression and function in guinea pig ventricular cardiomyocytes. We developed an electrophysiological model of these cells to simulate the effect of circadian variation in L-type calcium conductance. In our simulations, we found that there is a circadian pattern in the occurrence of early afterdepolarizations (EADs), which are abnormal depolarizations during the repolarization phase of a cardiac action potential that can trigger fatal ventricular arrhythmias. Specifically, the model produces EADs in the morning, but not at other times of day. We show that the model exhibits a codimension-2 Takens-Bogdanov bifurcation that serves as an organizing center for different types of EAD dynamics. We also simulated a two-dimensional spatial version of this model across a circadian cycle. We found that there is a circadian pattern in the breakup of spiral waves, which represents ventricular fibrillation in cardiac tissue. Specifically, the model produces spiral wave breakup in the morning, but not in the evening. Our computational study is the first, to our knowledge, to propose a link between circadian rhythms and EAD formation and suggests that the efficacy of drugs targeting EAD-mediated arrhythmias may depend on the time of day that they are administered.

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.

Multiple Dynamical Mechanisms of Phase-2 Early Afterdepolarizations in a Human Ventricular Myocyte Model: Involvement of Spontaneous SR Ca 2+ Release.

Early afterdepolarization (EAD) is known to cause lethal ventricular arrhythmias in long QT syndrome (LQTS). In this study, dynamical mechanisms of EAD formation in human ventricular myocytes (HVMs) were investigated using the mathematical model developed by ten Tusscher and Panfilov (Am J Physiol Heart Circ Physiol 291, 2006). We explored how the rapid (IKr) and slow (IKs) components of delayed-rectifier K+ channel currents, L-type Ca2+ channel current (ICaL), Na+/Ca2+ exchanger current (INCX), and intracellular Ca2+ handling via the sarcoplasmic reticulum (SR) contribute to initiation, termination and modulation of phase-2 EADs during pacing in relation to bifurcation phenomena in non-paced model cells. Parameter-dependent dynamical behaviors of the non-paced model cell were determined by calculating stabilities of equilibrium points (EPs) and limit cycles, and bifurcation points to construct bifurcation diagrams. Action potentials (APs) and EADs during pacing were reproduced by numerical simulations for constructing phase diagrams of the paced model cell dynamics. Results are summarized as follows: (1) A modified version of the ten Tusscher-Panfilov model with accelerated ICaL inactivation could reproduce bradycardia-related EADs in LQTS type 2 and β-adrenergic stimulation-induced EADs in LQTS type 1. (2) Two types of EADs with different initiation mechanisms, ICaL reactivation–dependent and spontaneous SR Ca2+ release–mediated EADs, were detected. (3) Termination of EADs (AP repolarization) during pacing depended on the slow activation of IKs. (4) Spontaneous SR Ca2+ releases occurred at higher Ca2+ uptake rates, attributable to the instability of steady-state intracellular Ca2+ concentrations. Dynamical mechanisms of EAD formation and termination in the paced model cell are closely related to stability changes (bifurcations) in dynamical behaviors of the non-paced model cell, but they are model-dependent. Nevertheless, the modified ten Tusscher-Panfilov model would be useful for systematically investigating possible dynamical mechanisms of EAD-related arrhythmias in LQTS.

Postpartum hormones oxytocin and prolactin cause pro-arrhythmic prolongation of cardiac repolarization in long QT syndrome type 2.

Women with long QT syndrome 2 (LQT2) have a particularly high postpartal risk for lethal arrhythmias. We aimed at investigating whether oxytocin and prolactin contribute to this risk by affecting repolarization. In female transgenic LQT2 rabbits (HERG-G628S, loss of IKr), hormone effects on QT/action potential duration (APD) were assessed (0.2-200 ng/L). Hormone effects (200 ng/L) on ion currents and cellular APD were determined in transfected cells and LQT2 cardiomyocytes. Hormone effects on ion channels were assessed with qPCR and western blot. Experimental data were incorporated into in silico models to determine the pro-arrhythmic potential. Oxytocin prolonged QTc and steepened QT/RR-slope in vivo and prolonged ex vivo APD75 in LQT2 hearts. Prolactin prolonged APD75 at high concentrations. As underlying mechanisms, we identified an oxytocin- and prolactin-induced acute reduction of IKs-tail and IKs-steady (-25.5%, oxytocin; -13.3%, prolactin, P < 0.05) in CHO-cells and LQT2-cardiomyocytes. IKr currents were not altered. This oxytocin-/prolactin-induced IKs reduction caused APD90 prolongation (+11.9%/+13%, P < 0.05) in the context of reduced/absent IKr in LQT2 cardiomyocytes. Hormones had no effect on IK1 and ICa,L in cardiomyocytes. Protein and mRNA levels of CACNA1C/Cav1.2 and RyR2 were enhanced by oxytocin and prolactin. Incorporating these hormone effects into computational models resulted in reduced repolarization reserve and increased propensity to pro-arrhythmic permanent depolarization, lack of capture and early afterdepolarizations formation. Postpartum hormones oxytocin and prolactin prolong QT/APD in LQT2 by reducing IKs and by increasing Cav1.2 and RyR2 expression/transcription, thereby contributing to the increased postpartal arrhythmic risk in LQT2.

Afterdepolarizations and triggered activity as a mechanism for clinical arrhythmias.

Afterdepolarizations cause triggered arrhythmias. One kind occurs after repolarization is complete, delayed afterdepolarizations (DADs). Another occurs as an interruption in repolarization, early afterdepolarizations (EADs). Afterdepolarizations initiate arrhythmias when they depolarize membrane potential to threshold potential for triggering action potentials. DADs usually occur mostly when Ca2+ in the sarcoplasmic reticulum (SR) is elevated. The SR leaks some of the Ca2+ into the myoplasm through Ca2+ release channels controlled by ryanodine receptors (RyR2) during diastole. The Na+ -Ca2+ exchanger extrudes elevated diastolic Ca2+ from the cell in exchange for Na+ (1 Ca2+ for 3 Na+ ) generating inward current causing DADs. DAD amplitude increases with decreasing cycle length, causing triggered activity during an increase in heart rate or during programmed electrical stimulation (PES). Coupling interval of the first triggered impulse is directly related to initiating cycle length. EADs are associated with an increased action potential duration (APD) causing long QT (LQT). EADs are caused by net inward currents (ICaL , INCX ) as a consequence. Hundreds of mutations can cause congenital LQT by altering repolarizing ion channels. Acquired LQT results from drug interaction with repolarizing ion channels. EAD-triggered ventricular tachycardia is polymorphic and called "torsade de pointes." Effects of PES on EAD-triggered activity is related to effects of cycle length on APD. Shortening cycle length prevents EADs by accelerating repolarization. Typical PES protocols inhibit formation of EADs which can be therapeutic.

Ca2 +-activated Cl− current is antiarrhythmic by reducing both spatial and temporal heterogeneity of cardiac repolarization

The role of Ca2+-activated Cl− current (ICl(Ca)) in cardiac arrhythmias is still controversial. It can generate delayed afterdepolarizations in Ca2+-overloaded cells while in other studies incidence of early afterdepolarization (EAD) was reduced by ICl(Ca). Therefore our goal was to examine the role of ICl(Ca) in spatial and temporal heterogeneity of cardiac repolarization and EAD formation.Experiments were performed on isolated canine cardiomyocytes originating from various regions of the left ventricle; subepicardial, midmyocardial and subendocardial cells, as well as apical and basal cells of the midmyocardium. ICl(Ca) was blocked by 0.5mmol/L 9-anthracene carboxylic acid (9-AC). Action potential (AP) changes were tested with sharp microelectrode recording. Whole-cell 9-AC-sensitive current was measured with either square pulse voltage-clamp or AP voltage-clamp (APVC). Protein expression of TMEM16A and Bestrophin-3, ion channel proteins mediating ICl(Ca), was detected by Western blot.9-AC reduced phase-1 repolarization in every tested cell. 9-AC also increased AP duration in a reverse rate-dependent manner in all cell types except for subepicardial cells. Neither ICl(Ca) density recorded with square pulses nor the normalized expressions of TMEM16A and Bestrophin-3 proteins differed significantly among the examined groups of cells. The early outward component of ICl(Ca) was significantly larger in subepicardial than in subendocardial cells in APVC setting. Applying a typical subepicardial AP as a command pulse resulted in a significantly larger early outward component in both subepicardial and subendocardial cells, compared to experiments when a typical subendocardial AP was applied.Inhibiting ICl(Ca) by 9-AC generated EADs at low stimulation rates and their incidence increased upon beta-adrenergic stimulation. 9-AC increased the short-term variability of repolarization also.We suggest a protective role for ICl(Ca) against risk of arrhythmias by reducing spatial and temporal heterogeneity of cardiac repolarization and EAD formation.

Myofilament protein dynamics modulate EAD formation in human hypertrophic cardiomyopathy

Patients with hypertrophic cardiomyopathy (HCM), a disease associated with sarcomeric protein mutations, often suffer from sudden cardiac death (SCD) resulting from arrhythmia. In order to advance SCD prevention strategies, our understanding of how sarcomeric mutations in HCM patients contribute to enhanced arrhythmogenesis needs to be improved. Early afterdepolarizations (EADs) are an important mechanism underlying arrhythmias associated with HCM-SCD. Although the ionic mechanisms underlying EADs have been studied in general, whether myofilament protein dynamics mechanisms also underlie EADs remains unknown. Thus, our goals were to investigate if myofilament protein dynamics mechanisms underlie EADs and to uncover how those mechanisms are affected by pacing rate, sarcomere length (SL), and different levels of HCM-induced myofilament remodeling. To achieve this, a mechanistically-based bidirectionally coupled human electrophysiology-force myocyte model under the conditions of HCM was constructed. HCM ionic remodeling included a reduced repolarization reserve, while HCM myofilament modeling involved altered thin filament activation. We found that the mechanoelectric feedback (MEF) on calcium dynamics in the bidirectionally coupled model, via Troponin C buffering of cytoplasmic Ca2+, was the myofilament mechanism underlying EADs. Incorporating MEF diminished the degree of repolarization reserve reduction necessary for EADs to emerge and increased the frequency of EAD occurrence, especially at faster pacing rates. Longer SLs and enhanced thin filament activation diminished the effects of MEF on EADs. Together these findings demonstrate that myofilament protein dynamics mechanisms play an important role in EAD formation.

Sex and regional differences in rabbit right ventricular L-type calcium current levels and mathematical modelling of arrhythmia vulnerability.

What is the central question of this study? Regional variations of ventricular L-type calcium current (ICa-L ) amplitude may underlie the increased arrhythmia risk in adult females. Current amplitude variations have been described for the left ventricle but not for the right ventricle. What is the main finding and its importance? Adult female rabbit right ventricular base myocytes exhibit elevated ICa-L compared with female apex or male myocytes. Oestrogen upregulated ICa-L in cultured female myocytes. Mathematical simulations modelling long QT syndrome type 2 demonstrated that elevated ICa-L prolonged action potentials and induced early after-depolarizations. Thus, ventricular arrhythmias in adult females may be associated with an oestrogen-induced upregulation of ICa-L . Previous studies have shown that adult rabbit left ventricular myocytes exhibit sex and regional differences in L-type calcium current (ICa-L ) levels that contribute to increased female susceptibility to arrhythmogenic early after-depolarizations (EADs). We used patch-clamp recordings from isolated adult male and female rabbit right ventricular myocytes to determine apex-base differences in ICa-L density and used mathematical modelling to examine the contribution of ICa-L to EAD formation. Current density measured at 0mV in female base myocytes was 67% higher than in male base myocytes and 55% higher than in female apex myocytes. No differences were observed between male and female apex myocytes, between male apex and base myocytes, or in the voltage dependences of ICa-L activation or inactivation. The role of oestrogen was investigated using cultured adult female right ventricular base myocytes. After 2days, 17β-estradiol (1nm) produced a 65% increase in ICa-L density compared with untreated control myocytes, suggesting an oestrogen-induced upregulation of ICa-L . Action potential simulations using a modified Luo-Rudy cardiomyocyte model showed that increased ICa-L density, at the level observed in female base myocytes, resulted in longer duration action potentials, and when combined with a 50% reduction of the rapidly inactivating delayed rectifier potassium current conductance to model long QT syndrome type 2, the action potential was accompanied by one or more EADs. Thus, we found higher levels of ICa-L in adult female right ventricle base myocytes and the upregulation of this current by oestrogen. Simulations of long QT syndrome type 2 showed that elevated ICa-L contributed to genesis of EADs.

Electrophysiology of Hypokalemia and Hyperkalemia.

Electrophysiology of Hypokalemia and Hyperkalemia.

Effects of Kinetics and State-Dependent Binding Properties of IKur Targeting Drugs on Atrial Electrophysiology

Background: The Kv1.5 potassium channel, which underlies the ultra-rapid delayed-rectifier current (IKur) and is predominantly expressed in atria vs. ventricles, has emerged as a promising target to treat atrial fibrillation (AF). However, while numerous Kv1.5-selective compounds have been screened, characterized, and tested in various animal models of AF, evidence of antiarrhythmic efficacy in humans is still lacking. Moreover, current guidelines for pre-clinical assessment of candidate drugs heavily rely on steady-state concentration-response curves or EC50 values, which can overlook adverse cardiotoxic events.Aim: We sought to investigate the effects of kinetics and state-dependent binding of IKur-targeting drugs on atrial electrophysiology in silico, and reveal the ideal therapeutic properties of IKur-blocking drugs that maximize efficacy and minimize pro-arrhythmic risk.Methods and results: A Markov model of IKur, based on experimental voltage-clamp data in atrial myocytes from patient samples in normal sinus rhythm (nSR) and chronic AF (cAF), was developed to describe Kv1.5 gating and state-specific Kv1.5-drug interactions and was incorporated into our human atrial cell models. We simulated 1Hz and 4Hz pacing protocols in drug-free conditions, and with [drug] equal to EC50. The effects of binding and unbinding kinetics were determined by examining permutations of the forward (Kon) and reverse (Koff) rates to the closed, open, and inactivated state of the Kv1.5 channel. We identified a subset of ideal drugs exhibiting anti-AF electrophysiological parameter changes in cAF (effective refractory period prolongation) at fast pacing rate, while having little effect in nSR (limited action potential prolongation and no early afterdepolarization formation) at normal heart rate.Conclusions: Our results highlight that accurately accounting for channel interaction with drugs, including kinetics and state-dependent binding, is critical for developing safer and more effective pharmacological anti-AF options.

Dynamical mechanisms of phase-2 early afterdepolarizations in human ventricular myocytes: insights from bifurcation analyses of two mathematical models.

We investigated mechanisms of phase-2 early afterdepolarization (EAD) by bifurcation analyses of human ventricular myocyte (HVM) models. EAD formation in paced HVMs basically depended on bifurcation phenomena in non-paced HVMs, but was strongly affected by intracellular ion concentrations in stationary and dynamic states. EAD generation did not necessarily require IKs.

Gabapentinoids Suppress Early Afterdepolarizations by Uncoupling α2δ-1 Subunits from CAV1.2 Channels: Implications for CAV Channel Gating-Modifiers as a New Class of Antiarrhythmics

Under certain pathological conditions, the repolarization phase of the cardiac action potential (AP) is interrupted by membrane potential oscillations called early afterdepolarizations (EADs), well-recognized triggers of deadly cardiac arrhythmias. The reactivation of L-type calcium current (ICa,L) plays a prominent role in EAD formation. In recent studies using the dynamic clamp technique, we have shown that a small depolarizing shift (<5mV) of the CaV1.2 steady-state activation potently suppresses EAD occurrence. Based on this findings, we hypothesized that pharmacological interventions that induce a depolarizing shift of macroscopic ICa,L activation (gating modifiers) can suppress EADs and their arrhythmogenic consequences. Gabapentinoids (gabapentin and pregabalin), drugs that selectively bind to α2δ-1 CaV subunits and are commonly used to treat epilepsy and neuropathic pain, are good candidates as Cav gating modifiers. We found that gabapentin induced a time-dependent depolarizing shift of the voltage-dependent activation of CaV1.2 channels expressed in Xenopus oocytes (α1C β3 α2δ-1; 1mM gabapentin, 2h incubation). Since α2δ-1 facilitates CaV1.2 voltage-dependent activation, these results suggest a functional uncoupling of α2δ-1 from a fraction of channels. Importantly, the maximal macroscopic conductance was unaltered, implying that CaV1.2 channel trafficking was not perturbed, in contrast to the effects shown in other CaV isoforms. We then studied gabapentinoids effect on EAD susceptibility in freshly-dissociated rabbit ventricular myocytes under EAD-promoting conditions (600μM H2O2). Strikingly, 1 mM pregabalin suppressed EAD occurrence and restored AP duration as predicted based on the depolarizing shift in voltage-dependent activation. Taken together, these results support the view that gabapentinoids, and more generally CaV1.2 gating modifiers, show promise in suppressing EAD-mediated arrhythmias and could constitute a novel class of antiarrhythmics.

Ephaptic Self-Attenuation Conceals Early Afterdepolarizations Associated with Long QT-3 Syndrome

Gain-of-function mutations in the cardiac voltage-gated sodium channel (Nav1.5) are associated with the long QT-3 (LQT3) syndrome. Early afterdepolarizations (EADs) are common in isolated myocyte models of LQT3, but rare in tissue and patients with LQT3 mutations. We have shown that Nav1.5 is densely expressed at the intercalated disk and narrowing intercellular separation can support an alternative form of cell-to-cell coupling known as ephaptic coupling (EpC). EpC occurs when sodium channels in the depolarizing cell decrease the intercellular cleft potential, depolarizing the apposing membrane from the extracellular rather than the intracellular domain. Critically, EpC models predict that decreasing intercellular separation slows conduction due to reduced sodium current driving force. Here, we test the novel hypothesis that ephaptic self-attenuation can “mask” the LQT3 phenotype by reducing the driving force and late sodium current that produces EADs.We tested our hypothesis both in a pharmacological LQT3 model in isolated guinea pig hearts experiments (n=3), paced at 500 ms basic cycle length using late sodium current agonist ATXII (7nM), and a computational model. Acute interstitial edema, induced by 20 g/l mannitol, increased intercellular cleft width but did not alter action potential duration (APD). ATXII prolonged APD by 80±11 ms, and ATXII plus mannitol prolonged APD by 165±7 ms and produced EADs. In a computational model incorporating EpC, a recent LRd formulation, and a sodium channel LQT3-associated mutant model, we show that for wide clefts, mutant myocytes consistently produce EADs, while for small clefts and wild-type myocytes, EADs are suppressed. For both large and small widths, the mutant sodium channel incompletely inactivates, but only for small cleft widths is late sodium current sufficiently reduced, suppressing EADs. These data demonstrate that intercellular edema in LQT3 underlies the formation of EADs presumably by an ephaptically-mediated mechanism.

Targeting the late component of the cardiac L-type Ca2+ current to suppress early afterdepolarizations.

Early afterdepolarizations (EADs) associated with prolongation of the cardiac action potential (AP) can create heterogeneity of repolarization and premature extrasystoles, triggering focal and reentrant arrhythmias. Because the L-type Ca(2+) current (ICa,L) plays a key role in both AP prolongation and EAD formation, L-type Ca(2+) channels (LTCCs) represent a promising therapeutic target to normalize AP duration (APD) and suppress EADs and their arrhythmogenic consequences. We used the dynamic-clamp technique to systematically explore how the biophysical properties of LTCCs could be modified to normalize APD and suppress EADs without impairing excitation-contraction coupling. Isolated rabbit ventricular myocytes were first exposed to H2O2 or moderate hypokalemia to induce EADs, after which their endogenous ICa,L was replaced by a virtual ICa,L with tunable parameters, in dynamic-clamp mode. We probed the sensitivity of EADs to changes in the (a) amplitude of the noninactivating pedestal current; (b) slope of voltage-dependent activation; (c) slope of voltage-dependent inactivation; (d) time constant of voltage-dependent activation; and (e) time constant of voltage-dependent inactivation. We found that reducing the amplitude of the noninactivating pedestal component of ICa,L effectively suppressed both H2O2- and hypokalemia-induced EADs and restored APD. These results, together with our previous work, demonstrate the potential of this hybrid experimental-computational approach to guide drug discovery or gene therapy strategies by identifying and targeting selective properties of LTCC.

Calcium Mediated Mechanism of Early Afterdepolarizations in LQT2 Ventricular Myocytes

Background: Loss of function mutations in hERG potassium channels underlie the long QT syndrome type 2 (LQT2). LQT2 is associated with fatal ventricular arrhythmias promoted by triggered activity in the form of early afterdepolarizations (EADs). However, the cellular mechanism of EAD formation remains unexplored.Methods: We have investigated the mechanism of EAD formation in LQT2 ventricular myocytes using a physiologically detailed computational model of calcium (Ca2+) cycling and membrane voltage dynamics. This model bridges the submicron scale of individual couplons of plasmalemmal L-type Ca2+ channels clusters and sarcoplasmic reticulum (SR) Ca2+ release units (CRUs) and the whole cell. We incorporate the novel experimental finding that ryanodine receptors (RyRs) Ca2+ release channels are remodeled in ventricular mycoytes isolated from LQT2 transgenic rabbits; RyRs are hyperphosphorylated leading to enhanced channel activity and hence increased Ca2+ leak.Results: Computer simulations with RyR hyperactivity modeled as an increased rate of channel opening show that hyperactivity is causally linked to EAD formation. Under stimulation with the β-adrenergic agonist isoproterenol (ISO), LQT2 myocytes with hyperactive RyRs exhibit EADs together with decreased SR load and Ca2+ transient (CaT) amplitude, while myocytes with normal RyR activity exhibit a prolonged action potential without reductions of SR load and CaT amplitude. Simulations show that RyR hyperactivity shortens RyR refractoriness at the CRU level, resulting in late aberrant Ca2+ releases during repolarization. Those releases promote onset of EADs by driving the forward mode Na+-Ca2+ exchanger NCX1 current, which slows repolarization and allows reactivation of L-type Ca2+ current. Modeling predictions are in good agreement with experimental observations, which show that EADs in ISO-stimulated LQT2 myocytes are accompanied by late Ca2+ releases together with decreased SR load and CaT amplitude, and that both late releases and EADs are abolished by inhibition of CaMKII.

Transient Outward K+ Current Underlies Heterogeneity of Action Potential Duration and Early Afterdepolarization from Right Ventricle in Transgenic Rabbit Model of Long QT Type 1

Introduction: Long QT syndrome type 1 (LQT1) is a congenital disease lacking slowly activating K+ channel (IKs), associated with polymorphic VT (pVT) and sudden cardiac death. Tissue heterogeneity has been proposed as an important factor to trigger and maintain pVT. We investigated ionic mechanisms that underlie regional differences in the formation of early afterdepolarization (EAD) using transgenic rabbit model of LQT1.Methods: The initiation of pVT under isoproterenol was mapped using optical mapping and myocytes isolated from RV and septum were studied using voltage clamp and confocal calcium imaging.Results: Optical mapping of LQT1 hearts showed pVT preferentially originating from right ventricle (RV) (16 of 18 pVTs), which was changed after perfusing transient outward K channel (Ito) blocker, 0.5 mM 4-aminopyridine (only 1 of 5 pVTs from RV, n=5 hearts). Myocytes isolated from RV demonstrated higher incidence of EADs under 50 nM isoproterenol (8 of 12 cells from RV vs. 2 of 11 cells from septum). Voltage clamp study highlighted regional differences of Ito (4.7 ± 0.5 in RV vs. 2.9 ± 0.7 pA/pF in septum at 0 mV) but other currents such as ICaL, IKr and RyR-mediated Ca2+ leak and SERCA-mediated Ca2+ uptake were same between RV and septum. Computer modeling study of rabbit action potential lacking IKs exhibit frequent EADs but reduction of Ito prevented EAD formation, verifying that Ito plays a major role in EAD formation by providing repolarizing currents during the plateau phase to maintain the membrane potential lower enough to re-activate ICa window current to form EADs.Conclusion: Regional differences of Ito in LQT1 rabbits underlie frequent pVTs originating mostly from RV myocytes with high incidence of EADs.

Intermittent Early Afterdepolarizations Caused by Bistability

Early afterdepolarization (EAD) is one of the main triggers for cardiac arrhythmias. The intracellular sodium concentration ([Na]i) is critical in regulating both intracellular calcium (Ca) homeostasis and membrane voltage (V). However, the role of [Na]i in the formation of early afterdepolarizations (EADs) is not completely understood. In this study, we applied mathematical modeling to show that slow [Na]i accumulation can lead to a novel form of aperiodic voltage dynamics where a sequence of action potentials (APs) with EADs occurs intermittently during regular periodic pacing. We find that these trains of EADs occur quasi-periodically and lead to intermittent EAD propagation and arrhythmias. The mechanism for these intermittent EADs can be traced to the subtle feedback between Na and Ca fluxes (especially via Na/Ca exchange (NCX) and Na/K-ATPase (IPump)) and their effect on the AP duration (APD). We analyzed the whole system by separating the slow [Na]i from the fast V-[Ca]i subsystem. We found that the fast subsystem exhibits bistability which leads to the observed EADs by forming hysteresis loops as [Na]i slowly accumulates and dissipates. That is, [Na]i can gradually decline during stable short APDs, but this gradually diminishes outward currents via IPump and INCX, which can result in abrupt APD prolongation with EADs. But that long APD gradually reverse the [Na]i decline, and shifts IPump and INCX more outward, which at some point abruptly prevents EADs and reverses APD prolongation. We argue that these intermittent EADs are robust and can occur at physiological [Na]i. Our study further provides a possible novel mechanism for the intermittency of cardiac arrhythmias.