IKr Blocker E-4031 Research Articles

Abstract 15219: Optogenetic “Printing” of Reentrant Arrhythmias in Human Induced Pluripotent Stem Cell Derived Cardiac Tissues

Introduction: Current models used to investigate reentrant arrhythmias allow for limited control of arrhythmia parameters and morphology. Here, we developed a method for the induction of morphologically and physiologically defined arrhythmias using human induced pluripotent stem cell derived cardiac cell sheets (hiPSC-CCSs) and optogenetic tools. Methods: One million hiPSC-cardiomyocytes expressing the optogenetic channel CoChR were used to create the hiPSC-CCSs. Rotor illumination patterns were generated either by computer-aided design or by utilizing previously recorded rotor patterns. A digital micro mirror device (DMD) and a 470nm LED were used to project five complete cycles of these rotor patterns onto the hiPSC-CCSs. Results: Optical projection of typical rotors led to repeated development of arrhythmias in all sampled tissues (n=11). The resulting arrhythmias resembled the projected rotor patterns in terms of directionality (clockwise or counterclockwise) and number of cores ,and were highly reproducible for each projection (Fig.1A). Interestingly, we identified a minimal and maximal frequency of the projected rotor that could induce arrhythmias (Fig.1B). Moreover, the frequency of the generated rotor could not be increased beyond a certain value despite further increases in the projected rotor frequency (Fig.1B). Lastly, to assess the effects of tissue EP properties, the process was repeated after treatment with the IKr blocker E-4031 (30nM). E-4031 treatment significantly reduced the minimal and maximal frequencies of the projected rotor that could still induce arrhythmia and slowed the maximal frequency of the resulting rotor (Fig.1B). Conclusions: We present a new method for the optical induction of reentrant arrhythmias allowing the generation of rotors rapidly, reproducibly, and with a predesigned morphology. This approach may allow for a paradigm shift in the way reentrant arrhythmias are studied.

Characterization of changes in the configuration of action potentials in the mouse, guinea pig, and pig sinoauricular node after application of channel blockers of the rapid and slow delayed rectifier potassium currents.

We hypothesized that the repolarization phase of action potentials (APs) in mammals with large body mass and high cardiac output could not be reliably controlled by only one of the delayed rectifier potassium IK current components. To test this hypothesis experimentally, we performed a comparative study of the response of AP phases to the rapid IKr channels blocker E-4031 and slow IKs blocker chromanol 293B in APs spontaneously generated in strips of sinoauricular (SA) tissue from mouse, guinea pig, and pig hearts. Application of a slow channels blocker chromanol 293B caused a decrease of Aps generation frequency in SA area strips from mouse, guinea-pig and pig by 5.3, 16, and 18% compared to the control. Treatment with the IKr blocker E-4031 caused a significant reduction of APs generation frequency in the mouse, guinea pig, and pig SA strips by 24, 26, and 36%, respectively, compared to the control values. These results suggest that the rapid IKr current is the key component responsible for AP generation in sinoauricular node cells of the pig heart.

Optogenetic sensors in the zebrafish heart: a novel in vivo electrophysiological tool to study cardiac arrhythmogenesis

Cardiac arrhythmias are among the most challenging human disorders to diagnose and treat due to their complex underlying pathophysiology. Suitable experimental animal models are needed to study the mechanisms causative for cardiac arrhythmogenesis. To enable in vivo analysis of cardiac cellular electrophysiology with a high spatial and temporal resolution, we generated and carefully validated two zebrafish models, one expressing an optogenetic voltage indicator (chimeric VSFP-butterfly CY) and the other a genetically encoded calcium indicator (GCaMP6f) in the heart.Methods: High-speed epifluorescence microscopy was used to image chimeric VSFP-butterfly CY and GCaMP6f in the embryonic zebrafish heart, providing information about the spatiotemporal patterning of electrical activation, action potential configuration and intracellular Ca2+ dynamics. Plotting VSFP or GCaMP6f signals on a line along the myocardial wall over time facilitated the visualization and analysis of electrical impulse propagation throughout the heart. Administration of drugs targeting the sympathetic nervous system or cardiac ion channels was used to validate sensitivity and kinetics of both zebrafish sensor lines. Using the same microscope setup, we imaged transparent juvenile casper fish expressing GCaMP6f, demonstrating the feasibility of imaging cardiac optogenetic sensors at later stages of development.Results: Isoproterenol slightly increased heart rate, diastolic Ca2+ levels and Ca2+ transient amplitudes, whereas propranolol caused a profound decrease in heart rate and Ca2+ transient parameters in VSFP-Butterfly and GCaMP6f embryonic fish. Ikr blocker E-4031 decreased heart rate and increased action potential duration in VSFP-Butterfly fish. ICa,L blocker nifedipine caused total blockade of Ca2+ transients in GCaMP6f fish and a reduced heart rate, altered ventricular action potential duration and disrupted atrial-ventricular electrical conduction in VSFP-Butterfly fish. Imaging of juvenile animals demonstrated the possibility of employing an older zebrafish model for in vivo cardiac electrophysiology studies. We observed differences in atrial and ventricular Ca2+ recovery dynamics between 3 dpf and 14 dpf casper fish, but not in Ca2+ upstroke dynamics.Conclusion: By introducing the optogenetic sensors chimeric VSFP-butterfly CY and GCaMP6f into the zebrafish we successfully generated an in vivo cellular electrophysiological readout tool for the zebrafish heart. Complementary use of both sensor lines demonstrated the ability to study heart rate, cardiac action potential configuration, spatiotemporal patterning of electrical activation and intracellular Ca2+ homeostasis in embryonic zebrafish. In addition, we demonstrated the first successful use of an optogenetic sensor to study cardiac function in older zebrafish. These models present a promising new research tool to study the underlying mechanisms of cardiac arrhythmogenesis.

\u03b2-adrenergic stimulation augments transmural dispersion of repolarization via modulation of delayed rectifier currents IKs and IKr in the human ventricle

Long QT syndrome (LQTS) is an inherited or drug induced condition associated with delayed repolarization and sudden cardiac death. The cardiac potassium channel, IKr, and the adrenergic-sensitive cardiac potassium current, IKs, are two primary contributors to cardiac repolarization. This study aimed to elucidate the role of β-adrenergic (β-AR) stimulation in mediating the contributions of IKr and IKs to repolarizing the human left ventricle (n = 18). Optical mapping was used to measure action potential durations (APDs) in the presence of the IKs blocker JNJ-303 and the IKr blocker E-4031. We found that JNJ-303 alone did not increase APD. However, under isoprenaline (ISO), both the application of JNJ-303 and additional E-4031 significantly increased APD. With JNJ-303, ISO decreased APD significantly more in the epicardium as compared to the endocardium, with subsequent application E-4031 increasing mid- and endocardial APD80 more significantly than in the epicardium. We found that β-AR stimulation significantly augmented the effect of IKs blocker JNJ-303, in contrast to the reduced effect of IKr blocker E-4031. We also observed synergistic augmentation of transmural repolarization gradient by the combination of ISO and E-4031. Our results suggest β-AR-mediated increase of transmural dispersion of repolarization, which could pose arrhythmogenic risk in LQTS patients.

Assessment of Testing Methods for Drug-Induced Repolarization Delay and Arrhythmias in an iPS Cell–Derived Cardiomyocyte Sheet: Multi-site Validation Study

A prospective comparison study across 3 independent research laboratories of a pure IKr blocker E-4031 was conducted by using the same batch of human iPS cell–derived cardiomyocytes in order to verify the utility and reliability of our original standard protocol. Field potential waveforms were recorded with a multi-electrode array system to measure the inter-spike interval and field potential duration. The effects of E-4031 at concentrations of 1 to 100 nM were sequentially examined every 10 min. In each facility, E-4031 significantly prolonged the field potential duration corrected by Fridericia’s formula and caused early afterdepolarizations occasionally resulting in triggered activities, whereas it tended to decrease the rate of spontaneous contraction. These results were qualitatively and quantitatively consistent with previous non-clinical in vitro and in vivo studies as well as clinical reports. There were inter-facility differences in some absolute values of the results, which were not observed when the values were normalized as percentage change. Information described in this paper may serve as a guide when predicting the drug-induced repolarization delay and arrhythmias with this new technology of stem cells.

The new antiarrhythmic drug vernakalant: ex vivo study of human atrial tissue from sinus rhythm and chronic atrial fibrillation

Vernakalant is a newly developed antiarrhythmic drug against atrial fibrillation (AF). However, its electrophysiological actions on human myocardium are unknown. Action potentials (APs) and ion currents were recorded in right atrial trabeculae and cardiomyocytes from patients in sinus rhythm (SR) and chronic AF. Vernakalant prolonged early repolarization in SR and AF, but late only in AF. AP amplitude (APA) and dV/dtmax were reduced in a concentration- and frequency-dependent manner with IC50 < 10 µM at >3 Hz. Effective refractory period was increased more than action potential duration (APD) in SR and AF. INa was blocked with IC50s of 95 and 84 µM for SR and AF, respectively (0.5 Hz). Vernakalant did not reduce outward potassium currents compared with time-matched controls. However, area under the current-time curve was reduced due to acceleration of current decline with IC50s of 19 and 12 µM for SR and AF, respectively. Vernakalant had less effect on APD than the IKr blocker E-4031, blocked IK,ACh, and had a small inhibitory effect on IK1 at 30 µM. L-Type Ca(2+) currents (SR) were reduced with IC50 of 84 µM. Rate-dependent block of Na(+) channels represents the main antiarrhythmic mechanism of vernakalant in the fibrillating atrium. Open channel block of early transient outward currents and IK,ACh could also contribute.

Abstract 136: Novel Disease Analysis Using Long QT Syndrome-Specific Induced Pluripotent Stem Cells

[Background] Although previous reports have indicated that long QT syndrome-specific iPS cells (LQTS-iPSCs)-derived cardiomyocytes recapitulated disease phenotypes, those patients were previously diagnosed with mutated channel profiles. In reality, most patients have no such specific information. To address whether iPSCs could be used for personalized disease characterization, we generated iPSCs from a sporadic LQTS patient with unknown disease cause. [Methods and Results] 1 We generated iPSCs from control (n = 2) and a patient with LQTS, and differentiated into cardiomyocytes through embryoid body (EB) formation. 2 Electrophysiological analysis of the LQTS-iPSCs-derived EBs using multi electrode array system revealed that the duration of the field potential (FPD) was markedly prolonged compared with the control (388.9 ± 44.3 msec vs 202.3 ± 16.3 msec, P&lt;0.01). 3 We tested several drugs affecting QT prolongation to clarify the electrophysiological properties. The IKr blocker E4031 significantly prolonged FPD (% change 1.21 ± 0.02, P&lt;0.01) and induced frequent severe arrhythmia, not only early-after depolarization (n = 8/16 vs n = 1/16) but also torsades de pointes-like arrhythmia, only in LQTS (n = 4/16 vs n = 0/16). The IKs blocker, chromanol 293B did not prolong FPD in LQTS but it significantly prolonged FPD in the control (% change 1.09 ± 0.04, NS vs 1.44 ± 0.07, P&lt;0.01), suggesting the involvement of IKs disturbance in the patient. Isoproterenol induced ventricular tachycardia-like arrhythmia only in LQTS, which was blocked by propranolol. These data strongly suggested a functional impairment in the patient’s IKs channel; genotype analysis for KCNQ1 gene revealed a novel heterozygous mutation, 1893delC. 4 Patch clamp analysis and immunostaining confirmed a dominant-negative role for 1893delC in IKs channel through a trafficking deficiency. [Conclusions] LQTS-iPSCs-derived cardiomyocytes recapitulated the disease phenotypes, and they can be utilized for identification of the disease cause and genotype. This study demonstrated that iPSCs could be useful to characterize the disease, drug responses, diagnosis and genotyping in patients with sporadic LQTS, which in turn may facilitate medical therapies in the clinical settings.

Disease characterization using LQTS-specific induced pluripotent stem cells

Long QT syndrome (LQTS) is an inheritable and life-threatening disease; however, it is often difficult to determine disease characteristics in sporadic cases with novel mutations, and more precise analysis is necessary for the successful development of evidence-based clinical therapies. This study thus sought to better characterize ion channel cardiac disorders using induced pluripotent stem cells (iPSCs). We reprogrammed somatic cells from a patient with sporadic LQTS and from controls, and differentiated them into cardiomyocytes through embryoid body (EB) formation. Electrophysiological analysis of the LQTS-iPSC-derived EBs using a multi-electrode array (MEA) system revealed a markedly prolonged field potential duration (FPD). The IKr blocker E4031 significantly prolonged FPD in control- and LQTS-iPSC-derived EBs and induced frequent severe arrhythmia only in LQTS-iPSC-derived EBs. The IKs blocker chromanol 293B did not prolong FPD in the LQTS-iPSC-derived EBs, but significantly prolonged FPD in the control EBs, suggesting the involvement of IKs disturbance in the patient. Patch-clamp analysis and immunostaining confirmed a dominant-negative role for 1893delC in IKs channels due to a trafficking deficiency in iPSC-derived cardiomyocytes and human embryonic kidney (HEK) cells. This study demonstrated that iPSCs could be useful to characterize LQTS disease as well as drug responses in the LQTS patient with a novel mutation. Such analyses may in turn lead to future progress in personalized medicine.

Human Heart Slices - a Novel Multicellular System Suitable for Electrophysiological and Pharmacological Studies

Electrophysiological and pharmacological data from the human heart are limited due to the absence of simple but representative experimental model systems of human cardiac tissue. Here, we describe and characterise a novel, simple and reproducible preparation suitable for studying human cardiac tissue at the multicellular level, the living human cardiac slice.Vibratome-cut slices (350 μm thick) were prepared from left ventricular transmural biopsies (5x5 mm) obtained from end-stage heart failure patients undergoing transplant or left ventricular assist device implantation. One biopsy was sufficient to generate >15 slices. Histology revealed that slices cut in parallel to the epicardial surface had predominantly longitudinal muscle fibre orientation. ATP/ADP and phosphocreatine/creatine ratios (3.32±0.61 & 1.37±0.31; n=5 slices/3 hearts) measured by HPLC were comparable to intact organ values.Electrical activity and its propagation in response to electrical stimulation (1Hz) were recorded using a multi-electrode array system. Field potential duration (FPD) remained stable and could be recorded for up to 8h.Mean FPD was 467±4 ms (n=42/10), similar to previously reported data using ventricular wedges.Longitudinal conduction velocity (CV) was faster than transversal CV (51±3 & 20±1 cm/sec, n=19/7, p<0.0001), with an anisotropic ratio of 2.5:1.Ikr blocker E4031 (1 μM) and Iks blocker Chromanol 293B (20 μM) prolonged FPD by 64±3% (n=20/7, p<0.001) and 22±2% (n=12/5, p<0.001) respectively, similar to previous results using intact papillary muscles. The non-specific K+ channel blocker 4-aminopyridine (1mM) prolonged FPD by 47±2% (n=6/2, p<0.001).Our results show that cardiac slices from human biopsies are a reproducible preparation that remains stable for several hours in vitro and has structural, electrophysiological, pharmacological and metabolic properties of native myocardium. Cardiac slices offer a novel multicellular system suitable for the study of heart failure and therapy.

Quantified proarrhythmic potential of selected human embryonic stem cell-derived cardiomyocytes

To improve proarrhythmic predictability of preclinical models, we assessed whether human ventricular-like embryonic stem cell-derived cardiomyocytes (hESC-CMs) can be selected following a standardized protocol. Also, we quantified their arrhythmogenic response and compared this to a contemporary used rabbit Purkinje fiber (PF) model. Multiple transmembrane action potentials (AP) were recorded from 164 hESC-CM clusters (9 different batches), and 12 isolated PFs from New Zealand White rabbits. AP duration (APD), early afterdepolarizations (EADs), triangulation (T), and short-term variability of repolarization (STV) were determined on application of the IKr blocker E-4031 (0.03/0.1/0.3/1 μM). Isoproterenol (0.1 μM) was used to assess adrenergic response. To validate the phenotype, RNA isolated from atrial- and ventricular-like clusters (n=8) was analyzed using low-density Taqman arrays. Based on initial experiments, slow beating rate (<50 bpm) and long APD (>200 ms) were used to select 31 ventricular-like clusters. E-4031 (1 μM) prolonged APD (31/31) and induced EADs only in clusters with APD90>300 ms (11/16). EADs were associated with increased T (1.6±0.2 vs 2.0±0.3⁎) and STV (2.7±1.5 vs 6.9±1.9⁎). Rabbit PF reacted in a similar way with regards to EADs (5/12), increased T (1.3±0.1 vs 1.9±0.4⁎), and STV (1.2±0.9 vs 7.1±5.6⁎). According to ROC values, hESC-CMs (STV 0.91) could predict EADs at least equivalent to PF (STV 0.69). Isoproterenol shortened APD and completely suppressed EADs. Gene expression analysis revealed that HCN1/2, KCNA5, and GJA5 were higher⁎ in atrial/nodal-like cells, whereas KCNJ2 and SCN1B were higher⁎ in ventricular-like cells (⁎P<0.05). Selection of hESC-CM clusters with a ventricular-like phenotype can be standardized. The proarrhythmic results are qualitatively and quantitatively comparable between hESC-CMs and rabbit PF. Our results indicate that additional validation of this new safety pharmacology model is warranted.

Potentiation of E‐4031‐induced torsade de pointes by HMR1556 or ATX‐II is not predicted by action potential short‐term variability or triangulation

Torsade de pointes (TdP) can be induced by a reduction in cardiac repolarizing capacity. The aim of this study was to assess whether IKs blockade or enhancement of INa could potentiate TdP induced by IKr blockade and to investigate whether short-term variability (STV) or triangulation of action potentials preceded TdP. Experiments were performed in open-chest, pentobarbital-anaesthetized, alpha 1-adrenoceptor-stimulated, male New Zealand White rabbits, which received three consecutive i.v. infusions of either the IKr blocker E-4031 (1, 3 and 10 nmol kg(-1) min(-1)), the IKs blocker HMR1556 (25, 75 and 250 nmol kg(-1) min(-1)) or E-4031 and HMR1556 combined. In a second study rabbits received either the same doses of E-4031, the INa enhancer, ATX-II (0.4, 1.2 and 4.0 nmol kg(-1)) or both of these drugs. ECGs and epicardial monophasic action potentials were recorded. HMR1556 alone did not cause TdP but increased E-4031-induced TdP from 25 to 80%. ATX-II alone caused TdP in 38% of rabbits, as did E-4031; 75% of rabbits receiving both drugs had TdP. QT intervals were prolonged by all drugs but the extent of QT prolongation was not related to the occurrence of TdP. No changes in STV were detected and triangulation was only increased after TdP occurred. Giving modulators of ion channels in combination substantially increased TdP but, in this model, neither STV nor triangulation of action potentials could predict TdP.

The effect of ATX-II on torsade de pointes induced by E-4031 in vivo

Torsade de pointes (TdP) is associated with long QT (LQT) syndromes which can be precipitated by altering cardiac potassium currents e.g. IKr (LQT2) or the cardiac sodium current INa (LQT3). The aim of this study was to test the hypothesis that ATX-II will potentiate E-4031-induced TdP. Experiments were performed in pentobarbital-anaesthetized α1-adrenoceptor-stimulated male New Zealand White rabbits. Rabbits were given increasing doses of either the INa opener ATX-II (n=6), the IKr blocker E-4031 (n=6), or both drugs in combination (n=6).

Antagonism by ranolazine of the pro-arrhythmic effects of increasing late INa in guinea pig ventricular myocytes.

The new anti-anginal drug ranolazine causes a slight (<10 milliseconds) prolongation of the QT interval, raising the concern that its use may be associated with an increased incidence of torsades de pointes ventricular tachyarrhythmias. The goal of this study was to show that ranolazine inhibits the late component of INa and attenuates prolongation of action potential duration when late INa is increased, both in the absence and presence of IK-blocking drugs. Currents and action potentials of guinea pig isolated ventricular myocytes were measured by whole-cell patch clamp. Sea anemone toxin (ATX)-II was used to increase late INa and mimic the effect of an SCN5A gene mutation. ATX-II (3-5 nmol/L) increased late INa by 5-fold; ranolazine attenuated this increase of late INa by up to 61 +/- 8%. ATX-II (10-20 nmol/L) increased action potential duration (APD) by > 1 seconds, and caused early afterdepolarizations; both actions were attenuated by ranolazine (0.1-30 micromol/L). Ranolazine (10 micromol/L) reduced by 89% the 13.6-fold increase in variability of APD caused by 10 nmol/L ATX-II. The effects of ATX-II (3 nmol/L) in combinations with either the IKr blocker E-4031 or the IKs blocker chromanol 293B to increase APD were attenuated 76 +/- 5% and 71 +/- 4%, respectively, by 10 micromol/L ranolazine. The results demonstrate that ranolazine reduces late INa and has an anti-arrhythmic effect when late INa is increased.

Three thiadiazinone derivatives, EMD 60417, EMD 66430, and EMD 66398, with class III antiarrhythmic activity but different electrophysiologic profiles.

The thiadiazinone derivatives EMD 60417, EMD 66430, and EMD 66398 were developed as class III antiarrhythmic agents. Their chemical structure is closely related to that of their calcium-sensitizing congener [+]-EMD 60263, and EMD 66398 possesses the methylsulfonylaminobenzoyl moiety present in the prototypical IKr blocker E-4031. We compared the electrophysiologic effects of these compounds with standard drugs (almokalant, E-4031, quinidine) in cardiac myocytes from guinea-pig ventricle and human atrium by whole-cell patch-clamp technique. The test compounds' class III action, which is related to impairment of K+ channel function, was confirmed by action potential measurements. EMD 60417, EMD 66430, EMD 66398, and almokalant (1 microM each) reversibly prolonged the action potential duration in guinea-pig myocytes. In the same cells, the rapidly activating component IKr of the delayed rectifier K+ current, which has been defined by its sensitivity to E-4031, was reduced by EMD 60417, EMD 66430, EMD 66398, and almokalant. Inhibition of IKr was concentration-dependent as determined by attenuation of tail currents. The slowly activating component IKs of the delayed rectifier K+ current was not affected. The inward rectifier K+ current IK1 was not influenced at potentials close to the reversal potential. Transient and sustained outward K+ currents (Ito, Iso) measured in human atrial myocytes were not altered by any EMD compound. L-type Ca2+ current was hardly affected at concentrations of 1-10 microM, but sodium current was decreased. Action potential prolongation by EMD 60417, EMD 66430, and EMD 66398 is due to block of IKr. INa is inhibited at higher concentrations by EMD 66430 and EMD 60417. EMD 66398 is more potent and selective for IKr than EMD 60417 and EMD 66430, and thus resembles E-4031 in structure and function.