Human induced pluripotent stem cell-derived atrial cardiomyocytes recapitulate contribution of the slowly activating delayed rectifier currents IKs to repolarization in the human atrium.

AIMS: Human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCM) could be a helpful tool to study the physiology and diseases of the human atrium. To fulfil this expectation, the electrophysiology of hiPSC-aCM should closely resemble the situation in the human atrium. Data on the contribution of the slowly activating delayed rectifier currents (IKs) to repolarization are lacking for both human atrium and hiPSC-aCM. METHODS AND RESULTS: Human atrial tissues were obtained from patients with sinus rhythm (SR) or atrial fibrillation (AF). Currents were measured in human atrial cardiomyocytes (aCM) and compared with hiPSC-aCM and used to model IKs contribution to action potential (AP) shape. Action potential was recorded by sharp microelectrodes. HMR-1556 (1 µM) was used to identify IKs and to estimate IKs contribution to repolarization. Less than 50% of hiPSC-aCM and aCM possessed IKs. Frequency of occurrence, current densities, activation/deactivation kinetics, and voltage dependency of IKs did not differ significantly between hiPSC-aCM and aCM, neither in SR nor AF. ß-Adrenoceptor stimulation with isoprenaline did not increase IKs neither in aCM nor in hiPSC-aCM. In tissue from SR, block of IKs with HMR-1556 did not lengthen the action potential duration, even when repolarization reserve was reduced by block of the ultra-rapid repolarizing current with 4-aminopyridine or the rapidly activating delayed rectifier potassium outward current with E-4031. CONCLUSION: I Ks exists in hiPSC-aCM with biophysics not different from aCM. As in adult human atrium (SR and AF), IKs does not appear to relevantly contribute to repolarization in hiPSC-aCM.

The electrophysiologic effects of KCNQ1 extend beyond expression of IKs: evidence from genetic and pharmacologic block.

AIMS: While variants in KCNQ1 are the commonest cause of the congenital long QT syndrome, we and others find only a small IKs in cardiomyocytes from human-induced pluripotent stem cells (iPSC-CMs) or human ventricular myocytes. METHODS AND RESULTS: We studied population control iPSC-CMs and iPSC-CMs from a patient with Jervell and Lange-Nielsen (JLN) syndrome due to compound heterozygous loss-of-function (LOF) KCNQ1 variants. We compared the effects of pharmacologic IKs block to those of genetic KCNQ1 ablation, using JLN cells, cells homozygous for the KCNQ1 LOF allele G643S, or siRNAs reducing KCNQ1 expression. We also studied the effects of two blockers of IKr, the other major cardiac repolarizing current, in the setting of pharmacologic or genetic ablation of KCNQ1: moxifloxacin, associated with a very low risk of drug-induced long QT, and dofetilide, a high-risk drug. In control cells, a small IKs was readily recorded but the pharmacologic IKs block produced no change in action potential duration at 90% repolarization (APD90). In contrast, in cells with genetic ablation of KCNQ1 (JLN), baseline APD90 was markedly prolonged compared with control cells (469 ± 20 vs. 310 ± 16 ms). JLN cells displayed increased sensitivity to acute IKr block: the concentration (µM) of moxifloxacin required to prolong APD90 100 msec was 237.4 [median, interquartile range (IQR) 100.6-391.6, n = 7] in population cells vs. 23.7 (17.3-28.7, n = 11) in JLN cells. In control cells, chronic moxifloxacin exposure (300 µM) mildly prolonged APD90 (10%) and increased IKs, while chronic exposure to dofetilide (5 nM) produced greater prolongation (67%) and no increase in IKs. However, in the siRNA-treated cells, moxifloxacin did not increase IKs and markedly prolonged APD90. CONCLUSION: Our data strongly suggest that KCNQ1 expression modulates baseline cardiac repolarization, and the response to IKr block, through mechanisms beyond simply generating IKs.

Cellular electrophysiological effects of the citrus flavonoid hesperetin in dog and rabbit cardiac ventricular preparations.

Recent experimental data shows that hesperetin, a citrus flavonoid, affects potassium channels and can prolong the QTc interval in humans. Therefore, in the present study we investigated the effects of hesperetin on various transmembrane ionic currents and on ventricular action potentials. Transmembrane current measurements and action potential recordings were performed by patch-clamp and the conventional microelectrode techniques in dog and rabbit ventricular preparations. At 10 µM concentration hesperetin did not, however, at 30 µM significantly decreased the amplitude of the IK1, Ito, IKr potassium currents. Hesperetin at 3-30 µM significantly and in a concentration-dependent manner reduced the amplitude of the IKs current. The drug significantly decreased the amplitudes of the INaL and ICaL currents at 30 µM. Hesperetin (10 and 30 µM) did not change the action potential duration in normal preparations, however, in preparations where the repolarization reserve had been previously attenuated by 100 nM dofetilide and 1 µg/ml veratrine, caused a moderate but significant prolongation of repolarization. These results suggest that hesperetin at close to relevant concentrations inhibits the IKs outward potassium current and thereby reduces repolarization reserve. This effect in certain specific situations may prolong the QT interval and consequently may enhance proarrhythmic risk.

Optimization of a cardiomyocyte model illuminates role of increased INa,L in repolarization reserve.

Cardiac ion currents may compensate for each other when one is compromised by a congenital or drug-induced defect. Such redundancy contributes to a robust repolarization reserve that can prevent the development of lethal arrhythmias. Most efforts made to describe this phenomenon have quantified contributions by individual ion currents. However, it is important to understand the interplay between all major ion-channel conductances, as repolarization reserve is dependent on the balance between all ion currents in a cardiomyocyte. Here, a genetic algorithm was designed to derive profiles of nine ion-channel conductances that optimize repolarization reserve in a mathematical cardiomyocyte model. Repolarization reserve was quantified using a previously defined metric, repolarization reserve current, i.e., the minimum constant current to prevent normal action potential repolarization in a cell. The optimization improved repolarization reserve current up to 84% compared to baseline in a human adult ventricular myocyte model and increased resistance to arrhythmogenic insult. The optimized conductance profiles were not only characterized by increased repolarizing current conductances but also uncovered a previously unreported behavior by the late sodium current. Simulations demonstrated that upregulated late sodium increased action potential duration, without compromising repolarization reserve current. The finding was generalized to multiple models. Ultimately, this computational approach, in which multiple currents were studied simultaneously, illuminated mechanistic insights into how the metric's magnitude could be increased and allowed for the unexpected role of late sodium to be elucidated.NEW & NOTEWORTHY Genetic algorithms are typically used to fit models or extract desired parameters from data. Here, we use the tool to produce a ventricular cardiomyocyte model with increased repolarization reserve. Since arrhythmia mitigation is dependent on multiple cardiac ion-channel conductances, study using a comprehensive, unbiased, and systems-level approach is important. The use of this optimization strategy allowed us to find robust profiles that illuminated unexpected mechanistic determinants of key ion-channel conductances in repolarization reserve.

Long-term QT prolongation in monkeys after doxorubicin administration at doses similar to breast cancer therapy.

Background: Studies in small animals and human patients have suggested that anthracyclines may prolong cardiac repolarization, or at least inhibit repolarization reserve, predisposing to QT prolongation and dangerous arrhythmias such as Torsades de pointes. This association in humans is difficult to confirm due to multiple confounding variables such as the presence of other medications and concurrent illness. Objectives: Identify a long-term association between anthracycline administration and repolarization prolongation in nonhuman primates, which can be measured as prolonged QT/QTc intervals on surface electrocardiogram. Methods: Five female African Green monkeys (AGMs) aged 13 ± 1 years received Doxorubicin (Dox) at doses similar to women treated for breast cancer (30-60 mg/m2/biweekly IV, total cumulative dose: 240 mg/m2) and underwent 12-lead electrocardiogram (ECG) before and 15 weeks after the final dose of Dox treatment. A blinded paired analysis was performed on ECG derived heart rate (HR), QRS, QT and QT corrected for HR (QTc) interval durations. Results: After Dox, all monkeys exhibited increased QT (BL: 323.2 ± 27.4 ms vs. Post-Dox: 366.4 ± 18.7 ms, p = 0.002) and QTc (BL: 440.2 ± 22.8 ms vs. Post-Dox: 500.8 ± 22.0 ms, p = 0.009) intervals, without any significant changes in HR or QRS duration (p = 0.92 and p = 0.47 respectively). Conclusions: AGMs treated with Dox exhibited long-term QT and QTc prolongation, along with the expected cardiotoxicity (LVEF decrease). While similar findings were shown in small animal studies, confounders make human association difficult to prove. Our finding provides a valuable intermediary step, showing direct effect of Dox on repolarization in nonhuman primates.

Optogenetic Modulation of Arrhythmia Triggers: Proof-of-Concept from Computational Modeling.

Introduction: Early afterdepolarizations (EADs) are secondary voltage depolarizations associated with reduced repolarization reserve (RRR) that can trigger lethal arrhythmias. Relating EADs to triggered activity is difficult to study, so the ability to suppress or provoke EADs would be experimentally useful. Here, we use computational simulations to assess the feasibility of subthreshold optogenetic stimulation modulating the propensity for EADs (cell-scale) and EAD-associated ectopic beats (organ-scale). Methods: We modified a ventricular ionic model by reducing rapid delayed rectifier potassium (0.25-0.1 × baseline) and increasing L-type calcium (1.0-3.5 × baseline) currents to create RRR conditions with varying severity. We ran simulations in models of single cardiomyocytes and left ventricles from post-myocardial infarction patient MRI scans. Optogenetic stimulation was simulated using either ChR2 (depolarizing) or GtACR1 (repolarizing) opsins. Results: In cell-scale simulations without illumination, EADs were seen for 164 of 416 RRR conditions. Subthreshold stimulation of GtACR1 reduced EAD incidence by up to 84.8% (25/416 RRR conditions; 0.1 µW/mm2); in contrast, subthreshold ChR2 excitation increased EAD incidence by up to 136.6% (388/416 RRR conditions; 50 µW/mm2). At the organ scale, we assumed simultaneous, uniform illumination of the epicardial and endocardial surfaces. GtACR1-mediated suppression (10-50 µW/mm2) and ChR2-mediated unmasking (50-100 µW/mm2) of EAD-associated ectopic beats were feasible in three distinct ventricular models. Conclusions: Our findings suggest that optogenetics could be used to silence or provoke both EADs and EAD-associated ectopic beats. Validation in animal models could lead to exciting new experimental regimes and potentially to novel anti-arrhythmia treatments. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00781-z.

Delayed KCNQ1/KCNE1 assembly on the cell surface helps IKs fulfil its function as a repolarization reserve in the heart.

KEY POINTS: In adult ventricular myocytes, the slow delayed rectifier (IKs ) channels are distributed on the surface sarcolemma, not t-tubules. In adult ventricular myocytes, KCNQ1 and KCNE1 have distinct cell surface and cytoplasmic pools. KCNQ1 and KCNE1 traffic from the endoplasmic reticulum to the plasma membrane by separate routes, and assemble into IKs channels on the cell surface. Liquid chromatography/tandem mass spectrometry applied to affinity-purified KCNQ1 and KCNE1 interacting proteins reveals novel interactors involved in protein trafficking and assembly. Microtubule plus-end binding protein 1 (EB1) binds KCNQ1 preferentially in its dimer form, and promotes KCNQ1 to reach the cell surface. An LQT1-associated mutation, Y111C, reduces KCNQ1 binding to EB1 dimer. ABSTRACT: Slow delayed rectifier (IKs ) channels consist of KCNQ1 and KCNE1. IKs functions as a 'repolarization reserve' in the heart by providing extra current for ventricular action potential shortening during ß-adrenergic stimulation. There has been much debate about how KCNQ1 and KCNE1 traffic in cells, where they associate to form IKs channels, and the distribution pattern of IKs channels relative to ß-adrenergic signalling complex. We used experimental strategies not previously applied to KCNQ1, KCNE1 or IKs , to provide new insights into these issues. 'Retention-using-selected-hook' experiments showed that newly translated KCNE1 constitutively trafficked through the conventional secretory path to the cell surface. KCNQ1 largely stayed in the endoplasmic reticulum, although dynamic KCNQ1 vesicles were observed in the submembrane region. Disulphide-bonded KCNQ1/KCNE1 constructs reported preferential association after they had reached cell surface. An in situ proximity ligation assay detected IKs channels in surface sarcolemma but not t-tubules of ventricular myocytes, similar to the reported location of adenylate cyclase 9/yotiao. Fluorescent protein-tagged KCNQ1 and KCNE1, in conjunction with antibodies targeting their extracellular epitopes, detected distinct cell surface and cytoplasmic pools of both proteins in myocytes. We conclude that, in cardiomyocytes, KCNQ1 and KCNE1 traffic by different routes to surface sarcolemma where they assemble into IKs channels. This mode of delayed channel assembly helps IKs fulfil its function of repolarization reserve. Proteomic experiments revealed a novel KCNQ1 interactor, microtubule plus-end binding protein 1 (EB1). EB1 dimer (active form) bound KCNQ1 and increased its surface level. An LQT1 mutation, Y111C, reduced KCNQ1 binding to EB1 dimer.

When Manual Analysis of 12-Lead ECG Holter Plays a Critical Role in Discovering Unknown Patterns of Increased Arrhythmogenic Risk: A Case Report of a Patient Treated with Tamoxifen and Subsequent Pneumonia in COVID-19.

Several medicines, including cancer therapies, are known to alter the electrophysiological function of ventricular myocytes resulting in abnormal prolongation and dispersion of ventricular repolarization (quantified by multi-lead QTc measurement). This effect could be amplified by other concomitant factors (e.g., combination with other drugs affecting the QT, and/or electrolyte abnormalities, such as especially hypokalemia, hypomagnesaemia, and hypocalcemia). Usually, this condition results in higher risk of torsade de point and other life-threatening arrhythmias, related to unrecognized unpaired cardiac ventricular repolarization reserve (VRR). Being VRR a dynamic phenomenon, QT prolongation might often not be identified during the 10-s standard 12-lead ECG recording at rest, leaving the patient at increased risk for life-threatening event. We report the case of a 49-year woman, undergoing tamoxifen therapy for breast cancer, which alteration of ventricular repolarization reserve, persisting also after correction of concomitant recurrent hypokalemia, was evidenced only after manual measurements of the corrected QT (QTc) interval from selected intervals of the 12-lead ECG Holter monitoring. This otherwise missed finding was fundamental to drive the discontinuation of tamoxifen, shifting to another "safer" therapeutic option, and to avoid the use of potentially arrhythmogenic antibiotics when treating a bilateral pneumonia in recent COVID-19.

Cardiac electrophysiological effects of ibuprofen in dog and rabbit ventricular preparations: possible implication to enhanced proarrhythmic risk.

Ibuprofen is a widely used nonsteroidal anti-inflammatory drug, which has recently been associated with increased cardiovascular risk, but its electrophysiological effects have not yet been properly studied in isolated cardiac preparations. We studied the effects of ibuprofen on action potential characteristics and several transmembrane ionic currents using the conventional microelectrode technique and the whole-cell configuration of the patch-clamp technique on cardiac preparations and enzymatically isolated ventricular myocytes. In dog (200 µM; n = 6) and rabbit (100 µM; n = 7) papillary muscles, ibuprofen moderately but significantly prolonged repolarization at 1 Hz stimulation frequency. In dog Purkinje fibers, repolarization was abbreviated and maximal rate of depolarization was depressed in a frequency-dependent manner. Levofloxacin (40 µM) alone did not alter repolarization, but augmented the ibuprofen-evoked repolarization lengthening in rabbit preparations (n = 7). In dog myocytes, ibuprofen (250 µM) did not significantly influence IK1, but decreased the amplitude of Ito and IKr potassium currents by 28.2% (60 mV) and 15.2% (20 mV), respectively. Ibuprofen also depressed INaL and ICa currents by 19.9% and 16.4%, respectively. We conclude that ibuprofen seems to be free from effects on action potential parameters at lower concentrations. However, at higher concentrations it may alter repolarization reserve, contributing to the observed proarrhythmic risk in patients.

Sex-specific IKAS activation in rabbit ventricles with drug-induced QT prolongation.

BACKGROUND: Female sex is a known risk factor for drug-induced long QT syndrome (diLQTS). We recently demonstrated a sex difference in apamin-sensitive small-conductance Ca2+-activated K+ current (IKAS) activation during ß-adrenergic stimulation. OBJECTIVE: The purpose of this study was to test the hypothesis that there is a sex difference in IKAS in the rabbit models of diLQTS. METHODS: We evaluated the sex difference in ventricular repolarization in 15 male and 22 female Langendorff-perfused rabbit hearts with optical mapping techniques during atrial pacing. HMR1556 (slowly activating delayed rectifier K+ current [IKs] blocker), E4031 (rapidly activating delayed rectifier K+ current [IKr] blocker) and sea anemone toxin (ATX-II, late Na+ current [INaL] activator) were used to simulate types 1-3 long QT syndrome, respectively. Apamin, an IKAS blocker, was then added to determine the magnitude of further QT prolongation. RESULTS: HMR1556, E4031, and ATX-II led to the prolongation of action potential duration at 80% repolarization (APD80) in both male and female ventricles at pacing cycle lengths of 300-400 ms. Apamin further prolonged APD80 (pacing cycle length 350 ms) from 187.8±4.3 to 206.9±7.1 (P=.014) in HMR1556-treated, from 209.9±7.8 to 224.9±7.8 (P=.003) in E4031-treated, and from 174.3±3.3 to 188.1±3.0 (P=.0002) in ATX-II-treated female hearts. Apamin did not further prolong the APD80 in male hearts. The Cai transient duration (CaiTD) was significantly longer in diLQTS than baseline but without sex differences. Apamin did not change CaiTD. CONCLUSION: We conclude that IKAS is abundantly increased in female but not in male ventricles with diLQTS. Increased IKAS helps preserve the repolarization reserve in female ventricles treated with IKs and IKr blockers or INaL activators.

A computational analysis of the effect of sevoflurane in a human ventricular cell model of long QT syndrome: Importance of repolarization reserve in the QT-prolonging effect of sevoflurane.

The slowly and rapidly activating delayed rectifier K+ channels (IKs and IKr, respectively) contribute to the repolarization of ventricular action potential in human heart and thereby determine QT interval on an electrocardiogram. Loss-of-function mutations in genes encoding IKs and IKr cause type 1 and type 2 long QT syndrome (LQT1 and LQT2, respectively), accompanied by a high risk of malignant ventricular arrhythmias and sudden cardiac death. This study was designed to investigate which cardiac electrophysiological conditions exaggerate QT-prolonging and arrhythmogenic effects of sevoflurane. We used the O'Hara-Rudy dynamic model to reconstruct human ventricular action potential and a pseudo-electrocardiogram, and simulated LQT1 and LQT2 phenotypes by decreasing conductances of IKs and IKr, respectively. Sevoflurane, but not propofol, prolonged ventricular action potential duration and QT interval in wild-type, LQT1 and LQT2 models. The QT-prolonging effect of sevoflurane was more profound in LQT2 than in wild-type and LQT1 models. The potent inhibitory effect of sevoflurane on IKs was primarily responsible for its QT-prolonging effect. In LQT2 model, IKs was considerably enhanced during excessive prolongation of ventricular action potential duration by reduction of IKr and relative contribution of IKs to ventricular repolarization was markedly elevated, which appears to underlie more pronounced QT-prolonging effect of sevoflurane in LQT2 model, compared with wild-type and LQT1 models. This simulation study clearly elucidates the electrophysiological basis underlying the difference in QT-prolonging effect of sevoflurane among wild-type, LQT1 and LQT2 models, and may provide important information for developing anesthetic strategies for patients with long QT syndrome in clinical settings.

Transgenic Rabbit Models in Proarrhythmia Research.

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

Validation of quantitative measure of repolarization reserve as a novel marker of drug induced proarrhythmia.

Repolarization reserve, the robustness of a cell to repolarize even when one of the repolarization mechanisms is failing, has been described qualitatively in terms of ionic currents, but has not been quantified by a generic metric that is applicable to drug screening. Prolonged repolarization leading to repolarization failure is highly arrhythmogenic. It may lead to ventricular tachycardia caused by triggered activity from early afterdepolarizations (EADs), or it may promote the occurrence of unidirectional conduction block and reentry. Both types of arrhythmia may deteriorate into ventricular fibrillation (VF) and death. We define the Repolarization Reserve Current (RRC) as the minimum constant current necessary to prevent normal repolarization of a cell. After developing and testing RRC for nine computational ionic models of various species, we applied it experimentally to atrial and ventricular human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM), and isolated guinea-pig ventricular cardiomyocytes. In simulations, repolarization was all-or-none with a precise, model-dependent critical RRC, resulting in a discrete shift in the Action Potential Duration (APD) - RRC relation, in the occurrence of EADs and repolarization failure. These data were faithfully reproduced in cellular experiments. RRC allows simple, fast, unambiguous quantification of the arrhythmogenic propensity in cardiac cells of various origins and species without the need of prior knowledge of underlying currents and is suitable for high throughput applications, and personalized medicine applications.

Left bundle branch area pacing is superior to right ventricular septum pacing concerning depolarization-repolarization reserve.

INTRODUCTION: Left bundle branch area pacing (LBBAP) has recently been reported to be a new physiological pacing strategy with clinical feasibility and safety. The present study aims to investigate depolarization-repolarization measures including QT interval, QT dispersion (QTD), and Tpeak-end interval (Tp Te ) in this novel LBBAP strategy. METHODS AND RESULTS: A total of 131 pacing-indicated patients were prospectively enrolled and randomized to the LBBAP group (n = 66) and right ventricular septum pacing (RVSP) group (n = 65). LBBAP was successfully achieved in 61 subjects with stable lead performance and comparable complications (ie, pocket hematoma, lead perforation, and dislodgement) compared with RVSP. Of the 61 patients with successful LBBAP, the mean LV peak activation time was 67.89 ± 6.80 ms, with the LBB potential mapped in 46 cases (75.4%). Electrocardiogram (ECG) indices were compared between these two groups before and after implantation. As a result, LBBAP yielded a narrower paced QRS duration (121.49 ± 9.87 ms vs 145.62 ± 8.89 ms; P < .001), shorter QT interval (434.16 ± 32.70 ms vs 462.66 ± 32.04 ms; P < .001), and QTc interval (472.44 ± 33.30 ms vs 499.65 ± 31.35 ms; P < .001), lower QTD (40.10 ± 8.68 ms vs 46.11 ± 10.85 ms; P = .001), and QTc D (43.57 ± 8.78 ms vs 49.86 ± 11.98 ms; P = .001), and shorter Tp Te (96.59 ± 10.76 ms vs 103.77 ± 10.16 ms; P < .001) than RVSP. However, Tp Te /QT ratio did not differ between these two groups (0.223 ± 0.026 vs 0.225 ± 0.022; P = .733). Furthermore, LBBAP displayed less increased QRS duration, QTc interval, QTD, QTc D, and a more shortened QT interval compared with RVSP (all P < .05). CONCLUSION: LBBAP proves to be a feasible and safe pacing procedure with better depolarization-repolarization reserve, which may predict lower risk of ventricular arrhythmia and sudden cardiac death.

Pharmacological enhancement of repolarization reserve in human induced pluripotent stem cells derived cardiomyocytes.

BACKGROUND: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for many applications including safety pharmacology. However, a deficiency or complete absence of several K+ currents suggests repolarization reserve is low in hiPSC-CMs. We determined whether a dual Ito and IKr activator can improve repolarization reserve in hiPSC-CMs resulting in a more electrophysiologically mature phenotype. METHODS AND RESULTS: Human iPSC were maintained on growth factor and differentiated into the cardiac phenotype by addition of selective Wnt molecules. Current and voltage clamp recordings in single cells were made using patch electrodes. Extracellular field potentials were made using a microelectrode array on hiPSC monolayers. Action potential recordings from hiPSC-CMs following application of an IKr inhibitor resulted in depolarization of the membrane potential and prolongation of the APD. A flattening of the T-wave was noted on the pseudo-ECG. In contrast, application of the IKr and Ito agonist, NS3623, resulted in hyperpolarization of the membrane, slowing of the spontaneous rate and shortening of the APD. Voltage clamp recording showed a significant increase in IKr; no enhancement of Ito in hiPSC-CMs was noted. AP clamp experiments revealed that IKr plays a role in both phase 3 repolarization and phase 4 depolarization. mRNA analysis revealed that KCNH2 is abundantly expressed in hiPSC-CM, consistent with electrophysiological recordings. CONCLUSIONS: Although NS3623 is a dual Ito and IKr activator in ventricular myocytes, application of this compound to hiPSC-CMs enhanced only IKr and no effect on Ito was noted. Our results suggest IKr enhancement can improve repolarization reserve in this cell type. The disconnect between a dramatic increase in Ito in adult myocytes versus the lack of effect in hiPSC-CMs suggest that the translation of pharmacological effects in hiPSC-CM to adult myocytes should be viewed with caution.

Chronic in vivo angiotensin II administration differentially modulates the slow delayed rectifier channels in atrial and ventricular myocytes.

BACKGROUND: In the heart, slow delayed rectifier channels provide outward currents (IKs) for action potential (AP) repolarization in a region- and context-dependent manner. In diseased hearts, chronic elevation of angiotensin II (Ang II) may remodel IKs in a region-dependent manner, contributing to atrial and ventricular arrhythmias of different mechanisms. OBJECTIVE: The purpose of this study was to study whether/how chronic in vivo Ang II administration remodels IKs in atrial and ventricular myocytes. METHODS: We used the guinea pig (GP) model whose myocytes express robust IKs. GPs were implanted with minipumps containing Ang II or vehicle. Treatment continued for 4-6 weeks. We used patch clamp, immunofluorescence/confocal microscopy, and immunoblots to evaluate changes in IKs function and to explore the underlying mechanisms. RESULTS: We confirmed the pathologic state of the heart after chronic Ang II treatment. IKs density was increased in atrial myocytes but decreased in ventricular myocytes in Ang II- vs vehicle-treated animals. The former was correlated with an increase in KCNQ1/KCNE1 colocalization in myocyte periphery, whereas the latter was correlated with a decrease in KCNQ1 protein level. Interestingly, these changes in IKs were not translated into expected alterations in AP duration or plateau voltage, indicating that other currents were involved. In atrial myocytes from Ang II-treated animals, the L-type Ca channel current was increased, contributing to AP plateau elevation and AP duration prolongation. CONCLUSION: IKs is differentially modulated by chronic in vivo Ang II administration between atrial and ventricular myocytes. Other currents remodeled by Ang II treatment also contribute to changes in action potentials.

Action Potential Prolongation, ß-Adrenergic Stimulation, and Angiotensin II as Co-factors in Sarcoplasmic Reticulum Instability.

Introduction: Increases in action potential duration (APD), genetic or acquired, and arrhythmias are often associated; nonetheless, the relationship between the two phenomena is inconstant, suggesting coexisting factors. ß-adrenergic activation increases sarcoplasmic reticulum (SR) Ca2+-content; angiotensin II (ATII) may increase cytosolic Ca2+ and ROS production, all actions stimulating RyRs opening. Here we test how APD interacts with ß-adrenergic and AT-receptor stimulation in facilitating spontaneous Ca2+ release events (SCR). Methods: Under "action potential (AP) clamp", guinea-pig cardiomyocytes (CMs) were driven with long (200 ms), normal (150 ms), and short (100 ms) AP waveforms at a CL of 500 ms; in a subset of CMs, all the 3 waveforms could be tested within the same cell. SCR were detected as inward current transients (ITI) following repolarization; ITI incidence and repetition within the same cycle were measured under increasing isoprenaline concentration ([ISO]) alone, or plus 100 nM ATII (30 min incubation+superfusion). Results: ITI incidence and repetition increased with [ISO]; at longer APs the [ISO]-response curve was shifted upward and ITI coupling interval was reduced. ATII increased ITI incidence more at low [ISO] and under normal (as compared to long) APs. Efficacy of AP shortening in suppressing ITI decreased in ATII-treated myocytes and at higher [ISO]. Conclusions: AP prolongation sensitized the SR to the destabilizing actions of ISO and ATII. Summation of ISO, ATII and AP duration effects had a "saturating" effect on SCR incidence, thus suggesting convergence on a common factor (RyRs stability) "reset" by the occurrence of spontaneous Ca2+ release events.

Effect of down-regulation of IKs repolarization-reserve on ventricular arrhythmogenesis in a guinea pig model of cardiac hypertrophy / 中南大学学报(医学版)

Objective:To observe the changes of rapidly activated delayed rectifier potassium channel (IKs) and slowly activated delayed rectifier potassium channel (IKs) in cardiac hypertrophy and to evaluate the effects of IKs and IKs blocker on the incidence ofventricular arrhythmias in guinea pigs with left ventricular hypertrophy (LVH).Methods:Guinea pigs were divided into a sham operation group and a left ventricular hypertrophy (LVH) group.LVH model was prepared.Whole cell patch-clamp technique was used to record IKr and IKs tail currents in a guinea pig model with LVH.The changes of QTc and the incidence rate of ventricular arrhythmias in LVH guinea pigs were observed by using the IKr and IKs blockers.Results:Compared with cardiac cells in the control group,the interventricular septal thickness at end systole (IVSs),left ventricular posterior wall thickness at end systole (LVPWs),QTc interval and cell capacitance in guinea pigs with LVH were significantly increased (P＜0.05);while IKs densities were significantly reduced [+60 mV:(0.36±0.03) pA/pF vs (0.58±0.05) pA/pF,P＜0.01].However,LVH exerted no significant effect on IKr densities.IKr blocker markedly prolonged the QTc interval (P＜0.01) and increased the incidence of ventricular arrhythmias in guinea pigs with LVH compared with the control guinea pigs.In contrast,IKs blocker produced modest increase in QTc interval in guinea pigs of control group with no increase in LVH animals.IKs blocker did not induce ventricular arrhythmias incidence in either control or LVH animals.Conclusion:The cardiac hypertrophy-induced arrhythmogenesis is due to the down-regulation of IKs.

Cardiac action potential repolarization revisited: early repolarization shows all-or-none behaviour.

In healthy mammalian hearts the action potential (AP) waveform initiates and modulates each contraction, or heartbeat. As a result, AP height and duration are key physiological variables. In addition, rate-dependent changes in ventricular AP duration (APD), and variations in APD at a fixed heart rate are both reliable biomarkers of electrophysiological stability. Present guidelines for the likelihood that candidate drugs will increase arrhythmias rely on small changes in APD and Q-T intervals as criteria for safety pharmacology decisions. However, both of these measurements correspond to the final repolarization of the AP. Emerging clinical evidence draws attention to the early repolarization phase of the action potential (and the J-wave of the ECG) as an additional important biomarker for arrhythmogenesis. Here we provide a mechanistic background to this early repolarization syndrome by summarizing the evidence that both the initial depolarization and repolarization phases of the cardiac action potential can exhibit distinct time- and voltage-dependent thresholds, and also demonstrating that both can show regenerative all-or-none behaviour. An important consequence of this is that not all of the dynamics of action potential repolarization in human ventricle can be captured by data from single myocytes when these results are expressed as 'repolarization reserve'. For example, the complex pattern of cell-to-cell current flow that is responsible for AP conduction (propagation) within the mammalian myocardium can change APD and the Q-T interval of the electrocardiogram alter APD stability, and modulate responsiveness to pharmacological agents (such as Class III anti-arrhythmic drugs).