Investigating the relevance of CYP2J2 inhibition for drugs known to cause intermediate to high risk torsades de pointes.

Cardiac cytochrome P450 2J2 (CYP2J2) metabolizes endogenous polyunsaturated fatty acid, arachidonic acid (AA), to bioactive regioisomeric epoxyeicosatrienoic acid (EET) metabolites. This endogenous metabolic pathway has been postulated to play a homeostatic role in cardiac electrophysiology. However, it is unknown if drugs that cause intermediate to high risk torsades de pointes (TdP) exhibit inhibitory effects against CYP2J2 metabolism of AA to EETs. In this study, we demonstrated that 11 out of 16 drugs screened with intermediate to high risk of TdP as defined by the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative are concurrently reversible inhibitors of CYP2J2 metabolism of AA, with unbound inhibitory constant (Ki,AA,u) values ranging widely from 0.132 to 19.9 µM. To understand the physiological relevancy of Ki,AA,u, the in vivo unbound drug concentration within human heart tissue (Cu,heart) was calculated via experimental determination of in vitro unbound partition coefficient (Kpuu) for 10 CYP2J2 inhibitors using AC16 human ventricular cardiomyocytes as well as literature-derived values of fraction unbound in plasma (fu,p) and plasma drug concentrations in clinical scenarios leading to TdP. Notably, all CYP2J2 inhibitors screened belonging to the high TdP risk category, namely vandetanib and bepridil, exhibited highest Kpuu values of 18.2 ± 1.39 and 7.48 ± 1.16 respectively although no clear relationship between Cu,heart and risk of TdP could eventually be determined. R values based on basic models of reversible inhibition as per FDA guidelines were calculated using unbound plasma drug concentrations (Cu,plasma) and adapted using Cu,heart which suggested that 4 out of 10 CYP2J2 inhibitors with intermediate to high risk of TdP demonstrate greatest potential for clinically relevant in vivo cardiac drug-AA interactions. Our results shed novel insights on the relevance of CYP2J2 inhibition in drugs with risk of TdP. Further studies ascertaining the role of CYP2J2 metabolism of AA in cardiac electrophysiology, characterizing inherent cardiac ion channel activities of drugs with risk of TdP as well as in vivo evidence of drug-AA interactions will be required prior to determining if CYP2J2 inhibition could be an alternative mechanism contributing to drug-induced TdP.

Use of high throughput ion channel profiling and statistical modeling to predict off-target arrhythmia risk - One pharma's experience and perspective.

INTRODUCTION: The use of high throughput patch clamp profiling to determine mixed ion channel-mediated arrhythmia risk was assessed using profiling data generated using proprietary internal and clinical reference compounds. We define the reproducibility of the platform and highlight inherent platform issues. The data generated was used to develop predictive models for cardiac arrhythmia risk, specifically Torsades de Pointes (TdP). METHODS: A retrospective analysis was performed using profiling data generated over a 3-year period, including patch clamp data from hERG, Cav1.2, and Nav1.5 (peak/late), together with hERG binding. RESULTS: Assay reproducibility was robust over the 3-year period examined. High throughput hERG patch IC50 values correlated well with GLP-hERG data (Pearson = 0.87). A disconnect between hERG binding and patch was observed for ∼10% compounds and trended with passive cellular permeability. hERG and Cav1.2 potency did not correlate for proprietary compounds, with more potent hERG compounds showing selectivity versus Cav1.2. For clinical compounds where hERG and Cav1.2 activity was more balanced, an analysis of TdP risk versus hERG/Cav1.2 ratio demonstrated low TdP probability when the hERG/Cav1.2 potency ratios were < 1. Modeling of clinical compound data revealed a lack of impact of the Nav1.5 (late) current in predicting TdP. Moreover, models using hERG binding data (ROC AUC = 0.876) showed an improved ability to predict TdP risk versus hERG patch clamp (ROC AUC = 0.787). DISCUSSION: The data highlight the value of high throughput patch clamp data in the prediction of TdP risk, as well as some potential limitations with this approach.

A Novel Mutation in the KCNH2 Gene Associatedwith Long QT Syndrome: A Case Report.

OBJECTIVE: Long QT syndrome is a cardiovascular disease with a prolonged QT interval. CASE REPORT: We report a 22-year-old woman presenting with frequent syncopal episodes two months after childbirth. Electrocardiography showed a sinus rhythm, QT interval prolongation, and Torsade de Pointes. Her mother had experienced an episode of syncope, but her father had not. Genetic analyses revealed that a new mutation in the KCNH2 gene, the c.2108dupA mutation (p.H703Qfs*20, exon8, M_000238), was found in the patient and in her mother and sister. CONCLUSION: The c.2108dupA mutation (p.H703Qfs*20, exon8, M_000238) is the first reported case of a KCNH2 mutation at this site.

Flavonoids and hERG channels: Friends or foes?

The cardiac action potential is regulated by several ion channels. Drugs capable to block these channels, in particular the human ether-à-go-go-related gene (hERG) channel, also known as KV11.1 channel, may lead to a potentially lethal ventricular tachyarrhythmia called "Torsades de Pointes". Thus, evaluation of the hERG channel off-target activity of novel chemical entities is nowadays required to safeguard patients as well as to avoid attrition in drug development. Flavonoids, a large class of natural compounds abundantly present in food, beverages, herbal medicines, and dietary food supplements, generally escape this assessment, though consumed in consistent amounts. Continuously growing evidence indicates that these compounds may interact with the hERG channel and block it. The present review, by examining numerous studies, summarizes the state-of-the-art in this field, describing the most significant examples of direct and indirect inhibition of the hERG channel current operated by flavonoids. A description of the molecular interactions between a few of these natural molecules and the Rattus norvegicus channel protein, achieved by an in silico approach, is also presented.

Cardiohemodynamic and Arrhythmogenic Effects of the Anti-Atrial Fibrillatory Compound Vanoxerine in Halothane-Anesthetized Dogs.

While vanoxerine (GBR-12909) is a synaptosomal dopamine uptake inhibitor, it also suppresses IKr, INa and ICa,L in vitro. Based on these profiles on ionic currents, vanoxerine has been developed as a candidate compound for treating atrial fibrillation. To investigate electropharmacological profiles, vanoxerine dihydrochloride was intravenously administered at 0.03 and 0.3 mg/kg to halothane-anesthetized dogs (n = 4), possibly providing subtherapeutic and therapeutic concentrations, respectively. The low dose increased the heart rate and cardiac output, whereas it prolonged the ventricular refractoriness. The high dose decreased the heart rate but increased the total peripheral vascular resistance, whereas it delayed the ventricular repolarization and increased the atrial refractoriness in addition to further enhancing the ventricular refractoriness. The extent of increase in the refractoriness in the atrium was 0.8 times of that in the ventricle. The high dose also prolonged the early and late repolarization periods of the ventricle as well as the terminal repolarization period. Meanwhile, no significant change was detected in the mean blood pressure, ventricular contraction, preload to the left ventricle, or the intra-atrial, intra-ventricular or atrioventricular conductions. The high dose can be considered to inhibit IKr, but it may not suppress INa or ICa in the in situ heart, partly explaining its poor atrial selectivity for increasing refractoriness. The prolongation of early repolarization period may reflect enhancement of net inward current, providing potential risk for intracellular Ca2+ overload. Thus, vanoxerine may provide both trigger and substrate toward torsade de pointes, which would make the drug less promising as an anti-atrial fibrillatory drug.

Revisiting the hERG safety margin after 20 years of routine hERG screening.

INTRODUCTION: It has been two decades since screening new molecules and potential clinical drug candidates against the hERG potassium channel became a routine part of safety pharmacology. The earliest heuristic for what was an adequate safety margin to separate molecules with a potential liability to cause the arrhythmia torsade de pointes (TdP) from those with no such liability emerged in 2002 and was determined to be a hERG IC50 value 30-fold above the therapeutic free plasma concentration (Webster, Leishman, & Walker, 2002). In the intervening years nonclinical and clinical ICH guidance has been introduced and intense scrutiny has been applied to the QT interval of the electrocardiogram in animals and man. Has the 30-fold heuristic stood the test of time? METHODS: The hERG margins between the IC50 value and the therapeutic unbound plasma concentrations were examined for 367 compounds. These margins were compared against the categories used by www.CredibleMeds.com to classify a drug's TdP risk. A subset of 336 of these drugs were compared against their US product labels with respect to black box warnings on QTc prolongation or TdP, warnings and precautions on QTc or TdP, and QTc language in the clinical pharmacology section. RESULTS: Against the CredibleMeds classification the means of the margins for Known, Conditional, or Possible Risk of TdP, and Not Listed (presumably no TdP liability) were 4.8, 28, 71 and 339, respectively. Against the US label language the means of margins for black boxes and warnings were 3.1 and 26, respectively. The average margins associated with, positive QTc outcome, negative QTc outcome and no QTc language were 16, 479 and 204, respectively. Based on ROC curves the optimal hERG margin thresholds to separate "Known risk of TdP" from "Not Listed" and, QTc prolongation positive from QTc negative were 37- and 50-fold, respectively. CONCLUSIONS: The observed optimal margin of 50-fold in the current study is not appreciably different from a previously reported 45-fold optimal margin (Gintant, 2011). The margin falls between the margins for negative (QTc outcome or no QTc language) and positive (positive QTc outcome, warnings or black boxes) compounds. The observed optimal margin of 37-fold in the current study is not appreciably different from the commonly used 30-fold optimal margin (Webster et al., 2002). This margin falls between those for drugs with a known or conditional TdP risk and those where it is at best a possible risk, and from the 240 drugs not listed on www.CredibleMeds.com. It is expected that there would be a small numerical difference (e.g. 37 vs. 50, or as previously published 30 vs. 45) between optimal cut-offs for the TdP liability and QTc prolongation predictions since some QTc positive drugs are described on CredibleMeds.com as having only a "Possible Risk of TdP" as they are not associated with TdP when used as directed. The fact that the margins in each category form distributions is also expected given biologic variability. However, we argue that a more consistent manner of assessing hERG potency and evaluating relevant exposures would be likely to reduce the spread in these distributions and make margins even more useful as a decision-making data point.

Cardiac Arrest Risk During Acute Infections: Systemic Inflammation Directly Prolongs QTc Interval via Cytokine-Mediated Effects on Potassium Channel Expression.

BACKGROUND: During acute infections, the risk of malignant ventricular arrhythmias is increased, partly because of a higher propensity to develop QTc prolongation. Although it is generally believed that QTc changes almost exclusively result from concomitant treatment with QT-prolonging antimicrobials, direct effects of inflammatory cytokines on ventricular repolarization are increasingly recognized. We hypothesized that systemic inflammation per se can significantly prolong QTc during acute infections, via cytokine-mediated changes in K+ channel expression. METHODS: We evaluated (1) the frequency of QTc prolongation and its association with inflammatory markers, in patients with different types of acute infections, during active disease and remission; (2) the prevalence of acute infections in a cohort of consecutive patients with Torsades de Pointes; (3) the relationship between K+ channel mRNA levels in ventricles and peripheral blood mononuclear cells and their changes in patients with acute infection over time. RESULTS: In patients with acute infections, regardless of concomitant QT-prolonging antimicrobial treatments, QTc was significantly prolonged but rapidly normalized in parallel to CRP (C-reactive protein) and cytokine level reduction. Consistently in the Torsades de Pointes cohort, concomitant acute infections were highly prevalent (30%), despite only a minority (25%) of these cases were treated with QT-prolonging antimicrobials. KCNJ2 K+ channel expression in peripheral blood mononuclear cell, which strongly correlated to that in ventricles, inversely associated to CRP and IL (interleukin)-1 changes in acute infection patients. CONCLUSIONS: During acute infections, systemic inflammation rapidly induces cytokine-mediated ventricular electrical remodeling and significant QTc prolongation, regardless concomitant antimicrobial therapy. Although transient, these changes may significantly increase the risk of life-threatening ventricular arrhythmia in these patients. It is timely and warranted to transpose these findings to the current coronavirus disease 2019 (COVID-19) pandemic, in which both increased amounts of circulating cytokines and cardiac arrhythmias are demonstrated along with a frequent concomitant treatment with several QT-prolonging drugs. Graphic Abstract: A graphic abstract is available for this article.

Experimental analysis of the onset mechanism of TdP reported in an LQT3 patient during pharmacological treatment with serotonin-dopamine antagonists against insomnia and nocturnal delirium.

Torsade de pointes (TdP) occurred in a long QT syndrome type 3 (LQT3) patient after switching perospirone to blonanserin. We studied how their electropharmacological effects had induced TdP in the LQT3 patient. Perospirone hydrochloride (n = 4) or blonanserin (n = 4) of 0.01, 0.1, and 1 mg/kg, i.v. was cumulatively administered to the halothane-anesthetized dogs over 10 min. The low dose of perospirone decreased total peripheral vascular resistance, but increased heart rate and cardiac output, facilitated atrioventricular conduction, and prolonged J-Tpeakc. The middle dose decreased mean blood pressure and prolonged repolarization period, in addition to those observed after the low dose. The high dose further decreased mean blood pressure with the reduction of total peripheral vascular resistance; however, it did not increase heart rate or cardiac output. It tended to delay atrioventricular conduction and further delayed repolarization with the prolongation of Tpeak-Tend, whereas J-Tpeakc returned to its baseline level. Meanwhile, each dose of blonanserin decreased total peripheral vascular resistance, but increased heart rate, cardiac output and cardiac contractility in a dose-related manner. J-Tpeakc was prolonged by each dose, but Tpeak-Tend was shortened by the middle and high doses. These results indicate that perospirone and blonanserin may cause the hypotension-induced, reflex-mediated increase of sympathetic tone, leading to the increase of inward Ca2+ current in the heart except that the high dose of perospirone reversed them. Thus, blonanserin may have more potential to produce intracellular Ca2+ overload triggering early afterdepolarization than perospirone, which might explain the onset of TdP in the LQT3 patient.

The Use of iPSC-Derived Cardiomyocytes and Optical Mapping for Erythromycin Arrhythmogenicity Testing.

Erythromycin is an antibiotic that prolongs the QT-interval and causes Torsade de Pointes (TdP) by blocking the rapid delayed rectifying potassium current (IKr) without affecting either the slow delayed rectifying potassium current (IKs) or inward rectifying potassium current (IK1). Erythromycin exerts this effect in the range of 1.5-100 µM. However, the mechanism of action underlying its cardiotoxic effect and its role in the induction of arrhythmias, especially in multicellular cardiac experimental models, remain unclear. In this study, the re-entry formation, conduction velocity, and maximum capture rate were investigated in a monolayer of human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes from a healthy donor and in a neonatal rat ventricular myocyte (NRVM) monolayer using the optical mapping method under erythromycin concentrations of 15, 30, and 45 µM. In the monolayer of human iPSC-derived cardiomyocytes, the conduction velocity (CV) varied up to 12 ± 9% at concentrations of 15-45 µM as compared with that of the control, whereas the maximum capture rate (MCR) declined substantially up to 28 ± 12% (p < 0.01). In contrast, the tests on the NRVM monolayer showed no significant effect on the MCR. The results of the arrhythmogenicity test provided evidence for a "window" of concentrations of the drug (15-30 µM) at which the probability of re-entry increased.

The influence of hERG1a and hERG1b isoforms on drug safety screening in iPSC-CMs.

The human Ether-à-go-go Related Gene (hERG) encodes the pore forming subunit of the channel that conducts the rapid delayed rectifier potassium current IKr. IKr drives repolarization in the heart and when IKr is dysfunctional, cardiac repolarization delays, the QT interval on the electrocardiogram (ECG) prolongs and the risk of developing lethal arrhythmias such as Torsade de Pointes (TdP) increases. TdP risk is incorporated in drug safety screening for cardiotoxicity where hERG is the main target since the IKr channels appear highly sensitive to blockage. hERG block is also included as an important read-out in the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative which aims to combine in vitro and in silico experiments on induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to screen for cardiotoxicity. However, the hERG channel has some unique features to consider for drug safety screening, which we will discuss in this study. The hERG channel consists of different isoforms, hERG1a and hERG1b, which individually influence the kinetics of the channel and the drug response in the human heart and in iPSC-CMs. hERG1b is often underappreciated in iPSC-CM studies, drug screening assays and in silico models, and the fact that its contribution might substantially differ between iPSC-CM and healthy but also diseased human heart, adds to this problem. In this study we show that the activation kinetics in iPSC-CMs resemble hERG1b kinetics using Cs+ as a charge carrier. Not including hERG1b in drug safety testing might underestimate the actual role of hERG1b in repolarization and drug response, and might lead to inappropriate conclusions. We stress to focus more on including hERG1b in drug safety testing concerning IKr.

Predicting critical drug concentrations and torsadogenic risk using a multiscale exposure-response simulator.

Torsades de pointes is a serious side effect of many drugs that can trigger sudden cardiac death, even in patients with structurally normal hearts. Torsadogenic risk has traditionally been correlated with the blockage of a specific potassium channel and a prolonged recovery period in the electrocardiogram. However, the precise mechanisms by which single channel block translates into heart rhythm disorders remain incompletely understood. Here we establish a multiscale exposure-response simulator that converts block-concentration characteristics from single cell recordings into three-dimensional excitation profiles and electrocardiograms to rapidly assess torsadogenic risk. For the drug dofetilide, we characterize the QT interval and heart rate at different drug concentrations and identify the critical concentration at the onset of torsades de pointes: For dofetilide concentrations of 2x, 3x, and 4x, as multiples of the free plasma concentration Cmax = 2.1 nM, the QT interval increased by +62.0%, +71.2%, and +82.3% compared to baseline, and the heart rate changed by -21.7%, -23.3%, and +88.3%. The last number indicates that, at the critical concentration of 4x, the heart spontaneously developed an episode of a torsades-like arrhythmia. Strikingly, this critical drug concentration is higher than the concentration estimated from early afterdepolarizations in single cells and lower than in one-dimensional cable models. Our results highlight the importance of whole heart modeling and explain, at least in part, why current regulatory paradigms often fail to accurately quantify the pro-arrhythmic potential of a drug. Our exposure-response simulator could provide a more mechanistic assessment of pro-arrhythmic risk and help establish science-based guidelines to reduce rhythm disorders, design safer drugs, and accelerate drug development.

Assessment of Multi-Ion Channel Block in a Phase I Randomized Study Design: Results of the CiPA Phase I ECG Biomarker Validation Study.

Balanced multi-ion channel-blocking drugs have low torsade risk because they block inward currents. The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative proposes to use an in silico cardiomyocyte model to determine the presence of balanced block, and absence of heart rate corrected J-Tpeak (J-Tpeak c) prolongation would be expected for balanced blockers. This study included three balanced blockers in a 10-subject-per-drug parallel design; lopinavir/ritonavir and verapamil met the primary end point of ΔΔJ-Tpeak c upper bound < 10 ms, whereas ranolazine did not (upper bounds of 8.8, 6.1, and 12.0 ms, respectively). Chloroquine, a predominant blocker of the potassium channel encoded by the ether-à-go-go related gene (hERG), prolonged ΔΔQTc and ΔΔJ-Tpeak c by ≥ 10 ms. In a separate crossover design, diltiazem (calcium block) did not shorten dofetilide-induced ΔQTc prolongation, but shortened ΔJ-Tpeak c and prolonged ΔTpeak -Tend . Absence of J-Tpeak c prolongation seems consistent with balanced block; however, small sample size (10 subjects) may be insufficient to characterize concentration-response in some cases.

Sex-Related Differences in Drug-Induced QT Prolongation and Torsades de Pointes: A New Model System with Human iPSC-CMs.

Numerous drugs have the potential to prolong the QT interval and may cause accidental cardiac arrest (torsades de pointes [TdP]). Women are at a higher risk than men for experiencing drug-induced TdP. Due to the lack of appropriate tools, few studies have investigated whether genetic differences between men and women have any effects on drug-induced proarrhythmia. Sex hormones are believed to play a predominant role in the induction of TdP. Recently, progress in induced pluripotent stem cell (iPSC) technologies has made it possible to utilize human iPSC-derived cardiomyocytes (hiPSC-CMs) to investigate the influence of both genetics and sex hormones on cardiac ion channel gene expression and cardiomyocyte function. In this study, we investigated genetic and hormonal effects on sex differences of drug-induced QT prolongation and TdP with hiPSC-CMs from healthy male and female donors. We found that despite batch variations in beating rates and field potential durations (FPD), female-derived hiPSC-CMs showed steeper slopes of FPD to interspike interval ratios and were more sensitive to IKr blocker-induced FPD prolongation. 17ß-estradiol increased FPD and 5α-dihydrotestosterone shortened FPD, but the addition of sex hormones had limited effect on the responses of hiPSC-CMs to IKr blockades. The differential expression of KCNE1 gene and reduced repolarization reserve in female-derived hiPSC-CMs compared with male-derived hiPSC-CMs may partially explain why females are more susceptible to proarrhythmias. Human iPSC-CMs can be a useful new model to study mechanisms of sex differences in cardiomyocyte repolarization processes and aid in the prediction of drug-induced proarrhythmias in both men and women.

Paradoxical Effects of Sodium-Calcium Exchanger Inhibition on Torsade de Pointes and Early Afterdepolarization in a Heart Failure Rabbit Model.

Calcium homeostasis plays an important role in development of early afterdepolarizations (EADs) and torsade de pointes (TdP). The role of sodium-calcium exchanger (NCX) inhibition in genesis of secondary Ca rise and EAD-TdP is still debated. Dual voltage and intracellular Ca optical mapping were conducted in 6 control and 9 failing rabbit hearts. After baseline electrophysiological and optical mapping studies, E4031 was given to simulate long QT syndrome. ORM-10103 was then administrated to examine the electrophysiological effects on EAD-TdP development. E4031 enhanced secondary Ca rise, EADs development, and TdP inducibility in both control and failing hearts. The results showed that ORM-10103 reduced premature ventricular beats but was unable to suppress the inducibility of TdP or EADs. The electrophysiological effects of ORM-10103 included prolongation of action potential duration (APD) and increased APD heterogeneity in failing hearts. ORM-10103 had a neutral effect on the amplitude of secondary Cai rise in control and heart failure groups. In this model, most EADs generated from long-short APD junction area. In conclusion, highly selective NCX inhibition with ORM-10103 reduced premature ventricular beat burden but was unable to suppress secondary Ca rise, EADs development, or inducibility of TdP. The possible electrophysiological mechanisms include APD prolongation and increased APD heterogeneity.

Dehydroevodiamine and hortiamine, alkaloids from the traditional Chinese herbal drug Evodia rutaecarpa, are IKr blockers with proarrhythmic effects in vitro and in vivo.

Evodiae fructus is a widely used herbal drug in traditional Chinese medicine. Evodia extract was found to inhibit hERG channels. The aim of the current study was to identify hERG inhibitors in Evodia extract and to investigate their potential proarrhythmic effects. Dehydroevodiamine (DHE) and hortiamine were identified as IKr (rapid delayed rectifier current) inhibitors in Evodia extract by HPLC-microfractionation and subsequent patch clamp studies on human embryonic kidney cells. DHE and hortiamine inhibited IKr with IC50s of 253.2±26.3nM and 144.8±35.1nM, respectively. In dog ventricular cardiomyocytes, DHE dose-dependently prolonged the action potential duration (APD). Early afterdepolarizations (EADs) were seen in 14, 67, 100, and 67% of cells after 0.01, 0.1, 1 and 10µM DHE, respectively. The proarrhythmic potential of DHE was evaluated in 8 anesthetized rabbits and in 8 chronic atrioventricular block (cAVB) dogs. In rabbits, DHE increased the QT interval significantly by 12±10% (0.05mg/kg/5min) and 60±26% (0.5mg/kg/5min), and induced Torsade de Pointes arrhythmias (TdP, 0.5mg/kg/5min) in 2 rabbits. In cAVB dogs, 0.33mg/kg/5min DHE increased QT duration by 48±10% (P<0.05*) and induced TdP in 2/4 dogs. A higher dose did not induce TdP. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), methanolic extracts of Evodia, DHE and hortiamine dose-dependently prolonged APD. At 3µM DHE and hortiamine induced EADs. hERG inhibition at submicromolar concentrations, APD prolongation and EADs in hiPSC-CMs and dose-dependent proarrhythmic effects of DHE at micromolar plasma concentrations in cAVB dogs should increase awareness regarding proarrhythmic effects of widely used Evodia extracts.

Ryanodine-receptor inhibition by dantrolene effectively suppresses ventricular arrhythmias in an ex vivo model of long-QT syndrome.

AIMS: A significant antiarrhythmic potential of ryanodine receptor inhibition was reported in experimental studies. The aim of the present study was to assess potential antiarrhythmic effects of dantrolene in an experimental whole-heart model of drug-induced long-QT syndrome (LQTS). METHODS: In 12 isolated rabbit hearts, long-QT-2-syndrome was simulated by infusion of erythromycin (300 µM). Twelve rabbit hearts were treated with veratridine (0.5 µM) to mimic long-QT-3-syndrome. RESULTS: Monophasic action potentials and ECG showed a significant prolongation of QT-interval (+71 ms, P < 0.01) and action potential duration (APD, +43 ms, P < 0.01) after infusion of erythromycin as compared with baseline. Similar results were obtained in veratridine-treated hearts (QT-interval: +43 ms, P < 0.01; APD: +36 ms, P < 0.01). Both erythromycin (+36 ms, P < 0.05) and veratridine (+38 ms) significantly increased dispersion of repolarization. Additional infusion of dantrolene (20 µM) did not significantly alter QT-interval and APD but resulted in a significant reduction of dispersion of repolarization (erythromycin group: -33 ms, P < 0.05; veratridine group: -29 ms, P < 0.05). Lowering of potassium concentration resulted in the occurrence of early afterdepolarizations (EAD) and polymorphic ventricular tachycardia (VT) in 9 of 12 erythromycin-treated hearts (175 episodes) and 8 of 12 veratridine-treated hearts (66 episodes). Additional infusion of dantrolene significantly reduced occurrence of polymorphic VT and resulted in occurrence of EAD and polymorphic VT in 1 of 12 erythromycin-treated hearts (18 episodes) and 1 of 12 veratridine-treated hearts (3 episodes). CONCLUSION: Inhibition of the ryanodine receptor by dantrolene significantly reduced occurrence of polymorphic VT in drug-induced LQTS. A significant reduction of spatial dispersion of repolarization represents a major antiarrhythmic mechanism. These results imply that dantrolene may represent a promising antiarrhythmic option in drug-induced LQTS.

Torsades de Pointes in Patients with Polymyalgia Rheumatica.

Polymyalgia rheumatica (PMR) represents the most common inflammatory rheumatic disease of the elderly. It is characterized by synovitis of proximal joints and extra-articular synovial structures, along with chronic high-grade systemic inflammation. PMR is closely related to giant cell arteritis (GCA), a large-vessel vasculitis that involves the major branches of the aorta, particularly the extracranial branches of carotid artery including temporal arteries. It is currently believed that PMR and GCA may represent different manifestations of the same disease process. Chronic systemic inflammation is presently recognized as one of the key pathogenic mechanisms underlying cardiovascular disease and associated complications, including cardiac arrhythmias and sudden death. In this regard, several studies demonstrated that besides promoting structural heart disease, inflammatory activation may also be per se arrhythmogenic, via cytokine-mediated effects on cardiac electrophysiology. In particular, increasing evidence points to inflammation as a novel risk factor for QTc prolongation and related life-threatening arrhythmias, specifically Torsade de Pointes (TdP). Starting from the report of two cases of TdP occurring in PMR patients with active disease and elevated circulating IL-6 levels, we here reviewed literature data regarding heart involvement and arrhythmic events in PMR/GCA, as well as TdP risk in inflammatory diseases. Potential underlying mechanisms were dissected, by focusing on the driving role of inflammatory activation.

Simulation study of the ionic mechanisms underlying Torsade de Pointes in a 2D cardiac tissue.

BACKGROUND: To understand the ionic mechanism behind the genesis of Torsade de Pointes (TdP) occurring with long QT syndrome 2 (LQTS2) in a remodelled transmural tissue. METHODS: The TP06 model is used to simulate the electrical activity of cells in a 2D transmural ventricular model. LQTS2 is realised by reducing the potassium current (IKr) to 0.5 in each cell. Each cell of the tissue is remodelled by increasing the conductance of calcium current (GCaL). The above two factors make the cells prone to early after depolarizations (EADs) development. The rise in GCaL that can develop a sustained TdP at normal pacing rate is determined from this study. A look at the calcium dynamics, sodium-calcium exchanger current (INaCa) and slow delayed rectifier potassium current (IKs) distribution maps of the tissue helps us in analysing the mechanism of TdP generation. RESULTS: A TdP type pattern at normal pacing rate is generated when GCaL is more than 3.5 times the control parameter. From the M-cell island, an adequate number of cells spontaneously release calcium from their sarcoplasmic reticulum leading to increased intracellular calcium and inward sodium current through the sodium calcium exchanger current (INaCa). These contribute to the development of EADs which create a depolarising wavefront that triggers TdP in the tissue. When GCaL is less than 3.5 times the control value, premature ventricular complexes (PVC) occur interspersed between normal beats. CONCLUSION: Normal pacing rates can induce a multi focal TdP when sufficient number of M-cells simultaneously undergo spontaneous calcium release (SCR) events.

Automated Patch-Clamp Methods for the hERG Cardiac Potassium Channel.

The human Ether-a-go-go Related Gene (hERG) product has been identified as a central ion channel underlying both familial forms of elongated QT interval on the electrocardiogram and drug-induced elongation of the same QT segment. Indeed, reduced function of this potassium channel involved in the repolarization of the cardiac action potential can produce a type of life-threatening cardiac ventricular arrhythmias called Torsades de Pointes (TdP). Therefore, hERG inhibitory activity of newly synthetized molecules is a relevant structure-activity metric for compound prioritization and optimization in medicinal chemistry phases of drug discovery. Electrophysiology remains the gold standard for the functional assessment of ion channel pharmacology. The recent years have witnessed automatization and parallelization of the manual patch-clamp technique, allowing higher throughput screening on recombinant hERG channels. However, the multi-well plate format of automatized patch-clamp does not allow visual detection of potential micro-precipitation of poorly soluble compounds. In this chapter we describe bench procedures for the culture and preparation of hERG-expressing CHO cells for recording on an automated patch-clamp workstation. We also show that the sensitivity of the assay can be improved by adding a surfactant to the extracellular medium.