Arrhythmic potency of human ether-a-go-go-related gene mutations L532P and N588K in a computational model of human atrial myocytes.

AIMS: Human ether-à-go-go-related gene (hERG) missense mutations N588K and L532P are both associated with atrial fibrillation (AF). However, the underlying gain-of-function mechanism is different. The aim of this computational study is to assess and understand the arrhythmogenic mechanisms of these genetic disorders on the cellular and tissue level as a basis for the improvement of therapeutic strategies. METHODS AND RESULTS: The IKr formulation of an established model of human atrial myocytes was adapted by using the measurement data of wild-type and mutant hERG channels. Restitution curves of the action potential duration and its slope, effective refractory period (ERP), conduction velocity, reentry wavelength (WL), and the vulnerable window (VW) were determined in a one-dimensional (1D) tissue strand. Moreover, spiral wave inducibility and rotor lifetime in a 2D tissue patch were evaluated. The two mutations caused an increase in IKr regarding both peak amplitude and current integral, whereas the duration during which IKr is active was decreased. The WL was reduced due to a shorter ERP. Spiral waves could be initiated by using mutation models as opposed to the control case. The frequency dependency of the VW was reversed. CONCLUSION: Both mutations showed an increased arrhythmogenicity due to decreased refractory time in combination with a more linear repolarization phase. The effects were more pronounced for mutation L532P than for N588K. Furthermore, spiral waves presented higher stability and a more regular pattern for L532P. These in silico investigations unveiling differences of mutations affecting the same ion channel may help to advance genotype-guided AF prevention and therapy strategies.

In-silico assessment of the dynamic effects of amiodarone and dronedarone on human atrial patho-electrophysiology.

AIMS: The clinical efficacy in preventing the recurrence of atrial fibrillation (AF) is higher for amiodarone than for dronedarone. Moreover, pharmacotherapy with these drugs is less successful in patients with remodelled substrate induced by chronic AF (cAF) and patients suffering from familial AF. To date, the reasons for these phenomena are only incompletely understood. We analyse the effects of the drugs in a computational model of atrial electrophysiology. METHODS AND RESULTS: The Courtemanche-Ramirez-Nattel model was adapted to represent cAF remodelled tissue and hERG mutations N588K and L532P. The pharmacodynamics of amiodarone and dronedarone were investigated with respect to their dose and heart rate dependence by evaluating 10 descriptors of action potential morphology and conduction properties. An arrhythmia score was computed based on a subset of these biomarkers and analysed regarding circadian variation of drug concentration and heart rate. Action potential alternans at high frequencies was observed over the whole dronedarone concentration range at high frequencies, while amiodarone caused alternans only in a narrow range. The total score of dronedarone reached critical values in most of the investigated dynamic scenarios, while amiodarone caused only minor score oscillations. Compared with the other substrates, cAF showed significantly different characteristics resulting in a lower amiodarone but higher dronedarone concentration yielding the lowest score. CONCLUSION: Significant differences exist in the frequency and concentration-dependent effects between amiodarone and dronedarone and between different atrial substrates. Our results provide possible explanations for the superior efficacy of amiodarone and may aid in the design of substrate-specific pharmacotherapy for AF.

Inhibition of cardiac Kir2.1-2.3 channels by beta3 adrenoreceptor antagonist SR 59230A.

Kir2.x channels form the molecular basis of cardiac I(K1) current and play a major role in cardiac electrophysiology. However, there is a substantial lack of selective Kir2 antagonists. We found the ß(3)-adrenoceptor antagonist SR59230A to be an inhibitor of Kir2.x channels. Therefore, we characterized the effects of SR59230A on Kir2.x and other relevant cardiac potassium channels. Cloned channels were expressed in the Xenopus oocyte expression system and measured with the double-microelectrode voltage clamp technique. SR59230A inhibited homomeric Kir2.1 channels with an IC(50) of 33µM. Homomeric Kir2.2 and Kir2.3 channels and Kir2.x heteromers were also inhibited by SR59230A with similar potency. In contrast, no relevant inhibitory effects of SR59230A were found in cardiac Kv1.5, Kv4.3 and KvLQT1/minK channels. In hERG channels, SR59230A only induced a weak inhibition at a high concentration. These findings establish SR59230A as a novel inhibitor of Kir2.1-2.3 channels with a favorable profile with respect to additional effects on other cardiac repolarizing potassium channels.

Impact of amiodarone and cisapride on simulated human ventricular electrophysiology and electrocardiograms.

AIMS: Amiodarone and cisapride are both known to prolong the QT interval, yet the two drugs have different effects on arrhythmia. Cisapride can cause torsades de pointes while amiodarone is found to be anti-arrhythmic. A computational model was used to investigate the action of these two drugs. METHODS AND RESULTS: In a biophysically detailed model, the ion current conductivities affected by both drugs were reduced in order to simulate the pharmacological effects in healthy and ischaemic cells. Furthermore, restitution curves of the action potential duration (APD), effective refractory period, conduction velocity, wavelength, and the vulnerable window were determined in a one-dimensional (1D) tissue strand. Moreover, cardiac excitation propagation was computed in a 3D model of healthy ventricles. The corresponding body surface potentials were calculated and standard 12-lead electrocardiograms were derived. Both cisapride and amiodarone caused a prolongation of the QT interval and the refractory period. However, cisapride did not significantly alter the conduction-related properties, such as e.g. the wavelength or vulnerable window, whereas amiodarone had a larger impact on them. It slightly flattened the APD restitution slope and furthermore reduced the conduction velocity and wavelength. CONCLUSION: Both drugs show similar prolongation of the QT interval, although they present different electrophysiological properties in the single-cell as well as in tissue simulations of cardiac excitation propagation. These computer simulations help to better understand the underlying mechanisms responsible for the initiation or termination of arrhythmias caused by amiodarone and cisapride.

Initial experience with robotic navigation for catheter ablation of paroxysmal and persistent atrial fibrillation.

BACKGROUND AND PURPOSE: Remote robotic navigation (RRN) technology has been developed to facilitate catheter ablation of symptomatic atrial fibrillation (AF). Here, we assess procedural parameters of AF ablation obtained during initial use of RRN compared with a control group treated with a manual ablation approach. METHODS: Consecutive patients with symptomatic paroxysmal or persistent AF were subjected to radiofrequency catheter ablation with RRN (Sensei X [Hansen Medical, Mountain View, CA]; n = 25; mean age, 60 ± 2.3 years) or using the standard manual technique (n = 61; mean age, 62 ± 1.4 years). A circumferential pulmonary vein isolation approach guided by 3-dimensional electroanatomical mapping was followed. RESULTS: Remote robotic navigation was associated with reduction of overall fluoroscopy time by 26%. In a case-control subgroup analysis comparing 25 patients with similar clinical characteristics from each group, mean fluoroscopy time was reduced by 22%. Acute isolation of pulmonary veins was achieved in 97% (RRN) and 96% (conventional ablation), respectively. Ablation times and frequency of adverse events were not significantly different among study groups. CONCLUSIONS: The early use of RRN resulted in a significant reduction of overall fluoroscopy time and was equally effective and safe compared with manual catheter ablation.

Reconstitution of defective protein trafficking rescues Long-QT syndrome in zebrafish.

Inherited cardiac arrhythmias are caused by genetic defects in ion channels and associated proteins. Mutations in these channels often do not affect their biophysical properties, but rather interfere with their trafficking to the cell membrane. Accordingly, strategies that could reroute the mutated channels to the membrane should be sufficient to restore the electrical properties of the affected cells, thereby suppressing the underlying arrhythmia. We identified here both, embryonic and adult zebrafish breakdance (bre) as a valuable model for human Long-QT syndrome. Electrocardiograms of adult homozygous bre mutants exhibit significant QT prolongation caused by delayed repolarization of the ventricle. We further show that the bre mutation (zERG(I59S)) disrupts ERG protein trafficking, thereby reducing the amount of active potassium channels on the cell membrane. Interestingly, improvement of channel trafficking by cisapride or dimethylsulfoxid is sufficient to reconstitute ERG channels on the cell membrane in a manner that suffices to suppress the Long-QT induced arrhythmia in breakdance mutant zebrafish. In summary, we show for the first time that therapeutic intervention can cure protein trafficking defects and the associated cardiac arrhythmia in vivo.

Cryoballoon pulmonary vein isolation-mediated rise of sinus rate in patients with paroxysmal atrial fibrillation.

BACKGROUND: Modulation of the cardiac autonomic nervous system by pulmonary vein isolation (PVI) influences the sinoatrial nodal rate. Little is known about the causes, maintenance and prognostic value of this phenomenon. We set out to explore the effects of cryoballoon PVI (cryo-PVI) on sinus rate and its significance for clinical outcome. METHODS AND RESULTS: We evaluated 110 patients with paroxysmal atrial fibrillation (AF), who underwent PVI using a second-generation 28 mm cryoballoon by pre-, peri- and postprocedural heart rate acquisition and analysis of clinical outcome. Ninety-one patients could be included in postinterventional follow-up, indicating that cryo-PVI resulted in a significant rise of sinus rate by 16.5% (+ 9.8 ± 0.9 beats/min, p < 0.001) 1 day post procedure compared to preprocedural acquisition. This effect was more pronounced in patients with initial sinus bradycardia (< 60 beats/min.) compared to patients with faster heart rate. Increase of rate was primarily driven by ablation of the right superior pulmonary vein and for a subset of patients, in whom this could be assessed, persisted ≥ 1 year after the procedure. AF recurrence was neither predicted by the magnitude of the initial rate, nor by the extent of rate change, but postprocedural sinus bradycardia was associated with higher recurrence of AF in the year post PVI. CONCLUSIONS: Cryo-PVI causes a significant rise of sinus rate that is more pronounced in subjects with previous sinus bradycardia. Patient follow-up indicates persistence of this effect and suggests an increased risk of AF recurrence in patients with postprocedural bradycardia.

Deficient zebrafish ether-à-go-go-related gene channel gating causes short-QT syndrome in zebrafish reggae mutants.

BACKGROUND: Genetic predisposition is believed to be responsible for most clinically significant arrhythmias; however, suitable genetic animal models to study disease mechanisms and evaluate new treatment strategies are largely lacking. METHODS AND RESULTS: In search of suitable arrhythmia models, we isolated the zebrafish mutation reggae (reg), which displays clinical features of the malignant human short-QT syndrome such as accelerated cardiac repolarization accompanied by cardiac fibrillation. By positional cloning, we identified the reg mutation that resides within the voltage sensor of the zebrafish ether-à-go-go-related gene (zERG) potassium channel. The mutation causes premature zERG channel activation and defective inactivation, which results in shortened action potential duration and accelerated cardiac repolarization. Genetic and pharmacological inhibition of zERG rescues recessive reg mutant embryos, which confirms the gain-of-function effect of the reg mutation on zERG channel function in vivo. Accordingly, QT intervals in ECGs from heterozygous and homozygous reg mutant adult zebrafish are considerably shorter than in wild-type zebrafish. CONCLUSIONS: With its molecular and pathophysiological concordance to the human arrhythmia syndrome, zebrafish reg represents the first animal model for human short-QT syndrome.

Biophysical properties of zebrafish ether-à-go-go related gene potassium channels.

The zebrafish is increasingly recognized as an animal model for the analysis of hERG-related diseases. However, functional properties of the zebrafish orthologue of hERG have not been analyzed yet. We heterologously expressed cloned ERG channels in Xenopus oocytes and analyzed biophysical properties using the voltage clamp technique. zERG channels conduct rapidly activating and inactivating potassium currents. However, compared to hERG, the half-maximal activation voltage of zERG current is shifted towards more positive potentials and the half maximal steady-state inactivation voltage is shifted towards more negative potentials. zERG channel activation is delayed and channel deactivation is accelerated significantly. However, time course of zERG conducted current under action potential clamp is highly similar to the human orthologue. In summary, we show that ERG channels in zebrafish exhibit biophysical properties similar to the human orthologue. Considering the conserved channel function, the zebrafish represents a valuable model to investigate human ERG channel related diseases.

[Cardiac arrhythmias in oncological diseases, during radiotherapy, chemotherapy]. / Rhythmusstörungen bei malignen Erkrankungen, unter Bestrahlung und Chemotherapie.

Patients with oncological diseases frequently show cardiac arrhythmias. This is explained by an increased risk in this specific patient cohort and is frequently associated with specific oncological therapies. So far, it is unclear how to deal with the occurrence of arrhythmias diagnostically and therapeutically, since the current clinical data do not provide satisfying answers to these questions. Clinical care of high-risk patients in specialized teams with a focus on cardio-oncology is recommended. Based on the current clinical studies and the position papers of the European Society of Cardiology (ESC), we give a brief overview of arrhythmias in malignant diseases and their therapies.

Kir2.x inward rectifier potassium channels are differentially regulated by adrenergic alpha1A receptors.

Inhibition of I(K1) currents by adrenergic alpha(1) receptors has been observed in cardiomyocytes and has been linked to arrhythmogenesis in an animal model. Both PKC-dependent and PKC-independent pathways have been implied in this regulation. The underlying molecular mechanisms, however, have not been elucidated to date. The molecular basis of native I(K1) current is mainly formed by Kir2.1 (KCNJ2), Kir2.2 (KCNJ12) and Kir2.3 (KCNJ4) channels that are differentially regulated by protein kinases. We therefore sought to investigate the role of those different Kir2.x channel subunits in this regulation and to identify the major signalling pathways involved. Adrenergic alpha(1A) receptors (the predominant cardiac isoform) were co-expressed with cloned Kir2.1, Kir2.2 and Kir2.3 channels in Xenopus oocytes and electrophysiological experiments were performed using two-microelectrode voltage clamp. Native I(K1) currents were measured with the whole-cell patch clamp technique in isolated rat ventricular cardiomyocytes. Activation of co-expressed adrenergic alpha(1A) receptors by phenylephrine induced differential effects in Kir2.x channels. No effect was noticed in Kir2.1 channels. However, a marked inhibitory effect was observed in Kir2.2 channels. This regulation was not attenuated by inhibitors of PKC, CamKII and PKA (chelerythrine, KN-93, KT-5720), and mutated Kir2.2 channels lacking functional phosphorylation sites for PKC and PKA exhibited the same effect as Kir2.2 wild-type channels. By contrast, the regulation could be suppressed by the general tyrosine kinase inhibitor genistein and by the src tyrosine kinase inhibitor PP2 indicating an essential role of src kinases. This finding was validated in rat ventricular cardiomyocytes where co-application of PP2 strongly attenuated the inhibitory regulation of I(K1) current by adrenergic alpha(1) receptors. The inactive analogue PP3 was tested as negative control for PP2 and did not reproduce the effects of PP2. In Kir2.3 channels, a marked inhibitory effect of alpha(1A) receptor activation was observed. This regulation could be attenuated by inhibition of PKC with chelerythrine or with Ro-32-0432, but not by tyrosine kinase inhibition with genistein. In summary, on the molecular level the inhibitory regulation of I(K1) currents by adrenergic alpha(1A) receptors is probably based on effects on Kir2.2 and Kir2.3 channels. Kir2.2 is regulated via src tyrosine kinase pathways independent of protein kinase C, whereas Kir2.3 is inhibited by protein kinase C-dependent pathways. Src tyrosine kinase pathways are essential for the inhibition of native I(K1) current by adrenergic alpha(1) receptors. This regulation may contribute to arrhythmogenesis under adrenergic stimulation.

Doxazosin induces apoptosis of cells expressing hERG K+ channels.

The antihypertensive drug doxazosin has been associated with an increased risk for congestive heart failure and cardiomyocyte apoptosis. Human ether-a-go-go-related gene (hERG) K(+) channels, previously shown to be blocked by doxazosin at therapeutically relevant concentrations, represent plasma membrane receptors for the antihypertensive drug. To elucidate the molecular basis for doxazosin-associated pro-apoptotic effects, cell death was studied in human embryonic kidney cells using three independent apoptosis assays. Doxazosin specifically induced apoptosis in hERG-expressing HEK cells, while untransfected control groups were insensitive to treatment with the antihypertensive agent. An unexpected biological mechanism has emerged: binding of doxazosin to its novel membrane receptor, hERG, triggers apoptosis, possibly representing a broader pathophysiological mechanism in drug-induced heart failure.

Inhibition of cardiac hERG potassium channels by tetracyclic antidepressant mianserin.

The antidepressant mianserin exhibits a tetracyclic structure that is different from typical tricyclic antidepressants (TCA) and that of selective serotonin reuptake inhibitors. In comparison to the older TCA, mianserin has been shown to have a superior risk profile regarding proarrhythmic effects, both in vitro and in vivo. However, the underlying molecular electrophysiological basis has not been elucidated to date. Therefore, we studied the effects of mianserin on cardiac hERG potassium channels, the predominant target of drug-induced proarrhythmia. HERG channels were expressed in the Xenopus oocyte expression system and in human embryonic kidney (HEK) cells and currents were measured with two-microelectrode voltage-clamp and whole-cell patch-clamp, respectively. Mianserin inhibited hERG currents in a dose-dependent manner with an IC(50) of 3.2 micromol/l in HEK cells. Onset of blockade was slow and the inhibitory effect was not reversible upon wash-out of the drug. In hERG channel mutants, Y652A and F656A, lacking aromatic residues in the S6 domain, the effect of mianserin was significantly reduced in comparison to the wild type. Mianserin inhibited hERG currents in the open and inactivated state, but not in the closed states. HERG inactivation kinetics were significantly altered by mianserin without marked effects on channel activation kinetics. The inhibitory effect was not frequency dependent. In conclusion, mianserin is a low-affinity hERG-blocking agent. However, taken together with the lack of APD-prolongation shown in other studies, mianserin seems to have a good safety profile. Lack of consistent QT prolonging effects of mianserin in previous studies may therefore be linked to additional effects such as inhibition of other cardiac ion channels. However, as demonstrated by clinical case reports, mianserin can induce proarrhythmic effects in susceptible patients. Therefore, in patients with complex co-medication (i.e., additional hERG-blocking agents) and in patients with risk factors for acquired long QT syndrome as well as in cases of overdose, adequate monitoring should be recommended.

Novel approach to discriminate left bundle branch block from nonspecific intraventricular conduction delay using pacing-induced functional left bundle branch block.

PURPOSE: Left bundle branch block (LBBB) has a predictive value for response to cardiac resynchronization therapy as reported by Zareba et al. (Circulation 123(10):1061-1072, 2011). However, based on ECG criteria, the discrimination between complete LBBB and nonspecific intraventricular conduction delay is challenging. We tested the hypothesis that discrimination can be performed using standard electrophysiological catheters and a simple stimulation protocol. METHODS: Fifty-nine patients were analyzed retrospectively. Patients were divided into groups of narrow QRS (n = 20), wide QRS of right bundle branch block (RBBB) morphology (n = 14), and wide QRS of LBBB morphology (n = 25). Using a diagnostic catheter placed in the coronary sinus, left ventricular activation was assessed during intrinsic conduction as well as during right ventricular (RV) stimulation. RESULTS: In patients with narrow QRS and RBBB, the Q-LV/QRS ratio was 0.43 ± 0.013 (n = 20) and 0.41 ± 0.026 (n = 14), respectively. In patients with LBBB morphology, the Q-LV/QRS split up into a group of patients with normal (0.43 ± 0.022, n = 7) and a group with delayed left ventricular activation (0.75 ± 0.016, n = 18). By direct comparison of the Q-LV/QRS ratio during intrinsic conduction with the Q-LV/QRS ratio during RV pacing leading to a functional LBBB, a clear distinction between a group of "true LBBB" and another group of "apparent LBBB"/nonspecific intraventricular conduction delay (NICD) could be generated. CONCLUSIONS: We present a novel and practical method that might facilitate discrimination between patients with apparent LBBB and true LBBB by comparing Q-LV/QRS ratios during intrinsic activation and during RV stimulation. Although this method can already be directly applied, validation by 3D electrical mapping and prospective correlation to cardiac resynchronization therapy (CRT) response will be required for further translation into clinical practice.

Activation of inwardly rectifying Kir2.x potassium channels by beta 3-adrenoceptors is mediated via different signaling pathways with a predominant role of PKC for Kir2.1 and of PKA for Kir2.2.

beta(3)-adrenoceptors have recently been shown to induce a complex modulation of intracellular signaling pathways including cyclic guanine monophosphate, cyclic adenosine monophosphate, nitric oxide, and protein kinases A and C. They are expressed in a broad variety of tissues including the myocardium, vascular smooth muscle, and endothelium. In those tissues, resting membrane potential is controlled mainly by inwardly rectifying potassium channels of the Kir2 family namely, Kir2.1 in the vascular smooth muscle, Kir2.1-2.3 in the myocardium, and Kir2.1-2.2 in the endothelium. In the present study, we investigated the possible modulation of Kir2 channel function by beta(3)-adrenoceptors in an expression system. Human-cloned beta(3)-adrenoceptors and Kir2.1 (KCNJ2), Kir2.2 (KCNJ12), and Kir2.3 (KCNJ4) channels were coexpressed in Xenopus oocytes, and currents were measured with double-microelectrode voltage clamp. Activation of beta(3)-adrenoceptors with isoproterenol resulted in markedly increased currents in Kir2.1 and in Kir2.2 potassium channels with EC50 values of 27 and 18 nM, respectively. In contrast, Kir2.3 currents were not modulated. Coapplication of specific inhibitors of protein kinase A (KT-5720) and calmodulin kinase II (KN-93) had no effects on the observed regulation in Kir2.1. However, coapplication of protein kinase C (PKC) inhibitors staurosporine and chelerythrine suppressed the observed effect. In Kir2.2, coapplication of KT-5720 reduced the effect of beta(3)-adrenoceptor activation. No differences in current increase after application of isoproterenol were observed between mutant Kir2.2 potassium channels lacking all functional PKC phosphorylation sites and Kir2.2 wild-type channels. In heteromeric Kir2.x channels, all types of heteromers were activated. The effect was most pronounced in Kir2.1/Kir2.2 and in Kir2.2/Kir2.3 channels. In summary, homomeric and heteromeric Kir2.x channels are activated by beta(3)-adrenoceptors via different protein kinase-dependent pathways: Kir2.1 subunits are modulated by PKC, whereas Kir2.2 is modulated by protein kinase A. In heteromeric composition, a marked activation of currents can be observed particularly with involvement of Kir2.2 subunits. This regulation may contribute to the hyperpolarizing effects of beta(3)-adrenoceptors in tissues that exhibit modulation by Kir2 channel function.

Anticholinergic antiparkinson drug orphenadrine inhibits HERG channels: block attenuation by mutations of the pore residues Y652 or F656.

The anticholinergic antiparkinson drug orphenadrine is an antagonist at central and peripheral muscarinic receptors. Orphenadrine intake has recently been linked to QT prolongation and Torsade-de-Pointes tachycardia. So far, inhibitory effects on I (Kr) or cloned HERG channels have not been examined. HERG channels were heterologously expressed in a HEK 293 cell line and in Xenopus oocytes and HERG current was measured using the whole cell patch clamp and the double electrode voltage clamp technique. Orphenadrine inhibits cloned HERG channels in a concentration dependent manner, yielding an IC(50) of 0.85 microM in HEK cells. Onset of block is fast and reversible upon washout. Orphenadrine does not alter the half-maximal activation voltage of HERG channels. There is no shift of the half-maximal steady-state-inactivation voltage. Time constants of direct channel inactivation are not altered significantly and there is no use-dependence of block. HERG blockade is attenuated significantly in mutant channels lacking either of the aromatic pore residues Y652 and F656. In conclusion, we show that the anticholinergic agent orphenadrine is an antagonist at HERG channels. These results provide a novel molecular basis for the reported proarrhythmic side effects of orphenadrine.

Regulation of cardiac inwardly rectifying potassium current IK1 and Kir2.x channels by endothelin-1.

To elucidate the ionic mechanism of endothelin-1 (ET-1)-induced focal ventricular tachyarrhythmias, the regulation of I(K1) and its main molecular correlates, Kir2.1, Kir2.2 and Kir2.3 channels, by ET-1 was investigated. Native I(K1) in human atrial cardiomyocytes was studied with whole-cell patch clamp. Human endothelin receptors were coexpressed with human Kir2.1, Kir2.2 and Kir2.3 channels in Xenopus oocytes. Currents were measured with a two-microelectrode voltage clamp. In human cardiomyocytes, ET-1 induced a marked inhibition of I(K1) that could be suppressed by the protein kinase C (PKC) inhibitor staurosporine. To investigate the molecular mechanisms underlying this regulation, we studied the coupling of ET(A) receptors to homomeric and heteromeric Kir2.1, Kir2.2 and Kir2.3 channels in the Xenopus oocyte expression system. ET(A) receptors coupled functionally to Kir2.2 and Kir2.3 channels but not to Kir2.1 channels. In Kir2.2 channels lacking functional PKC phosphorylation sites, the inhibitory effect was abolished. The inhibition of Kir2.3 currents could be suppressed by the PKC inhibitors staurosporine and chelerythrine. The coupling of ET(A) receptors to heteromeric Kir2.1/Kir2.2 and Kir2.2/Kir2.3 channels resulted in a strong inhibition of currents comparable with the effect observed in Kir2.2 homomers. Surprisingly, in heteromeric Kir2.1/Kir2.3 channels, no effect was observed. ET-1 inhibits human cardiac I(K1) current via a PKC-mediated phosphorylation of Kir2.2 channel subunits and additional regulatory effects on Kir2.3 channels. This mechanism may contribute to the intrinsic arrhythmogenic potential of ET-1.

Orange flavonoid hesperetin modulates cardiac hERG potassium channel via binding to amino acid F656.

BACKGROUND AND AIMS: Hesperetin belongs to the flavonoid subgroup classified as citrus flavonoids and is the main flavonoid in oranges. A high dietary intake of flavonoids has been associated with a significant reduction in cardiovascular mortality. HERG potassium channels play a major role in cardiac repolarisation and represent the most important pharmacologic target of both antiarrhythmic and proarrhythmic drugs. METHODS AND RESULTS: We used the two-microelectrode voltage-clamp technique to analyse inhibitory effects of hesperetin on hERG potassium channels heterologously expressed in Xenopus oocytes. Hesperetin blocked hERG potassium channels in a concentration dependent manner. Onset of block was fast and completely reversible upon wash-out. There was no significant effect of hesperetin on channel kinetics. Affinity of hesperetin to mutant F656A hERG channel was significantly decreased compared to WT hERG, indicating a binding site in the channel pore cavity. In contrast, affinity of hesperetin to Y652A hERG was not different from the affinity to WT hERG. CONCLUSION: We found an antagonist of cardiac hERG channels that modulates hERG currents by accessing the aromatic pore binding site, particularly amino acid phe-656. Regarding high hesperetin concentrations found in oranges and the increasing consumption of oranges and orange juice in Europe, potential effects of hesperetin on cardiac electrophysiology in vivo deserve further investigation.

Novel algorithm for accelerated electroanatomic mapping and prediction of earliest activation of focal cardiac arrhythmias using mathematical optimization.

BACKGROUND: Premature beats (PBs) are a common finding in patients suffering from structural heart disease, but they can also be present in healthy individuals. Catheter ablation represents a suitable therapeutic approach. However, the exact localization of the origin can be challenging, especially in cases of low PB burden during the procedure. OBJECTIVE: The aim of this study was to develop an automated mapping algorithm on the basis of the hypothesis that mathematical optimization would significantly accelerate the localization of earliest activation. METHODS: The algorithm is based on iterative regression analyses. When acquiring local activation times (LATs) within a 3-dimensional anatomic map of the corresponding heart chamber, this algorithm is able to identify that exact position where a next LAT measurement adds maximum information about the predicted site of origin. Furthermore, on the basis of the acquired LAT measurements, the algorithm is able to predict earliest activation with high accuracy. RESULTS: A systematic retrospective analysis of the mapping performance comparing the operator with simulated search processes by the algorithm within 17 electroanatomic maps of focal spreading arrhythmias revealed a highly significant reduction of necessary LAT measurements from 55 ± 8.8 to 10 ± 0.51 (n = 17; P < .0001). CONCLUSION: On the basis of mathematical optimization, we developed an algorithm that is able to reduce the number of LAT measurements necessary to locate the site of earliest activation. This algorithm might significantly accelerate the mapping procedure by guiding the operator to the optimal position for the next LAT measurement. Furthermore, the algorithm would be able to predict the site of origin with high accuracy early during the mapping procedure.

Inhibition of inwardly rectifying Kir2.x channels by the novel anti-cancer agent gambogic acid depends on both pore block and PIP2 interference.

The caged xanthone gambogic acid (GA) is a novel anti-cancer agent which exhibits anti-proliferative, anti-inflammatory and cytotoxic effects in many types of cancer tissues. In a recent phase IIa study, GA exhibits a favourable safety profile. However, limited data are available concerning its interaction with cardiac ion channels. Heteromeric assembly of Kir2.x channels underlies the cardiac inwardly rectifying IK1 current which is responsible for the stabilization of the diastolic resting membrane potential. Inhibition of the cardiac IK1 current may lead to ventricular arrhythmia due to delayed afterdepolarizations. Compared to Kv2.1, hERG and Kir1.1, a slow, delayed inhibition of Kir2.1 channels by GA in a mammalian cell line was reported before but no data exist in literature concerning action of GA on homomeric Kir2.2 and Kir2.3 and heteromeric Kir2.x channels. Therefore, the aim of this study was to provide comparative data on the effect of GA on homomeric and heteromeric Kir2.x channels. Homomeric and heteromeric Kir2.x channels were heterologously expressed in Xenopus oocytes, and the two-microelectrode voltage-clamp technique was used to record Kir2.x currents. To investigate the mechanism of the channel inhibition by GA, alanine-mutated Kir2.x channels with modifications in the channels pore region or at phosphatidylinositol 4,5-bisphosphate (PIP2)-binding sites were employed. GA caused a slow inhibition of homomeric and heteromeric Kir2.x channels at low micromolar concentrations (with IC50 Kir2.1/2.2 < Kir2.2 < Kir2.2/2.3 < Kir2.3 < Kir2.1 < Kir2.1/2.3). The effect did not reach saturation within 60 min and was not reversible upon washout for 30 min. The inhibition showed no strong voltage dependence. We provide evidence for a combination of direct channel pore blockade and a PIP2-dependent mechanism as a molecular basis for the observed effect. We conclude that Kir2.x channel inhibition by GA may be relevant in patients with pre-existing cardiac disorders such as chronic heart failure or certain rhythm disorders and recommend a close cardiac monitoring for those patients when treated with GA.