Eberhard P. Scholz

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.

QTc Prolongation by Grapefruit Juice and Its Potential Pharmacological Basis HERG Channel Blockade by Flavonoids

Background—A high intake of dietary flavonoids, which are abundant in fruits, vegetables, tea, and wine, is known to reduce cardiovascular mortality. The effects of flavonoids on cardiac electrophysiology, which theoretically may have both antiarrhythmic and proarrhythmic consequences, have not been studied systematically to date. Methods and Results—We screened a broad spectrum of flavonoids for their inhibitory activity on HERG channels by using heterologous expression in Xenopus oocytes. At a concentration of 1 mmol/L, 10 compounds caused a significant inhibition of HERG currents, whereas 11 other flavonoids had no effect. The IC50 value for HERG block by naringenin, the most potent inhibitor, was 102.3 &mgr;mol/L in Xenopus oocytes and 36.5 &mgr;mol/L in HEK cells. To demonstrate the physiological relevance of these findings, we studied the effects of pink grapefruit juice, which contains large amounts of naringenin glycosides (>1000 &mgr;mol/L), in human volunteers. In 10 persons, we observed a peak QTc prolongation of 12.5±4.2 ms 5 hours after oral ingestion of 1 L of grapefruit juice. This effect was significant (P=0.02). Conclusions—We found a significant QTc prolongation by grapefruit juice in healthy volunteers, probably caused by block of HERG channels by flavonoids. These findings reveal new perspectives on the potential for dietary modification of cardiac electrophysiology.

Human Cardiac Inwardly-Rectifying K+ Channel Kir2.1b Is Inhibited by Direct Protein Kinase C-Dependent Regulation in Human Isolated Cardiomyocytes and in an Expression System

Background—Protein kinases A (PKA) and C (PKC) are activated in ischemic preconditioning and heart failure, conditions in which patients develop arrhythmias. The native inward rectifier potassium current (IK1) plays a central role in the stabilization of the resting membrane potential and the process of arrhythmogenesis. This study investigates the functional relationship between PKC and IK1. Methods and Results—In whole-cell patch-clamp experiments with isolated human atrial cardiomyocytes, the IK1 was reduced by 41% when the nonspecific activator of PKC phorbol 12 myristate 13-acetate (PMA; 100 nmol/L) was applied. To investigate the effects of PKC on cloned channel underlying parts of the native IK1, we expressed Kir2.1b heterologously in Xenopus oocytes and measured currents with the double-electrode voltage-clamp technique. PMA decreased the current by an average of 68%, with an IC50 of 0.68 nmol/L. The inactive compound 4-&agr;-PMA was ineffective. Thymeleatoxin and 1-oleolyl-2-acetyl-sn-glycerol, 2 specific activators of PKC, produced effects similar to those of PMA. Inhibitors of PKC, ie, staurosporine and chelerytrine, could inhibit the PMA effect (1 nmol/L) significantly. After mutation of the PKC phosphorylation sites (especially S64A and T353A), PMA became ineffective. Conclusions—The human IK1 in atrial cardiomyocytes and one of its underlying ion channels, the Kir2.1b channel, is inhibited by PKC-dependent signal transduction pathways, possibly contributing to arrhythmogenesis in patients with structural heart disease in which PKC is activated.

Regulation of two-pore-domain (K2P) potassium leak channels by the tyrosine kinase inhibitor genistein

Two‐pore‐domain potassium (K2P) channels mediate potassium background (or ‘leak’) currents, controlling excitability by stabilizing membrane potential below firing threshold and expediting repolarization. Inhibition of K2P currents permits membrane potential depolarization and excitation. As expected for key regulators of excitability, leak channels are under tight control from a plethora of stimuli. Recently, signalling via protein tyrosine kinases (TKs) has been implicated in ion channel modulation. The objective of this study was to investigate TK regulation of K2P channels.

TASK1 (K2P3.1) K+ channel inhibition by endothelin-1 is mediated through Rho kinase-dependent phosphorylation

BACKGROUND AND PURPOSE TASK1 (K2P3.1) two‐pore‐domain K+ channels contribute substantially to the resting membrane potential in human pulmonary artery smooth muscle cells (hPASMC), modulating vascular tone and diameter. The endothelin‐1 (ET‐1) pathway mediates vasoconstriction and is an established target of pulmonary arterial hypertension (PAH) therapy. ET‐1‐mediated inhibition of TASK1 currents in hPASMC is implicated in the pathophysiology of PAH. This study was designed to elucidate molecular mechanisms underlying inhibition of TASK1 channels by ET‐1.

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.

Selective noradrenaline reuptake inhibitor atomoxetine directly blocks hERG currents

Background and purpose:  Atomoxetine is a selective noradrenaline reuptake inhibitor, recently approved for the treatment of attention‐deficit/hyperactivity disorder. So far, atomoxetine has been shown to be well tolerated, and cardiovascular effects were found to be negligible. However, two independent cases of QT interval prolongation, associated with atomoxetine overdose, have been reported recently. We therefore analysed acute and subacute effects of atomoxetine on cloned human Ether‐à‐Go‐Go‐Related Gene (hERG) channels.

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 IK1 and its main molecular correlates, Kir2.1, Kir2.2 and Kir2.3 channels, by ET-1 was investigated. Native IK1 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 IK1 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 ETA receptors to homomeric and heteromeric Kir2.1, Kir2.2 and Kir2.3 channels in the Xenopus oocyte expression system. ETA 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 ETA 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 IK1 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.

Inhibition of cardiac HERG currents by the DNA topoisomerase II inhibitor amsacrine: mode of action

The topoisomerase II inhibitor amsacrine is used in the treatment of acute myelogenous leukemia. Although most anticancer drugs are believed not to cause acquired long QT syndrome (LQTS), concerns have been raised by reports of QT interval prolongation, ventricular fibrillation and death associated with amsacrine treatment. Since blockade of cardiac human ether‐a‐go‐go‐related gene (HERG) potassium currents is an important cause of acquired LQTS, we investigated the acute effects of amsacrine on cloned HERG channels to determine the electrophysiological basis for its proarrhythmic potential. HERG channels were heterologously expressed in human HEK 293 cells and Xenopus laevis oocytes, and the respective potassium currents were recorded using patch‐clamp and two‐microelectrode voltage‐clamp electrophysiology. Amsacrine blocked HERG currents in HEK 293 cells and Xenopus oocytes in a concentration‐dependent manner, with IC50 values of 209.4 nM and 2.0 μM, respectively. HERG channels were primarily blocked in the open and inactivated states, and no additional voltage dependence was observed. Amsacrine caused a negative shift in the voltage dependence of both activation (−7.6 mV) and inactivation (−7.6 mV). HERG current block by amsacrine was not frequency dependent. The S6 domain mutations Y652A and F656A attenuated (Y652A) or abolished (F656A, Y652A/F656A) HERG current blockade, indicating that amsacrine binding requires a common drug receptor within the pore‐S6 region. In conclusion, these data demonstrate that the anticancer drug amsacrine is an antagonist of cloned HERG potassium channels, providing a molecular mechanism for the previously reported QTc interval prolongation during clinical administration of amsacrine.

Cardiovascular Ion Channels as a Molecular Target of Flavonoids

Flavonoids are a class of naturally occurring polyphenols abundant in edibles and beverages of plant origin. Epidemiological studies consistently associate high flavonoid intake with a reduced risk for the development of cardiovascular diseases. So far these beneficial effects have been mainly attributed to nonspecific antioxidant and antiinflammatory properties. However, there is an increasing body of evidence that flavonoids specifically target molecular structures including cardiovascular ion channels. Playing a pivotal role in the regulation of vascular tone and cardiac electric activity, ion channels represent a major target for the induction of antihypertensive and cardioprotective effects. Thus, pharmacological properties of flavonoids on cardiovascular ion channels, ion currents and tissue preparations are being increasingly addressed in experimental studies. Whereas it has become clear that cardiovascular ion channels represent an important molecular target of flavonoids, the published data have not yet been systematically reviewed.