Claudia Seyler

Genetic suppression of atrial fibrillation using a dominant-negative ether-a-go-go–related gene mutant

BACKGROUND Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Gene therapy-dependent modulation of atrial electrophysiology may provide a more specific alternative to pharmacological and ablative treatment strategies. OBJECTIVE We hypothesized that genetic inactivation of atrial repolarizing ether-a-go-go-related gene (ERG) K(+) currents using a dominant-negative mutant would provide rhythm control in AF. METHODS Ten domestic swine underwent pacemaker implantation and were subjected to atrial burst pacing to induce persistent AF. Animals were then randomized to receive either AdCERG-G627S to suppress ERG/I(Kr) currents or green fluorescent protein (AdGFP) as control. Adenoviruses were applied using a novel hybrid technique combining atrial virus injection and epicardial electroporation to increase transgene expression. RESULTS In pigs treated with AdCERG-G627S, the onset of persistent AF was prevented (n = 2) or significantly delayed compared with AdGFP controls (12 ± 2.1 vs. 6.2 ± 1.3 days; P < .001) during 14-day follow-up. Effective refractory periods were prolonged in the AdCERG-G627S group compared with AdGFP animals (221.5 ± 4.7 ms vs. 197.0 ± 4.7 ms; P < .006). Impairment of left ventricular ejection fraction (LVEF) during AF was prevented by AdCERG-G627S application (LVEF(CERG-G627S) = 62.1% ± 4.0% vs. LVEF(GFP) = 30.3% ± 9.1%; P < .001). CONCLUSION Inhibition of ERG function using atrial AdCERG-G627S gene transfer suppresses or delays the onset of persistent AF by prolongation of atrial refractoriness in a porcine model. Targeted gene therapy represents an alternative to pharmacological or ablative treatment of AF.

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.

Carvedilol targets human K2P3.1 (TASK1) K+ leak channels

BACKGROUND AND PURPOSE Human K2P3.1 (TASK1) channels represent potential targets for pharmacological management of atrial fibrillation. K2P channels control excitability by stabilizing membrane potential and by expediting repolarization. In the heart, inhibition of K2P currents by class III antiarrhythmic drugs results in action potential prolongation and suppression of electrical automaticity. Carvedilol exerts antiarrhythmic activity and suppresses atrial fibrillation following cardiac surgery or cardioversion. The objective of this study was to investigate acute effects of carvedilol on human K2P3.1 (hK2P3.1) channels.

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.

Modulation of K2P2.1 and K2P10.1 K+ channel sensitivity to carvedilol by alternative mRNA translation initiation

The β‐receptor antagonist carvedilol blocks a range of ion channels. K2P2.1 (TREK1) and K2P10.1 (TREK2) channels are expressed in the heart and regulated by alternative translation initiation (ATI) of their mRNA, producing functionally distinct channel variants. The first objective was to investigate acute effects of carvedilol on human K2P2.1 and K2P10.1 channels. Second, we sought to study ATI‐dependent modulation of K2P K+ current sensitivity to carvedilol.

Therapeutic targeting of two-pore-domain potassium (K2P) channels in the cardiovascular system

The improvement of treatment strategies in cardiovascular medicine is an ongoing process that requires constant optimization. The ability of a therapeutic intervention to prevent cardiovascular pathology largely depends on its capacity to suppress the underlying mechanisms. Attenuation or reversal of disease-specific pathways has emerged as a promising paradigm, providing a mechanistic rationale for patient-tailored therapy. Two-pore-domain K(+) (K(2P)) channels conduct outward K(+) currents that stabilize the resting membrane potential and facilitate action potential repolarization. K(2P) expression in the cardiovascular system and polymodal K2P current regulation suggest functional significance and potential therapeutic roles of the channels. Recent work has focused primarily on K(2P)1.1 [tandem of pore domains in a weak inwardly rectifying K(+) channel (TWIK)-1], K(2P)2.1 [TWIK-related K(+) channel (TREK)-1], and K(2P)3.1 [TWIK-related acid-sensitive K(+) channel (TASK)-1] channels and their role in heart and vessels. K(2P) currents have been implicated in atrial and ventricular arrhythmogenesis and in setting the vascular tone. Furthermore, the association of genetic alterations in K(2P)3.1 channels with atrial fibrillation, cardiac conduction disorders and pulmonary arterial hypertension demonstrates the relevance of the channels in cardiovascular disease. The function, regulation and clinical significance of cardiovascular K(2P) channels are summarized in the present review, and therapeutic options are emphasized.

Inhibition of cardiac two-pore-domain K+ (K2P) channels by the antiarrhythmic drug vernakalant – Comparison with flecainide

The mixed ion channel blocker, vernakalant (RSD1235), is effective in rapid conversion of atrial fibrillation (AF) to sinus rhythm (SR). Suppression of cardiac two-pore-domain potassium (K2P) channels causes action potential prolongation and has recently been proposed as a novel antiarrhythmic strategy. The objective of this study was to investigate acute effects of vernakalant on human K2P2.1 (TREK-1) and K2P3.1 (TASK-1) channels to provide a more complete picture of its antiarrhythmic mechanism of action. The class IC antiarrhythmic drug flecainide was studied as a comparator agent. Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record K2P currents from Xenopus oocytes and Chinese hamster ovary (CHO) cells. Vernakalant inhibited cardiac K2P2.1 channels expressed in Xenopus oocytes and in CHO cells. The IC50 value obtained from mammalian cells (13.3 µM) was close to the range of vernakalant levels reported in patients (2-8 µM), indicating potential clinical significance of K2P2.1 blockade. Open rectification characteristics and current-voltage relationships of K2P2.1 currents were not affected by vernakalant. Vernakalant did not significantly reduce K2P3.1 currents. Finally, the class I antiarrhythmic drug flecainide had no effect on K2P2.1 or K2P3.1 channels. In conclusion, the recently developed antiarrhythmic drug vernakalant targets human K2P2.1 K(+) background channels. This previously unrecognized inhibitory property adds to the multichannel blocking profile of vernakalant and extends the mechanistic basis for its anti-fibrillatory effect.

TREK-1 (K2P2.1) K+ channels are suppressed in patients with atrial fibrillation and heart failure and provide therapeutic targets for rhythm control

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Concomitant heart failure (HF) poses a particular therapeutic challenge and is associated with prolonged atrial electrical refractoriness compared with non-failing hearts. We hypothesized that downregulation of atrial repolarizing TREK-1 (K2P2.1) K+ channels contributes to electrical remodeling during AF with HF, and that TREK-1 gene transfer would provide rhythm control via normalization of atrial effective refractory periods in this AF subset. In patients with chronic AF and HF, atrial TREK-1 mRNA levels were reduced by 82% (left atrium) and 81% (right atrium) compared with sinus rhythm (SR) subjects. Human findings were recapitulated in a porcine model of atrial tachypacing-induced AF and reduced left ventricular function. TREK-1 mRNA (−66%) and protein (−61%) was suppressed in AF animals at 14-day follow-up compared with SR controls. Downregulation of repolarizing TREK-1 channels was associated with prolongation of atrial effective refractory periods versus baseline conditions, consistent with prior observations in humans with HF. In a preclinical therapeutic approach, pigs were randomized to either atrial Ad-TREK-1 gene therapy or sham treatment. Gene transfer effectively increased TREK-1 protein levels and attenuated atrial effective refractory period prolongation in the porcine AF model. Ad-TREK-1 increased the SR prevalence to 62% during follow-up in AF animals, compared to 35% in the untreated AF group. In conclusion, TREK-1 downregulation and rhythm control by Ad-TREK-1 transfer suggest mechanistic and potential therapeutic significance of TREK-1 channels in a subgroup of AF patients with HF and prolonged atrial effective refractory periods. Functional correction of ionic remodeling through TREK-1 gene therapy represents a novel paradigm to optimize and specify AF management.

Central role of PKCα in isoenzyme-selective regulation of cardiac transient outward current Ito and Kv4.3 channels

The transient outward current I(to) is an important determinant of the early repolarization phase. I(to) and its molecular basis Kv4.3 are regulated by adrenergic pathways including protein kinase C. However, the exact regulatory mechanisms have not been analyzed yet. We here analyzed isoenzyme specific regulation of Kv4.3 and I(to) by PKC. Kv4.3 channels were expressed in Xenopus oocytes and currents were measured with double electrode voltage clamp technique. Patch clamp experiments were performed in isolated rat cardiomyocytes. Unspecific PKC stimulation with PMA resulted in a reduction of Kv4.3 current. Similar effects could be observed after activation of conventional PKC isoforms by TMX. Both effects were reversible by pharmacological inhibition of the conventional PKC isoenzymes (Gö6976). In contrast, activation of the novel PKC isoforms (ingenol) did not significantly affect Kv4.3 current. Whereas TMX-induced PKC activation was not attenuated inhibition of PKCβ, inhibition of PKCα with HBDDE prevented inhibitory effects of both PMA and TMX. Accordingly, stimulatory effects of PMA and TMX could be mimicked by the α-isoenzyme selective PKC activator iripallidal. Further evidence for the central role of PKCα was provided with the use of siRNAs. We found that PKCα siRNA but not PKCβ siRNA abolished the TMX induced effect. In isolated rat cardiomyocytes, PMA dependent I(to) reduction could be completely abolished by pharmacologic inhibition of PKCα. In summary we show that PKCα plays a central role in protein kinase C dependent regulation of Kv4.3 current and native I(to). These results add to the current understanding of isoenzyme selective ion channel regulation by protein kinases.