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Pharmacological cardioversion of atrial fibrillation (AF) is frequently inefficacious. AP30663, a small conductance Ca2+ activated K+ (KCa2) channel blocker, prolonged the atrial effective refractory period in preclinical studies and subsequently converted AF into normal sinus rhythm. This first-in-human study evaluated the safety and tolerability, and pharmacokinetic (PK) and pharmacodynamic (PD) effects were explored. Forty-seven healthy male volunteers (23.7 ± 3.0 years) received AP30663 intravenously in ascending doses. Due to infusion site reactions, changes to the formulation and administration were implemented in the latter 24 volunteers. Extractions from a 24-hour continuous electrocardiogram were used to evaluate the PD effect of AP30663. Data were analyzed with a repeated measure analysis of covariance, noncompartmental analysis, and concentration-effect analysis. In total, 33 of 34 adverse events considered related to AP30663 exposure were related to the infusion site, mild in severity, and temporary in nature, although full recovery took up to 110 days. After formulation and administration changes, the local infusion site reaction remained, but the median duration was shorter despite higher dose levels. AP30663 displayed a less than dose proportional increase in peak plasma concentration (Cmax) and a terminal half-life of around 5 hours. In healthy volunteers, no effect of AP30663 was observed on electrocardiographic parameters, other than a concentration-dependent effect on the corrected QT Fridericia’s formula interval (+18.8 ± 4.3 ms for the highest dose level compared with time matched placebo). In conclusion, administration of AP30663, a novel KCa2 channel inhibitor, was safe and well-tolerated systemically in humans, supporting further development in patients with AF undergoing cardioversion.

Aims Acute myocardial infarction (AMI) is associated with intracellular Ca2+ build-up. In healthy ventricles, small conductance Ca2+-activated K+ (SK) channels are present but do not participate in repolarization. However, SK current is increased in chronic myocardial infarction and heart failure, and recently, SK channel inhibition was demonstrated to reduce arrhythmias in AMI rats. Hence, we hypothesized that SK channel inhibitors (NS8593 and AP14145) could reduce arrhythmia development during AMI in a porcine model.Methods and results Twenty-seven pigs were randomized 1:1:1 to control, NS8593, or AP14145. Haemodynamic and electrophysiological parameters [electrocardiogram (ECG) and monophasic action potentials (MAP)] were continuously recorded. A balloon was placed in the mid-left anterior descending artery, blinded to treatment. Infusion lasted from 10 min before occlusion until 30 min after. Occlusion was maintained for 1 h, followed by 2 h of reperfusion. Upon occlusion, cardiac output dropped similarly in all groups, while blood pressure remained stable. Heart rate decreased in the NS8593 and AP14145 groups. QRS duration increased upon occlusion in all groups but more prominently in AP14145-treated pigs. Inhibition of SK channels did not affect QT interval. Infarct MAP duration shortened comparably in all groups. Ventricular fibrillation developed in 4/9 control-, 4/9 AP14145-, and 2/9 NS8593-treated pigs. Ventricular tachycardia was rarely observed in either group, whereas ventricular extrasystoles occurred comparably in all groups.Conclusion Inhibition of SK channels was neither beneficial nor detrimental to ventricular arrhythmia development in the setting of AMI in this porcine model.

The mechanisms underlying atrial-selective prolongation of effective refractory period (ERP) and suppression of atrial fibrillation (AF) by NS8593 and UCL1684, small conductance calcium-activated potassium (SK) channel blockers, are poorly defined. The purpose of the study was to confirm the effectiveness of these agents to suppress AF and to probe the underlying mechanisms. Transmembrane action potentials and pseudoelectrocardiograms were recorded from canine isolated coronary-perfused canine atrial and ventricular wedge preparations. Patch clamp techniques were used to record sodium channel current (INa) in atrial and ventricular myocytes and human embryonic kidney cells. In both atria and ventricles, NS8593 (3-10 µM) and UCL1684 (0.5 µM) did not significantly alter action potential duration, suggesting little to no SK channel inhibition. Both agents caused atrial-selective: (1) prolongation of ERP secondary to development of postrepolarization refractoriness, (2) reduction of Vmax, and (3) increase of diastolic threshold of excitation (all are sodium-mediated parameters). NS8593 and UCL1684 significantly reduced INa density in human embryonic kidney cells as well as in atrial but not in ventricular myocytes at physiologically relevant holding potentials. NS8593 caused a shift of steady-state inactivation to negative potentials in atrial but not ventricular cells. NS8593 and UCL1684 prevented induction of acetylcholine-mediated AF in 6/6 and 8/8 preparations, respectively. This anti-AF effect was associated with strong rate-dependent depression of excitability. The SK channel blockers, NS8593 and UCL1684, are effective in preventing the development of AF due to potent atrial-selective inhibition of INa, causing atrial-selective prolongation of ERP secondary to induction of postrepolarization refractoriness.

Background Hypokalemia reduces the cardiac repolarization reserve. This prolongs the QT-interval and increases the risk of ventricular arrhythmia; a risk that is exacerbated by administration of classical class 3 anti-arrhythmic agents. Small conductance Ca2+-activated K+-channels (KCa2) are a promising new atrial selective target for treatment of atrial fibrillation. Under physiological conditions KCa2 plays a minor role in ventricular repolarization. However, this might change under hypokalemia because of concomitant increases in ventriculay -60r intracellur Ca2+. Purpose To study the effects of pharmacological KCa2 channel inhibition by the compounds AP14145, ICA, or AP30663 under hypokalemic conditions as compared to dofetilide and hypokalemia alone time-matched controls (TMC). Methods The current at +10 mV was compared in HEK293 cells stably expressing KCa2.3 perfused first with normo- and then hypokalemic solutions (4 mM K+ and 2.5 mM K+, respectively). Guinea pig hearts were isolated and perfused with normokalemic (4 mM K+) Krebs-Henseleit solution, followed by perfusion with drug or vehicle control. The perfusion was then changed to hypokalemic solution (2.5 mM K+) in presence of drug. 30 animals were randomly assigned to 5 groups: ICA, AP14145, AP30663, dofetilide, or TMC. QT-interval, the interval from the peak to the end of the T wave (Tp–Te), ventricular effective refractory period (VERP), arrhythmia score, and ventricular fibrillation (VF) incidence were recorded. Results Hypokalemia slightly increased KCa2.3 current compared to normokalemia. Application of KCa2 channel inhibitors and dofetilide prolonged the QT interval corrected for heart rate. Dofetilide, but none of the KCa2 channel inhibitors increased Tp–Te during hypokalemia. During hypokalemia 4/6 hearts in the TMC group developed VF (two spontaneously, two by S1S2 stimulation) whereas 5/6 hearts developed VF in the dofetilide group (two spontaneously, three by S1S2 stimulation). In comparison, 0/6, 1/6, and 1/6 hearts developed VF when treated with the KCa2 channel inhibitors AP30663, ICA, or AP14145, respectively. Conclusion Hypokalemia was associated with an increased incidence of VF, an effect that also seen in the presence of dofetilide. In comparison, the structurally and functionally different KCa2 channel inhibitors, ICA, AP14145, and AP30663 protected the heart from hypokalemia induced VF. These results support that KCa2 inhibition may be associated with a better safety and tolerability profile than dofetilide.

Significance Spider venom is a rich source of peptides, many targeting ion channels. We assessed a venom peptide, Hm1a, as a potential targeted therapy for Dravet syndrome, the genetic epilepsy linked to a mutation in the gene encoding the sodium channel alpha subunit NaV1.1. Cell-based assays showed Hm1a was selective for hNaV1.1 over other sodium and potassium channels. Utilizing a mouse model of Dravet syndrome, Hm1a restored inhibitory neuron function and significantly reduced seizures and mortality in heterozygote mice. Evidence from the structure of Hm1a and modeling suggest Hm1a interacts with NaV1.1 inactivation domains, providing a structural correlate of the functional mechanisms. This proof-of-concept study provides a promising strategy for future drug development in genetic epilepsy and other neurogenetic disorders., Dravet syndrome is a catastrophic, pharmacoresistant epileptic encephalopathy. Disease onset occurs in the first year of life, followed by developmental delay with cognitive and behavioral dysfunction and substantially elevated risk of premature death. The majority of affected individuals harbor a loss-of-function mutation in one allele of SCN1A, which encodes the voltage-gated sodium channel NaV1.1. Brain NaV1.1 is primarily localized to fast-spiking inhibitory interneurons; thus the mechanism of epileptogenesis in Dravet syndrome is hypothesized to be reduced inhibitory neurotransmission leading to brain hyperexcitability. We show that selective activation of NaV1.1 by venom peptide Hm1a restores the function of inhibitory interneurons from Dravet syndrome mice without affecting the firing of excitatory neurons. Intracerebroventricular infusion of Hm1a rescues Dravet syndrome mice from seizures and premature death. This precision medicine approach, which specifically targets the molecular deficit in Dravet syndrome, presents an opportunity for treatment of this intractable epilepsy.

Background and Purpose Small conductance calcium-activated potassium (KCa2) channels represent a promising atrial-selective target for treatment of atrial fibrillation. Here, we establish the mechanism of KCa2 channel inhibition by the new compound AP14145. Experimental Approach Using site-directed mutagenesis, binding determinants for AP14145 inhibition were explored. AP14145 selectivity and mechanism of action were investigated by patch-clamp recordings of heterologously expressed KCa2 channels. The biological efficacy of AP14145 was assessed by measuring atrial effective refractory period (AERP) prolongation in anaesthetized rats, and a beam walk test was performed in mice to determine acute CNS-related effects of the drug. Key Results AP14145 was found to be an equipotent negative allosteric modulator of KCa2.2 and KCa2.3 channels (IC50 = 1.1 ± 0.3 μM). The presence of AP14145 (10 μM) increased the EC50 of Ca2+ on KCa2.3 channels from 0.36 ± 0.02 to 1.2 ± 0.1 μM. The inhibitory effect strongly depended on two amino acids, S508 and A533 in the channel. AP14145 concentration-dependently prolonged AERP in rats. Moreover, AP14145 (10 mg·kg−1) did not trigger any apparent CNS effects in mice. Conclusions and Implications AP14145 is a negative allosteric modulator of KCa2.2 and KCa2.3 channels that shifted the calcium dependence of channel activation, an effect strongly dependent on two identified amino acids. AP14145 prolonged AERP in rats and did not trigger any acute CNS effects in mice. The understanding of how KCa2 channels are inhibited, at the molecular level, will help further development of drugs targeting KCa2 channels.

Background and Purpose PDE1, a subfamily of cyclic nucleotide PDEs consisting of three isoforms, PDE1A, PDE1B and PDE1C, has been implicated in the regulation of vascular tone. The PDE1 isoform(s) responsible for tone regulation is unknown. This study used isoform-preferring PDE1 inhibitors, Lu AF58027, Lu AF64196, Lu AF66896 and Lu AF67897, to investigate the relative contribution of PDE1 isoforms to regulation of vascular tone. Experimental Approach In rat mesenteric arteries, expression and localization of Pde1 isoforms were determined by quantitative PCR and in situ hybridization, and physiological impact of PDE1 inhibition was evaluated by isometric tension recordings. Key Results In rat mesenteric arteries, Pde1a mRNA expression was higher than Pde1b and Pde1c. In situ hybridization revealed localization of Pde1a to vascular smooth muscle cells (VSMCs) and only minor appearance of Pde1b and Pde1c. The potency of the PDE1 inhibitors at eliciting relaxation showed excellent correlation with their potency at inhibiting PDE1A. Thus, Lu AF58027 was the most potent at inhibiting PDE1A and was also the most potent at eliciting relaxation in mesenteric arteries. Inhibition of NOS with l-NAME, soluble GC with ODQ or PKG with Rp-8-Br-PET-cGMP all attenuated the inhibitory effect of PDE1 on relaxation, whereas PKA inhibition with H89 had no effect. Conclusions and Implications Pde1a is the dominant PDE1 isoform present in VSMCs, and relaxation mediated by PDE1A inhibition is predominantly driven by enhanced cGMP signalling. These results imply that isoform-selective PDE1 inhibitors are powerful investigative tools allowing examination of physiological and pathological roles of PDE1 isoforms.

Background and purpose Phosphodiesterase enzymes (PDE1-11) break down cyclic nucleotides and are important in the regulation of the cardiovascular system. The purpose of the present study was to investigate the effect on the cardiovascular system of two novel selective PDE1 inhibitors, Lu AF41228 and Lu AF58027. Experimental approach We used rat mesenteric small arteries (internal diameters of 200-300 µm), RT-PCR and measurement of isometric wall tension. The effect of Lu AF41228 and Lu AF58027 on heart rate and blood pressure was assessed in both anesthetized and conscious male rats. Key results Nanomolar concentrations of Lu AF41228 and Lu AF58027 inhibited PDE1A, PDE1B, and PDE1C enzyme activity, while micromolar concentrations were required to observe inhibitory effects at other PDEs. RT-PCR revealed expression of PDE1A, PDE1B, and PDE1C in rat brain, heart, and aorta, but only PDE1A and PDE1B in mesenteric arteries. In rat isolated mesenteric arteries contracted with phenylephrine or U46619, Lu AF41228 and Lu AF58027 induced concentration-dependent relaxations which were markedly reduced by inhibitors of guanylate cyclase, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), and adenylate cyclase, SQ22536 (9-(tetrahydro-2-furanyl)-9H-purin-6-amine, 9-THF-Ade), and in preparations without endothelium. In anesthetized rats Lu AF41228 and Lu AF58027 dose-dependently lowered mean blood pressure and increased heart rate. In conscious rats with telemetric pressure transducers, repeated dosing of Lu AF41228 lowered mean arterial blood pressure 10-15 mmHg and increased heart rate. Conclusions and implications The findings that these novel PDE1 inhibitors induce vasodilation and blood pressure lowering suggest a potential use of these vasodilators in the treatment of hypertension and vasospasm.

Due to their fast kinetic properties, Kv3.1 voltage gated potassium channels are important in setting and controlling firing frequency in neurons and pivotal in generating high frequency firing of interneurons. Pharmacological activation of Kv3.1 channels may possess therapeutic potential for treatment of epilepsy, hearing disorders, schizophrenia and cognitive impairments. Here we thoroughly investigate the selectivity and positive modulation of the two small molecules, EX15 and RE01, on Kv3 channels. Selectivity studies, conducted in Xenopus laevis oocytes confirmed a positive modulatory effect of the two compounds on Kv3.1 and to a minor extent on Kv3.2 channels. RE01 had no effect on the Kv3.3 and Kv3.4 channels, whereas EX15 had an inhibitory impact on the Kv3.4 mediated current. Voltage-clamp experiments in monoclonal hKv3.1b/HEK293 cells (34 °C) revealed that the two compounds indeed induced larger currents and faster activation kinetics. They also decrease the speed of deactivation and shifted the voltage dependence of activation, to a more negative activation threshold. Application of action potential clamping and repetitive stimulation protocols of hKv3.1b expressing HEK293 cells revealed that EX15 and RE01 significantly increased peak amplitude, half width and decay time of Kv3.1 mediated currents, even during high-frequency action potential clamping (250 Hz). In rat hippocampal slices, EX15 and RE01 increased neuronal excitability in fast-spiking interneurons in dentate gyrus. Action potential frequency was prominently increased at minor depolarizing steps, whereas more marginal effects of EX15 and RE01 were observed after stronger depolarizations. In conclusion, our results suggest that EX15 and RE01 positive modulation of Kv3.1 and Kv3.2 currents facilitate increased firing frequency in fast-spiking GABAergic interneurons.

Atrial fibrillation (AF) is the most common type of arrhythmia. Current pharmacological treatment for AF is moderately effective and/or increases the risk of serious ventricular adverse effects. To avoid ventricular adverse effects, a new target has been considered, the small conductance calcium-activated K+ channels (KCa2.X, SK channels). In the heart, KCa2.X channels are functionally more important in atria compared to ventricles, and pharmacological inhibition of the channel confers atrial selective prolongation of the cardiac action potential and converts AF to sinus rhythm in animal models of AF. Whether antiarrhythmic drugs (AADs) recommended for treating AF target KCa2.X channels is unknown. To this end, we tested a large number of AADs on the human KCa2.2 and KCa2.3 channels to assess their effect on this new target using automated whole-cell patch clamp. Of the AADs recommended for treatment of AF only dofetilide and propafenone inhibited hKCa2.X channels, with no subtype selectivity. The calculated IC50 were 90 ± 10 µmol/l vs 60 ± 10 µmol/l for dofetilide and 42 ± 4 µmol/ l vs 80 ± 20 µmol/l for propafenone (hKCa2.3 vs hKCa2.2). Whether this inhibition has clinical importance for their antiarrhythmic effect is unlikely, as the calculated IC50 values are very high compared to the effective free therapeutic plasma concentration of the drugs when used for AF treatment, 40,000-fold for dofetilide and 140- fold higher for propafenone.

Aims To describe the effects of the KCa2 channel inhibitor AP30663 in pigs regarding tolerability, cardiac electrophysiology, pharmacokinetics, atrial functional selectivity, effectiveness in cardioversion of tachy-pacing induced vernakalant-resistant atrial fibrillation (AF), and prevention of reinduction of AF. Methods and Results Six healthy pigs with implanted pacemakers and equipped with a Holter monitor were used to compare the effects of increasing doses (0, 5, 10, 15, 20, and 25 mg/kg) of AP30663 on the right atrial effective refractory period (AERP) and on various ECG parameters, including the QT interval. Ten pigs with implanted neurostimulators were long-term atrially tachypaced (A-TP) until sustained vernakalant-resistant AF was present. 20 mg/kg AP30663 was tested to discover if it could successfully convert vernakalant-resistant AF to sinus rhythm (SR) and protect against reinduction of AF. Seven anesthetized pigs were used for pharmacokinetic experiments. Two pigs received an infusion of 20 mg/kg AP30663 over 60 min while five pigs received 5 mg/kg AP30663 over 30 min. Blood samples were collected before, during, and after infusion on AP30663. AP30663 was well-tolerated and prominently increased the AERP in pigs with little effect on ventricular repolarization. Furthermore, it converted A-TP induced AF that had become unresponsive to vernakalant, and it prevented reinduction of AF in pigs. Both a >30 ms increase of the AERP and conversion of AF occurred in different pigs at a free plasma concentration level of around 1.0–1.4 µM of AP30663, which was achieved at a dose level of 5 mg/kg. Conclusion AP30663 has shown properties in animals that would be of clinical interest in man.

Dose is an important parameter in terms of both efficacy and adverse effects in pharmacological treatment of atrial fibrillation (AF). Both of the class III antiarrhythmics dofetilide and amiodarone have documented anti-AF effects. While dofetilide has dose-related ventricular side effects, amiodarone primarily has adverse non-cardiac effects. Pharmacological inhibition of small conductance Ca(2+)-activated K(+) (SK) channels has recently been reported to be antiarrhythmic in a number of animal AF models. In a Langendorff model of acutely induced AF on guinea pig hearts, it was investigated whether a combination of the SK channel blocker N-(pyridin-2-yl)-4-(pyridin-2-yl)thiazol-2-amine (ICA) together with either dofetilide or amiodarone provided a synergistic effect. The duration of AF was reduced with otherwise subefficacious concentrations of either dofetilide or amiodarone when combined with ICA, also at a subefficacious concentration. At a concentration level effective as monotherapy, dofetilide produced a marked increase in the QT interval. This QT prolonging effect was absent when combined with ICA at non-efficacious monotherapy concentrations. The results thereby reveal that combination of subefficacious concentrations of an SK channel blocker and either dofetilide or amiodarone can maintain anti-AF properties, while the risk of ventricular arrhythmias is reduced.

Background and Purpose: Prolongation of cardiac action potentials is considered antiarrhythmic in the atria but can be proarrhythmic in ventricles if the current carried by Kv11.1-channels (IKr) is inhibited. The current mediated by KCa2-channels, IKCa, is considered a promising new target for treatment of atrial fibrillation (AF). Selective inhibitors of IKr (dofetilide) and IKCa (AP14145) were used to compare the effects on ventricular and atrial repolarization. Ondansetron, which has been reported to be a potent blocker of both IKr and IKCa, was included to examine its potential atrial antiarrhythmic properties. Experimental Approach: The expression of KCa2- and Kv11.1-channels in the guinea pig heart was investigated using quantitative polymerase chain reaction (qPCR). Whole-cell patch clamp technique was used to investigate the effects of dofetilide, AP14145, and ondansetron on IKCa and/or IKr. The effect of dofetilide, AP14145, and ondansetron on atrial and ventricular repolarization was investigated in isolated hearts. A novel atrial paced in vivo guinea pig model was further validated using AP14145 and dofetilide. Key Results: AP14145 increased the atrial effective refractory period (AERP) without prolonging the QT interval with Bazett’s correction for heart rate (QTcB) both ex vivo and in vivo. In contrast, dofetilide increased QTcB and, to a lesser extent, AERP in isolated hearts and prolonged QTcB with no effects on AERP in the in vivo guinea pig model. Ondansetron did not inhibit IKCa, but did inhibit IKr in vitro. Ondansetron prolonged ventricular, but not atrial repolarization ex vivo. Conclusion and Implications: IKCa inhibition by AP14145 selectively increases atrial repolarization, whereas IKr inhibition by dofetilide and ondansetron increases ventricular repolarization to a larger extent than atrial repolarization.

The fastspiking parvalbumin expressing basketcells (FS BC) and the neurogliaform interneurons (NGF IN) innervate somatic and dendritic compartments of postsynaptic pyramidal neurons, respectively. Here we have used paired recordings of each type of interneuron with a postsynaptic pyramidal neuron in layer II-IV of the adult rat prefrontal cortex (PFC) to characterize the interneuron action potentials (AP), evoked IPSC characteristics and response to the GAT-1 inhibitor NNC 711 and the N-type Ca-channel inhibitor ω-conotoxin GVIA. GABA released from FS BCs were insensitive to ω-conotoxin GVIA (0.5 μM), the postsynaptic response did not desensitize and the IPSC was unchanged in NNC 711 (2 μM). Conversely, IPSCs generated by APs from NGF INs were 80 % inhibited by ω-conotoxin GVIA, displayed a strong degree of desensitization and the decay-time of the IPSCs doubled in NNC 711. We used these pharmacological differences to identify the source of GABA in paradigms of synaptic overspill induced by extracellular stimulation. The decaytime of the electrically evoked event doubled in NNC 711 and this increase was reversed by subsequent application of ω-conotoxin GVIA, suggesting that the source of GABA responding to NNC 711 originate from axons that terminate on pyramidal neuron dendrites and employ N-type Ca-channels for release.

Small-conductance Ca2+-activated potassium (SK) channels are relative newcomers within the field of cardiac electrophysiology. In recent years, an increased focus has been given to these channels because they might constitute a relatively atrial-selective target. This review will give a general introduction to SK channels followed by their proposed function in the heart under normal and pathophysiological conditions. It is revealed how antiarrhythmic effects can be obtained by SK channel inhibition in a number of species in situations of atrial fibrillation. On the contrary, the beneficial effects of SK channel inhibition in situations of heart failure are questionable and still needs investigation. The understanding of cardiac SK channels is rapidly increasing these years, and it is hoped that this will clarify whether SK channel inhibition has potential as a new anti–atrial fibrillation principle.

Genome-wide association studies have shown that the common single nucleotide polymorphism rs6800541 located in SCN10A, encoding the voltage-gated Nav1.8 sodium channel, is associated with PR-interval prolongation and atrial fibrillation (AF). Single nucleotide polymorphism rs6800541 is in high linkage disequilibrium with the nonsynonymous variant in SCN10A, rs6795970 (V1073A, r(2)=0.933). We therefore sought to determine whether common and rare SCN10A variants are associated with early onset AF.SCN10A was sequenced in 225 AF patients in whom there was no evidence of other cardiovascular disease or dysfunction (lone AF). In an association study of the rs6795970 single nucleotide polymorphism variant, we included 515 AF patients and 2 control cohorts of 730 individuals free of AF and 6161 randomly sampled individuals. Functional characterization of SCN10A variants was performed by whole-cell patch-clamping. In the lone AF cohort, 9 rare missense variants and 1 splice site donor variant were detected. Interestingly, AF patients were found to have higher G allele frequency of rs6795970, which encodes the alanine variant at position 1073 (described from here on as A1073, odds ratio =1.35 [1.16-1.54]; P=2.3×10(-5)). Both of the common variants, A1073 and P1092, induced a gain-of-channel function, whereas the rare missense variants, V94G and R1588Q, resulted in a loss-of-channel function.The common variant A1073 is associated with increased susceptibility to AF. Both rare and common variants have effect on the function of the channel, indicating that these variants influence susceptibility to AF. Hence, our study suggests that SCN10A variations are involved in the genesis of AF.

Background— Genome-wide association studies have shown that the common single nucleotide polymorphism rs6800541 located in SCN10A , encoding the voltage-gated Na v 1.8 sodium channel, is associated with PR-interval prolongation and atrial fibrillation (AF). Single nucleotide polymorphism rs6800541 is in high linkage disequilibrium with the nonsynonymous variant in SCN10A , rs6795970 (V1073A, r 2 =0.933). We therefore sought to determine whether common and rare SCN10A variants are associated with early onset AF. Methods and Results— SCN10A was sequenced in 225 AF patients in whom there was no evidence of other cardiovascular disease or dysfunction (lone AF). In an association study of the rs6795970 single nucleotide polymorphism variant, we included 515 AF patients and 2 control cohorts of 730 individuals free of AF and 6161 randomly sampled individuals. Functional characterization of SCN10A variants was performed by whole-cell patch-clamping. In the lone AF cohort, 9 rare missense variants and 1 splice site donor variant were detected. Interestingly, AF patients were found to have higher G allele frequency of rs6795970, which encodes the alanine variant at position 1073 (described from here on as A1073, odds ratio =1.35 [1.16−1.54]; P =2.3×10 −5 ). Both of the common variants, A1073 and P1092, induced a gain-of-channel function, whereas the rare missense variants, V94G and R1588Q, resulted in a loss-of-channel function. Conclusions— The common variant A1073 is associated with increased susceptibility to AF. Both rare and common variants have effect on the function of the channel, indicating that these variants influence susceptibility to AF. Hence, our study suggests that SCN10A variations are involved in the genesis of AF.

PDE1, a subfamily of cyclic nucleotide PDEs consisting of three isoforms, PDE1A, PDE1B and PDE1C, has been implicated in the regulation of vascular tone. The PDE1 isoform(s) responsible for tone regulation is unknown. This study used isoform-preferring PDE1 inhibitors, Lu AF58027, Lu AF64196, Lu AF66896 and Lu AF67897, to investigate the relative contribution of PDE1 isoforms to regulation of vascular tone.In rat mesenteric arteries, expression and localization of Pde1 isoforms were determined by quantitative PCR and in situ hybridization, and physiological impact of PDE1 inhibition was evaluated by isometric tension recordings.In rat mesenteric arteries, Pde1a mRNA expression was higher than Pde1b and Pde1c. In situ hybridization revealed localization of Pde1a to vascular smooth muscle cells (VSMCs) and only minor appearance of Pde1b and Pde1c. The potency of the PDE1 inhibitors at eliciting relaxation showed excellent correlation with their potency at inhibiting PDE1A. Thus, Lu AF58027 was the most potent at inhibiting PDE1A and was also the most potent at eliciting relaxation in mesenteric arteries. Inhibition of NOS with l-NAME, soluble GC with ODQ or PKG with Rp-8-Br-PET-cGMP all attenuated the inhibitory effect of PDE1 on relaxation, whereas PKA inhibition with H89 had no effect.Pde1a is the dominant PDE1 isoform present in VSMCs, and relaxation mediated by PDE1A inhibition is predominantly driven by enhanced cGMP signalling. These results imply that isoform-selective PDE1 inhibitors are powerful investigative tools allowing examination of physiological and pathological roles of PDE1 isoforms.

Small conductance calcium-activated potassium (KUsing site-directed mutagenesis, binding determinants for AP14145 inhibition were explored. AP14145 selectivity and mechanism of action were investigated by patch-clamp recordings of heterologously expressed KAP14145 was found to be an equipotent negative allosteric modulator of KAP14145 is a negative allosteric modulator of K

The nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels broadly involved in regulating neurotransmission in the central nervous system (CNS) by conducting cation currents through the membrane of neurons. Many different nAChR subtypes exist with each their functional characteristics, expression pattern and pharmacological profile. The focus of the present MiniReview is on the heteromeric α4β2 nAChR, as activity at this subtype contributes to cognitive functioning through interactions with multiple neurotransmitter systems and is implicated in various CNS disorders, for example schizophrenia and Alzheimer's disease. Additionally, the α4β2 nAChR provides an extra layer of molecular complexity by existing in two different stoichiometries determined by the subunit composition. Such findings have founded the rationale that pharmacological modulation of the α4β2 nAChR may be used as a treatment approach in various CNS disorders. As subtype-selective agonists and other cholinergic ligands have only shown limited therapeutic success, the focus of recent drug development endeavours has largely shifted to positive allosteric modulators (PAMs). By potentiating the action of an agonist through binding to an allosteric site, a PAM can enhance cholinergic neurotransmission, thus compensating for compromised neuronal communication in a pathophysiological setting. The pharmacological advantages of PAMs compared to other types of ligands include minimal interference with spatial and temporal aspects of neurotransmission as well as higher subtype selectivity, hypothetically resulting in high clinical efficacy with minimal adverse effects. In this MiniReview, we describe the currently identified compounds, which potentiate the effects of agonists at the α4β2 nAChR. The potential clinical advantages and concerns of PAMs are discussed in the light of the role of α4β2 nAChRs as key regulators of fast synaptic transmission.

Large-conductance Ca(2+)-activated K(+) channels (BK, KCa1.1, MaxiK) are important regulators of urinary bladder function and may be an attractive therapeutic target in bladder disorders. In this study, we established a high-throughput fluorometric imaging plate reader-based screening assay for BK channel activators and identified a small-molecule positive modulator, NS19504 (5-[(4-bromophenyl)methyl]-1,3-thiazol-2-amine), which activated the BK channel with an EC50 value of 11.0 ± 1.4 µM. Hit validation was performed using high-throughput electrophysiology (QPatch), and further characterization was achieved in manual whole-cell and inside-out patch-clamp studies in human embryonic kidney 293 cells expressing hBK channels: NS19504 caused distinct activation from a concentration of 0.3 and 10 µM NS19504 left-shifted the voltage activation curve by 60 mV. Furthermore, whole-cell recording showed that NS19504 activated BK channels in native smooth muscle cells from guinea pig urinary bladder. In guinea pig urinary bladder strips, NS19504 (1 µM) reduced spontaneous phasic contractions, an effect that was significantly inhibited by the specific BK channel blocker iberiotoxin. In contrast, NS19504 (1 µM) only modestly inhibited nerve-evoked contractions and had no effect on contractions induced by a high K(+) concentration consistent with a K(+) channel-mediated action. Collectively, these results show that NS19504 is a positive modulator of BK channels and provide support for the role of BK channels in urinary bladder function. The pharmacologic profile of NS19504 indicates that this compound may have the potential to reduce nonvoiding contractions associated with spontaneous bladder overactivity while having a minimal effect on normal voiding.

Aims The purpose of this study was to investigate the functional role of G-protein-coupled inward rectifier potassium (GIRK) channels in the cardiac ventricle. Methods and results Immunofluorescence experiments demonstrated that GIRK4 was localized in outer sarcolemmas and t-tubules in GIRK1 knockout (KO) mice, whereas GIRK4 labelling was not detected in GIRK4 KO mice. GIRK4 was localized in intercalated discs in rat ventricle, whereas it was expressed in intercalated discs and outer sarcolemmas in rat atrium. GIRK4 was localized in t-tubules and intercalated discs in human ventricular endocardium and epicardium, but absent in mid-myocardium. Electrophysiological recordings in rat ventricular tissue ex vivo showed that the adenosine A1 receptor agonist N 6-cyclopentyladenosine (CPA) and acetylcholine (ACh) shortened action potential duration (APD), and that the APD shortening was reversed by either the GIRK channel blocker tertiapin-Q, the adenosine A1 receptor antagonist DPCPX or by the muscarinic M2 receptor antagonist AF-DX 116. Tertiapin-Q prolonged APD in the absence of the exogenous receptor activation. Furthermore, CPA and ACh decreased the effective refractory period and the effect was reversed by either tertiapin-Q, DPCPX or AF-DX 116. Receptor activation also hyperpolarized the resting membrane potential, an effect that was reversed by tertiapin-Q. In contrast, tertiapin-Q depolarized the resting membrane potential in the absence of the exogenous receptor activation. Conclusion Confocal microscopy shows that among species GIRK4 is differentially localized in the cardiac ventricle, and that it is heterogeneously expressed across human ventricular wall. Electrophysiological recordings reveal that GIRK current may contribute significantly to ventricular repolarization and thereby to cardiac electrical stability.

Objective: Tesofensine is a novel triple monoamine reuptake inhibitor which is in development for the treatment of obesity. Preclinical and clinical data suggest that appetite suppression is an important mechanism by which tesofensine exerts its robust weight reducing effect. Notably, the strong hypophagic response to tesofensine treatment is demonstrated to be linked to central stimulation of noradrenergic and dopaminergic neurotransmission. The sympathomimetic mode of action of tesofensine may also associate with the elevated heart rate and blood pressure observed in clinical settings, and we therefore sought experimentally to address this issue. Design and Methods: The anorexigenic and cardiovascular effects of tesofensine were studied simultaneously in telemetrized conscious rats in a combined real-time food intake and cardiovascular telemetry monitoring system. Results: Acute administration of tesofensine caused a dose-dependent hypophagic effect as well as increased heart rate and blood pressure. Interestingly, combined treatment with metoprolol (b1 adrenoceptor blocker, 10-20 mg/kg, p.o.) fully prevented the cardiovascular sympathetic effects of tesofensine while leaving the robust inhibitory efficacy on food intake unaffected. Similarly, the angiotensin AT1 receptor antagonist telmisartan (1.0-3.0 mg/kg, p.o.) did not interfere with the anti-obesity effects of tesofensine, however, telmisartan only partially reversed the increase in systolic blood pressure and had no effect on the elevated heart rate induced by tesofensine. Conclusion: These data suggests that tesofensine causes elevations in heart rate and blood pressure by increasing sympathetic activity, and that different adrenoceptor subtypes may be responsible for the anti-obesity and cardiovascular effects of tesofensine.

Background and Purpose Strong implications in major neurological diseases make the neuronal α4β2 nicotinic ACh receptor (nAChR) a highly interesting drug target. In this study, we present a detailed electrophysiological characterization of NS9283, a potent positive allosteric modulator acting selectively at 3α:2β stoichiometry of α2* and α4* nAChRs. Experimental Approach The whole-cell patch-clamp technique equipped with an ultra-fast drug application system was used to perform electrophysiological characterization of NS9283 modulatory actions on human α4β2 nAChRs stably expressed in HEK293 cells (HEK293-hα4β2). Key Results NS9283 was demonstrated to increase the potency of ACh-evoked currents in HEK293-hα4β2 cells by left-shifting the concentration–response curve ∼60-fold. Interestingly, this modulation did not significantly alter maximal efficacy levels of ACh. Further, NS9283 did not affect the rate of desensitization of ACh-evoked currents, was incapable of reactivating desensitized receptors and only moderately slowed recovery from desensitization. However, NS9283 strongly decreased the rate of deactivation kinetics and also modestly decreased the rate of activation. This resulted in a left-shift of the ACh window current of (α4)3(β2)2 nAChRs in the presence of NS9283. Conclusions and Implications This study demonstrates that NS9283 increases responsiveness of human (α4)3(β2)2 nAChR to ACh with no change in maximum efficacy. We propose that this potentiation is due to a significant slowing of deactivation kinetics. In summary, the mechanism of action of NS9283 bears high resemblance to that of benzodiazepines at the GABAA receptor and to our knowledge, NS9283 constitutes the first nAChR compound of this class.

Supplemental Digital Content is available in the text., Background Evidence has emerged that small-conductance Ca2+-activated K+ (SK) channels constitute a new target for treatment of atrial fibrillation (AF). SK channels are predominantly expressed in the atria as compared with the ventricles. Various marketed antiarrhythmic drugs are limited by ventricular adverse effects and efficacy loss as AF progresses. Methods and Results A total of 43 pigs were used for the studies. AF reversion in conscious long-term tachypaced pigs: Pigs were subjected to atrial tachypacing (7 Hz) until they developed sustained AF that could not be reverted by vernakalant 4 mg/kg (18.8±3.3 days of atrial tachypacing). When the SK channel inhibitor AP14145 was tested in these animals, vernakalant-resistant AF was reverted to sinus rhythm, and reinduction of AF by burst pacing (50 Hz) was prevented in 8 of 8 pigs. Effects on refractory period and AF duration in open chest pigs: The effects of AP14145 and vernakalant on the effective refractory periods and acute burst pacing-induced AF were examined in anaesthetized open chest pigs. Both vernakalant and AP14145 significantly prolonged atrial refractoriness and reduced AF duration without affecting the ventricular refractoriness or blood pressure in pigs subjected to 7 days atrial tachypacing, as well as in sham-operated control pigs. Conclusions SK currents play a role in porcine atrial repolarization, and pharmacological inhibition of these with AP14145 demonstrates antiarrhythmic effects in a vernakalant-resistant porcine model of AF. These results suggest SK channel blockers as potentially interesting anti-AF drugs.

Aims: Diminished repolarization reserve contributes to the arrhythmogenic substrate in many disease states. Pharmacological activation of K + channels has been suggested as a potential antiarrhythmic therapy in such conditions. Having previously demonstrated that IK1 and IKr can modulate cardiac conduction, we tested here the effects of pharmacological IKATP and IKs activation on cardiac conduction and its dependence on the sodium current (INa). Methods and Results: Bath electrocardiograms (ECGs) recorded from Langendorff-perfused guinea pig ventricles revealed QRS prolongation during IKATP activation by pinacidil but not during IKs activation by R-L3 relative to control. In contrast, when INa was partially blocked by flecainide, R-L3 but not pinacidil prolonged the QRS relative to flecainide alone. Conduction velocity (h) was quantified by optical mapping during epicardial pacing. Both longitudinal (hL) and transverse (hT) h were reduced by pinacidil (by 10 ± 1a nd 9± 3%, respectively) and R-L3 (by 11 ± 2% and 15 ± 4%, respectively). Flecainide decreased hL by 33 ± 4% and hT by 36 ± 5%. Whereas pinacidil did not further slow h relative to flecainide alone, R-L3 decreased both hL and hT. Conclusion: Pharmacological activation of IKATP and IKs slows cardiac conduction; however, they demonstrate diverse effects on h dependence on INa blockade. These findings may have significant implications for the use of K + channel activators as antiarrhythmic drugs and for patients with Na + channel abnormalities or being treated with Na + channel blockers.

BACKGROUND AND PURPOSE Positive allosteric modulation of α4β2 nicotinic acetylcholine (nACh) receptors could add a new dimension to the pharmacology and therapeutic approach to these receptors. The novel modulator NS9283 was therefore tested extensively.

Angiotensin II (AngII) is a key peptide in cardiovascular homeostasis and is a ligand for the Angiotensin II type 1 and 2 seven transmembrane receptors (AT(1)R and AT(2)R). The AT(1) receptor is a seven-transmembrane (7TM) G protein-coupled receptor (GPCR) mediating the majority of the physiological functions of AngII. The AT(1)R mediates its effects through both G protein-dependent and independent signaling, which can be separated by functionally selective agonists. In the present study we investigate the effect of AngII and the β-arrestin biased agonist [SII]AngII on ischemia-reperfusion injury in rat hearts. Isolated hearts mounted in a Langendorff perfused rat heart preparations showed that preconditioning with [SII]AngII reduced the infarct size induced by global ischemia from 46±8.4% to 22±3.4%. In contrast, neither preconditioning with AngII nor postconditioning with AngII or [SII]AngII had a protective effect. Together these results demonstrate a cardioprotective effect of simultaneous blockade of G protein signaling and activation of G protein independent signaling through AT(1) receptors.

Kv7.1 (KCNQ1) channels are regulators of several physiological processes including vasodilatation, repolarization of cardiomyocytes, and control of secretory processes. A number of Kv7.1 pore mutants are sensitive to extracellular potassium. We hypothesized that extracellular potassium also modulates wild-type Kv7.1 channels. The Kv7.1 currents were measured in Xenopus laevis oocytes at different concentrations of extracellular potassium (1–50 mM). As extracellular potassium was elevated, Kv7.1 currents were reduced significantly more than expected from theoretical calculations based on the Goldman-Hodgkin-Katz flux equation. Potassium inhibited the steady-state current with an IC50 of 6.0 ± 0.2 mM. Analysis of tail-currents showed that potassium increased the fraction of channels in the inactivated state. Similarly, the recovery from inactivation was slowed by potassium, suggesting that extracellular potassium stabilizes an inactivated state in Kv7.1 channels. The effect of extracellular potassium was absent in noninactivating Kv7.1/KCNE1 and Kv7.1/KCNE3 channels, further supporting a stabilized inactivated state as the underlying mechanism. Interestingly, coexpression of Kv7.1 with KCNE2 did not attenuate the inhibition by potassium. In a number of other Kv channels, including Kv1.5, Kv4.3, and Kv7.2–5 channels, currents were only minimally reduced by an increase in extracellular potassium as expected. These results show that extracellular potassium modulates Kv7.1 channels and suggests that physiological changes in potassium concentrations may directly control the function of Kv7.1 channels. This may represent a novel regulatory mechanism of excitability and of potassium transport in tissues expressing Kv7.1 channels.

Background: Ischemic postconditioning (PostC), i.e. brief ischemia-reperfusion cycles before full reperfusion, is protective against cardiac ischemia/reperfusion (I/R) injury. Inhibition of the Na+/H+ exchanger NHE1 and delayed intracellular pH-normalization have been proposed to underlie protection by PostC. Methods and Results: We used Langendorff perfused rat hearts exposed to 35 min global ischemia to show that 15 min acidic (pH 6.5) treatment at onset of reperfusion decreased infarct size and functional deterioration at least to the same extent as PostC. In contrast, NHE1 inhibition by EIPA was detrimental. To evaluate HL-1 atrial cardiomyocytes as a cellular model for PostC, we exposed the cells to simulated ischemia/reperfusion (I/R) mimicking that in perfused hearts. Necrosis and apoptosis induced by I/R were unaffected by 15 min of pH 6.0 at onset of reperfusion. I/R increased the activity of c-Jun N-terminal Kinase 1/2 (JNK1/2) and Akt, but not of p38 MAPK, with no further effect of acidic reperfusion or EIPA. Conclusion: In rat hearts, 15 min acidic reperfusion improves myocardial performance at least as much as does PostC, whereas NHE1 inhibition is detrimental. In contrast, in HL-1 cardiomyocytes, acidic reperfusion or NHE1 inhibition affect neither survival nor JNK1/2-, Akt-, and p38 MAPK activity after I/R, pointing to different mechanisms of damage and protection in these systems.

Human ether-à-go-go-related gene 1 (hERG1) channels conduct the rapid delayed rectifier K+ current, I(Kr), an important determinant of action potential repolarization in mammals, including humans. Reduced I(Kr) function caused by mutations in KCNH2 or drug block of hERG1 channels prolongs the QT interval of the electrocardiogram and increases the risk of ventricular fibrillation and sudden cardiac death. Several activators of hERG1 channels have been discovered in recent years. These compounds shorten the duration of cardiac action potentials and have been proposed as a new therapeutic approach for the treatment of acquired or congenital long QT syndrome. We defined previously the mechanism of action of 1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643), a compound that increases hERG1 currents by shifting the voltage-dependence of inactivation to more positive potentials. Here, we use scanning mutagenesis of hERG1 and functional characterization of 56 mutant channels heterologously expressed in Xenopus laevis oocytes to define the molecular determinants of the binding site for NS1643. Most point mutations did not alter response to the drug; however, 10 mutant channels had reduced sensitivity, and F619A and I567A exhibited enhanced activation by the drug. Some of these residues form a cluster and, together with molecular modeling, suggest that NS1643 binds to a pocket near the extracellular ends of the S5/S6 segments of two adjacent hERG1 channel subunits. This putative binding site differs from the sites described previously for two other hERG1 activators, (3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid (RPR260243) and 2-(4-[2-(3,4-dichloro-phenyl)-ethyl]-phenylamino)-benzoic acid (PD-118057).

BACKGROUND AND PURPOSE K v 11.1 channels are involved in regulating cellular excitability in various tissues including brain, heart and smooth muscle. In these tissues, at least two isoforms, K v 11.1a and K v 11.1b, with different kinetics, are expressed. K v 11.1 activators are potential therapeutic agents, but their effects have only been tested on the K v 11.1a isoform. In this study, the effects of two different K v 11.1 activators, NS1643 and RPR260243, were characterized on K v 11.1a and K v 11.1b channels. EXPERIMENTAL APPROACH K v 11.1a and K v 11.1b channels were expressed in Xenopus laevis oocytes, and currents were measured using two-electrode voltage clamp. I/V curves and channel kinetics were measured before and after application of 30 μM NS1643 or 10 μM RPR260243. KEY RESULTS NS1643 increased steady-state currents through Kv11.1b several fold more than through K v 11.1a channels, without affecting EC 50 values. NS1643 increased activation rates and decreased rates of inactivation, recovery from inactivation and deactivation for both channels. Except for activation, where effect of NS1643 was comparable, relative changes were greater for K v 11.1b than for K v 11.1a. RPR260243 increased steady-state currents only through Kv11.1a channels, but slowed the process of deactivation for both channels primarily by decreasing time constant of slow deactivation. This effect was greater on Kv11.1b than on K v 11.1a. Effects of both compounds on heteromeric K v 11.1a/K v 11.1b channels were similar to those on K v 11.1a. CONCLUSIONS AND IMPLICATIONS Both NS1643 and RPR260243 displayed differential effects on K v 11.1a and K v 11.1b channels, the effects being relatively more pronounced on K v 11.1b channels. This affirms the importance of testing the effect of K v 11.1 activators on different channel isoforms.

Background and purpose The compound NS5806 increases the transient outward current (I(to)) in canine ventricular cardiomyocytes and slows current decay. In human and canine ventricle, I(to) is thought to be mediated by K(V)4.3 and various ancillary proteins, yet, the exact subunit composition of I(to) channels is still debated. Here we characterize the effect of NS5806 on heterologously expressed putative I(to) channel subunits and other potassium channels. Experimental approach Cloned K(V)4 channels were co-expressed with KChIP2, DPP6, DPP10, KCNE2, KCNE3 and K(V)1.4 in Xenopus laevis oocytes or CHO-K1 cells. Key results NS5806 increased K(V)4.3/KChIP2 peak current amplitudes with an EC(50) of 5.3 +/- 1.5microM and significantly slowed current decay. KCNE2, KCNE3, DPP6 and DPP10 modulated K(V)4.3 currents and the response to NS5806, but current decay was slowed only in complexes containing KChIP2. The effect of NS5806 on K(V)4.2 was similar to that on K(V)4.3, and current decay was only slowed in presence of KChIP2. However, for K(V)4.1, the slowing of current decay by NS5806 was independent of KChIP2. K(V)1.4 was strongly inhibited by 10 microM NS5806 and K(V)1.5 was inhibited to a smaller extent. Effects of NS5806 on kinetics of currents generated by K(V)4.3/KChIP2/DPP6 with K(V)1.4 in oocytes could reproduce those on cardiac I(to) in canine ventricular myocytes. K(V)7.1, K(V)11.1 and K(ir)2 currents were unaffected by NS5806. Conclusion and implications NS5806 modulated K(V)4 channel gating depending on the presence of KChIP2, suggesting that NS5806 can potentially be used to address the molecular composition as well as the physiological role of cardiac I(to).

The cardiac action potential can be divided into five distinct phases designated phases 0-4. The exact shape of the action potential comes about primarily as an orchestrated function of ion channels. The present review will give an overview of ion channels involved in generating the cardiac action potential with special emphasis on potassium channels involved in phase 3 repolarization. In humans, these channels are primarily K(v)11.1 (hERG1), K(v)7.1 (KCNQ1) and K(ir)2.1 (KCNJ2) being the responsible alpha-subunits for conducting I(Kr), I(Ks) and I(K1). An account will be given about molecular components, biophysical properties, regulation, interaction with other proteins and involvement in diseases. Both loss and gain of function of these currents are associated with different arrhythmogenic diseases. The second part of this review will therefore elucidate arrhythmias and subsequently focus on newly developed chemical entities having the ability to increase the activity of I(Kr), I(Ks) and I(K1). An evaluation will be given addressing the possibility that this novel class of compounds have the ability to constitute a new anti-arrhythmic principle. Experimental evidence from in vitro, ex vivo and in vivo settings will be included. Furthermore, conceptual differences between the short QT syndrome and I(Kr) activation will be accounted for.

At present, transgenes in Caenorhabditis elegans are generated by injecting DNA into the germline. The DNA assembles into a semistable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double-strand break in noncoding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single-copy insertions can be obtained in less than 2 weeks by injecting approximately 20 worms. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single-copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines.

The cardiac action potential is the result of an orchestrated function of a number of different ion channels. Action potential repolarisation in humans relies on three potassium current components named I(Kr), I(Ks) and I(K1) with party overlapping functions. The ion channel alpha-subunits conducting these currents are hERG1 (Kv11.1), KCNQ1 (Kv7.1) and Kir2.1. Loss-of-function in any of these currents can result in long QT syndrome. Long QT is a pro-arrhythmic disease with increased risk of developing lethal ventricular arrhythmias such as Torsade de Pointes and ventricular fibrillation. In addition to congenital long QT, acquired long QT can also constitute a safety risk. Especially unintended inhibition of the hERG1 channel constitutes a major concern in the development of new drugs. Based on this knowledge is has been speculated whether activation of the hERG1 channel could be anti-arrhythmic and thereby constitute a new principle in treatment of cardiac arrhythmogenic disorders. The first hERG1 channel agonist was reported in 2005 and a limited number of such compounds are now available. In the present text we review results obtained by hERG1 channel activation in a number of cardiac relevant settings from in vitro to in vivo. It is demonstrated how the principle of hERG1 channel activation under certain circumstances can constitute a new anti-arrhythmogenic principle. Finally, important conceptual differences between the short QT syndrome and the hERG1 channel activation, are evaluated.

The long QT syndrome is characterized by a prolongation of the QT interval measured on the surface electrocardiogram. Prolonging the QT interval increases the risk of dangerous ventricular fibrillations, eventually leading to sudden cardiac death. Pharmacologically induced QT interval prolongations are most often caused by antagonizing effects on the repolarizing cardiac current called IKr. In humans IKr is mediated by the human ether-a-go-go related gene (hERG) potassium channel. We recently presented NS3623, a compound that selectively activates this channel. The present study was dedicated to examining the in vivo effects of NS3623. Injection of 30 mg/kg NS3623 shortened the corrected QT interval by 25 +/- 4% in anaesthetized guinea pigs. Accordingly, 50 mg/kg of NS3623 shortened the QT interval by 30 +/- 6% in conscious guinea pigs. Finally, pharmacologically induced QT prolongation by a hERG channel antagonist (0.15 mg/kg E-4031) could be reverted by injection of NS3623 (50 mg/kg) in conscious guinea pigs. In conclusion, the present in vivo study demonstrates that injection of the hERG channel agonist NS3623 results in shortening of the QTc interval as well as reversal of a pharmacologically induced QT prolongation in both anaesthetized and conscious guinea pigs.

Aims Impaired repolarization in cardiac myocytes can lead to long QT syndrome (LQTS), with delayed repolarization and increased susceptibility to Torsades de Pointes (TdP) arrhythmias. Current pharmacological treatment of LQTS is often inadequate. This study sought to evaluate the antiarrhythmic effect of a novel compound (NS1643) that activates the rapid delayed-rectifier K+ current, I Kr, in two rabbit models of acquired LQTS. Methods and results We used two clinically relevant in vivo rabbit models of TdP in which we infused NS1643 or vehicle: (i) three-week atrioventricular block with ventricular bradypacing; (ii) dofetilide-induced I Kr inhibition in methoxamine-sensitized rabbits. In addition, we studied effects on ionic currents in cardiomyocytes with I Kr suppressed by bradycardia remodelling or dofetilide exposure. Bradypaced rabbits developed QT interval prolongation, spontaneous ventricular ectopy, and TdP. Infusion of NS1643 completely suppressed arrhythmic activity and shortened the QT interval; vehicle had no effect. NS1643 also suppressed ventricular tachyarrhythmias caused by infusion of dofetilide to methoxamine-sensitized rabbits, and reversed dofetilide-induced QT prolongation. NS1643 increased I Kr in cardiomyocytes isolated from normal and bradycardia-remodelled rabbits by approximately 75% and 50%, respectively ( P < 0.001 for each). Similarly, NS1643 restored I Kr suppressed by 5 nmol/L dofetilide (tail current 0.28 ± 0.03 pA/pF pre-dofetilide, 0.20 ± 0.01 pA/pF in the presence of dofetilide, 0.27 ± 0.02 pA/pF after adding NS1643 to dofetilide-containing solution, P < 0.01). Conclusion Pharmacological activation of I Kr reverses acquired LQTS and TdP caused by bradycardic remodelling and I Kr-blocking drugs. I Kr-activating drug therapy could be a potentially interesting treatment approach for LQTS.

The short QT syndrome is a newly discovered pro-arrhythmic condition, which may cause ventricular fibrillation and sudden death. Short QT can originate from the apparent gain-of-function mutation N588K in the hERG potassium channel that conducts repolarising I(Kr) current. The present study describes a profound biophysical characterization of HERG-N588K revealing both loss-of-function and gain-of-function properties of the mutant. Experiments were conducted after heterologous expression in both Xenopus laevis oocytes and mammalian cells and at both room temperature and at 37 degrees C. Also the impact of the beta-subunits KCNE2 was investigated. The most prominent loss-of-function property of HERG-N588K was reduced tail currents but also the activation properties was compromised. Based on these biophysical results we suggest that the general view of HERG-N588K being a gain-of-function is modified to a mixed gain- and loss-of-function mutation. This might also have impact on the pathological picture of the HERG-N588K channels ability to trigger arrhythmic events.