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Aims: Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are increasingly used to treat type 2 diabetes and obesity. Albeit cardiovascular outcomes generally improve, treatment with GLP-1 RAs is associated with increased heart rate, the mechanism of which is unclear., Methods and Results: We employed a large animal model, the female landrace pig, and used multiple in vivo and ex vivo approaches including pharmacological challenges, electrophysiology, and high-resolution mass spectrometry to explore how GLP-1 elicits an increase in heart rate. In anaesthetized pigs, neither cervical vagotomy, adrenergic blockers (alpha, beta, or combined alpha-beta blockade), ganglionic blockade (hexamethonium), nor inhibition of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (ivabradine) abolished the marked chronotropic effect of GLP-1. GLP-1 administration to isolated perfused pig hearts also increased heart rate, which was abolished by GLP-1 receptor blockade. Electrophysiological characterization of GLP-1 effects in vivo and in isolated perfused hearts localized electrical modulation to the atria and conduction system. In isolated sinus nodes, GLP-1 administration shortened the action potential cycle length of pacemaker cells and shifted the site of earliest activation. The effect was independent of HCN blockade. Collectively, these data support a direct effect of GLP-1 on GLP-1 receptors within the heart. Consistently, single nucleus RNA sequencing showed GLP-1 receptor expression in porcine pacemaker cells. Quantitative phosphoproteomics analyses of sinus node samples revealed that GLP-1 administration leads to phosphorylation changes of calcium cycling proteins of the sarcoplasmic reticulum, known to regulate heart rate., Conclusion: GLP-1 has direct chronotropic effects on the heart mediated by GLP-1 receptors in pacemaker cells of the sinus node, inducing changes in action potential morphology and the leading pacemaker site through a calcium signalling response characterized by PKA-dependent phosphorylation of Ca2+ cycling proteins involved in pacemaking. Targeting the pacemaker calcium clock may be a strategy to lower heart rate in people treated with GLP-1 RAs., Competing Interests: Conflict of interest: J.J.H. has been on advisory boards for Novo Nordisk. The other authors report no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Aims: AP30663 is a novel compound under development for pharmacological conversion of atrial fibrillation by targeting the small conductance Ca 2+ activated K + (K Ca 2) channel. The aim of this extension phase 1 study was to test AP30663 at higher single doses compared to the first-in-human trial., Methods: Sixteen healthy male volunteers were randomized into 2 cohorts: 6- and 8-mg/kg intravenous single-dose administration of AP30663 vs. placebo. Safety, pharmacokinetic and pharmacodynamic data were collected., Results: AP30663 was associated with mild and transient infusion site reactions with no clustering of other adverse events but with an estimated maximum mean QTcF interval prolongation of 45.2 ms (95% confidence interval 31.5-58.9) in the 6 mg/kg dose level and 50.4 ms (95% confidence interval 36.7-64.0) with 8 mg/kg. Pharmacokinetics was dose proportional with terminal half-life of around 3 h., Conclusion: AP30663 in doses up to 8 mg/kg was associated with mild and transient infusion site reactions and an increase of the QTcF interval. Supporting Information support that the QTc effect may be explained by an off-target inhibition of the I Kr channel., (© 2023 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Existing antiarrhythmic drugs to treat atrial fibrillation (AF) have incomplete efficacy, contraindications and adverse effects, including proarrhythmia. AP30663, an inhibitor of the K Ca 2 channel, has demonstrated AF efficacy in animals; however, its efficacy in humans with AF is unknown. Here we conducted a phase 2 trial in which patients with a current episode of AF lasting for 7 days or less were randomized to receive an intravenous infusion of 3 or 5 mg kg -1 AP30663 or placebo. The trial was prematurely discontinued because of slow enrollment during the coronavirus disease 2019 pandemic. The primary endpoint of the trial was cardioversion from AF to sinus rhythm within 90 min from the start of the infusion, analyzed with Bayesian statistics. Among 59 patients randomized and included in the efficacy analyses, the primary endpoint occurred in 42% (5 of 12), 55% (12 of 22) and 0% (0 of 25) of patients treated with 3 mg kg -1 AP30663, 5 mg kg -1 AP30663 or placebo, respectively. Both doses demonstrated more than 99.9% probability of superiority over placebo, surpassing the prespecified 95% threshold. The mean time to cardioversion, a secondary endpoint, was 47 (s.d. = 23) and 41 (s.d. = 24) minutes for 3 mg kg -1 and 5 mg kg -1 AP30663, respectively. AP30663 caused a transient increase in the QTcF interval, with a maximum mean effect of 37.7 ms for the 5 mg kg -1 dose. For both dose groups, no ventricular arrhythmias occurred and adverse event rates were comparable to the placebo group. AP30663 demonstrated AF cardioversion efficacy in patients with recent-onset AF episodes. K Ca 2 channel inhibition may be an attractive mechanism for rhythm control of AF that should be studied further in randomized trials. ClinicalTrials.gov registration: NCT04571385 ., (© 2023. The Author(s).)

The Coronavirus envelope (E) protein is a small structural protein with ion channel activity that plays an important role in virus assembly, budding, immunopathogenesis and disease severity. The viroporin E is also located in Golgi and ER membranes of infected cells and is associated with inflammasome activation and immune dysregulation. Here we evaluated in vitro antiviral activity, mechanism of action and in vivo efficacy of BIT225 for the treatment of SARS-CoV-2 infection. BIT225 showed broad-spectrum direct-acting antiviral activity against SARS-CoV-2 in Calu3 and Vero cells with similar potency across 6 different virus strains. BIT225 inhibited ion channel activity of E protein but did not inhibit endogenous currents or calcium-induced ion channel activity of TMEM16A in Xenopus oocytes. BIT225 administered by oral gavage for 12 days starting 12 hours before infection completely prevented body weight loss and mortality in SARS-CoV-2 infected K18 mice (100% survival, n = 12), while all vehicle-dosed animals reached a mortality endpoint by Day 9 across two studies (n = 12). When treatment started at 24 hours after infection, body weight loss, and mortality were also prevented (100% survival, n = 5), while 4 of 5 mice maintained and increased body weight and survived when treatment started 48 hours after infection. Treatment efficacy was dependent on BIT225 dose and was associated with significant reductions in lung viral load (3.5 log10), virus titer (4000 pfu/ml) and lung and serum cytokine levels. These results validate viroporin E as a viable antiviral target and support the clinical study of BIT225 for treatment and prophylaxis of SARS-CoV-2 infection., Competing Interests: GE, AT and MM are employees of Biotron Limited; KK and SB are consultants of Biotron Limited. MMR is co-founder, member of the Board of Directors and minority shareholder of Synklino., (Copyright: © 2023 Ewart et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Small-conductance (SK) calcium-activated potassium channels are a promising treatment target in atrial fibrillation. However, the functional properties that differentiate SK inhibitors remain poorly understood. The objective of this study was to determine how two unrelated SK channel inhibitors, apamin and AP14145, impact SK channel function in excised inside-out single-channel recordings. Surprisingly, both apamin and AP14145 exert much of their inhibition by inducing a class of very-long-lived channel closures (apamin: τ c,vl  = 11.8 ± 7.1 s, and AP14145: τ c,vl  = 10.3 ± 7.2 s), which were never observed under control conditions. Both inhibitors also induced changes to the three closed and two open durations typical of normal SK channel gating. AP14145 shifted the open duration distribution to favor longer open durations, whereas apamin did not alter open-state kinetics. AP14145 also prolonged the two shortest channel closed durations (AP14145: τ c,s  = 3.50 ± 0.81 ms, and τ c,i  = 32.0 ± 6.76 ms versus control: τ c,s  = 1.59 ± 0.19 ms, and τ c,i  = 13.5 ± 1.17 ms), thus slowing overall gating kinetics within bursts of channel activity. In contrast, apamin accelerated intraburst gating kinetics by shortening the two shortest closed durations (τ c,s  = 0.75 ± 0.10 ms and τ c,i  = 5.08 ± 0.49 ms) and inducing periods of flickery activity. Finally, AP14145 introduced a unique form of inhibition by decreasing unitary current amplitude. SK channels exhibited two clearly distinguishable amplitudes (control: A high  = 0.76 ± 0.03 pA, and A low  = 0.54 ± 0.03 pA). AP14145 both reduced the fraction of patches exhibiting the higher amplitude (AP14145: 4/9 patches versus control: 16/16 patches) and reduced the mean low amplitude (0.38 ± 0.03 pA). Here, we have demonstrated that both inhibitors introduce very long channel closures but that each also exhibits unique effects on other components of SK gating kinetics and unitary current. The combination of these effects is likely to be critical for understanding the functional differences of each inhibitor in the context of cyclical Ca 2+ -dependent channel activation in vivo., Competing Interests: Declaration of interests B.H.B. is an employee of, and has financial interest in, Acesion Pharma ApS, a developer of SK targeting compounds including AP14145., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: Genotype-positive patients who suffer from the cardiac channelopathy Long QT Syndrome (LQTS) may display a spectrum of clinical phenotypes, with often unknown causes. Therefore, there is a need to identify factors influencing disease severity to move towards an individualized clinical management of LQTS. One possible factor influencing the disease phenotype is the endocannabinoid system, which has emerged as a modulator of cardiovascular function. In this study, we aim to elucidate whether endocannabinoids target the cardiac voltage-gated potassium channel K V 7.1/KCNE1, which is the most frequently mutated ion channel in LQTS., Methods: We used two-electrode voltage clamp, molecular dynamics simulations and the E4031 drug-induced LQT2 model of ex-vivo guinea pig hearts., Findings: We found a set of endocannabinoids that facilitate channel activation, seen as a shifted voltage-dependence of channel opening and increased overall current amplitude and conductance. We propose that negatively charged endocannabinoids interact with known lipid binding sites at positively charged amino acids on the channel, providing structural insights into why only specific endocannabinoids modulate K V 7.1/KCNE1. Using the endocannabinoid ARA-S as a prototype, we show that the effect is not dependent on the KCNE1 subunit or the phosphorylation state of the channel. In guinea pig hearts, ARA-S was found to reverse the E4031-prolonged action potential duration and QT interval., Interpretation: We consider the endocannabinoids as an interesting class of hK V 7.1/KCNE1 channel modulators with putative protective effects in LQTS contexts., Funding: ERC (No. 850622), Canadian Institutes of Health Research, Canada Research Chairs and Compute Canada, Swedish National Infrastructure for Computing., Competing Interests: Declaration of interests A patent application (#62/032,739) including a description of the interaction of charged lipophilic compounds with the K(V)7.1 channel has been submitted by the University of Miami with S.I.L. identified as one of the inventors. The other authors have no conflict of interest to declare., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Adenosine, an endogenous nucleoside, plays a critical role in maintaining homeostasis during stressful situations, such as energy deprivation or cellular damage. Therefore, extracellular adenosine is generated locally in tissues under conditions such as hypoxia, ischemia, or inflammation. In fact, plasma levels of adenosine in patients with atrial fibrillation (AF) are elevated, which also correlates with an increased density of adenosine A 2A receptors (A 2A Rs) both in the right atrium and in peripheral blood mononuclear cells (PBMCs). The complexity of adenosine-mediated effects in health and disease requires simple and reproducible experimental models of AF. Here, we generate two AF models, namely the cardiomyocyte cell line HL-1 submitted to Anemonia toxin II (ATX-II) and a large animal model of AF, the right atrium tachypaced pig (A-TP). We evaluated the density of endogenous A 2A R in those AF models. Treatment of HL-1 cells with ATX-II reduced cell viability, while the density of A 2A R increased significantly, as previously observed in cardiomyocytes with AF. Next, we generated the animal model of AF based on tachypacing pigs. In particular, the density of the key calcium regulatory protein calsequestrin-2 was reduced in A-TP animals, which is consistent with the atrial remodelling shown in humans suffering from AF. Likewise, the density of A 2A R in the atrium of the AF pig model increased significantly, as also shown in the biopsies of the right atrium of subjects with AF. Overall, our findings revealed that these two experimental models of AF mimicked the alterations in A 2A R density observed in patients with AF, making them attractive models for studying the adenosinergic system in AF.

Background: Obstructive sleep apnea (OSA) creates a complex substrate for atrial fibrillation (AF), which is refractory to many clinically available pharmacological interventions. We investigated atrial antiarrhythmogenic properties and ventricular electrophysiological safety of small-conductance Ca 2+ -activated K + (SK)-channel inhibition in a porcine model for obstructive respiratory events., Methods: In spontaneously breathing pigs, obstructive respiratory events were simulated by intermittent negative upper airway pressure (INAP) applied via a pressure device connected to the intubation tube. INAP was applied for 75 s, every 10 min, three times before and three times during infusion of the SK-channel inhibitor AP14145. Atrial effective refractory periods (AERP) were acquired before (pre-INAP), during (INAP) and after (post-) INAP. AF-inducibility was determined by a S1S2 atrial pacing protocol. Ventricular arrhythmicity was evaluated by heart rate adjusted QT-interval duration (QT-paced) and electromechanical window (EMW) shortening., Results: During vehicle infusion, INAP transiently shortened AERP (pre-INAP: 135 ± 10 ms vs. post-INAP 101 ± 11 ms; p = .008) and increased AF-inducibility. QT-paced prolonged during INAP (pre-INAP 270 ± 7 ms vs. INAP 275 ± 7 ms; p = .04) and EMW shortened progressively throughout INAP and post-INAP (pre-INAP 80 ± 4 ms; INAP 59 ± 6 ms, post-INAP 46 ± 10 ms). AP14145 prolonged baseline AERP, partially prevented INAP-induced AERP-shortening and reduced AF-susceptibility. AP14145 did not alter QT-paced at baseline (pre-AP14145 270 ± 7 ms vs. AP14145 268 ± 6 ms, p = .83) or QT-paced and EMW-shortening during INAP., Conclusion: In a pig model for obstructive respiratory events, the SK-channel-inhibitor AP14145 prevented INAP-associated AERP-shortening and AF-susceptibility without impairing ventricular electrophysiology. Whether SK-channels represent a target for OSA-related AF in humans warrants further study., (© 2022 The Authors. Journal of Cardiovascular Electrophysiology published by Wiley Periodicals LLC.)

Viroporins are ion channels found in many viruses, where they contribute to virus life cycle and thereby pathogenesis. Viroporin targeting is a known, yet largely unexplored, therapeutic strategy so far only used in Influenza A with the drugs amantadine and rimantadine. In this review, we seek to utilize the inhibition by amantadine of the viroporin Protein E in SARS-CoV-2 in an attempt to treat COVID-19 in its early stages. We are executing a double-blinded placebo-controlled trial based on promising in vivo and in vitro work as a stepping-stone for establishing a therapeutic antiviral regime: blocking of viroporins.

Background: Atrial Fibrillation (AF) is the most prevalent sustained cardiac arrhythmia, responsible for considerable morbidity and mortality. The heterogenic and complex pathogenesis of AF remains poorly understood, which contributes to the current limitation in effective treatments. We aimed to identify rare genetic variants associated with AF in patients with familial AF. Methods and results: We performed whole exome sequencing in a large family with familial AF and identified a rare variant in the gene CACNA1A c.5053G > A which co-segregated with AF. The gene encodes for the protein variants Ca V 2.1-V1686M, and is important in neuronal function. Functional characterization of the CACNA1A, using patch-clamp recordings on transiently transfected mammalian cells, revealed a modest loss-of-function of Ca V 2.1-V1686M. Conclusion: We identified a rare loss-of-function variant associated with AF in a gene previously linked with neuronal function. The results allude to a novel link between dysfunction of an ion channel previously associated with neuronal functions and increased risk of developing AF., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vad, Yan, Denti, Ahlberg, Refsgaard, Bomholtz, Santos, Rasmussen, Haunsø, Svendsen, Christophersen, Schmitt, Olesen and Bentzen.)

The dire need for COVID-19 treatments has inspired strategies of repurposing approved drugs. Amantadine has been suggested as a candidate, and cellular as well as clinical studies have indicated beneficial effects of this drug. We demonstrate that amantadine and hexamethylene-amiloride (HMA), but not rimantadine, block the ion channel activity of Protein E from SARS-CoV-2, a conserved viroporin among coronaviruses. These findings agree with their binding to Protein E as evaluated by solution NMR and molecular dynamics simulations. Moreover, we identify two novel viroporins of SARS-CoV-2; ORF7b and ORF10, by showing ion channel activity in a X. laevis oocyte expression system. Notably, amantadine also blocks the ion channel activity of ORF10, thereby providing two ion channel targets in SARS-CoV-2 for amantadine treatment in COVID-19 patients. A screen of known viroporin inhibitors on Protein E, ORF7b, ORF10 and Protein 3a from SARS-CoV-2 revealed inhibition of Protein E and ORF7b by emodin and xanthene, the latter also blocking Protein 3a. This illustrates a general potential of well-known ion channel blockers against SARS-CoV-2 and specifically a dual molecular basis for the promising effects of amantadine in COVID-19 treatment. We therefore propose amantadine as a novel, cheap, readily available and effective way to treat COVID-19., (© 2021. The Author(s).)

Aims: Pharmacological termination of atrial fibrillation (AF) remains a challenge due to limited efficacy and potential ventricular proarrhythmic effects of antiarrhythmic drugs. SK channels are proposed as atrial-specific targets in the treatment of AF. Here, we investigated the effects of the new SK channel inhibitor AP14145., Methods and Results: Eight goats were implanted with pericardial electrodes for induction of AF (30 days). In an open-chest study, the atrial conduction velocity (CV) and effective refractory period (ERP) were measured during pacing. High-density mapping of both atrial free-walls was performed during AF and conduction properties were assessed. All measurements were performed at baseline and during AP14145 infusion [10 mg/kg/h (n = 1) or 20 mg/kg/h (n = 6)]. At an infusion rate of 20 mg/kg/h, AF terminated in five of six goats. AP14145 profoundly increased ERP and reduced CV during pacing. AP14145 increased spatiotemporal instability of conduction at short pacing cycle lengths. Atrial fibrillation cycle length and pathlength (AF cycle length × CV) underwent a strong dose-dependent prolongation. Conduction velocity during AF remained unchanged and conduction patterns remained complex until the last seconds before AF termination, during which a sudden and profound organization of fibrillatory conduction occurred., Conclusion: AP14145 provided a successful therapy for termination of persistent AF in goats. During AF, AP14145 caused an ERP and AF cycle length prolongation. AP14145 slowed CV during fast pacing but did not lead to a further decrease during AF. Termination of AF was preceded by an abrupt organization of AF with a decline in the number of fibrillation waves., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: Atrial dilation is an important risk factor for atrial fibrillation (AF) and animal studies have found that acute atrial dilation shortens the atrial effective refractory period (AERP) and increases the risk of AF. Stretch activated ion channels (SACs) and calcium channels play a role in this. The expression profile and calcium dependent activation makes the small conductance calcium activated K + channel (K Ca 2.x) a candidate for coupling stretch induced increases in intracellular calcium through K + -efflux and thereby shortening of atrial refractoriness., Objectives: We hypothesized that K Ca 2.x channel inhibitors can prevent the stretch induced shortening of AERP and protect the heart from AF., Methods: The effect of K Ca 2 channel inhibitor (N-(pyridin-2-yl)-4-(pyridin-2-yl)thiazol-2-amine (ICA) 1 µM) was investigated using the isolated perfused rabbit heart preparation. To stretch the left atrium (LA) a balloon was inserted and inflated. AERP and action potential duration (APD) were recorded before and after atrial stretch. AF was induced by burst pacing the LA at different degrees of atrial stretch., Results: Stretching of the LA by increasing the balloon pressure from 0 to 20 mmHg shortened the AERP by 8.6 ± 1 ms. In comparison, the K Ca 2 inhibitor ICA significantly attenuated the stretch induced shortening of AERP to 2.5 ± 1.1 ms. Total AF duration increased linearly with atrial balloon pressure. This relationship was not found in the presence of ICA. ICA lowered the incidence of AF induction and total AF duration., Conclusion: The K Ca 2 channel inhibitor ICA attenuates the acute stretch induced shortening of AERP and decreases stretch induced vulnerability to AF., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Marine tunicates produce defensive amino-acid-derived metabolites, including 2-(3,5-diiodo-4-methoxyphenyl)ethan-1-amine (DIMTA), but their mechanisms of action are rarely known. Using an assay-guided approach, we found that out of the many different sensory cells in the mouse dorsal root ganglion (DRG), DIMTA selectively affected low-threshold cold thermosensors. Whole-cell electrophysiology experiments using DRG cells, channels expressed in Xenopus oocytes, and human cell lines revealed that DIMTA blocks several potassium channels, reducing the magnitude of the afterhyperpolarization and increasing the baseline intracellular calcium concentration [Ca 2+ ] i of low-threshold cold thermosensors. When injected into mice, DIMTA increased the threshold of cold sensation by >3 °C. DIMTA may thus serve as a lead in the further design of compounds that inhibit problems in the cold-sensory system, such as cold allodynia and other neuropathic pain conditions.

Delineating human cardiac pathologies and their basic molecular mechanisms relies on research conducted in model organisms. Yet translating findings from preclinical models to humans present a significant challenge, in part due to differences in cardiac protein expression between humans and model organisms. Proteins immediately determine cellular function, yet their large-scale investigation in hearts has lagged behind those of genes and transcripts. Here, we set out to bridge this knowledge gap: By analyzing protein profiles in humans and commonly used model organisms across cardiac chambers, we determine their commonalities and regional differences. We analyzed cardiac tissue from each chamber of human, pig, horse, rat, mouse, and zebrafish in biological replicates. Using mass spectrometry-based proteomics workflows, we measured and evaluated the abundance of approximately 7,000 proteins in each species. The resulting knowledgebase of cardiac protein signatures is accessible through an online database: atlas.cardiacproteomics.com. Our combined analysis allows for quantitative evaluation of protein abundances across cardiac chambers, as well as comparisons of cardiac protein profiles across model organisms. Up to a quarter of proteins with differential abundances between atria and ventricles showed opposite chamber-specific enrichment between species; these included numerous proteins implicated in cardiac disease. The generated proteomics resource facilitates translational prospects of cardiac studies from model organisms to humans by comparisons of disease-linked protein networks across species., Competing Interests: The authors have declared that no competing interests exist.

Activation of the voltage-gated Kv7 channels holds therapeutic promise in several neurological and psychiatric disorders, including epilepsy, schizophrenia, and depression. Here, we present a pharmacological characterization of Lu AA41178, a novel, pan-selective Kv7.2-7.5 opener, using both in vitro assays and a broad range of in vivo assays with relevance to epilepsy, schizophrenia, and depression. Electrophysiological characterization in Xenopus oocytes expressing human Kv7.2-Kv7.5 confirmed Lu AA41178 as a pan-selective opener of Kv7 channels by significantly left-shifting the activation threshold. Additionally, Lu AA41178 was tested in vitro for off-target effects, demonstrating a clean Kv7-selective profile, with no impact on common cardiac ion channels, and no potentiating activity on GABAA channels. Lu AA41178 was evaluated across preclinical in vivo assays with relevance to neurological and psychiatric disorders. In the maximum electroshock seizure threshold test and PTZ seizure threshold test, Lu AA41178 significantly increased the seizure thresholds in mice, demonstrating anticonvulsant efficacy. Lu AA41178 demonstrated antipsychotic-like activity by reducing amphetamine-induced hyperlocomotion in mice as well as lowering conditioned avoidance responses in rats. In the mouse forced swim test, a model with antidepressant predictivity, Lu AA41178 significantly reduced immobility. Additionally, behavioral effects typically observed with Kv7 openers was also characterized. In vivo assays were accompanied by plasma and brain exposures, revealing minimum effective plasma levels <1000 ng/ml. Lu AA41178, a potent opener of neuronal Kv7 channels demonstrate efficacy in assays of epilepsy, schizophrenia and depression and might serve as a valuable tool for exploring the role of Kv7 channels in both neurological and psychiatric disorders., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Pharmacological cardioversion of atrial fibrillation (AF) is frequently inefficacious. AP30663, a small conductance Ca 2+ activated K + (K Ca 2) 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 (C max ) 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 K Ca 2 channel inhibitor, was safe and well-tolerated systemically in humans, supporting further development in patients with AF undergoing cardioversion., (© 2020 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Aim: We aimed to assess the ability of natural and modified polyunsaturated fatty acids (PUFAs) to shorten QT interval in ex-vivo and in-vivo guinea pig hearts., Methods: The effect of one natural (docosahexaenoic acid [DHA]) and three modified (linoleoyl glycine [Lin-GLY], docosahexaenoyl glycine [DHA-GLY], N-arachidonoyl taurine [N-AT]) PUFAs on ventricular action potential duration (APD) and QT interval was studied in a E4031 drug-induced long QT2 model of ex-vivo guinea pig hearts. The effect of DHA-GLY on QT interval was also studied in in-vivo guinea pig hearts upon intravenous administration. The effect of modified PUFAs on I Ks was studied using Xenopus laevis oocytes expressing human KCNQ1 and KCNE1., Results: All tested PUFAs shortened ADP and QT interval in ex-vivo guinea pig hearts, however, with different ability in restoring baseline APD/QT interval with specific modified PUFAs being most efficacious. Despite comparable ability in activating the human KCNQ1/KCNE1 channel, Lin-GLY was not as effective in shortening APD/QT interval as DHA-GLY in ex-vivo hearts. By constructing a guinea pig-like KCNE1, we found Lin-GLY to induce less activating effect compared with DHA-GLY on human KCNQ1 co-expressed with guinea pig-like KCNE1. Docosahexaenoyl glycine was studied in more detail and was found to shorten QT interval in in-vivo guinea pig hearts., Conclusion: Our results show that specific PUFAs shorten QT interval in guinea pig hearts. The tendency of modified PUFAs with pronounced I Ks channel activating effect to better restore QT interval suggests that modifying PUFAs to target the I Ks channel is a means to improve the QT-shortening effect., (© 2020 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Background: Adenosine leads to atrial action potential (AP) shortening through activation of adenosine 1 receptors (A 1 -R) and subsequent opening of G-protein-coupled inwardly rectifying K + channels. Extracellular production of adenosine is drastically increased during stress and ischemia., Objective: The aim of this study was to address whether the pharmacological blockade of endogenous production of adenosine and of its signaling prevents atrial fibrillation (AF)., Methods: The role of A 1 -R activation on atrial action potential duration, refractoriness, and AF vulnerability was investigated in rat isolated beating heart preparations (Langendorff) with an A 1 -R agonist [2-chloro- N 6 -cyclopentyladenosine (CCPA), 50 nM] and antagonist [1-butyl-3-(3-hydroxypropyl)-8-(3-noradamantyl)xanthine (PSB36), 40 nM]. Furthermore, to interfere with the endogenous adenosine release, the ecto-5'-nucleotidase (CD73) inhibitor was applied [5'-(α,β-methylene) diphosphate sodium salt (AMPCP), 500 μM]. Isolated trabeculae from human right atrial appendages (hRAAs) were used for comparison., Results: As expected, CCPA shortened AP duration at 90% of repolarization (APD 90 ) and effective refractory period (ERP) in rat atria. PSB36 prolonged APD 90 and ERP in rat atria, and CD73 inhibition with AMPCP prolonged ERP in rats, confirming that endogenously produced amount of adenosine is sufficiently high to alter atrial electrophysiology. In human atrial appendages, CCPA shortened APD 90 , while PSB36 prolonged it. Rat hearts treated with CCPA are prone to AF. In contrast, PSB36 and AMPCP prevented AF events and reduced AF duration (vehicle, 11.5 ± 2.6 s; CCPA, 40.6 ± 16.1 s; PSB36, 6.5 ± 3.7 s; AMPCP, 3.0 ± 1.4 s; P < 0.0001)., Conclusion: A 1 -R activation by intrinsic adenosine release alters atrial electrophysiology and promotes AF. Inhibition of adenosine pathway protects atria from arrhythmic events., (Copyright © 2020 Soattin, Lubberding, Bentzen, Christ and Jespersen.)

Our heart is comprised of many different cell types that all contribute to cardiac function. An important step in deciphering the molecular complexity of our heart is to decipher the molecular composition of the various cardiac cell types. Here we set out to delineate a comprehensive protein expression profile of the two most prevalent cell types in the heart: cardiomyocytes and cardiac fibroblasts. To this end, we isolated cardiomyocytes and fibroblasts from rat hearts and combined state-of-the-art flow cytometry with high-resolution mass spectrometry to investigate their proteome profiles right after isolation. We measured and quantified 5240 proteins in cardiomyocytes and 6328 proteins in cardiac fibroblasts. In addition to providing a global protein profile for these cardiac cell types, we also present specific findings, such as unique expression of ion channels and transcription factors for each cell type. For instance, we show that the sodium channel Scn7a and the cation channel Trpm7 are expressed in fibroblasts but not in cardiomyocytes, which underscores the importance of investigating the endogenous cell host prior to functional studies. Our dataset represents a valuable resource on protein expression profiles in these two primary cardiac cells types., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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 Ca 2+ -activated K + -channels (K Ca 2) are a promising new atrial selective target for treatment of atrial fibrillation. Under physiological conditions K Ca 2 plays a minor role in ventricular repolarization. However, this might change under hypokalemia because of concomitant increases in ventriculay -60r intracellur Ca 2+ ., Purpose: To study the effects of pharmacological K Ca 2 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 K Ca 2.3 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 K Ca 2.3 current compared to normokalemia. Application of K Ca 2 channel inhibitors and dofetilide prolonged the QT interval corrected for heart rate. Dofetilide, but none of the K Ca 2 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 K Ca 2 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 K Ca 2 channel inhibitors, ICA, AP14145, and AP30663 protected the heart from hypokalemia induced VF. These results support that K Ca 2 inhibition may be associated with a better safety and tolerability profile than dofetilide., (Copyright © 2020 Diness, Abildgaard, Bomholtz, Skarsfeldt, Edvardsson, Sørensen, Grunnet and Bentzen.)

Aims: Small conductance Ca 2+ -activated K + channels (SK channels, K Ca 2) are a new target for treatment of atrial fibrillation (AF). AP30663 is a small molecule inhibitor of K Ca 2 channels that is currently in clinical development for treatment of AF. The aim of this study is to present the electrophysiological profile and mechanism of action of AP30663 and its efficacy in prolonging atrial refractoriness in rodents, and by bioinformatic analysis investigate if genetic variants in KCNN2 or KCNN3 influence the expression level of these in human heart tissue., Methods and Results: Whole-cell and inside-out patch-clamp recordings of heterologously expressed K Ca 2 channels revealed that AP30663 inhibits K Ca 2 channels with minor effects on other relevant cardiac ion channels. AP30663 modulates the K Ca 2.3 channel by right-shifting the Ca 2+ -activation curve. In isolated guinea pig hearts AP30663 significantly prolonged the atrial effective refractory period (AERP) with minor effects on the QT-interval corrected for heart rate. Similarly, in anaesthetized rats 5 and 10 mg/kg of AP30663 changed the AERP to 130.7±5.4% and 189.9±18.6 of baseline values. The expression quantitative trait loci analyses revealed that the genome wide association studies for AF SNP rs13376333 in KCNN3 is associated with increased mRNA expression of KCNN3 in human atrial appendage tissue., Conclusions: AP30663 is a novel negative allosteric modulator of K Ca 2 channels that concentration-dependently prolonged rodent atrial refractoriness with minor effects on the QT-interval. Moreover, AF associated SNPs in KCNN3 influence KCNN3 mRNA expression in human atrial tissue. These properties support continued development of AP30663 for treatment of AF in man., (Copyright © 2020 Bentzen, Bomholtz, Simó-Vicens, Folkersen, Abildgaard, Speerschneider, Muthukumarasamy, Edvardsson, Sørensen, Grunnet and Diness.)

Background: Inhibition of K Ca 2 channels, conducting I KCa , can convert atrial fibrillation (AF) to sinus rhythm and protect against its induction. I KCa inhibition has been shown to possess functional atrial selectivity with minor effects on ventricles. Under pathophysiological conditions with ventricular remodeling, however, inhibiting I KCa can exhibit both proarrhythmic and antiarrhythmic ventricular effects. The aim of this study was to evaluate the effects of the I KCa inhibitor AP14145, when given before or after the I Kr blocker dofetilide, on cardiac function and ventricular proarrhythmia markers in pigs with or without left ventricular dysfunction (LVD)., Methods: Landrace pigs were randomized into an AF group (n = 6) and two control groups: SHAM1 (n = 8) and SHAM2 (n = 4). AF pigs were atrially tachypaced (A-TP) for 43 ± 4 days until sustained AF and LVD developed. A-TP and SHAM1 pigs received 20 mg/kg AP14145 followed by 100 µg/kg dofetilide whereas SHAM2 pigs received the same drugs in the opposite order. Proarrhythmic markers such as short-term variability of QT (STV QT ) and RR (STV RR ) intervals, and the number of premature ventricular complexes (PVCs) were measured at baseline and after administration of drugs. The influence on cardiac function was assessed by measuring cardiac output, stroke volume, and relevant echocardiographic parameters., Results: I KCa inhibition by AP14145 did not increase STV QT or STV RR in any of the pigs. I Kr inhibition by dofetilide markedly increased STV QT in the A-TP pigs, but not in SHAM operated pigs. Upon infusion of AP14145 the number of PVCs decreased or remained unchanged both when AP14145 was infused after baseline and after dofetilide. Conversely, the number of PVCs increased or remained unchanged upon dofetilide infusion. Neither AP14145 nor dofetilide affected relevant echocardiographic parameters, cardiac output, or stroke volume in any of the groups., Conclusion: I KCa inhibition with AP14145 was not proarrhythmic in healthy pigs, or in the presence of LVD resulting from A-TP. In pigs already challenged with 100 µg/kg dofetilide there were no signs of proarrhythmia when 20 mg/kg AP14145 were infused. K Ca 2 channel inhibition did not affect cardiac function, implying that K Ca 2 inhibitors can be administered safely also in the presence of LV dysfunction., (Copyright © 2020 Citerni, Kirchhoff, Olsen, Sattler, Grunnet, Edvardsson, Bentzen and Diness.)

Background: Atrial fibrillation (AF) is characterized by electrical and structural remodeling. Irregular and/or fast atrio-ventricular (AV) conduction during AF can result in AV dyssynchrony, tachymyopathy, pressure and volume overload with subsequent dilatation, valve regurgitation, and ventricular dysfunction with progression to heart failure. Objective: To gain further insight into the myocardial pathophysiological changes induced by right atrial tachypacing (A-TP) in a large animal model. Methods: A total of 28 Landrace pigs were randomized as 14 into AF-induced A-TP group and 14 pigs to control group. AF pigs were tachypaced for 43 ± 4 days until in sustained AF. Functional remodeling was investigated by echocardiography (after cardioversion to sinus rhythm). Structural remodeling was quantified by histological preparations with picrosirius red and immunohistochemical stainings. Results: A-TP resulted in decreased left ventricular ejection fraction (LVEF) accompanied by increased end-diastolic and end-systolic left atrium (LA) volume and area. In addition, A-TP was associated with mitral valve (MV) regurgitation, diastolic dysfunction and increased atrial and ventricular fibrotic extracellular matrix (ECM). Conclusions: A-TP induced AF with concomitant LV systolic and diastolic dysfunction, increased LA volume and area, and atrial and ventricular fibrosis., (Copyright © 2020 Citerni, Kirchhoff, Olsen, Sattler, Gentilini, Forni, Zannoni, Grunnet, Edvardsson, Bentzen and Diness.)

Aims: To describe the effects of the K Ca 2 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., (Copyright © 2020 Diness, Kirchhoff, Speerschneider, Abildgaard, Edvardsson, Sørensen, Grunnet and Bentzen.)

Atrial Fibrillation (AF) is the most common cardiac arrhythmia. Its pathogenesis is complex and poorly understood. Whole exome sequencing of Danish families with AF revealed a novel four nucleotide deletion c.1041&#95;1044del in CLCN2 shared by affected individuals. We aimed to investigate the role of genetic variation of CLCN2 encoding the inwardly rectifying chloride channel ClC-2 as a risk factor for the development of familiar AF. The effect of the CLCN2 variant was evaluated by electrophysiological recordings on transiently transfected cells. We used quantitative PCR to assess CLCN2 mRNA expression levels in human atrial and ventricular tissue samples. The nucleotide deletion CLCN2 c.1041&#95;1044del results in a frame-shift and premature stop codon. The truncated ClC-2 p.V347fs channel does not conduct current. Co-expression with wild-type ClC-2, imitating the heterozygote state of the patients, resulted in a 50% reduction in macroscopic current, suggesting an inability of truncated ClC-2 protein to form channel complexes with wild type channel subunits. Quantitative PCR experiments using human heart tissue from healthy donors demonstrated that CLCN2 is expressed across all four heart chambers. Our genetic and functional data points to a possible link between loss of ClC-2 function and an increased risk of developing AF.

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 (I Kr ) is inhibited. The current mediated by K Ca 2-channels, I KCa , is considered a promising new target for treatment of atrial fibrillation (AF). Selective inhibitors of I Kr (dofetilide) and I KCa (AP14145) were used to compare the effects on ventricular and atrial repolarization. Ondansetron, which has been reported to be a potent blocker of both I Kr and I KCa , was included to examine its potential atrial antiarrhythmic properties. Experimental Approach: The expression of K Ca 2- and K v 11.1-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 I KCa and/or I Kr . 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 I KCa , but did inhibit I Kr in vitro . Ondansetron prolonged ventricular, but not atrial repolarization ex vivo . Conclusion and Implications: I KCa inhibition by AP14145 selectively increases atrial repolarization, whereas I Kr inhibition by dofetilide and ondansetron increases ventricular repolarization to a larger extent than atrial repolarization.

A variety of polycyclic pyridines have been proposed as inhibitors of the small conductance calcium-activated potassium (SK) channel. To this group belongs 2,6-bis(2-benzimidazolyl)pyridine (BBP), a commercially and readily available small organic compound which has earlier been described in a broad range of chemical and biological uses. Here, we show how BBP can also be used as a potent and specific SK channel blocker in vitro . The potency of BBP was measured using automatic patch clamp on all three SK channel subtypes, resulting in similar IC 50 of 0.4 μM. We also assessed the selectivity of BBP on a panel of calcium-activated and voltage-activated potassium channels using two-electrode voltage clamp, automatic and manual patch clamp. BBP did not have any effect on IK, K ir 2.1, K ir 3.1+K ir 3.4, K v 1.5, K v 4.3/K CHIP 2 and K v 7.1/KCNE1 currents and was 4.8-fold and 46-fold more potent on all SK channel subtypes vs. BK and hERG channels, respectively. Moreover, we were able to identify H491 as a critical amino acid for the pharmacological effect of BBP on the SK channel. From a medicinal chemistry perspective, BBP could be used as a starting point for the design of new and improved SK inhibitors.

Optically pumped magnetometers are becoming a promising alternative to cryogenically-cooled superconducting magnetometers for detecting and imaging biomagnetic fields. Magnetic field detection is a completely non-invasive method, which allows one to study the function of excitable human organs with a sensor placed outside the human body. For instance, magnetometers can be used to detect brain activity or to study the activity of the heart. We have developed a highly sensitive miniature optically pumped magnetometer based on cesium atomic vapor kept in a paraffin-coated glass container. The magnetometer is optimized for detection of biological signals and has high temporal and spatial resolution. It is operated at room- or human body temperature and can be placed in contact with or at a mm-distance from a biological object. With this magnetometer, we detected the heartbeat of an isolated guinea-pig heart, which is an animal widely used in biomedical studies. In our recordings of the magnetocardiogram, we can detect the P-wave, QRS-complex and T-wave associated with the cardiac cycle in real time. We also demonstrate that our device is capable of measuring the cardiac electrographic intervals, such as the RR- and QT-interval, and detecting drug-induced prolongation of the QT-interval, which is important for medical diagnostics.

Aim: The zebrafish has emerged as a novel model for investigating cardiac physiology and pathology. The aim of this study was to investigate the atrium-specific ion channels responsible for shaping the atrial cardiac action potential in zebrafish., Methods: Using quantitative polymerase chain reaction, we assessed the expression level of atrium-specific potassium channels. The functional role of these channels was studied by patch clamp experiments on isolated atrial and ventricular cardiomyocytes and by optical mapping of explanted adult zebrafish hearts. Finally, surface ECGs were recorded to establish possible in vivo roles of atrial ion channels., Results: In isolated adult zebrafish hearts, we identified the expression of kcnk3, kcnk9, kcnn1, kcnn2, kcnn3, kcnj3 and kcnj5, the genes that encode the atrium-specific K 2P , K Ca 2.x and K ir 3.1/4 (K ACh ) ion channels. The electrophysiological data indicate that the acetylcholine-activated inward-rectifying current, I KACh, plays a major role in the zebrafish atrium, whereas K 2P 3.1/9.1 and K Ca 2.x channels do not appear to be involved in regulating the action potential in the zebrafish heart., Conclusion: We demonstrate that the acetylcholine-activated inward-rectifying current (I KACh ) current plays a major role in the zebrafish atrium and that the zebrafish could potentially be a cost-effective and reliable model for pharmacological testing of atrium-specific I KACh modulating compounds., (© 2018 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Background: Dysfunction of Na V 1.5 encoded by SCN5A accounts for approximately half of the channelopathic SIDS cases. We investigated the functional effect of two gene variants identified in the same patient, one in SCN5A and one in SCN1Bb. The aim of the study was to risk stratify the proband's family., Methods: The family was referred for cardiovascular genetic evaluation to assess familial risk of cardiac disease. Functional analysis of the identified variants was performed with patch-clamp electrophysiology in HEK293 cells., Results: A 16-month-old healthy boy died suddenly in the context of nonspecific illness and possible fever. Postmortem genetic testing revealed variants in the SCN5A and SCN1Bb genes. The proband's father carries the same variants but is asymptomatic. Electrophysiological analysis of the Na V 1.5&#95;1281X truncation revealed complete loss-of-function of the channel. Coexpression of Na V 1.5 with Na V β1b significantly increased I Na density when compared to Na V 1.5 alone. The Na V β1b &#95;V268I variant abolished this I Na density increase. Moreover, it shifted the activation curve toward more depolarized potentials., Conclusions: Genetic variation of both sodium channel and its modifiers may contribute to sudden unexplained death in childhood. However, the asymptomatic father suggests that genetic variation of these genes is not sufficient to cause sudden death or clinically detectable SCN5A phenotypes., (© 2018 Wiley Periodicals, Inc.)

Aim: We investigated the effect of variants in genes encoding sodium channel modifiers SNTA1 and GPD1L found in early onset atrial fibrillation (AF) patients., Patients & Methods: Genetic screening in patients with early onset lone AF revealed three variants in GPD1L and SNTA1 in three AF patients. Functional analysis was performed by patch-clamp electrophysiology., Results: Co-expression of GPD1L or its p.A326E variant with Na V 1.5 did not alter I Na density or current kinetics. SNTA1 shifted the peak-current by -5 mV. The SNTA1-p.A257G variant significantly increased I Na . SNTA1-p.P74L did not produce functional changes., Conclusion: Although genetic variation of sodium channel modifiers may contribute to development of AF at a molecular level, it is unlikely a monogenic cause of the disease.

Background and Purpose: K v 7.4 and K v 7.5 channels are regulators of vascular tone. 4-Aminopyridine (4-AP) is considered a broad inhibitor of voltage-gated potassium (K V ) channels, with little inhibitory effect on K v 7 family members at mmol concentrations. However, the effect of 4-AP on K v 7 channels has not been systematically studied. The aim of this study was to investigate the pharmacological activity of 4-AP on K v 7.4 and K v 7.5 channels and characterize the effect of 4-AP on rat resistance arteries., Experimental Approach: Voltage clamp experiments were performed on Xenopus laevis oocytes injected with cRNA encoding KCNQ4 or KCNQ5, HEK cells expressing K v 7.4 channels and on rat, freshly isolated mesenteric artery smooth muscle cells. The effect of 4-AP on tension, membrane potential, intracellular calcium and pH was assessed in rat mesenteric artery segments., Key Results: 4-AP increased the K v 7.4-mediated current in oocytes and HEK cells but did not affect K v 7.5 current. 4-AP also enhanced native mesenteric artery myocyte K + current at sub-mmol concentrations. When applied to NA-preconstricted mesenteric artery segments, 4-AP hyperpolarized the membrane, decreased [Ca 2+ ] i and caused concentration-dependent relaxations that were independent of 4-AP-mediated changes in intracellular pH. Application of the K v 7 channel blocker XE991 and BK Ca channel blocker iberiotoxin attenuated 4-AP-mediated relaxation. 4-AP also inhibited the NA-mediated signal transduction to elicit a relaxation., Conclusions and Implications: These data show that 4-AP is able to relax NA-preconstricted rat mesenteric arteries by enhancing the activity of K v 7.4 and BK Ca channels and attenuating NA-mediated signalling., (© 2017 The British Pharmacological Society.)

Background and Purpose: Small conductance calcium-activated potassium (K Ca 2) channels represent a promising atrial-selective target for treatment of atrial fibrillation. Here, we establish the mechanism of K Ca 2 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 K Ca 2 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 K Ca 2.2 and K Ca 2.3 channels (IC 50  = 1.1 ± 0.3 μM). The presence of AP14145 (10 μM) increased the EC 50 of Ca 2+ on K Ca 2.3 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 K Ca 2.2 and K Ca 2.3 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 K Ca 2 channels are inhibited, at the molecular level, will help further development of drugs targeting K Ca 2 channels., (© 2017 The British Pharmacological Society.)

Background: Evidence has emerged that small-conductance Ca 2+ -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., (© 2017 The Authors.)

Background: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Valvular heart disease is a strong predictor, yet the underlying molecular mechanisms are unknown., Objective: The purpose of this study was to investigate the prevalence of somatic variants in AF candidate genes in an AF patient population undergoing surgery for mitral valve regurgitation (MVR) to determine whether these patients are genetically predisposed to AF., Methods: DNA was extracted from blood and left atrial tissue from 44 AF patients with MVR. Using next-generation sequencing, we investigated 110 genes using the HaloPlex Target Enrichment System. MuTect software was used for identification of somatic point variants. We functionally characterized selected variants using electrophysiologic techniques., Results: No somatic variants were identified in the cardiac tissue. Thirty-three patients (75%) had a rare germline variation in ≥1 candidate genes. Fourteen variants were novel. Fifteen variants were predicted damaging or likely damaging in ≥6 in silico predictions. We identified rare variants in genes never directly associated with AF: KCNE4, SCN4B, NEURL1, and CAND2. Interestingly, 7 patients (16%) had variants in genes involved in cellular potassium handling. The variants KCNQ1 (p.G272S) and KCNH2 (p.A913V) resulted in gain of function due to faster activation (KCNQ1) and slowed deactivation kinetics (KCNQ1, KCNH2)., Conclusion: We did not find any somatic variants in patients with AF and MVR. Surprisingly, we found that our cohort of non-lone AF patients might, like lone AF patients, be predisposed to AF by rare germline variants. Our findings emphasize the extent of still unknown factors in the pathogenesis of AF., (Copyright © 2017 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

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 (K Ca 2.X, SK channels). In the heart, K Ca 2.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 K Ca 2.X channels is unknown. To this end, we tested a large number of AADs on the human K Ca 2.2 and K Ca 2.3 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 hK Ca 2.X channels, with no subtype selectivity. The calculated IC 50 were 90±10µmol/l vs 60±10µmol/l for dofetilide and 42±4µmol/l vs 80±20µmol/l for propafenone (hK Ca 2.3 vs hK Ca 2.2). Whether this inhibition has clinical importance for their antiarrhythmic effect is unlikely, as the calculated IC 50 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

About 300 loss-of-function mutations in the I Ks channel have been identified in patients with Long QT syndrome and cardiac arrhythmia. How specific mutations cause arrhythmia is largely unknown and there are no approved I Ks channel activators for treatment of these arrhythmias. We find that several Long QT syndrome-associated I Ks channel mutations shift channel voltage dependence and accelerate channel closing. Voltage-clamp fluorometry experiments and kinetic modeling suggest that similar mutation-induced alterations in I Ks channel currents may be caused by different molecular mechanisms. Finally, we find that the fatty acid analogue N-arachidonoyl taurine restores channel gating of many different mutant channels, even though the mutations are in different domains of the I Ks channel and affect the channel by different molecular mechanisms. N-arachidonoyl taurine is therefore an interesting prototype compound that may inspire development of future I Ks channel activators to treat Long QT syndrome caused by diverse I Ks channel mutations., Competing Interests: SIL: A patent application (62/032,739) based on these results has been submitted by the University of Miami with SIL and HPL identified as inventors. HPL: A patent application (62/032,739) based on these results has been submitted by the University of Miami with SIL and HPL identified as inventors. The other authors declare that no competing interests exist.

Aim: Polyunsaturated fatty acids have been reported to reduce neuronal excitability, in part by promoting inactivation of voltage-gated sodium and calcium channels. Effects on neuronal potassium channels are less explored and experimental data ambiguous. The aim of this study was to investigate anti-excitable effects of polyunsaturated fatty acids on the neuronal M-channel, important for setting the resting membrane potential in hippocampal and dorsal root ganglion neurones., Methods: Effects of fatty acids and fatty acid analogues on mouse dorsal root ganglion neurones and on the human KV 7.2/3 channel expressed in Xenopus laevis oocytes were studied using electrophysiology., Results: Extracellular application of physiologically relevant concentrations of the polyunsaturated fatty acid docosahexaenoic acid hyperpolarized the resting membrane potential (-2.4 mV by 30 μm) and increased the threshold current to evoke action potentials in dorsal root ganglion neurones. The polyunsaturated fatty acids docosahexaenoic acid, α-linolenic acid and eicosapentaenoic acid facilitated opening of the human M-channel, comprised of the heteromeric human KV 7.2/3 channel expressed in Xenopus oocytes, by shifting the conductance-vs.-voltage curve towards more negative voltages (by -7.4 to -11.3 mV by 70 μm). Uncharged docosahexaenoic acid methyl ester and monounsaturated oleic acid did not facilitate opening of the human KV 7.2/3 channel., Conclusions: These findings suggest that circulating polyunsaturated fatty acids, with a minimum requirement of multiple double bonds and a charged carboxyl group, dampen excitability by opening neuronal M-channels. Collectively, our data bring light to the molecular targets of polyunsaturated fatty acids and thus a possible mechanism by which polyunsaturated fatty acids reduce neuronal excitability., (© 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

In isolated human atrial cardiomyocytes, inhibition of K2P3.1 K(+) channels results in action potential (action potential duration (APD)) prolongation. It has therefore been postulated that K2P3.1 (KCNK3), together with K2P9.1 (KCNK9), could represent novel drug targets for the treatment of atrial fibrillation (AF). However, it is unknown whether these findings in isolated cells translate to the whole heart. The purposes of this study were to investigate the expression levels of KCNK3 and KCNK9 in human hearts and two relevant rodent models and determine the antiarrhythmic potential of K2P3.1 inhibition in isolated whole-heart preparations. By quantitative PCR, we found that KCNK3 is predominantly expressed in human atria whereas KCNK9 was not detectable in heart human tissue. No differences were found between patients in AF or sinus rhythm. The expression in guinea pig heart resembled humans whereas rats displayed a more uniform expression of KCNK3 between atria and ventricle. In voltage-clamp experiments, ML365 and A293 were found to be potent and selective inhibitors of K2P3.1, but at pH 7.4, they failed to prolong atrial APD and refractory period (effective refractory period (ERP)) in isolated perfused rat and guinea pig hearts. At pH 7.8, which augments K2P3.1 currents, pharmacological channel inhibition produced a significant prolongation of atrial ERP (11.6 %, p = 0.004) without prolonging ventricular APD but did not display a significant antiarrhythmic effect in our guinea pig AF model (3/8 hearts converted on A293 vs 0/7 hearts in time-matched controls). These results suggest that when K2P3.1 current is augmented, K2P3.1 inhibition leads to atrial-specific prolongation of ERP; however, this ERP prolongation did not translate into significant antiarrhythmic effects in our AF model.

The inwardly rectifying potassium current (IK 1) conducted through Kir2.X channels contribute to repolarization of the cardiac action potential and to stabilization of the resting membrane potential in cardiomyocytes. Our aim was to investigate the effect of the recently discovered IK 1 inhibitor PA-6 on action potential repolarization and refractoriness in isolated rat hearts. Transiently transfected HEK-293 cells expressing IK 1 were voltage-clamped with ramp protocols. Langendorff-perfused heart experiments were performed on male Sprague-Dawley rats, effective refractory period, Wenckebach cycle length, and ventricular effective refractory period were determined following 200 nmol/L PA-6 perfusion. 200 nmol/L PA-6 resulted in a significant time-latency in drug effect on the IK 1 current expressed in HEK-293 cells, giving rise to a maximal effect at 20 min. In the Langendorff-perfused heart experiments, PA-6 prolonged the ventricular action potential duration at 90% repolarization (from 41.8 ± 6.5 msec to 72.6 ± 21.1 msec, 74% compared to baseline, P < 0.01, n = 6). In parallel, PA-6 significantly prolonged the ventricular effective refractory period compared to baseline (from 34.8 ± 4.6 msec to 58.1 ± 14.7 msec, 67%, P < 0.01, n = 6). PA-6 increased the short-term beat-to-beat variability and ventricular fibrillation was observed in two of six hearts. Neither atrial ERP nor duration of atrial fibrillation was altered following PA-6 application. The results show that pharmacological inhibition of cardiac IK 1 affects ventricular action potential repolarization and refractoriness and increases the risk of ventricular arrhythmia in isolated rat hearts., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Small-conductance Ca(2+)-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.