Inhibition Of Potassium Channels Research Articles

First-in-Human Study to Assess the Safety, Pharmacokinetics, and Pharmacodynamics of BMS-986308: A Renal Outer Medullary Potassium Channel Inhibitor.

In patients with heart failure (HF) who respond inadequately to loop diuretic therapy, BMS-986308, an oral, selective, reversible renal outer medullary potassium channel (ROMK) inhibitor may represent an effective diuretic with a novel mechanism of action. We present data from the first-in-human study aimed to assess the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) following single ascending doses of BMS-986308 in healthy adult participants. Forty healthy participants, aged from 20 to 55 years, and body mass index (BMI) from 19.8 to 31.6 kg/m2 were assigned to 1 of 5 dose cohorts (1, 3, 10, 30, and 100 mg) and randomized (6:2) to receive BMS-986308 oral solution or matching placebo. Following administration, BMS-986308 was rapidly absorbed with a median time to maximum concentration (Tmax) of 1.00 to 1.75 h and exhibiting a mean terminal half-life (t1/2) of approximately 13 h. Dose proportionality was evident in BMS-986308 area under the concentration-time curve (AUC), while maximum concentration (Cmax) was slightly greater than dose-proportional. We observed that urine output (or diuresis; mL) and urinary sodium excretion (or natriuresis; mmol) increased in a dose-dependent manner, starting at a minimum pharmacologically active dose of 30 mg. The largest mean changes from baseline in diuresis and natriuresis occurred in both the 6- and -24 h post-dose period following administration of 100 mg (1683.0 mL and 2055.3 mL, and 231.7 mmol and 213.7 mmol, respectively; ***P < 0.001). Overall, single-dose BMS-986308 was found to be safe, well-tolerated, with an excellent PK profile, and substantial diuretic and natriuretic activity.

Tarantula toxin inhibits potassium channels by the pore occlusion

Tarantula toxin inhibits potassium channels by the pore occlusion

Diversely evolved xibalbin variants from remipede venom inhibit potassium channels and activate PKA-II and Erk1/2 signaling

BackgroundThe identification of novel toxins from overlooked and taxonomically exceptional species bears potential for various pharmacological applications. The remipede Xibalbanus tulumensis, an underwater cave-dwelling crustacean, is the only crustacean for which a venom system has been described. Its venom contains several xibalbin peptides that have an inhibitor cysteine knot (ICK) scaffold.ResultsOur screenings revealed that all tested xibalbin variants particularly inhibit potassium channels. Xib1 and xib13 with their eight-cysteine domain similar to spider knottins also inhibit voltage-gated sodium channels. No activity was noted on calcium channels. Expanding the functional testing, we demonstrate that xib1 and xib13 increase PKA-II and Erk1/2 sensitization signaling in nociceptive neurons, which may initiate pain sensitization. Our phylogenetic analysis suggests that xib13 either originates from the common ancestor of pancrustaceans or earlier while xib1 is more restricted to remipedes. The ten-cysteine scaffolded xib2 emerged from xib1, a result that is supported by our phylogenetic and machine learning-based analyses.ConclusionsOur functional characterization of synthesized variants of xib1, xib2, and xib13 elucidates their potential as inhibitors of potassium channels in mammalian systems. The specific interaction of xib2 with Kv1.6 channels, which are relevant to treating variants of epilepsy, shows potential for further studies. At higher concentrations, xib1 and xib13 activate the kinases PKA-II and ERK1/2 in mammalian sensory neurons, suggesting pain sensitization and potential applications related to pain research and therapy. While tested insect channels suggest that all probably act as neurotoxins, the biological function of xib1, xib2, and xib13 requires further elucidation. A novel finding on their evolutionary origin is the apparent emergence of X. tulumensis-specific xib2 from xib1. Our study is an important cornerstone for future studies to untangle the origin and function of these enigmatic proteins as important components of remipede but also other pancrustacean and arthropod venoms.

Insights into the antifungal mechanism of Bacillus subtilis cyclic lipopeptide iturin A mediated by potassium ion channel

Fungal infections pose severe and potentially lethal threats to plant, animal, and human health. Ergosterol has served as the primary target for developing antifungal medications. However, many antifungal drugs remain highly toxic to humans due to similarity in cell membrane composition between fungal and animal cells. Iturin A, lipopeptide produced by Bacillus subtilis, efficiently inhibit various fungi, but demonstrated safety in oral administration, indicating the existence of targets different from ergosterol. To pinpoint the exact antifungal target of iturin A, we used homologous recombination to knock out and overexpress erg3, a key gene in ergosterol synthesis. Saccharomyces cerevisiae and Aspergillus carbonarius were transformed using the LiAc/SS-DNNPEG and Agrobacterium-mediated transformation (AMT), respectively. Surprisingly, increasing ergosterol content did not augment antifungal activity. Furthermore, iturin A's antifungal activity against S. cerevisiae was reduced while it pre-incubation with voltage-gated potassium (Kv) channel inhibitor, indicating that Kv activation was responsible for cell death. Iturin A was found to activate the Kv protein, stimulating K+ efflux from cell. In vitro tests confirmed interaction between iturin A and Kv protein. This study highlights Kv as one of the precise targets of iturin A in its antifungal activity, offering a novel target for the development of antifungal medications.

Discovery of BMS-986308: A Renal Outer Medullary Potassium Channel Inhibitor for the Treatment of Heart Failure.

Recent literature reports highlight the importance of the renal outer medullary potassium (ROMK) channel in renal sodium and potassium homeostasis and emphasize the potential impact that ROMK inhibitors could have as a novel mechanism diuretic in heart failure patients. A series of piperazine-based ROMK inhibitors were designed and optimized to achieve excellent ROMK potency, hERG selectivity, and ADME properties, which led to the identification of compound 28 (BMS-986308). BMS-986308 demonstrated efficacy in the volume-loaded rat diuresis model as well as promising in vitro and in vivo profiles and was therefore advanced to clinical development.

Structure-Activity Relationship Studies in a Series of Xanthine Inhibitors of SLACK Potassium Channels.

Gain-of-function mutations in the KCNT1 gene, which encodes the sodium-activated potassium channel known as SLACK, are associated with the rare but devastating developmental and epileptic encephalopathy known as epilepsy of infancy with migrating focal seizures (EIMFS). The design of small molecule inhibitors of SLACK channels represents a potential therapeutic approach to the treatment of EIMFS, other childhood epilepsies, and developmental disorders. Herein, we describe a hit optimization effort centered on a xanthine SLACK inhibitor (8) discovered via a high-throughput screen. Across three distinct regions of the chemotype, we synthesized 58 new analogs and tested each one in a whole-cell automated patch-clamp assay to develop structure-activity relationships for inhibition of SLACK channels. We further evaluated selected analogs for their selectivity versus a variety of other ion channels and for their activity versus clinically relevant SLACK mutants. Selectivity within the series was quite good, including versus hERG. Analog 80 (VU0948578) was a potent inhibitor of WT, A934T, and G288S SLACK, with IC50 values between 0.59 and 0.71 µM across these variants. VU0948578 represents a useful in vitro tool compound from a chemotype that is distinct from previously reported small molecule inhibitors of SLACK channels.

ML365 ameliorates postoperative cognitive impairment in aged mice by inhibiting NLRP3 inflammasome activation in the hippocampus

The aim of this study was to examine the effects of ML365, a two-pore potassium channel (K2P) inhibitor, on postoperative cognitive impairment (POCD). A mouse model of POCD was constructed by subjecting aged C57BL/6 mice to exploratory laparotomy. Changes in cognitive function were assessed using the Morris water maze test. Western blotting and qPCR were used to detect hippocampal NLRP3, Caspase-1 and IL-1β expression levels on days 3 and 7 post-surgery. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) expression level was also assessed by western blotting. Pathological changes and nerve damage in the hippocampal CA1 and CA3 regions were detected by H&E staining, while the concentration of malondialdehyde (MDA) in the plasma was measured. We found that pretreatment with ML365 (administered intraperitoneally at a dose of 10 mg/kg) 30 min prior to exploratory laparotomy effectively ameliorated POCD in mice. ML365 pretreatment also reduced NLRP3, Caspase-1, ASC and IL-1β expression levels in the hippocampus, improved POCD-induced pathological changes in the hippocampal CA1 and CA3 areas of aged mice, and decreased levels of plasma MDA and oxidative stress. Together, our findings indicate that ML365 can alleviate POCD in mice by inhibiting NLRP3 inflammasome activation in the hippocampus.

Inhibition of voltage-gated potassium channel by aripiprazole in rabbit coronary arterial smooth muscle cells

Aripiprazole, a third-generation antipsychotic, has been widely used to treat schizophrenia. In this study, we evaluated the effect of aripiprazole on voltage-gated potassium (Kv) channels in rabbit coronary arterial smooth muscle cells using the patch clamp technique. Aripiprazole reduced the Kv current in a concentration-dependent manner with a half-maximal inhibitory concentration of 0.89 ± 0.20 μM and a Hill coefficient of 1.30 ± 0.25. The inhibitory effect of aripiprazole on Kv channels was voltage-dependent, and an additional aripiprazole-induced decrease in the Kv current was observed in the voltage range of full channel activation. The decay rate of Kv channel inactivation was accelerated by aripiprazole. Aripiprazole shifted the steady-state activation curve to the right and the inactivation curve to the left. Application of a repetitive train of pulses (1 and 2 Hz) promoted inhibition of the Kv current by aripiprazole. Furthermore, the recovery time constant from inactivation increased in the presence of aripiprazole. Pretreatment of Kv1.5 subtype inhibitor reduced the inhibitory effect of aripiprazole. However, pretreatment with Kv 7 and Kv2.1 subtype inhibitors did not change the degree of aripiprazole-induced inhibition of the Kv current. We conclude that aripiprazole inhibits Kv channels in a concentration-, voltage-, time-, and use (state)-dependent manner by affecting the gating properties of the channels.

Abstract 7121: The novel WEE1i, APR-1051, is a potentially well tolerated and effective treatment for cyclin E-overexpressing cancers

Abstract Previous studies have demonstrated that WEE1 inhibitors (WEE1i) are a promising cancer therapeutic through synthetic lethality with Cyclin E overexpression. One challenge for two of the leading WEE1i, AZD1775 and ZN-c3, has been the observed off-targeting of PLK family members, namely, PLK1, PLK2 and PLK3. In addition, the therapeutic application of these inhibitors in clinical trials has been limited by adverse hematological effects, including anemia. Herein, we describe the testing of a novel WEE1i (APR-1051) that, based on preclinical studies: 1) demonstrates reduced off-targeting of PLK1, PLK2 and PLK3, and 2) has the ability to suppress the growth of CCNE1-overexpressing xenografted tumors while causing little impact on red blood cell and reticulocyte levels. APR-1051 has an IC50 of 2.2 nM for WEE1 in vitro and has shown the ability to limit the proliferation of various cancer cell lines in culture. Interestingly, the genotoxicity of APR-1051 is suppressed by increasing concentrations of PLK1 inhibitors, suggesting that off-targeting of PLK1 by other WEE1i may compromise efficacy of this targeted cancer treatment in addition to the risk of producing PLK1i-associated adverse effects. APR-1051 demonstrated effectiveness in suppressing the growth of Cyclin E-overexpressing breast and ovarian cancer cells lines, thus pinpointing Cyclin E as a potential biomarker for APR-1051 treatment. Importantly, dose and scheduling of APR-1051 that causes significant suppression of CCNE1-amplified high-grade serious ovarian cancer tumors in mice is well tolerated, with red blood cell and platelet counts remaining within non-pathogenic ranges after a 28-day treatment period. In addition, inhibition WEE1 by APR-1051 occurs at an IC50 that is 200-fold lower on average than the IC50 of hERG potassium channel inhibition, implying that APR-1051 will potentially exhibit low cardiotoxicity. APR-1051 is now progressing through IND-enabling studies. Together, these findings underscore the potential of APR-1051 as a novel WEE1i for the treatment of Cyclin E-overexpressing cancers. Citation Format: Molly M. Hansbarger, Stephen J. Rocca, Sonia Ritzmann, Joseph Vacca, Bridget Tokiwa, Jonathan Weinstein, Henry Zabierek, Justin Frye, Michael Carleton, Eric J. Brown, Oren Gilad. The novel WEE1i, APR-1051, is a potentially well tolerated and effective treatment for cyclin E-overexpressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7121.

Cardiotoxicity Assessment through a Polymer-Based Cantilever Platform: An Integrated Electro-Mechanical Screening Approach.

Preclinical drug screening for cardiac toxicity has traditionally relied on observing changes in cardiomyocytes' electrical activity, primarily through invasive patch clamp techniques or non-invasive microelectrode arrays (MEA). However, relying solely on field potential duration (FPD) measurements for electrophysiological assessment can miss the full spectrum of drug-induced toxicity, as different drugs affect cardiomyocytes through various mechanisms. A more comprehensive approach, combining field potential and contractility measurements, is essential for accurate toxicity profiling, particularly for drugs targeting contractile proteins without affecting electrophysiology. However, previously proposed platform has significant limitations in terms of simultaneous measurement. The novel platform addresses these issues, offering enhanced, non-invasive evaluation of drug-induced cardiotoxicity. It features eight cantilevers with patterned strain sensors and MEA, enabling real-time monitoring of both cardiomyocyte contraction force and field potential. This system can detect minimum cardiac contraction force of ≈2 µN and field potential signals with 50µm MEA diameter, using the same cardiomyocytes in measurements of two parameters. Testing with six drugs of varied mechanisms of action, the platform successfully identifies these mechanisms and accurately assesses toxicity profiles, including drugs not inhibiting potassium channels. This innovative approach presents a comprehensive, non-invasive method for cardiac function assessment, poised to revolutionize preclinical cardiotoxicity screening.

Unveiling the antinociceptive mechanisms of Methyl-2-(4-chloro-phenyl)-5-benzoxazoleacetate: insights from nociceptive assays in mice.

Methyl-2-(4-chloro- phenyl)-5-benzoxazoleacetate (MCBA), a synthetic benzoxazole derivative with established antipsoriatic efficacy, was investigated for potential antinociceptive effects. This study employs various nociceptive assays in mice to elucidate MCBA's antinociceptive mechanisms. MCBA's antinociceptive potential was tested against various nociception models induced by formalin, glutamate, capsaicin, a transient receptor potential vanilloid 1 (TRPV1) receptor agonist, and phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator. It was then assessed using the hot plate test and examined within the acetic acid-induced writhing test. During the acetic acid-induced writhing test, MCBA was pre-challenged against selective receptor antagonists such as naloxone, caffeine, atropine, yohimbine, ondansetron, and haloperidol. It was also pre-challenged with ATP-sensitive potassium channel inhibitor (glibenclamide) to further elucidate its antinociceptive mechanism. The results showed that oral administration of MCBA led to a dose-dependent and significant inhibition (p < 0.05) of nociceptive effects across all evaluated models at doses of 60, 120, and 240 mg/kg. Moreover, the efficacy of MCBA's antinociceptive potential was significantly counteracted (p < 0.0001) by specific antagonists: (i) directed at adenosinergic, alpha-2 adrenergic, and cholinergic receptors using caffeine, yohimbine, and atropine, respectively; and (ii) targeting ATP-sensitive potassium channels, employing glibenclamide. Antagonists aimed at opioidergic and serotoninergic receptors (naloxone and ondansetron, respectively) had poor utility in inhibiting antinociceptive activity. Conversely, the dopaminergic receptor antagonist haloperidol potentiated locomotor abnormalities associated with MCBA treatment. MCBA-induced antinociception involves modulation of glutamatergic-, TRVP1 receptors- and PKC-signaling pathways. It impacts adenosinergic, alpha-2 adrenergic, and cholinergic receptors and opens ATP-sensitive potassium channels.

Sensitive Detection of Ciguatoxins Using a Neuroblastoma Cell-Based Assay with Voltage-Gated Potassium Channel Inhibitors.

Ciguatoxins (CTXs) are neurotoxins responsible for ciguatera poisoning (CP), which affects more than 50,000 people worldwide annually. The development of analytical methods to prevent CP is a pressing global issue, and the N2a assay is one of the most promising methods for detecting CTXs. CTXs are highly toxic, and an action level of 0.01 μg CTX1B equivalent (eq)/kg in fish has been proposed. It is desirable to further increase the detection sensitivity of CTXs in the N2a assay to detect such low concentrations reliably. The opening of voltage-gated sodium channels (NaV channels) and blocking of voltage-gated potassium channels (KV channels) are thought to be involved in the toxicity of CTXs. Therefore, in this study, we developed an assay that could detect CTXs with higher sensitivity than conventional N2a assays, using KV channel inhibitors as sensitizing reagents for N2a cells. The addition of the KV channel inhibitors 4-aminopyridine and tetraethylammonium chloride to N2a cells, in addition to the traditional sensitizing reagents ouabain and veratridine, increased the sensitivity of N2a cells to CTXs by up to approximately 4-fold. This is also the first study to demonstrate the influence of KV channels on the toxicity of CTXs in a cell-based assay.

HRAS-Mutant Cardiomyocyte Model of Multifocal Atrial Tachycardia.

Germline HRAS gain-of-function pathogenic variants cause Costello syndrome (CS). During early childhood, 50% of patients develop multifocal atrial tachycardia, a treatment-resistant tachyarrhythmia of unknown pathogenesis. This study investigated how overactive HRAS activity triggers arrhythmogenesis in atrial-like cardiomyocytes (ACMs) derived from human-induced pluripotent stem cells bearing CS-associated HRAS variants. HRAS Gly12 mutations were introduced into a human-induced pluripotent stem cells-ACM reporter line. Human-induced pluripotent stem cells were generated from patients with CS exhibiting tachyarrhythmia. Calcium transients and action potentials were assessed in induced pluripotent stem cell-derived ACMs. Automated patch clamping assessed funny currents. HCN inhibitors targeted pacemaker-like activity in mutant ACMs. Transcriptomic data were analyzed via differential gene expression and gene ontology. Immunoblotting evaluated protein expression associated with calcium handling and pacemaker-nodal expression. ACMs harboring HRAS variants displayed higher beating rates compared with healthy controls. The hyperpolarization activated cyclic nucleotide gated potassium channel inhibitor ivabradine and the Nav1.5 blocker flecainide significantly decreased beating rates in mutant ACMs, whereas voltage-gated calcium channel 1.2 blocker verapamil attenuated their irregularity. Electrophysiological assessment revealed an increased number of pacemaker-like cells with elevated funny current densities among mutant ACMs. Mutant ACMs demonstrated elevated gene expression (ie, ISL1, TBX3, TBX18) related to intracellular calcium homeostasis, heart rate, RAS signaling, and induction of pacemaker-nodal-like transcriptional programming. Immunoblotting confirmed increased protein levels for genes of interest and suppressed MAPK (mitogen-activated protein kinase) activity in mutant ACMs. CS-associated gain-of-function HRASG12 mutations in induced pluripotent stem cells-derived ACMs trigger transcriptional changes associated with enhanced automaticity and arrhythmic activity consistent with multifocal atrial tachycardia. This is the first human-induced pluripotent stem cell model establishing the mechanistic basis for multifocal atrial tachycardia in CS.

In Silico Human Cardiomyocyte Action Potential Modeling: Exploring Ion Channel Input Combinations.

In silico modeling offers an opportunity to supplement and accelerate cardiac safety testing. With in silico modeling, computational simulation methods are used to predict electrophysiological interactions and pharmacological effects of novel drugs on critical physiological processes. The O'Hara-Rudy's model was developed to predict the response to different ion channel inhibition levels on cardiac action potential duration (APD) which is known to directly correlate with the QT interval. APD data at 30% 60% and 90% inhibition were derived from the model to delineate possible ventricular arrhythmia scenarios and the marginal contribution of each ion channel to the model. Action potential values were calculated for epicardial, myocardial, and endocardial cells, with action potential curve modeling. This study assessed cardiac ion channel inhibition data combinations to consider when undertaking in silico modeling of proarrhythmic effects as stipulated in the Comprehensive in Vitro Proarrhythmia Assay (CiPA). As expected, our data highlight the importance of the delayed rectifier potassium channel (IKr) as the most impactful channel for APD prolongation. The impact of the transient outward potassium channel (Ito) inhibition on APD was minimal while the inward rectifier (IK1) and slow component of the delayed rectifier potassium channel (IKs) also had limited APD effects. In contrast, the contribution of fast sodium channel (INa) and/or L-type calcium channel (ICa) inhibition resulted in substantial APD alterations supporting the pharmacological relevance of in silico modeling using input from a limited number of cardiac ion channels including IKr, INa, and ICa, at least at an early stage of drug development.

Analysis of drug-induced and spontaneous cardioversions reveals similar patterns leading to termination of atrial fibrillation.

The mechanisms leading to the conversion of atrial fibrillation (AF) to sinus rhythm are poorly understood. This study describes the dynamic behavior of electrophysiological parameters and conduction patterns leading to spontaneous and pharmacological AF termination. Five independent groups of goats were investigated: (1) spontaneous termination of AF, and drug-induced terminations of AF by various potassium channel inhibitors: (2) AP14145, (3) PA-6, (4) XAF-1407, and (5) vernakalant. Bi-atrial contact mapping was performed during an open chest surgery and intervals with continuous and discrete atrial activity were determined. AF cycle length (AFCL), conduction velocity and path length were calculated for each interval, and the final conduction pattern preceding AF termination was evaluated. AF termination was preceded by a sudden episode of discrete activity both in the presence and absence of an antiarrhythmic drug. This episode was accompanied by substantial increases in AFCL and conduction velocity, resulting in prolongation of path length. In 77% ± 4% of all terminations the conduction pattern preceding AF termination involved medial to lateral conduction along Bachmann's bundle into both atria, followed by anterior to posterior conduction. This finding suggests conduction block in the interatrial septum and/or pulmonary vein area as final step of AF termination. AF termination is preceded by an increased organization of fibrillatory conduction. The termination itself is a sudden process with a critical role for the interplay between spatiotemporal organization and anatomical structure.

The adhesion G-protein-coupled receptor mayo/CG11318 controls midgut development in Drosophila

Adhesion G-protein-coupled receptors (aGPCRs) form a large family of cell surface molecules with versatile tasks in organ development. Many aGPCRs still await their functional and pharmacological deorphanization. Here, we characterized the orphan aGPCR CG11318/mayo of Drosophila melanogaster and found it expressed in specific regions of the gastrointestinal canal and anal plates, epithelial specializations that control ion homeostasis. Genetic removal of mayo results in tachycardia, which is caused by hyperkalemia of the larval hemolymph. The hyperkalemic effect can be mimicked by a raise in ambient potassium concentration, while normal potassium levels in mayoKO mutants can be restored by pharmacological inhibition of potassium channels. Intriguingly, hyperkalemia and tachycardia are caused non-cell autonomously through mayo-dependent control of enterocyte proliferation in the larval midgut, which is the primary function of this aGPCR. These findings characterize the ancestral aGPCR Mayo as a homeostatic regulator of gut development.

Inhibition of the KCa2 potassium channel in atrial fibrillation: a randomized phase 2 trial

Existing antiarrhythmic drugs to treat atrial fibrillation (AF) have incomplete efficacy, contraindications and adverse effects, including proarrhythmia. AP30663, an inhibitor of the KCa2 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. KCa2 channel inhibition may be an attractive mechanism for rhythm control of AF that should be studied further in randomized trials. ClinicalTrials.gov registration: NCT04571385.

Artemisinin combined with low dose quinidine suppresses ventricular tachyarrhythmia in a model of Brugada syndrome

Abstract Background The mechanism of Brugada syndrome (BrS) is unclear. One of the mechanisms has been suggested that uneven dispersion of Ito channel between epicardium and endocardium induced ventricular arrhythmia. Artemisinin, which originates from a Chinese herb and is used widely as an antimalarial drug, is reported to inhibit the Ito channel in a canine experimental model previously. Objective We aimed to investigate the effect of artemisinin, alone and in combination with low dose quinidine, on the suppression of ventricular tachyarrhythmia in BrS in a canine experimental model. Methods We recorded transmural pseudo-ECG and epicardial/endocardial action potentials (APs) from coronary-perfused canine right ventricular wedge preparation. We used acetylcholine (3 μM), calcium channel blocker verapamil (1 μM), and Ito agonist NS5806 (6 – 10 μM) to mimic the BrS model. We induced ventricular arrhythmia and then perfused low dose quinidine (1 – 2 μM) alone and additionally perfused artemisinin (100 – 150 μM) to suppress the arrhythmia. We evaluated ventricular arrhythmia inducibility and measured AP duration, J wave area, notch index, and T wave dispersion. Results The provocation agents induced prominent J wave in all sample models. In 6 sample models out of 8 for artemisinin alone group, ventricular arrhythmia was induced. Artemisinin suppressed ventricular arrhythmia and recovered the AP dome in all preparations. J wave area and epicardial notch index were also decreased after artemisinin perfusion. The low dose quinidine (1 – 2 μM) did not completely suppressed the ventricular arrhythmia in 6 out of 6 sample models. However, additional artemisinin (100 – 200 μM) infusion suppressed the ventricular arrhythmia in all sample models and restored the AP dome. J wave area and epicardial notch index were also decreased. Conclusions Our findings suggest that artemisinin can alleviate ventricular arrhythmia in the BrS wedge preparation model. Combining artemisinin with low dose quinidine may further suppress ventricular arrhythmia by inhibiting potassium channels, including Ito channel. Artemisinin might reduce the therapeutic dose of quinidine by additional inhibition of potassium channels.

Unravelling the signaling pathway downstream of the neurokinin receptor in atrium: discovering novel anti-arrhythmic targets for treatment of atrial fibrillation

Abstract Background Stimulation of neurokinin receptor (NKR) 3 is anti-fibrillatory by prolonging the action potential duration (APD) of atrial cardiomyocytes via inhibition of a background potassium current (1). The NKR is a Gq/11 protein-coupled receptor, but the downstream intracellular pathway and the end-effector potassium (K+)-channel in atrium are unknown. We aimed at unravelling the components of this pathway in atrial cardiomyocytes. Methods Rabbit (New Zealand White) atrial cardiomyocytes were isolated from Langendorff-perfused hearts by enzymatic dissociation. Human immortalized atrial myocytes (HiAMs) were developed, and kindly provided by our collaborators (2). Adult human atrial cardiomyocytes were obtained by enzymatic dissociation from left atrial appendages (LAA) routinely removed from atrial fibrillation (AF) patients undergoing minimally invasive surgical pulmonary vein isolation in the clinics. Action potentials (APs) and K+ currents were recorded at 36.5°C with the amphotericin-B-perforated patch clamp technique. RNA was isolated from rabbit atrial tissue and sequenced on an Illumina HiSeq4000 platform. Results In rabbit atrial cardiomyocytes, stimulation of NKR3 prolonged APD by ∼50% and inhibited an background outward K+-current by ∼45%. Inhibition of phosphokinase C (PKC) did not prevent APD prolongation by NKR3 stimulation, but application of a diacylglycerol (DAG) analog inhibited an outward K+-current by ∼40% indicating that DAG, but not PKC, is downstream of NKR3. Inhibition of phosphatidylcholine-specific phospholipase C (PC-PLC), but not of phosphoinositide-specific PLC (PI-PLC), reduced APD increase by NKR3 stimulation by five-fold, indicating PC-PLC as the intracellular signaling molecule. In HiAMs, direct PLC activation prolonged APD by 34%. RNA sequencing analysis revealed potential end-targets of the NKR3 pathway in rabbit. Inhibition of small conductance potassium (KCNN2/KCNN3) or TASK-1 (KCNK3) channels (prolonging APD by ∼15%) did not abolish the APD prolongation effect by subsequent NKR3 stimulation (∼50%), indicating that these channels are not the end-effector K+-channels inhibited by NKR3 stimulation. Inhibition of constitutive KACh channels had little effect on APD at baseline. Nevertheless, APD shortening by agonist-induced KACh channel activity could be completely reversed by NKR3 stimulation, indicating that increased outward KACh current can be fully counterbalanced by the reduction in K+ background current. In human atrial cardiomyocytes from AF patients, stimulation of NKR3 prolonged APD by 25%, Conclusions PC-PLC and DAG, but not PI-PLC and PKC, are intracellularly downstream of NKR3 in atrial rabbit cardiomyocytes. Stimulation of the NKR pathway may have an anti-arrhythmic potential in patients with AF.

Proteasome inhibition potentiates Kv1.3 potassium channel expression as therapeutic target in drug-sensitive and -resistant human melanoma cells

Primary and acquired therapy resistance is a major problem in patients with BRAF-mutant melanomas being treated with BRAF and MEK inhibitors (BRAFI, MEKi). Therefore, development of alternative therapy regimes is still required. In this regard, new drug combinations targeting different pathways to induce apoptosis could offer promising alternative approaches. Here, we investigated the combination of proteasome and Kv1.3 potassium channel inhibition on chemo-resistant, BRAF inhibitor-resistant as well as sensitive human melanoma cells. Our experiments demonstrated that all analyzed melanoma cell lines were sensitive to proteasome inhibitor treatment at concentrations that are not toxic to primary human fibroblasts. To further reduce proteasome inhibitor-associated side effects, and to foster apoptosis, potassium channels, which are other targets to induce pro-apoptotic effects in cancer cells, were blocked. In support, combined exposure of melanoma cells to proteasome and Kv1.3 channel inhibitor resulted in synergistic effects and significantly reduced cell viability. On the molecular level, enhanced apoptosis correlated with an increase of intracellular Kv1.3 channels and pro-apoptotic proteins such as Noxa and Bak and a reduction of anti-apoptotic proteins. Thus, use of combined therapeutic strategies triggering different apoptotic pathways may efficiently prevent the outgrowth of drug-resistant and -sensitive BRAF-mutant melanoma cells. In addition, this could be the basis for an alternative approach to treat other tumors expressing mutated BRAF such as non-small-cell lung cancer.