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BACKGROUND AND PURPOSE Drug development requires the testing of new chemical entities for adverse effects. For cardiac safety screening, improved assays are urgently needed. Isolated adult cardiomyocytes (CM) and human embryonic stem cell-derived cardiomyocytes (hESC-CM) could be used to identify pro-arrhythmic compounds. In the present study, five assays were employed to investigate their sensitivity and specificity for evaluating the pro-arrhythmic properties of IKr blockers, using moxifloxacin (safe compound) and dofetilide or E-4031 (unsafe compounds). EXPERIMENTAL APPROACH Assays included the anaesthetized remodelled chronic complete AV block (CAVB) dog, the anaesthetized methoxamine-sensitized unremodelled rabbit, multi-cellular hESC-CM clusters, isolated CM obtained from CAVB dogs and isolated CM obtained from the normal rabbit. Arrhythmic outcome was defined as Torsade de Pointes (TdP) in the animal models and early afterdepolarizations (EADs) in the cell models. KEY RESULTS At clinically relevant concentrations (5-12 µM), moxifloxacin was free of pro-arrhythmic properties in all assays with the exception of the isolated CM, in which 10 µM induced EADs in 35% of the CAVB CM and in 23% of the rabbit CM. At supra-therapeutic concentrations (≥100 µM), moxifloxacin was pro-arrhythmic in the isolated rabbit CM (33%), in the hESC-CM clusters (18%), and in the methoxamine rabbit (17%). Dofetilide and E-4031 induced EADs or TdP in all assays (50-83%), and the induction correlated with a significant increase in beat-to-beat variability of repolarization. CONCLUSION AND IMPLICATIONS Isolated cardiomyocytes lack specificity to discriminate between TdP liability of the IKr blocking drugs moxifloxacin and dofetilide or E4031. [ABSTRACT FROM AUTHOR]

Purpose: In healthy hearts, ventricular gap junctions are composed by Connexin43 proteins (Cx43) and are localized in the intercalated disk (ID) where they enable appropriate electrical and metabolic coupling. During pathophysiological conditions, Cx43 is heterogeneously downregulated whereas the activity of CaM/CaMKII signaling increases. It is unclear if CaM/CaMKII affects the expression or the trafficking of Cx43. We analyzed several different models to assess the influence of CaM/CaMKII inhibition on Cx43 gap junctions and conduction properties of the heart.Methods: AC3-I mice, in which CaMKII was genetically inhibited, were subjected to pressure overload (16 wks, TAC vs sham) to evaluate the effect of chronic inhibition of CaMKII under pathological conditions. Conduction velocity (CV) was measured by high-resolution optical mapping and epicardial mapping on Langendorff perfused rabbit and AC3I mouse hearts respectively. Intercellular communication between cultured neonatal rat cardiomyocytes (CMs) was assessed by a dye transfer assay. Subcellular expression of junctional Cx43 from rabbit ventricles and cultured dog cardiomyocytes was evaluated by Triton X-100-based fractionation and Western blot (WB). Subcellular distribution of Cx43 in perfused rabbit and mouse ventricles was evaluated by immunohistochemistry.Results: In mice, CV was augmented in AC3-I sham (n=11, p<0.05 vs. WT) and preserved after TAC in the AC3-I mice (n=11), but impaired in TAC-WT mice (-20%). The expression of Cx43 was preserved after TAC surgery in AC3-I mice though arrhythmias were still present as was increased fibrosis. W7 (CaM inhibitor, 10 μM) significantly increased CV (+8%, n=4, p<0.05 vs. control) in rabbits while susceptibility to arrhythmias decreased. Immuno-confocal microscopy revealed enlarged Cx43 cluster sizes in the ID of W7 treated rabbit hearts. Total amount of Cx43 did not change by W7 (n=5), whereas junctional Cx43 increased (+10%, n=5, p<0.05). These data were confirmed in cultured dog cardiomyocytes both via treatment with W7 and KN93, (CaMKII inhibitor, 1-10 μM). Dye coupling in rat CMs improved (+37.5%, n=7, p<0.05 vs. control).Conclusion: Both acute and chronic CaM/CaMKII inhibition positively affects conduction properties and localization of Cx43 in ID. Only in absence of pathological fibrosis this reduced the susceptibility for arrhythmias. [ABSTRACT FROM AUTHOR]

Pronounced beat-to-beat variability in cardiac repolarization duration (BVR) is associated with increased arrhythmic risk in patients, animal models and isolated cardiomyocytes. However, the mechanisms linking an enhanced BVR to arrhythmogenicity is unknown and may differ between single cells and the intact heart, since cellular coupling is known to attenuate repolarization variability and suppress early afterdepolarizations. We hypothesized that in dogs with chronic AV-block (CAVB) and reduced repolarization strength, beat-to-beat variability in preload, only present in the intact heart, is required to increase BVR.Methods: Endocardial left ventricular monophasic action potential duration (LVMAPD80) was recorded in acute (AAVB, n=6) and CAVB dogs (n=7), before and after a challenge with the IKr blocker dofetilide. We used atrial and ventricular stimulation to provide either a constant or an alternating preload pattern, which was verified by PV-loop determined mechanical parameters. The effect of the stretch activated channel blocker streptomycin on baseline BVR and arrhythmic response to dofetilide was evaluated in a second CAVB group (n=9). Short-term variability of LVMAPD was used to quantify BVR. Arrhythmic outcome was quantified by combining the number of ectopic beats, episodes of TdP and defibrillations into a single arrhythmia score (AS).Results: Pro-arrhythmic remodeling (>3weeks AV-block) increased BVR during alternating preload, (0.45±0.14 AAVB vs 2.2±1.1 ms CAVB, p<0.01), while no change in BVR was seen at constant preload (0.35±0.12 AAVB vs 0.32±0.14 ms CAVB, NS).At AAVB, reduction of repolarization reserve by dofetilide did not induce TdP (AS 1[1-2], median[IQR]), but was pro-arrhythmic in CAVB (AS 27[9-62], p<0.05 vs AAVB). Variation of preload did not alter arrhythmic outcome at CAVB (AS 16[7-37] constant vs 47[2-57] alternating, NS), but was present in BVR determined prior to arrhythmias (2.5±1.4 constant vs 5.9±4.5 ms alternating, p=0.08).In the second group, the increase in BVR at baseline by alternating preload (0.3±0.03 vs 1.0±0.8 ms , p<0.01) was almost abolished by streptomycin (0.5±0.2 ms alternating, p<0.05 vs baseline alternating, NS vs baseline constant). Furthermore, arrhythmia score after dofetilide was reduced after streptomycin pre-treatment (AS 4[2-13], p=0.05).Conclusions: In the anesthetized CAVB dog BVR is a marker of pro-arrhythmic remodeling and critically reduced repolarization reserve, and originates from altered response to changes in preload. There is involvement of stretch activated channels both in the mechanisms of BVR and of drug-induced arrhythmia. [ABSTRACT FROM PUBLISHER]

Background and Purposes: Reduced inward rectifier potassium channel (K ir 2.1) functioning is associated with heart failure and may cause Andersen-Tawil Syndrome, among others characterized by ventricular arrhythmias. Most heart failure or Andersen-Tawil Syndrome patients are treated with β-adrenoceptor antagonists (β-blockers) or sodium channel blockers; however, these do not specifically address the inward rectifier current (I K1 ) nor aim to improve resting membrane potential stability. Consequently, additional pharmacotherapy for heart failure and Andersen-Tawil Syndrome treatment would be highly desirable. Acute propafenone treatment at low concentrations enhances I K1 current, but it also exerts many off-target effects. Therefore, discovering and exploring new I K1 -channel openers is necessary., Experimental Approach: Effects of propafenone and 10 additional propafenone analogues were analysed. Currents were measured by single-cell patch-clamp electrophysiology. K ir 2.1 protein expression levels were determined by western blot analysis and action potential characteristics were further validated in human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMCs). Molecular docking was performed to obtain detailed information on drug-channel interactions., Key Results: Analogues GPV0019, GPV0057 and GPV0576 strongly increased the outward component of I K1 while not affecting the K ir 2.1 channel expression levels. GPV0057 did not block I Kr at concentrations below 0.5 μmol L -1 nor Na V 1.5 current below 1 μmol L -1 . Moreover, hiPSC-CMC action potential duration was also not affected by GPV0057 at 0.5 and 1 μmol L -1 . Structure analysis indicates a mechanism by which GPV0057 might enhance K ir 2.1 channel activation., Conclusion and Implications: GPV0057 has a strong efficiency towards increasing I K1 , which makes it a good candidate to address I K1 deficiency-associated diseases., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

The functioning of the human heart relies on complex electrical and communication systems that coordinate cardiac contractions and sustain rhythmicity. One of the key players contributing to this intricate system is the K IR 2.1 potassium ion channel, which is encoded by the KCNJ2 gene. K IR 2.1 channels exhibit abundant expression in both ventricular myocytes and Purkinje fibers, exerting an important role in maintaining the balance of intracellular potassium ion levels within the heart. And by stabilizing the resting membrane potential and contributing to action potential repolarization, these channels have an important role in cardiac excitability also. Either gain- or loss-of-function mutations, but also acquired impairments of their function, are implicated in the pathogenesis of diverse types of cardiac arrhythmias. In this review, we aim to elucidate the system functions of K IR 2.1 channels related to cellular electrical signaling, communication, and their contributions to cardiovascular disease. Based on this knowledge, we will discuss existing and new pharmacological avenues to modulate their function., (© 2024. The Author(s).)

Introduction: 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., Methods: 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., Results: 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., Conclusion: 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., Competing Interests: Author JM was employed by Xention Ltd. Authors JD and US were employed by Acesion Pharma. The remaining 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 © 2024 van Hunnik, Sobota, Zeemering, Opacic, Scaf, D’Alessandro, Oyaert, Kuiper, Diness, Sørensen, Milnes, van der Heyden, Jespersen, Schotten and Verheule.)

Introduction: Medical undergraduate students receive limited education on scholarly publishing. However, publishing experiences during this phase are known to influence study and career paths. The medical bachelor Honours Program (HP) at Utrecht University initiated a hands-on writing and publishing course, which resulted in nine reviews published in internationally peer reviewed academic journals. We wanted to share the project set-up, explore the academic development of the participating students and determine the impact of the reviews on the scientific community., Methods: Thirty-one out of 50 alumni completed a digital retrospective questionnaire on for example, development of skills and benefit for their studies and career. Publication metrics of the HP review papers were retrieved from Web of Science., Results: This hands-on project provides a clear teaching method on academic writing and scholarly publishing in the bachelor medical curriculum. Participants were able to obtain and improve writing and publishing skills. The output yielded well-recognized scientific papers and valuable learning experiences. 71% of the participating students published at least one additional paper following this project, and 55% of the students indicated the project influenced their academic study and/or career path. Nine manuscripts were published in journals with an average impact factor of 3.56 and cited on average 3.73 times per year., Discussion: This course might inspire other medical educators to incorporate similar projects successfully into their curriculum. To this end, a number of recommendations with regard to supervision, time investment and group size are given., (© 2024. The Author(s).)

Background: The rapid delayed rectifier potassium current (I Kr ) is important for cardiac repolarization and is most often involved in drug-induced arrhythmias. However, accurately measuring this current can be challenging in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes because of its small current density. Interestingly, the ion channel conducting I Kr , hERG channel, is not only permeable to K + ions but also to Cs + ions when present in equimolar concentrations inside and outside of the cell. Methods: In this study, I hERG was measured from Chinese hamster ovary (CHO)-hERG cells and hiPSC-CM using either Cs + or K + as the charge carrier. Equimolar Cs + has been used in the literature in manual patch-clamp experiments, and here, we apply this approach using automated patch-clamp systems. Four different (pre)clinical drugs were tested to compare their effects on Cs + - and K + -based currents. Results: Using equimolar Cs + solutions gave rise to approximately ten-fold larger hERG conductances. Comparison of Cs + - and K + -mediated currents upon application of dofetilide, desipramine, moxifloxacin, or LUF7244 revealed many similarities in inhibition or activation properties of the drugs studied. Using equimolar Cs + solutions gave rise to approximately ten-fold larger hERG conductances. In hiPSC-CM, the Cs + -based conductance is larger compared to the known K + -based conductance, and the Cs + hERG conductance can be inhibited similarly to the K + -based conductance. Conclusion: Using equimolar Cs + instead of K + for I hERG measurements in an automated patch-clamp system gives rise to a new method by which, for example, quick scans can be performed on effects of drugs on hERG currents. This application is specifically relevant when such experiments are performed using cells which express small I Kr current densities in combination with small membrane capacitances., Competing Interests: Author FS was employed by Nanion Technologies GmbH. The remaining 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Bloothooft, Verbruggen, Seibertz, Heyden, Voigt and de Boer.)

Background: An electrical storm of Torsade de Pointes arrhythmias (TdP) can be reproducibly induced in the anesthetized chronic AV-block (CAVB) dog by infusion of the I Kr -blocker dofetilide. Earlier studies showed that these arrhythmias 1) arise from locations with high spatial dispersion in repolarization (SDR) and 2) can be suppressed by high-rate pacing. We examined whether suppression of TdP by high-rate pacing is established through a decrease in SDR in the CAVB dog. Methods: Dofetilide (25 μg/kg in 5 min) was administered to 5 anesthetized CAVB dogs to induce TdP arrhythmias. During the experiments, animals were continuously paced from the right ventricular apex at 50 beats/minute (RVA50). Upon TdP occurrence and conversion, RVA pacing was consecutively set to 100, 80 and 60 beats/minute for 2 min, referred to as pacing blocks. To determine the additional anti-arrhythmic effects of HRP over defibrillation alone, the number of arrhythmic events and SDR at RVA100 were compared to data from three previously conducted experiments, in which dogs underwent the same experimental protocol but were paced at RVA60 upon TdP occurrence (RVA60 retro ). In all experiments, recordings included surface electrocardiogram and mapping by 56 intramural needles, each recording four electrograms, evenly inserted into the ventricular walls and septum. For each pacing block, the number of ectopic beats (EB), and TdP severity were scored. SDR was quantified as the average difference in repolarization time within four squared needles (SDR cubic ). Results: In 4 out of 5 animals, pacing at RVA100 suppressed TdP occurrence. One dog could not be converted by defibrillation after the initial TdP. Compared to RVA50, pacing at RVA100, but not RVA80 and RVA60, significantly reduced the TdP score (78 ± 33 vs . 0 ± 0, p < 0.05 and vs . 12.5 ± 25 and 25 ± 50, both p > 0.05). The reduction in TdP score was reflected by a significant decrease in SDR cubic (125 ± 46 ms before TdP vs . 49 ± 18 ms during RVA100, p < 0.05), and SDR was smaller than in the RVA60 retro animals (101 ± 52 ms, p < 0.05 vs . RVA100). Conclusion: In CAVB dogs, high-rate pacing effectively suppresses TdP, which, at least in part, results from a spatial homogenization of cardiac repolarization, as reflected by a decrease in SDR., 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Loen, Van Weperen, Beekman, Van Bavel, Meijborg, Van der Waal, Coronel, van der Heyden and Vos.)

Aims: An automated method for determination of short-term variability (STV) of repolarization on intracardiac electrograms (STV-ARIauto) has previously been developed for arrhythmic risk monitoring by cardiac implantable devices, and has proved effective in predicting ventricular arrhythmias (VA) and guiding preventive high-rate pacing (HRP) in a canine model. Current study aimed to assess (i) STV-ARIauto in relation to VA occurrence and secondarily (ii-a) to confirm the predictive capacity of STV from the QT interval and (ii-b) explore the effect of HRP on arrhythmic outcomes in a porcine model of acute myocardial infarction (MI)., Methods and Results: Myocardial infarction was induced in 15 pigs. In 7/15 pigs, STV-QT was assessed at baseline, occlusion, 1 min before VA, and just before VA. Eight of the 15 pigs were additionally monitored with an electrogram catheter in the right ventricle, underwent echocardiography at baseline and reperfusion, and were randomized to paced or control group. Paced group received atrial pacing at 20 beats per min faster than sinus rhythm 1 min after occlusion. Short-term variability increased prior to VA in both STV modalities. The percentage change in STV from baseline to successive timepoints correlated well between STV-QT and STV-ARIauto. High-rate pacing did not improve arrhythmic outcomes and was accompanied by a stronger decrease in ejection fraction., Conclusion: STV-ARIauto values increase before VA onset, alike STV-QT in a porcine model of MI, indicating imminent arrhythmias. This highlights the potential of automatic monitoring of arrhythmic risk by cardiac devices through STV-ARIauto and subsequently initiates preventive strategies. Continuous HRP during onset of acute MI did not improve arrhythmic outcomes., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)