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.

BACKGROUND AND PURPOSEDrug interference with normal hERG protein trafficking substantially reduces the channel density in the plasma membrane and thereby poses an arrhythmic threat. The chemical substructures important for hERG trafficking inhibition were investigated using pentamidine as a model drug. Furthermore, the relationship between acute ion channel block and correction of trafficking by dofetilide was studied. EXPERIMENTAL APPROACHhERG and KIR2.1 trafficking in HEK293 cells was evaluated by Western blot and immunofluorescence microscopy after treatment with pentamidine and six pentamidine analogues, and correction with dofetilide and four dofetilide analogues that displayed different abilities to inhibit IKr. Molecular dynamics simulations were used to address mode, number and type of interactions between hERG and dofetilide analogues. KEY RESULTSStructural modifications of pentamidine differentially affected plasma membrane levels of hERG and KIR2.1. Modification of the phenyl ring or substituents directly attached to it had the largest effect, affirming the importance of these chemical residues in ion channel binding. PA-4 had the mildest effects on both ion channels. Dofetilide corrected pentamidine-induced hERG, but not KIR2.1 trafficking defects. Dofetilide analogues that displayed high channel affinity, mediated by pi-pi stacks and hydrophobic interactions, also restored hERG protein levels, whereas analogues with low affinity were ineffective. CONCLUSIONS AND IMPLICATIONSDrug-induced trafficking defects can be minimized if certain chemical features are avoided or 'synthesized out'; this could influence the design and development of future drugs. Further analysis of such features in hERG trafficking correctors may facilitate the design of a non-blocking corrector for trafficking defective hERG proteins in both congenital and acquired LQTS. AbbreviationsER, endoplasmic reticulum; hERG, human ether-a-go-go-related gene; IK1, cardiac inward rectifying K + current; IKr, rapid component of the delayed rectifier K

Background and purpose:Pentamidine is a drug used in treatment of protozoal infections. Pentamidine treatment may cause sudden cardiac death by provoking cardiac arrhythmias associated with QTc prolongation and U-wave alterations. This proarrhythmic effect was linked to inhibition of hERG trafficking, but not to acute block of ion channels contributing to the action potential. Because the U-wave has been linked to the cardiac inward rectifier current (IK1), we examined the action and mechanism of pentamidine-mediated IK1 block. Experimental approach: Patch clamp measurements of IK1 were made on cultured adult canine ventricular cardiomyocytes, KIR2.1-HEK293 cells and KIR2.x inside-out patches. Pentamidine binding to cytoplasmic amino acid residues of KIR2.1 channels was studied by molecular modelling. Key results: Pentamidine application (24 h) decreased IK1 in cultured canine cardiomyocytes and KIR2.1-HEK293 cells under whole cell clamp conditions. Pentamidine inhibited IK1 in KIR2.1-HEK293 cells 10 min after application. When applied to the cytoplasmic side under inside-out patch clamp conditions, pentamidine block of IK1 was acute (IC50 = 0.17 mM). Molecular modelling predicted pentamidine-channel interactions in the cytoplasmic pore region of KIR2.1 at amino acids E224, D259 and E299. Mutation of these conserved residues to alanine reduced pentamidine block of IK1. Block was independent of the presence of spermine. KIR2.2, and KIR2.3 based IK1 was also sensitive to pentamidine blockade. Conclusions and implications: Pentamidine inhibits cardiac IK1 by interacting with three negatively charged amino acids in the cytoplasmic pore region. Our findings may provide new insights for development of specific IK1 blocking compounds.

Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Young Talent Programme, CVON Introduction Chronic kidney disease (CKD) is represented by a diminished filtration capacity of the kidneys. End stage renal disease patients need dialysis treatment to remove waste and toxins from the circulation. However, endogenously produced uremic toxins (UTs) cannot always be filtered during dialysis. UTs are one of the CKD-related factors already linked to maladaptive and pathophysiological remodeling of the heart. Importantly, 50% of the deaths in dialysis patients is cardiovascular related, with sudden cardiac death predominating. However, the mechanisms responsible still remain poorly understood. Purpose To assess the potentially increased pro-arrhythmic risk of pre-identified UTs at clinically relevant concentrations: 1) acutely in isolated adult dog ventricular cardiomyocytes (ADCMs) and human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), 2) chronically in iPSC-CMs and 3) both conditions in human embryonic kidney (HEK) cells transfected with constructs that generate different ion channels, using optical and manual electrophysiological in vitro approaches. Methods and results Using the voltage sensitive membrane dye FluoVolt and manual patch clamp, action potential duration (APD) prolongation, considered as a pro-arrhythmic parameter, was assessed in ADCMs and iPSC-CMs. Acute exposure of indoxyl sulfate (IS), kynurenine (KYN) or kynurenic acid (KYNA) induced no alterations in APD in ADCMs and iPSC-CMs. iPSC-CMs, although having rather immature electrophysiological characteristics, showed significant APD prolongation when exposed chronically (48h) to those UTs. Manual patch clamp on HEK293 cells was performed to investigate individual ion currents to reveal underlying mechanisms. The repolarizing current IKr, often most sensitive and responsible for APD alterations, was not acutely affected by IS, KYN, or KYNA. However, chronic exposure (48h) to IS or KYNA significantly decreased IKr at clinically relevant concentrations. Finally, the protein level of the ion channel conducting IKr was determined in HEK293 cells, to further unveil the mechanism behind the effects of UTs on channels conducting IKr. Chronic exposure (48-72h) of the three UTs decreased total ion channel expression at pathological concentrations, which could be responsible for the decreased IKr and prolonged APD. Conclusion The results show the proof of concept to identify potentially pro-arrhythmogenic UTs and their mode of action, allowing the provision of a clinically relevant overview of (patho)physiological concentrations of UTs in both acute and chronic exposure.

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