No abstract

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.

BACKGROUND AND PURPOSE Ca2+ leak from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR2s) contributes to cardiomyocyte dysfunction. RyR2 Ca 2+ leak has been related to RyR2 phosphorylation. In these conditions, JTV519 (K201), a 1,4-benzothiazepine derivative and multi-channel blocker, stabilizes RyR2s and decrease SR Ca 2+ leak. We investigated whether JTV519 stabilizes RyR2s without increasing RyR2 phosphorylation in mice and in non-failing human myocardium and explored underlying mechanisms. EXPERIMENTAL APPROACH SR Ca2+ leak was induced by ouabain in murine cardiomyocytes. [Ca 2+ ]-transients, SR Ca 2+ load and RyR2-mediated Ca 2+ leak (sparks/waves) were quantified, with or without JTV519 (1 mmol·L-1 ). Contribution of Ca 2+-/calmodulin-dependent kinase II (CaMKII) was assessed by KN-93 and Western blot (RyR2-Ser 2814 phosphorylation). Effects of JTV519 on contractile force were investigated in non-failing human ventricular trabeculae. ], Ca 2+ leak was significantly reduced by JTV519, but it had no effect on fractional Ca 2+ release or Ca 2+ wave propagation velocity. In human muscle, JTV519 was negatively inotropic at baseline but significantly enhanced ouabain-induced force and reduced its deleterious effects on diastolic function. 2+ removal varies among species between 7% (rats), 28% (rabbits) and up to about 50% (failing human hearts) (Dipla et al., 1999;Bers, 2001). KEY RESULTS Ouabain CONCLUSIONS AND IMPLICATIONSLeak of Ca 2+ from the SR through RyR2s during diastole has been implicated in arrhythmias and contractile dysfunction in heart failure (Marx et al., 2000;Lindner et al., 2002), myocardial ischaemia (Hirose et al., 2008), atrial fibrillation (Vest et al., 2005) and congenital arrhythmias (Mohamed et al., 2007). Several mechanisms have been proposed to explain RyR2 dysfunction, including altered RyR2 gating due to point mutations in the RyR2 molecule (Cerrone et al., 2005), alterations in RyR2 phosphorylation (Witcher et al., 1991;Wehrens et al., 2004b;Xiao et al., 2005) MethodsThe use of donor hearts and research protocols for human samples were approved by the local ethics committee (ref. nr. 20-277 ex 08/09). Investigations on human tissue conform with the principles outlined in the Declaration of Helsinki. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (McGrath et al., 2010). Animals were treated according to the Guidelines for the Care and Use of Laboratory Animals (National Institute of Health, USA). Cell isolationAdult FVB/N mice (11-16 weeks old; total = 43) provided by the Abt. f. Labortierkunde, Medical University of Vienna, Austria, were used for all experiments. Ventricular cardiomyocytes were isolated as previously described (Heinzel et al., 2002;Sedej et al., 2010). Mice were anesthetized with isoflu- . Only quiescent, rod-shaped ventricular cardiomyocytes were used for experiments. Confocal [Ca 2+ ] i measurementsCells were studied in a perfusion chamber mounted on the stage of an ...