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1.
Nat Commun ; 12(1): 7151, 2021 12 09.
Article in English | MEDLINE | ID: mdl-34887420

ABSTRACT

The heterozygous Phospholamban p.Arg14del mutation is found in patients with dilated or arrhythmogenic cardiomyopathy. This mutation triggers cardiac contractile dysfunction and arrhythmogenesis by affecting intracellular Ca2+ dynamics. Little is known about the physiological processes preceding induced cardiomyopathy, which is characterized by sub-epicardial accumulation of fibrofatty tissue, and a specific drug treatment is currently lacking. Here, we address these issues using a knock-in Phospholamban p.Arg14del zebrafish model. Hearts from adult zebrafish with this mutation display age-related remodeling with sub-epicardial inflammation and fibrosis. Echocardiography reveals contractile variations before overt structural changes occur, which correlates at the cellular level with action potential duration alternans. These functional alterations are preceded by diminished Ca2+ transient amplitudes in embryonic hearts as well as an increase in diastolic Ca2+ level, slower Ca2+ transient decay and longer Ca2+ transients in cells of adult hearts. We find that istaroxime treatment ameliorates the in vivo Ca2+ dysregulation, rescues the cellular action potential duration alternans, while it improves cardiac relaxation. Thus, we present insight into the pathophysiology of Phospholamban p.Arg14del cardiomyopathy.


Subject(s)
Calcium-Binding Proteins/genetics , Calcium/metabolism , Cardiomyopathy, Dilated/genetics , Etiocholanolone/analogs & derivatives , Zebrafish/metabolism , Animals , Calcium-Binding Proteins/metabolism , Cardiomyopathy, Dilated/metabolism , Cardiomyopathy, Dilated/physiopathology , Disease Models, Animal , Echocardiography , Etiocholanolone/administration & dosage , Female , Gene Knock-In Techniques , Humans , Male , Myocardial Contraction , Myocardium/metabolism , Sequence Deletion , Zebrafish/genetics
2.
Expert Rev Cardiovasc Ther ; 14(2): 245-53, 2016.
Article in English | MEDLINE | ID: mdl-26560188

ABSTRACT

Drug-induced Torsade de Pointes arrhythmia is a life-threatening adverse effect feared by pharmaceutical companies. For the last decade, the cardiac safety guidelines have imposed human ether-a-go-go-related gene channel blockade and prolongation of QT interval as surrogates for proarrhythmic risk propensity of a new chemical entity. Suffering from a lack of specificity, this assessment strategy led to a great amount of false positive outcomes. Therefore, this review will discuss new pharmaceutical strategies: the cardiac safety proposal that recently emerged, the Comprehensive in vitro Proarrhythmia Assay, combining in vitro assays that integrate effects on main cardiac ion channels, with computational models of human ventricular action potential as well as assays using human stem cell-derived cardiomyocytes for an improved prediction of drug's proarrhythmic liability, alternative pharmacological perspectives as well as the current treatment of drug-induced long QT syndrome.


Subject(s)
Arrhythmias, Cardiac/prevention & control , Long QT Syndrome/prevention & control , Torsades de Pointes/prevention & control , Action Potentials/drug effects , Animals , Arrhythmias, Cardiac/chemically induced , Computer Simulation , Electrocardiography , Heart Ventricles/physiopathology , Humans , Ion Channels/metabolism , Long QT Syndrome/chemically induced , Myocytes, Cardiac/metabolism , Torsades de Pointes/chemically induced
3.
Br J Pharmacol ; 165(2): 467-78, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21718297

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 I(Kr) 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 I(Kr) blocking drugs moxifloxacin and dofetilide or E4031.


Subject(s)
Anti-Arrhythmia Agents/pharmacology , Aza Compounds/pharmacology , Myocytes, Cardiac/drug effects , Phenethylamines/pharmacology , Piperidines/pharmacology , Potassium Channel Blockers/pharmacology , Pyridines/pharmacology , Quinolines/pharmacology , Sulfonamides/pharmacology , Torsades de Pointes/chemically induced , Action Potentials/drug effects , Animals , Cell Line , Disease Models, Animal , Dogs , Embryonic Stem Cells/cytology , Female , Fluoroquinolones , Heart/drug effects , Heart/physiopathology , Heart Block/physiopathology , Humans , Methoxamine , Moxifloxacin , Myocytes, Cardiac/physiology , Rabbits , Torsades de Pointes/physiopathology , Ventricular Remodeling/drug effects
4.
Acta Physiol (Oxf) ; 199(3): 243-56, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20331539

ABSTRACT

Inward rectifier currents based on K(IR)2.x subunits are regarded as essential components for establishing a stable and negative resting membrane potential in many excitable cell types. Pharmacological inhibition, null mutation in mice and dominant positive and negative mutations in patients reveal some of the important functions of these channels in their native tissues. Here we review the complex mammalian expression pattern of K(IR)2.x subunits and relate these to the outcomes of functional inhibition of the resultant channels. Correlations between expression and function in muscle and bone tissue are observed, while we recognize a discrepancy between neuronal expression and function.


Subject(s)
Potassium Channels, Inwardly Rectifying/genetics , Potassium Channels, Inwardly Rectifying/physiology , Animals , Humans , Mice , Mice, Knockout , Mutation/physiology , Phenotype , Potassium Channel Blockers/pharmacology , Potassium Channels, Inwardly Rectifying/biosynthesis , Potassium Channels, Inwardly Rectifying/drug effects
5.
Br J Pharmacol ; 159(7): 1532-41, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20180941

ABSTRACT

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 (I(K1)), we examined the action and mechanism of pentamidine-mediated I(K1) block. EXPERIMENTAL APPROACH: Patch clamp measurements of I(K1) were made on cultured adult canine ventricular cardiomyocytes, K(IR)2.1-HEK293 cells and K(IR)2.x inside-out patches. Pentamidine binding to cytoplasmic amino acid residues of K(IR)2.1 channels was studied by molecular modelling. KEY RESULTS: Pentamidine application (24 h) decreased I(K1) in cultured canine cardiomyocytes and K(IR)2.1-HEK293 cells under whole cell clamp conditions. Pentamidine inhibited I(K1) in K(IR)2.1-HEK293 cells 10 min after application. When applied to the cytoplasmic side under inside-out patch clamp conditions, pentamidine block of I(K1) was acute (IC(50)= 0.17 microM). Molecular modelling predicted pentamidine-channel interactions in the cytoplasmic pore region of K(IR)2.1 at amino acids E224, D259 and E299. Mutation of these conserved residues to alanine reduced pentamidine block of I(K1). Block was independent of the presence of spermine. K(IR)2.2, and K(IR)2.3 based I(K1) was also sensitive to pentamidine blockade. CONCLUSIONS AND IMPLICATIONS: Pentamidine inhibits cardiac I(K1) by interacting with three negatively charged amino acids in the cytoplasmic pore region. Our findings may provide new insights for development of specific I(K1) blocking compounds.


Subject(s)
Antiprotozoal Agents/pharmacology , Cytoplasm/drug effects , Pentamidine/pharmacology , Potassium Channels, Inwardly Rectifying/antagonists & inhibitors , Animals , Blotting, Western , Cell Line , Cytoplasm/metabolism , Dogs , Humans , Mutation , Patch-Clamp Techniques , Potassium Channels, Inwardly Rectifying/genetics
6.
Neth Heart J ; 16(3): 106-9, 2008.
Article in English | MEDLINE | ID: mdl-18345333

ABSTRACT

During cardiac maturation, increased exposure of the heart to circulating catecholamines correlates with increased conduction velocity and growth of the heart. We used an in vitro approach to study the underlying mechanisms of adrenergic stimulation induced changes in conduction velocity. By combining functional measurements and molecular techniques, we were able to demonstrate that the increased conduction velocity after beta-adrenergic stimulation is probably not caused by changes in intercellular coupling. Instead, RT-PCR experiments and action potential measurements have shown an increased excitability that may well explain the observed increase in conduction velocity. Apart from being relevant to cardiac maturation, our findings are relevant in the context of stem cells and cardiac repair. Preconditioning of stem cell derived cardiomyocytes may help to enhance electrical maturation of de novo generated cardiomyocytes and consequently reduce their proarrhythmogenic potential. (Neth Heart J 2008;16:106-9.).

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