RESUMO
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.
Assuntos
Antiarrítmicos/farmacologia , Compostos Aza/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Fenetilaminas/farmacologia , Piperidinas/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Piridinas/farmacologia , Quinolinas/farmacologia , Sulfonamidas/farmacologia , Torsades de Pointes/induzido quimicamente , Potenciais de Ação/efeitos dos fármacos , Animais , Linhagem Celular , Modelos Animais de Doenças , Cães , Células-Tronco Embrionárias/citologia , Feminino , Fluoroquinolonas , Coração/efeitos dos fármacos , Coração/fisiopatologia , Bloqueio Cardíaco/fisiopatologia , Humanos , Metoxamina , Moxifloxacina , Miócitos Cardíacos/fisiologia , Coelhos , Torsades de Pointes/fisiopatologia , Remodelação Ventricular/efeitos dos fármacosRESUMO
BACKGROUND AND PURPOSE: The high predisposition to Torsade de Pointes (TdP) in dogs with chronic AV-block (CAVB) is well documented. The anti-arrhythmic efficacy and mode of action of Ca(2+) channel antagonists, flunarizine and verapamil against TdP were investigated. EXPERIMENTAL APPROACH: Mongrel dogs with CAVB were selected based on the inducibility of TdP with dofetilide. The effects of flunarizine and verapamil were assessed after TdP and in different experiments to prevent dofetilide-induced TdP. Electrocardiogram and ventricular monophasic action potentials were recorded. Electrophysiological parameters and short-term variability of repolarization (STV) were determined. In vitro, flunarizine and verapamil were added to determine their effect on (i) dofetilide-induced early after depolarizations (EADs) in canine ventricular myocytes (VM); (ii) diastolic Ca(2+) sparks in RyR2(R4496+/+) mouse myocytes; and (iii) peak and late I(Na) in SCN5A-HEK 293 cells. KEY RESULTS: Dofetilide increased STV prior to TdP and in VM prior to EADs. Both flunarizine and verapamil completely suppressed TdP and reversed STV to baseline values. Complete prevention of TdP was achieved with both drugs, accompanied by the prevention of an increase in STV. Suppression of EADs was confirmed after flunarizine. Only flunarizine blocked late I(Na). Ca(2+) sparks were reduced with verapamil. CONCLUSIONS AND IMPLICATIONS: Robust anti-arrhythmic efficacy was seen with both Ca(2+) channel antagonists. Their divergent electrophysiological actions may be related to different additional effects of the two drugs.
Assuntos
Antiarrítmicos/uso terapêutico , Flunarizina/uso terapêutico , Fenetilaminas/toxicidade , Sulfonamidas/toxicidade , Torsades de Pointes/induzido quimicamente , Torsades de Pointes/tratamento farmacológico , Verapamil/uso terapêutico , Animais , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Cães , Humanos , Lidocaína/administração & dosagem , Lidocaína/uso terapêutico , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Verapamil/administração & dosagemRESUMO
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.
Assuntos
Antiprotozoários/farmacologia , Citoplasma/efeitos dos fármacos , Pentamidina/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Animais , Western Blotting , Linhagem Celular , Citoplasma/metabolismo , Cães , Humanos , Mutação , Técnicas de Patch-Clamp , Canais de Potássio Corretores do Fluxo de Internalização/genéticaRESUMO
BACKGROUND & PURPOSE: The therapeutically available quinolone antibiotic moxifloxacin has been used as a positive control for prolonging the QT interval in both clinical and non-clinical studies designed to assess the potential of new drugs to delay cardiac repolarization. Despite moxifloxacin prolonging QT, it has not been shown to cause torsades de pointes arrhythmias (TdP). Azithromycin is a macrolide antibiotic that has rarely been associated, clinically, with cases of proarrhythmia. As there is a lack of clinical data available, the cardiac safety of these drugs was assessed in a TdP-susceptible animal model by evaluating their repolarization and proarrhythmia effects. EXPERIMENTAL APPROACH & KEY RESULTS: In transfected HEK cells, the IC(50)s for I (hERG) were 45+/-6 and 856+/-259 microg ml(-1) for moxifloxacin and azithromycin, respectively. Intravenous administration of 2 and 8 mg kg(-1) moxifloxacin (total peak-plasma concentrations 4.6+/-1.5 and 22.9+/-6.8 microg ml(-1)) prolonged the QT(c) in 6 anaesthetized dogs with chronic AV block by 7+/-3 and 21+/-19%, respectively. Similar intravenous doses of azithromycin (total peak-plasma concentrations 5.4+/-1.3 and 20.8+/-4.9 microg ml(-1)) had no electrophysiological effects in the same dogs. The reference compound, dofetilide (25 microg kg(-1) i.v.) caused QT(c) prolongation (29+/-15%) and TdP in all dogs. Beat-to-beat variability of repolarization (BVR), quantified as short-term variability of the left ventricular monophasic action potential duration, was only increased after dofetilide (1.8+/-0.7 to 3.8+/-1.5 ms; P<0.05). CONCLUSION & IMPLICATIONS: As neither moxifloxacin nor azithromycin caused TdP or an increase in the BVR, we conclude that both drugs can be used safely in clinical situations.
Assuntos
Anestesia , Antibacterianos/toxicidade , Arritmias Cardíacas/induzido quimicamente , Compostos Aza/toxicidade , Azitromicina/toxicidade , Bloqueio Cardíaco/fisiopatologia , Quinolinas/toxicidade , Animais , Antiarrítmicos/farmacologia , Arritmias Cardíacas/fisiopatologia , Cães , Relação Dose-Resposta a Droga , Estimulação Elétrica , Eletroencefalografia/efeitos dos fármacos , Eletrofisiologia , Fluoroquinolonas , Frequência Cardíaca/efeitos dos fármacos , Síndrome do QT Longo/induzido quimicamente , Síndrome do QT Longo/fisiopatologia , Moxifloxacina , Fenetilaminas/farmacologia , Sulfonamidas/farmacologia , Torsades de Pointes/induzido quimicamente , Torsades de Pointes/fisiopatologiaRESUMO
BACKGROUND: Myocardial stress and strain are considered primary mechanical stimuli for hypertrophic remodeling. Their values and significance in the intact beating heart during chronic overload remain poorly characterized. METHODS AND RESULTS: Left-ventricular (LV) dimensions (echocardiography) and pressure (invasive) were simultaneously recorded in anesthetized dogs at sinus rhythm (SR), acute and 1, 2, 6, 12 weeks of atrioventricular block (AVB), leading to structural, electrical and contractile remodeling. Mechanical load of the myocardium was quantified as myofiber stress (sigma(f)), being force along myofiber orientation per cross-sectional area, and natural myofiber strain (e(f)), being change in natural logarithm of myofiber length (l) divided by its reference length: e(f) = ln(l/l(ref)). Time courses of sigma(f) and e(f) were calculated from LV pressure and dimensions, using a validated mathematical model of cardiac mechanics. End-diastolic sigmaf increased from 2.0 +/- 0.1 kPa at SR to 3.4 +/- 0.3 kPa at acute AVB, remaining elevated for > 6 weeks. Systolic sigma(f) was not affected by AVB. Ejection strain rose instantly upon AVB, reaching a maximum at 2 weeks: 0.24 +/- 0.02 vs. 0.10 +/- 0.01 at SR. The increase of myofiber stroke work (sigma(f)-e(f) loop area) from 3.1 +/- 0.3 at SR to 6.0 +/- 0.5 kJ/m(3)/beat at 1 week AVB was attributed mainly to an increase of strain during ejection. Stroke work and ejection strain remained elevated up to 12 weeks. The rate of LV-mass increase was maximal (2.2 +/- 0.4 g/day) at 1 week AVB. CONCLUSIONS: Serial mechanical phenotyping is feasible in the intact anesthetized dog with chronic ventricular overload. Our new approach yields values of mechanical load that are comparable to those found in isolated myocardium by others. In chronic AVB, both end-diastolic myofiber stress and ejection strain are increased. Early increases of both parameters coincide with peak hypertrophic growth, suggesting their important role for mechanotransduction. Peak systolic sigmaf is likely not important for hypertrophy in this model, since it does not change throughout the experiment.
Assuntos
Diástole , Bloqueio Cardíaco/fisiopatologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Animais , Fenômenos Biomecânicos , Cães , Feminino , Hemodinâmica , Masculino , Fenótipo , Estresse Mecânico , Remodelação VentricularRESUMO
Overdrive stimulation of reentrant ventricular tachycardias (VT) may result in entrainment and/or termination of these arrhythmias. We investigated whether surface ECG criteria of entrainment can also be observed in nonreentrant VT. For this purpose ouabain-induced tachycardias were used that are considered to be based on delayed afterdepolarizations. In nine conscious dogs, having surgically induced complete AV block, pacing was performed using trains of 20 stimuli from a site distant to the origin of the VT. The pacing intervals were shortened in steps of 5-10 msec, until complete capture from the pacing site or termination of the VT was obtained. During stimulation variable fusion was seen and complete capture of the ventricles from the pacing site occurred just after a slight decrease in pacing cycle length (20 +/- 10 msec). Overdrive stimulation resulted only in 1 out of 58 stimulation trains in termination of VT. Following stimulation it was observed that: (1) The length of the first postpacing interval was significantly longer (P less than 0.001) than both the mean prepacing VT cycle length and interstimulus interval; (2) A change in QRS configuration occurred after 57% of the stimulation trains; (3) The VT accelerated slightly in comparison to the prepacing rate (P less than 0.05); and (4) The length of the first postpacing interval and the mean R-R interval of the VT postpacing were directly related to the interstimulus interval (r = 0.82 and 0.97, respectively). In conclusion, overdrive stimulation of ouabain-induced arrhythmias did not result in entrainment or in termination of the tachycardia. Instead, other responses were seen that may be of help in differentiating between arrhythmias caused by delayed afterdepolarizations and reentry.