RESUMEN
INTRODUCTION: It has been proposed that proarrhythmia assessment for safety pharmacology testing includes the use of human pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) to detect drug-induced changes in cardiac electrophysiology. This study measured the actions of diverse agents on action potentials (AP) and ion currents recorded from hiPSC-CM. METHODS: During AP experiments, the hiPSC-CM were paced at 1Hz during a baseline period, and when increasing concentrations of test compound were administered at 4-minute intervals. AP parameters, including duration (APD60 and APD90), resting membrane potential, rate of rise, and amplitude, were measured throughout the entire experiment. Voltage clamp experiments with E-4031 and nifedipine were similarly conducted. RESULTS: E-4031 produced a dose-dependent prolongation of cardiac action potential and blocked the hERG/IKr current with an IC50 of 17nM. At 3nM, dofetilide significantly increased APD90. Astemizole significantly increased APD60 and APD90 at 30nM. Terfenadine significantly increased APD90 at concentrations greater than 10nM. Fexofenadine, a metabolite of terfenadine, did not produce any electrophysiologic changes in cardiac action potentials. Flecainide produced a dose-dependent prolongation of the cardiac action potential at 1 and 3µM. Acute exposure to nifedipine significantly decreased APD60 and APD90 and produced a dose-dependent block of calcium current with an IC50 of 0.039µM. Verapamil first shortened APD60 and APD90 in a dose-dependent manner, until a compensating increase in APD90, presumably via hERG blockade, was observed at 1 and 3µM. Following a chronic exposure (20-24h) to clinically relevant levels of pentamidine, a significant increase in action potential duration was accompanied by early afterdepolarizations (EADs). DISCUSSION: These experiments show the ability of AP measured from hiPSC-CM to record the interactions of various ion channels via AP recording and avoid the limitations of using several single ion channel assays in a noncardiac tissue.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Células Madre Pluripotentes Inducidas/citología , Canales Iónicos/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Adulto , Astemizol/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Flecainida/farmacología , Humanos , Miocitos Cardíacos/metabolismo , Nifedipino/farmacología , Pentamidina/farmacología , Fenetilaminas/farmacología , Piperidinas/farmacología , Piridinas/farmacología , Sulfonamidas/farmacología , Terfenadina/análogos & derivados , Terfenadina/farmacología , Verapamilo/farmacologíaRESUMEN
Contractility studies in isolated feline myocytes have demonstrated that sphingosine, a metabolite stimulated by tumor necrosis factor (TNF) binding, decreases intracellular calcium release and depresses inotropic activity. This study investigated the electrophysiologic effects of sphingosine in isolated cat myocytes as well as the cardiodynamic consequence of TNF, sphingosine, and its metabolic precursors in vivo. In cat myocytes, sphingosine markedly decreased action potential duration, lowered action potential plateau, and inhibited L-type calcium current (I(Ca-L)). After administration of TNF, sphingomyelin, C2-ceramide, or sphingosine, only C2-ceramide and sphingosine depressed cardiac function in normal rats. Negative inotropic effects of C2-ceramide were attenuated by N-oleoylethanolamine (NOE), a ceramidase inhibitor that blocks sphingosine formation. Rats pretreated with NOE before undergoing 30 min of acute regional myocardial ischemia followed by 150 min of reperfusion exhibited improved survival. Most deaths could be attributed to acute pump failure accompanied by bradycardia. Myocardial infarct size and peak serum TNF were not different between NOE- and vehicle-treated groups (3,908 +/- 1097 pg/ml and 3,027 +/- 846 pg/ml, respectively). These results indicate that sphingosine exerts direct inhibitory effects on the action potential and I(Ca-L) in isolated feline myocytes, consistent with previously reported sphingosine activity on I(Ca-L) in isolated rat myocytes. The in vivo study suggests that reducing sphingosine production with N-oleoylethanolamine attenuates cardiodepression and can improve overall survival after ischemic injury. Clearly, agents that modulate sphingosine production limit cardiodepression and may provide a therapeutic benefit in clinical conditions of myocardial inflammatory injury.