RESUMEN
The authors wish to make the following corrections to this paper [...].
RESUMEN
Side effects on cardiac ion channels causing lethal arrhythmias are one major reason for drug withdrawals from the market. Field potential (FP) recording from cardiomyocytes, is a well-suited tool to assess such cardiotoxic effects of drug candidates in preclinical drug development, but it is currently limited to the spontaneous beating of the cardiomyocytes and manual analysis. Herein, we present a novel optogenetic cardiotoxicity screening system suited for the parallel automated frequency-dependent analysis of drug effects on FP recorded from human-induced pluripotent stem cell-derived cardiomyocytes. For the expression of the light-sensitive cation channel Channelrhodopsin-2, we optimised protocols using virus transduction or transient mRNA transfection. Optical stimulation was performed with a new light-emitting diode lid for a 96-well FP recording system. This enabled reliable pacing at physiologically relevant heart rates and robust recording of FP. Thereby we detected rate-dependent effects of drugs on Naâº, Ca2+ and K⺠channel function indicated by FP prolongation, FP shortening and the slowing of the FP downstroke component, as well as generation of afterdepolarisations. Taken together, we present a scalable approach for preclinical frequency-dependent screening of drug effects on cardiac electrophysiology. Importantly, we show that the recording and analysis can be fully automated and the technology is readily available using commercial products.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Moduladores del Transporte de Membrana/toxicidad , Miocitos Cardíacos/efectos de los fármacos , Optogenética/métodos , Pruebas de Toxicidad/métodos , Potenciales de Acción , Cardiotoxicidad , Línea Celular , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/fisiología , Canales Iónicos/metabolismo , Miocitos Cardíacos/fisiología , Optogenética/instrumentación , Pruebas de Toxicidad/instrumentaciónRESUMEN
The need for predictive, in vitro cardiac safety screening drives further development of automated, high-throughput-compatible drug evaluation based on cardiac cell preparations. Recently, pluripotent stem cells are evaluated as a new, more predictive model for cardiovascular risk assessment pertaining to in vitro assays. We present a new screening platform, the CardioExcyte 96, a hybrid instrument that combines impedance (cell contractility) with extracellular field potential (EFP) recordings. The electrophysiological measurements are noninvasive, label free and have a temporal resolution of 1 ms. This hybrid technology addresses the lack of easy-to-use high-throughput screening for in vitro assays and permits the reliable investigation of short- and long-term pharmacological effects. Several models of cardiomyocyte preparations were successfully validated for use with the CardioExcyte96. Furthermore, the pharmacological effects of a number of reference compounds were evaluated. Compound effects on cell monolayers of human-induced pluripotent stem cell-derived cardiomyocytes are evaluated using a quasi-simultaneous hybrid recording mode that combines impedance and EFP readouts. A specialized software package for rapid data handling and real-time analysis was developed, which allows for comprehensive investigation of the cellular beat signal. Combining impedance readouts of cell contractility and EFP (microelectrode array-like) recordings, the system opens up new possibilities in the field of in vitro cardiac safety assessment.