Abstract
Side effects on cardiac ion channels are one major reason for new drugs to fail during preclinical evaluation. Herein we propose a simple optogenetic screening tool measuring extracellular field potentials (FP) from paced cardiomyocytes to identify drug effects over the whole physiological heart range, which is essential given the rate-dependency of ion channel function and drug action. Human induced pluripotent stem cell-derived cardiomyocytes were transduced with an adeno-associated virus to express Channelrhodopsin2 and plated on micro-electrode arrays. Global pulsed illumination (470 nm, 1 ms, 0.9 mW/mm2) was applied at frequencies from 1 to 2.5 Hz, which evoked FP simultaneously in all cardiomyocytes. This synchronized activation allowed averaging of FP from all electrodes resulting in one robust FP signal for analysis. Field potential duration (FPD) was ~25% shorter at 2.5 Hz compared to 1 Hz. Inhibition of hERG channels prolonged FPD only at low heart rates whereas Ca2+ channel block shortened FPD at all heart rates. Optogenetic pacing also allowed analysis of the maximum downstroke velocity of the FP to detect drug effects on Na+ channel availability. In principle, the presented method is well scalable for high content cardiac toxicity screening or personalized medicine for inherited cardiac channelopathies.
Highlights
Side effects on cardiac ion channels are one major reason for new drugs to fail during preclinical evaluation
One of the most common cardiotoxic effects of drugs is delayed cardiac repolarization resulting in prolonged action potential (AP), which clinically manifests in the electrocardiogram as a long QT interval and is called long QT-syndrome (LQTS)
We used commercially available, purified cardiomyocytes derived from hiPSC and inscribed light sensitivity by transduction with associated virus (AAV) for expressing the non-selective cation channel ChR2 (H134R)[42] fused to the red fluorescence protein mCherry (Fig. 1A,B)
Summary
OPEN Frequency-dependent drug screening using optogenetic stimulation of human iPSC-derived. The specific advantages of this technology are the easy-to-use commercially available instrumentation and the lack of dye loading or high intensity illumination, which enables the long-term analysis over days in the incubator This could be an important aspect for drug screening assays as it was shown before that tyrosine kinase inhibitors used in cancer therapy and some IKr inhibitors show additional pro-arrhythmic effects on the late Na+ current only after long term application[26, 27]. We report a novel technology for drug screening on human cardiomyocytes at the whole range of physiological heat rates using optogenetic pacing[34] on MEA This enables artefact-free and synchronized stimulation of cardiomyocytes with light pulses, which allows averaging of FP from the single recording electrodes for objective analysis. Data were analysed and represented with Graph Pad Prism (Version 5.01, GraphPad Software)
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