Cardiotoxicity screening with simultaneous optogenetic pacing, voltage imaging and calcium imaging

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• 作者：Graham T. Dempsey^a ; Khuram W. Chaudhary^b ; ^{khuram.w.chaudhary@gsk.com" class="auth_mail" title="E-mail the corresponding author} ; Nicholas Atwater^a ; Cuong Nguyen^a ; Barry S. Brown^b ; John D. McNeish^c ; Adam E. Cohen^d ; ^e ; ^f ; Joel M. Kralj^a
• 关键词：CiPA ; Comprehensive in vitro Proarrhythmia Assay ; TdP ; Torsades de Pointes ; AP ; action potential ; CT ; Ca2  ; + transient ; hiPSC ; human induced pluripotent stem cell ; CM ; cardiomyocyte ; CaViar ; Ca2  ; + and voltage indicator ; CheRiff ; channelrhodopsin variant ; EAD ; early after depolarization ; ECG ; electrocardiogram ; hERG ; human Ether-à ; -go-go-Related Gene ; MEA ; multi-electrode array ; Arch ; Archaerhodopsin ; VSD ; voltage sensitive dye
• 刊名：Journal of Pharmacological and Toxicological Methods
• 出版年：2016
• 出版时间：September-October 2016
• 年：2016
• 卷：81
• 期：Complete
• 页码：240-250
• 全文大小：1592 K

文摘

The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative seeks an in vitro test to accurately predict clinical Torsades de Pointes (TdP). We developed a cardiotoxicity assay incorporating simultaneous measurement of the action potential (AP) waveform and Ca^2 + transient (CT) in human iPSC-derived cardiomyocytes (CMs). Concurrent optogenetic pacing provided a well-controlled electrophysiological background.

Methods

We used the Optopatch platform for all-optical electrophysiology (Hochbaum et al., 2014). In a monolayer culture, a subset of cells expressed a genetically encoded, calcium and voltage reporter, CaViar (Hou, Kralj, Douglass, Engert, & Cohen, 2014), while others expressed a channelrhodopsin variant, CheRiff. Optical pacing of CheRiff-expressing cells synchronized the syncytium. We screened 12 compounds (11 acute, 1 chronic) to identify electrophysiological (AP rise time, AP50, AP90, beat rate) and CT effects in spontaneously beating and paced cultures (1 Hz, 2 Hz).

Results

CaViar reported spontaneous and paced APs and CTs with high signal-to-noise ratio and low phototoxicity. Quinidine, flecainide, E-4031, digoxin and cisapride prolonged APs, while verapamil and nifedipine shortened APs. Early after depolarizations (EADs) were elicited by quinidine, flecainide and cisapride. All but four compounds (amiodarone, chromanol, nifedipine, verapamil) prolonged AP rise time. Nifedipine and verapamil decreased CT amplitude, while digoxin increased CT amplitude. Pentamidine prolonged APs after chronic exposure.

Discussion

The Optopatch platform provides a robust assay to measure APs and CTs in hiPSC-CMs. This addresses the CiPA mandate and will facilitate comparisons of cell-based assays to human clinical data.