The use of ratiometric fluorescence measurements of the voltage sensitive dye Di-4-ANEPPS to examine action potential characteristics and drug effects on human induced pluripotent stem cell-derived cardiomyocytes

Hortigon-Vinagre, M.P., Zamora, V., Burton, F.L., Green, J., Gintant, G.A. and Smith, G.L. (2016) The use of ratiometric fluorescence measurements of the voltage sensitive dye Di-4-ANEPPS to examine action potential characteristics and drug effects on human induced pluripotent stem cell-derived cardiomyocytes. Toxicological Sciences, 154(2), pp. 320-331. (doi:10.1093/toxsci/kfw171) (PMID:27621282) (PMCID:PMC5139069)

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Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and higher throughput platforms have emerged as potential tools to advance cardiac drug safety screening. This study evaluated the use of high bandwidth photometry applied to voltage-sensitive fluorescent dyes (VSDs) to assess drug-induced changes in action potential characteristics of spontaneously active hiPSC-CM. Human iPSC-CM from 2 commercial sources (Cor.4U and iCell Cardiomyocytes) were stained with the VSD di-4-ANEPPS and placed in a specialized photometry system that simultaneously monitors 2 wavebands of emitted fluorescence, allowing ratiometric measurement of membrane voltage. Signals were acquired at 10 kHz and analyzed using custom software. Action potential duration (APD) values were normally distributed in cardiomyocytes (CMC) from both sources though the mean and variance differed significantly (APD90: 229 ± 15 ms vs 427 ± 49 ms [mean ± SD, P < 0.01]; average spontaneous cycle length: 0.99 ± 0.02 s vs 1.47 ± 0.35 s [mean ± SD, P < 0.01], Cor.4U vs iCell CMC, respectively). The 10–90% rise time of the AP (Trise) was ∼6 ms and was normally distributed when expressed as 1/T2riseTrise2 in both cell preparations. Both cell types showed a rate dependence analogous to that of adult human cardiac cells. Furthermore, nifedipine, ranolazine, and E4031 had similar effects on cardiomyocyte electrophysiology in both cell types. However, ranolazine and E4031 induced early after depolarization-like events and high intrinsic firing rates at lower concentrations in iCell CMC. These data show that VSDs provide a minimally invasive, quantitative, and accurate method to assess hiPSC-CM electrophysiology and detect subtle drug-induced effects for drug safety screening while highlighting a need to standardize experimental protocols across preparations.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Burton, Dr Francis and Zamora Rodriguez, Dr Victor and Smith, Professor Godfrey
Authors: Hortigon-Vinagre, M.P., Zamora, V., Burton, F.L., Green, J., Gintant, G.A., and Smith, G.L.
Subjects:?? action potential duration ??
?? drug screening ??
?? human induced pluripotent stem cell-derived cardiomyocytes ??
?? methods ??
?? stem cells ??
?? voltage sensitive dye ??
College/School:College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
Journal Name:Toxicological Sciences
Publisher:Oxford University Press
ISSN:1096-6080
ISSN (Online):1096-0929
Published Online:11 September 2016
Copyright Holders:Copyright © 2016 The Authors
First Published:First published in Toxicological Sciences 154(2):320-331
Publisher Policy:Reproduced under a Creative Commons License

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