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. 2016 Dec;154(2):320-331.
doi: 10.1093/toxsci/kfw171. Epub 2016 Sep 11.

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

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Free PMC article

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

M P Hortigon-Vinagre et al. Toxicol Sci. .
Free PMC article

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/[Formula: see text] 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.

Keywords: action potential duration; drug screening; human induced pluripotent stem cell-derived cardiomyocytes; methods; stem cells; voltage sensitive dye.

Figures

FIG. 1
FIG. 1
Classification of “cellular arrhythmias”. Exemplar records of EAD-like events recorded from hiPSC-CM with distinct characteristics. Type A events are single transient depolarizations during the plateau phase of the AP. Type B events are oscillating or multiple depolarizing events during the plateau phase. Type C events are rapid depolarizing events during the late repolarization phase of the AP. Tachycardia-like events are sustained high frequency spontaneous activity (>2 Hz).
FIG. 2
FIG. 2
Baseline electrophysiological properties. Baseline action potential properties of Cor.4U cardiomyocytes (n = 168) and iCell Cardiomyocytes (n = 160) using the voltage-sensitive dye di-4-ANEPPS in combination with the optical platform CellOPTIQ. (A) Representative recordings of spontaneous activity in cellular monolayers in Cor.4U cells (upper panel) and iCell Cardiomyocytes (lower panel). Averaged APs from each cell population are also shown (right panels). (B) Histograms showing the distribution of values measured from individual wells from iCell Cardiomyocytes (n = 159) and Cor.4U cardiomyocytes (n = 161) along with the best-fit normal distribution and the associated mean and standard deviation values for the following parameters: (i) cycle length: iCell Cardiomyocytes = 1.5 ± 0.5 s; Cor.4U = 0.99 ± 0.14 s. (ii) APD90: iCell Cardiomyocytes = 427 ± 49; Cor.4U = 229 ± 15 ms. (iii) diastolic interval: iCell Cardiomyocytes = 1.07 ± 0.44 s; Cor.4U = 0.76 ± 0.13 s.
FIG. 3
FIG. 3
Rate dependence of action potential duration. (A) Example AP signals from iCell Cardiomyocytes in response to field stimulation at 0.6, 1, 2, and 3 Hz. (B) Mean APD90 (±SEM, n = 10) data describing the rate dependence of APD90 based on recordings from Cor.4U (grey points) and iCell (green points) cardiomyocytes in response to the range of stimulation rates described above. The solid lines through the data are a best-fit single exponential fits to the data: APD = APD0 [1 − exp (DI/tau)], where APD0 is the APD at maximum diastolic interval (DI). (C) Normalization of APD90 values are shown in panel B relative to APDmax at the longest DI. Solid line is the best-fit exponential: APD = 1 − exp(DI/0.12). (D) Relationship between average (±SD) of APD90 and cycle length (CL) during stimulation (solid circles) and during spontaneous activity (open circles) in Cor.4U cells. The solid line is the best-fit double exponential function: APD90 = APD0 + A1.exp (CL − CL0)/tau1 + A2.exp (CL − CL0)/tau2; where APD0 = 1.28 s, CL0 = 0.007, A1 = −1.45, tau1 = 0.0013, A2 = −1069, tau2 = 0.021; r2 = 0.93, P < .01. (E) Relationship between average (±SD) of APD90 and CL during stimulation (solid circles) and during spontaneous activity (open circles) in iCell Cardiomyocytes. The solid line is the best-fit double exponential function: APD90 = APD0+A1.exp (CL − CL0)/tau1 + A2.exp (CL − CL0)/tau2; where APD0 = 0.745, CL0 = 0.780, A1 = −0.177, t1 = 0.0007;A2 = −404.9, tau2 = 0.0027; r2 = 0.95 P<.01. (F) Average baseline repolarization profile for the 2 cell types corrected for differences in spontaneous rates (mean ± SD).
FIG. 4
FIG. 4
Action potential rise time characteristics. (A) Trise distribution profile from averaged recordings of di-4-ANEPPS ratio recordings from Cor.4U (n = 168) and iCell Cardiomyocytes (n = 160). Results expressed as median ± SD. iCell Cardiomyocytes = 5.7 ± 1.3 ms; Cor.4U = 6.45 ± 2.2 ms. Transforming the data to UVI (1/Trise)2 results in a normal distribution of the data. Results expressed as mean ± SD. iCell Cardiomyocytes = 0.029 ± 0.01 ms−1; Cor.4U = 0.024 ± 0.01 ms−1. (B) Indicated mean and standard deviation values are based on the best fit normal distribution.
FIG. 5
FIG. 5
Effect of nifedipine on action potential characteristics. (A) Example recordings from cells exposed to nifedipine. (B) The average relative (±SD, n = 4) change of APD50, APD90, and CL in response to nifedipine in Cor.4U cells with values corrected for differences in spontaneous rate. (C) Relative change of APD50, APD90, and CL in response to nifedipine in iCell Cardiomyocytes with values corrected for differences in spontaneous rate.
FIG. 6
FIG. 6
Effect of ranolazine on action potential characteristics. (A) Example recordings from cells exposed to ranolazine. (B) The average relative (±SD, n = 4) change of APD50, APD90, and CL in response to ranolazine in Cor.4U cells with values corrected for differences in spontaneous rate. (C) Relative change of APD50, APD90, and CL in response to ranolazine in iCell Cardiomyocytes with values corrected for differences in spontaneous rate. Note in some instances SD limits were obscured by point size.
FIG. 7
FIG. 7
Effect of E4031 on action potential characteristics. (A) Example recordings from cells exposed to E4031. (B) The average relative (±SD, n = 4) change of APD50, APD90, and CL in response to E4031 in Cor.4U cells with values corrected for differences in spontaneous rate. (C) Relative change of APD50, APD90, and CL in response to E4031 in iCell Cardiomyocytes with values corrected for differences in spontaneous rate. Note in some instances SD limits are obscured by point size.
FIG. 8
FIG. 8
Repolarization profiles (nifedipine, ranolazine, and E4031). Average rate corrected repolarization profiles for Cor.4U and iCell Cardiomyocytes in response to increasing concentrations of (i) nifedipine, (ii) ranolazine, and (iii) E4031 (mean ± SD, n = 4).
FIG. 9
FIG. 9
Incidence of cellular arrhythmias (Ranolazine and E4031). Summary of the presence, incidence, and type of cellular arrhythmias observed in Cor.4U and iCell Cardiomyocytes following ranolazine and E4031. (A) Presence and type of cellular arrhythmia (with exemplars). (B) Plot of drug concentration versus incidence of cellular arrhythmia with each point representing incidence in 20 s recording from a single well.

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