On-chip spatiotemporal electrophysiological analysis of human stem cell derived cardiomyocytes enables quantitative assessment of proarrhythmia in drug development

Abstract

We examined a simultaneous combined spatiotemporal field potential duration (FPD) and cell-to-cell conduction time (CT) in lined-up shaped human embryonic stem cell-derived cardiomyocytes (hESC-CMs) using an on-chip multielectrode array (MEA) system to evaluate two origins of lethal arrhythmia, repolarization and depolarization. The repolarization index, FPD, was prolonged by E-4031 and astemizole, and shortened by verapamil, flecainide and terfenadine at 10 times higher than therapeutic plasma concentrations of each drug, but it did not change after lidocaine treatment up to 100 μM. CT was increased by astemizol, flecainide, terfenadine, and lidocaine at equivalent concentrations of Nav1.5 IC₅₀, suggesting that CT may be an index of cardiac depolarization because the increase in CT (i.e., decrease in cell-to-cell conduction speed) was relevant to Nav1.5 inhibition. Fluctuations (short-term variability; STV) of FPD and CT, STV_FPD and STV_CT also discriminated between torsadogenic and non-torsadogenic compounds with significant increases in their fluctuation values, enabling precise prediction of arrhythmogenic risk as potential new indices.

Figure 1

Relationship of the external field potential and action potential of cells. (A) Micrograph shows an example of electrophysiological measurement set-up of external field potential (FP: with 10 μm multi electrode array) and action potential (AP: patch clamp) of single cardiomyocytes. (B) Examples of action potentials of three types of single cardiomyocytes (atrial (left), ventricle (center), sinus node (right)), and their time differential plots (−dV/dt). (C) Typical FP signal of a single cardiomyocyte on a 10 μm microelectrode. (D) Examples of FPs of three types of cardiomyocytes.

Figure 2

Experimental designs of temporal and spatial fluctuation measurement. (A) Preparation of linearly-craved MEA chip with collagen coating. A schematic drawing of MEA chip for the lined-up hESC-CMs. (B) Time-course of cell culture and drug administration. FP waveforms were recorded during 10 min of drug exposure, and last 50 waveforms of obtained data were analyzed to calculate cFPD, STV_cFPD, CT and STV_CT. (C) Assay parameters measured in the multi-electrode array (MEA) system. cFPD: field potential duration corrected by Bazett’s formula, CT: conduction time which is time difference of first peak from that of an adjacent channel, STV_cFPD: Short term variability of cFPD. The image of fluctuations of cFPD and CT was shown as Poincaré plottings. (D) Representative change in lined-up hESC-CMs with administration of E-4031 (hERG blocker).

Figure 3

Relationship of FPD or cFPD and inter-spike interval (ISI). Scatter plots of ISI and FPD (A), cFPD corrected by Bazett’s formulae (FPD_cB) (B), and cFPD corrected by Fridericia’s formulae (FPD_cF) (C) in line-up hESC-CMs. All data from vehicle control was plotted. Bazett’s and Fridericia’s formulae for correcting FPD are given as FPD/(ISI)^1/2 and FPD/(RR)^1/3, respectively.

Figure 4

Effects of the arrhythmic drugs to the repolarization function and its temporal fluctuation on the line-shaped model using hESC-CMs. Dose-dependent changes of the field potential duration corrected by beating frequency using Bazett’s formula (cFPD, blue filled circles and lines) and its short-term variability (STV_cFPD, red open triangles and lines) are plotted on DMSO as a vehicle control (A), E-4031 as a I_Kr blocker (B), verapamil as a Cav1.2 blocker (C), lidocine and flecainide as I_Na blockers (D and E), astemizole and terfenadine as multi-ion channel blockers (F and G). cFPD and STV_cFPD were normalized to control condition. Symbols and error bars indicate mean values and standerd error (SE). 10% cFPD prolongation (cFPD > 1.1) and 90% STV_cFPD increase (STV_cFPD > 1.9) are shown in blue or red dashed lines, respectively. The green, orange, and purple bars show hERG, Cav1.2, and Nav1.5 IC₅₀ of each drug, respectively.

Figure 5

Effects of arrhythmic drugs on conductivity and its temporal fluctuation in the line-shaped model using hESC-CMs. Dose-dependent changes in the conduction time (CT) between two neighboring electrodes with a 150 μm of inter-electrode distance and its short-term variability (STV_CT, red open triangles and lines) are plotted for DMSO as a vehicle control (A), E-4031 as a I_Kr blocker (B), verapamil as a Cav1.2 blocker (C), lidocine and flecainide as I_Na blockers (D and E), astemizole and terfenadine as multi-ion channel blockers (F and G). CT and STV_CT were normalized to control condition. Symbols and error bars indicate mean values and standard error (SE). A 40% CT increase (CT > 1.4) and 130% STV_cFPD increase (STV_cFPD > 2.3) are shown iby blue and red dashed lines, respectively. The green, orange, and purple bars show hERG, Cav1.2, and Nav1.5 IC₅₀ of each drug, respectively.

Figure 6

Summary of the effects of overdosed cardiotoxic compounds on cFPD, CT, and their STV of hESC-CMs. cFPD vs. STV_cFPD (A), CT vs. STV_CT (B) and STV_cFPD vs. STV_CT (C). All parameters were normalized to the control condition before addition of drugs. The concentration of the compounds were higher than therapeutic C_max. E-4031; 1 μM, astemizole (Ast): 1 μM, flecainide (Fle): 10² μM, terfenadine (Ter): 10 μM, lldocaine (Lid): 10² μM, verapamil (Ver): 10 μM. Red symbols: positive compounds; purple symbols: false negative compounds; green symbols: negative compounds; blue symbols: vehicle. Square symbols: I_Kr, or I_Ca blocker; triangle symbols: I_Na blockers; diamond symbols: multi-ion channel blockers. Red dashed line in (A) shows a 1.9 risk value in the cluster model shown by Kaneko et al.. The red dashed lines in (B and C) were determined by the maximum differences from the line to the nearest point in each negative and positive compound data (equal) (Excel 2013 solver).