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

Figures

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  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.

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  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, STVcFPD, CT and STVCT. (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, STVcFPD: 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).

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  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 (FPDcB) (B), and cFPD corrected by Fridericia’s formulae (FPDcF) (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.

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  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 (STVcFPD, red open triangles and lines) are plotted on DMSO as a vehicle control (A), E-4031 as a IKr blocker (B), verapamil as a Cav1.2 blocker (C), lidocine and flecainide as INa blockers (D and E), astemizole and terfenadine as multi-ion channel blockers (F and G). cFPD and STVcFPD were normalized to control condition. Symbols and error bars indicate mean values and standerd error (SE). 10% cFPD prolongation (cFPD > 1.1) and 90% STVcFPD increase (STVcFPD > 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.

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  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 (STVCT, red open triangles and lines) are plotted for DMSO as a vehicle control (A), E-4031 as a IKr blocker (B), verapamil as a Cav1.2 blocker (C), lidocine and flecainide as INa blockers (D and E), astemizole and terfenadine as multi-ion channel blockers (F and G). CT and STVCT 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% STVcFPD increase (STVcFPD > 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.

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  Figure 6. Summary of the effects of overdosed cardiotoxic compounds on cFPD, CT, and their STV of hESC-CMs. cFPD vs. STVcFPD (A), CT vs. STVCT (B) and STVcFPD vs. STVCT (C). All parameters were normalized to the control condition before addition of drugs. The concentration of the compounds were higher than therapeutic Cmax. 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: IKr, or ICa blocker; triangle symbols: INa 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.40. 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).