Action potential variability in human pluripotent stem cell-derived cardiomyocytes obtained from healthy donors

Abstract

Human pluripotent stem cells (PSC) have been used for disease modelling, after differentiation into the desired cell type. Electrophysiologic properties of cardiomyocytes derived from pluripotent stem cells are extensively used to model cardiac arrhythmias, in cardiomyopathies and channelopathies. This requires strict control of the multiple variables that can influence the electrical properties of these cells. In this article, we report the action potential variability of 780 cardiomyocytes derived from pluripotent stem cells obtained from six healthy donors. We analyze the overall distribution of action potential (AP) data, the distribution of action potential data per cell line, per differentiation protocol and batch. This analysis indicates that even using the same cell line and differentiation protocol, the differentiation batch still affects the results. This variability has important implications in modeling arrhythmias and imputing pathogenicity to variants encountered in patients with arrhythmic diseases. We conclude that even when using isogenic cell lines to ascertain pathogenicity to variants associated to arrythmias one should use cardiomyocytes derived from pluripotent stem cells using the same differentiation protocol and batch and pace the cells or use only cells that have very similar spontaneous beat rates. Otherwise, one may find phenotypic variability that is not attributable to pathogenic variants.

Keywords: action potential (AP); cardiomyocytes; cell lines; differentiation batches; differentiation methods; healthy donors; iPSC (induced pluripotent stem cell); variability.

FIGURE 1

Density distribution of action potential parameters from PSC-derived cardiomyocytes. (A) Maximum diastolic potentials (MDP), (B) action potential amplitudes (APA), (C) maximum dV/dt (dV/dt max), (D) action potential duration at 90% repolarization (APD90) and (E) cycle length are shown for the whole dataset, comprising 780 electrophysiological recordings.

FIGURE 2

Comparison of electrophysiologic parameters between ES and iPS-derived cardiomyocytes. (A) Maximum diastolic potentials (MDP), (B) action potential amplitudes (APA), (C) maximum dV/dt (dV/dt max), (D) action potential duration at 90% repolarization (APD90) and (E) cycle length are shown. It is important to note that differentiation protocols varied between the two cell types, and herefore the significance of the differences may be attributable to the varying protocols used for differentiation. # indicates p < 0.05 using Student’s t-test.

FIGURE 3

Comparison of electrophysiologic parameters of cardiomyocytes differentiated from individual cell lines. (A) Maximum diastolic potentials (MDP), (B) action potential amplitudes (APA), (C) maximum dV/dt (dV/dt max), (D) action potential duration at 90% repolarization (APD90) and (E) cycle length are shown. # indicates p < 0.05 using one-way ANOVA followed by Tukey’s post test. Significant differences were observed in the following parameters: MDP (red: 1 vs. 3, orange: 4 vs. 2, 3 and 6), APA (red: 1 vs. 2, 4 and 5, green: 3 vs. 2, 4, 5 and 6, pink: 5 vs. 1, 2 and 3), dV/dt max (red: 1 vs. 2, 4 and 5, green: 3 vs. 2, 4 and 5, purple: 6 vs. 2, 4 and 5), APD90 (red: 1 vs. 3, 5 and 6, blue: 2 vs. 3, 5 and 6, green: 3 vs. all lines, orange: 4 vs. 3, 5 and 6, pink: 5 vs. all lines), cycle length (red: 1 vs. all lines, blue: 2 vs. 1, 3 and 5, green: 3 vs. 1, 2, 4 and 6, orange: 4 vs. 1, 3 and 5, pink: 5 vs. 1, 2, 4 and 6).

FIGURE 4

Correlation of action potential variables. Scatter plots of action potential amplitudes (APA) and maximum dV/dt (dV/dt max) as a function of maximum diastolic potential (MDP) are shown in (A,B), respectively. (C) shows a scatter plot of action potential duration at 90% repolarization (APD90) as a function of cycle length. All correlations are statistically significant although correlation coefficients are low. Heteroscedasticity increases with cycle length especially above 2,000 ms.

FIGURE 5

Effect of differentiation batch on action potential parameters. Box plots of (A–C) maximum diastolic potentials (MDP), (D–F) action potential amplitudes (APA), (G–I) maximum dV/dt (dV/dt max), (J–L) action potential duration at 90% repolarization (APD90) and (M–O) cycle length are shown for three different cell lines. For line 1, all batches used Kattman’s differentiation protocol. For lines 2 and 4, Lian’s protocol was used for all batches.

FIGURE 6

Comparison of action potential variables using two differentiation protocols in the same cell line. Box plots of (A) maximum diastolic potentials (MDP), (B) action potential amplitudes (APA), (C) maximum dV/dt (dV/dt max), (D) action potential duration at 90% repolarization (APD90) and (E) cycle length for cell line 2 using Lian’s and a commercial protocol. Red # indicates p < 0.05 using Student’s t-test.

FIGURE 7

Action potential duration at 90% repolarization (APD90) under spontaneous and pacing conditions. Box plots in (A) shows significant differences in APD90 between PSC-derived cardiomyocytes beating spontaneously and paced at 1 Hz. In (B), using only spontaneous cycle lengths varying between 0.9 and 1.1 Hz, similar values for APD90 are obtained under both conditions.

FIGURE 8

(A) Maximum diastolic potentials (MDP), (B) action potential amplitudes (APA), (C) maximum dV/dt (dV/dt max), (D) action potential duration at 90% repolarization (APD90) and (E) cycle length as a function of differentiation time. Cardiomyocytes derived from PSC differentiated for less or more than 30 days show significant increases in APA, dV/dt max and APD90 with longer differentiation times.