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. 2022 Apr 8;119(1):167–182. doi: 10.1093/cvr/cvac059

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© The Author(s) 2022. Published by Oxford University Press on behalf of the European Society of Cardiology.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Electrical maturation of hiPSC-CMs in 3D cardiac microtissues with hiPSC-derived non-myocytes reveals functional effects of p.R225W SCN5A mutation. (A) Schematic of the microtissue formation using hiPSC-CMs from four lines (hiPSC^corr/R225W, hiPSC^W156X/R225W, hiPSC^corr/corr, hiPSC^WT/WT) and ECs and CFs from hiPSC^WT/WT. The percentage of each cell type is indicated. (B) Schematic of 21-day MT culture protocol until single-cell dissociation for analysis. (C) Representative I_Na traces recorded in hiPSC^corr/R225W-, hiPSC^W156X/R225W-, hiPSC^corr/corr-, and hiPSC^WT/WT-CMs dissociated from MTs, corresponding to the voltage steps reported on the right. In the inset, the current at −40 mV normalized to the peak current, to compare the fraction of open channels around the V_1/2 of activation. (D) Activation (AC) and inactivation (IC) curve of I_Na recorded in hiPSC^corr/R225W- (black; AC: V_1/2 = −38.1 ± 1.2 mV; IC: V_1/2 = −80.6 ± 1.1 mV; n = 16), hiPSC^W156X/R225W- (red; AC: V_1/2 = −32.4 ± 1.2 mV*, n = 11; IC: V_1/2 = −77.3 ± 1.4 mV, n = 9), hiPSC^corr/corr- (blue; AC: V_1/2 = −39.3 ± 0.9 mV, n = 17; IC: V_1/2 = −77.9 ± 1.2 mV; n = 16), and hiPSC^WT/WT- (light blue; AC: V_1/2 = −36.5 ± 0.9 mV, n = 8; IC: V_1/2 = −80.8 ± 1.3 mV, n = 7) CMs showing a rightward shift in I_Na activation in hiPSC^W156X/R225W-CMs. *P < 0.05 vs. the other lines with one-way ANOVA and Fisher’s post hoc test. Experiments = 3. (E) Mean voltage-density plot of I_Na recorded in hiPSC^corr/R225W- (black), hiPSC^W156X/R225W- (red), hiPSC^corr/corr- (blue), and hiPSC^WT/WT- (light blue) CMs, showing a reduction in current density in hiPSC^W156X/R225W- and hiPSC^corr/R225W-CMs compared with the corrected and WT line and a shift in the activation of hiPSC^W156X/R225W-CMs. From −45 to −20 mV: P < 0.05 for hiPSC^W156X/R225W and for hiPSC^corr/corr vs. the other lines with two-way ANOVA repeated measures. Experiments = 3. (F) Representative AP traces recorded with dynamic clamp from hiPSC^corr/R225W- (black), hiPSC^W156X/R225W- (red), hiPSC^corr/corr- (blue), and hiPSC^WT/WT- (light blue) CMs paced at 1 Hz. The inset indicates the respective derivative trace of the AP after the stimulus. (G) Box plot of mean V_max of APs recorded in hiPSC^corr/R225W- (black, n = 23), hiPSC^W156X/R225W- (red, n = 22), hiPSC^corr/corr- (blue, n = 17), and hiPSC^WT/WT- (light blue, n = 20) CMs. Dots: single values, lines: median, squares: mean, error bars: 1.5× IQR. *P < 0.05 vs. the other lines with one-way ANOVA and Fisher’s post hoc test. Experiments = 4.