R534C mutation in hERG causes a trafficking defect in iPSC-derived cardiomyocytes from patients with type 2 long QT syndrome

Figures

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  Figure 1. Differentiation and electrophysiology of iPSC-derived cardiomyocytes. (a) Schematic diagram demonstrating the main steps of the differentiation procedure. (b) Representative action potential recordings of spontaneously contracting ventricular-like cardiomyocytes. Note the red line that marks the end of phase 3 for CTRL-iPSC and the green line that marks the end of phase 3 for LQTS2-iPSC1 and LQTS2-iPSC2. (c,d) Our analysis demonstrates that action potential duration of LQTS2-iPSC1, 2 and CRISPR was significantly longer than that of CTRL-iPSC, as was the triangulation of action potentials (e), implying a longer duration of phase 3. CTRL-iPSC (n = 116); LQTS2-iPSC1 (n = 59); LQTS2-iPSC2 (n = 77); LQTS2-CRISPR (n = 20) from 8 independent differentiations for each cell line. Box plots represent 1st quartile, median and 3rd quartile. Whiskers represent minimum and maximum values. “+” represents the mean for each cell line. (f) Treatment with E4031 caused APD prolongation only in CTRL-iPSC, indicating that hERG does not contribute to repolarization in LQTS2- and CRISPR-iPSC (n = 4). Bars represent mean and SEM. *p < 0.05, ****p < 0.0001.

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  Figure 2. Patch clamp of iPSC-derived cardiomyocytes. (a) Representative patch clamp recordings of potassium currents before and after treatment with E4031 in all groups. These currents were subtracted to obtain IKr (E4031-sensitive IK). (b) I-V graph showing IKr current densities for all cell lines. Control cardiomyocytes had significantly higher currents at 10, 20 and 30 mV compared to LQTS2 cardiomyocytes (*p < 0.05). (c) Average IKr tail current normalized to the maximal current following repolarization to −40 mV in CTRL-iPSC, LQTS2-iPSC1 and LQTS2-iPSC2. Voltage protocol used for patch clamp recordings of the representative currents is also shown.

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  Figure 3. Characterization of hERG trafficking defect in LQTS2-iPSC cardiomyocytes. (a) Immunostaining for sarcomeric α-actinin (red) and hERG (green) in hiPSC-CM. Control cardiomyocytes exhibit membrane and intracellular hERG localization. A higher magnification of the membrane region marked by the dashed rectangle is shown on the right. Observe that, in LQTS2-iPSCs and LQTS2-CRISPR, hERG staining is reduced when compared to control. Quantification of hERG fluorescence intensity signal in the membrane (b) and intracellular space (c). CTRL-iPSC (n = 25); LQTS2-iPSC1 (n = 22); LQTS2-iPSC2 (n = 20); LQTS2-CRISPR (n = 17). Box plots represent 1st quartile, median and 3rd quartile. Whiskers represent minimum and maximum values. **p < 0.01, ****p < 0.0001. Scale bar: 10 µm.