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, 13 (2), 394-404

Phenotype-Based High-Throughput Classification of Long QT Syndrome Subtypes Using Human Induced Pluripotent Stem Cells

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Phenotype-Based High-Throughput Classification of Long QT Syndrome Subtypes Using Human Induced Pluripotent Stem Cells

Daisuke Yoshinaga et al. Stem Cell Reports.

Abstract

For long QT syndrome (LQTS), recent progress in genome-sequencing technologies enabled the identification of rare genomic variants with diagnostic, prognostic, and therapeutic implications. However, pathogenic stratification of the identified variants remains challenging, especially in variants of uncertain significance. This study aimed to propose a phenotypic cell-based diagnostic assay for identifying LQTS to recognize pathogenic variants in a high-throughput manner suitable for screening. We investigated the response of LQT2-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) following IKr blockade using a multi-electrode array, finding that the response to IKr blockade was significantly smaller than in Control-iPSC-CMs. Furthermore, we found that LQT1-iPSC-CMs and LQT3-iPSC-CMs could be distinguished from Control-iPSC-CMs by IKs blockade and INa blockade, respectively. This strategy might be helpful in compensating for the shortcomings of genetic testing of LQTS patients.

Keywords: genome editing; induced pluripotent stem cell; long QT syndrome; multi-electrode array; phenotype-based diagnosis.

Figures

Figure 1
Figure 1
Introduction of iPSC Lines Used in This Study (A) Summary of topologies of mutations of iPSC lines used in this study. LQT1A344Aspl carries a heterozygous KCNQ1 mutation (c.1032C > A, p.A344Aspl); LQT2A422T and LQT2A422T-corr are the isogenic pair harboring the heterozygous KCNH2 mutation (c.1264G > A, p.A422T) and the corrected sequence, respectively; LQT2G601S carries a heterozygous KCNH2 mutation (c.1801G > A, p.G601S); and LQT3N406K and LQT3corr are the isogenic pair harboring the heterozygous SCN5A mutation (c.1218C > A, p.N406K) and the corrected sequence, respectively. (B) Sequence analysis of PCR-amplified genomic DNA of the isogenic pair of LQT2A422T and LQT2 A422T-corr and of LQT3N406K and LQT3corr, respectively. The gene-corrected cell lines harbor several silent mutations (white arrow head) to avoid further digestion by CRISPR/Cas9.
Figure 2
Figure 2
Functional Analysis of iPSC-CMs Using MEA Following IKr Blockade (A) Representative traces of FP in Control-, LQT2A422T-, LQT2A422T-corr-, and LQT2G601S-iPSC-CMs. (B) FPDc baseline data in Control-, LQT2A422T-, LQT2corr-, and LQT2G601S-iPSC-CMs (independent experiments, n = 45, 35, 12, and 9 from independent differentiation experiments, n = 16, 11, 6, and 3, respectively; mean ± SEM; p = 0.001; one-way ANOVA). p < 0.05, Fisher's LSD post hoc test. (C) Representative traces of the FPD following administration of 30 nmol/L (red), 100 nmol/L (green), and 300 nmol/L (blue) E4031. (D) Averaged FPDc ratio before and after E4031 treatment (%ΔFPDc) in Control-, LQT2A422T-, LQT2A422T-corr-, LQT2G601S-, and LQT1A344Aspl-iPSC-CMs. FPDc prolongation upon treatment with 100 and 300 nmol/L E4031 was smaller in LQT2A422T- and LQT2G601S-iPSC-CMs than in Control-, LQT2corr-, or LQT1A344Aspl-iPSC-CMs (independent experiments, n = 23, 24, 12, 9, and 12 from independent differentiation experiments, n = 8, 8, 6, 3, and 3 in Control-, LQT2A422T-, LQT2A422T-corr-, LQT2G601S-, and LQT1A344Aspl-iPSC-CMs, respectively; mean ± SEM; p = 0.025; two-way repeated measures ANOVA). p < 0.05, Fisher's LSD post hoc test. (E) Examples of EADs. EAD documented in a sample of LQT2A422T-iPSC-CMs before E4031 treatment (upper) and following 30 nmol/L E4031 treatment (lower). (F) Percentage of samples in which EADs occurred in Control-, LQT2A422T-, LQT2A422T-corr-, and LQT2G601S-iPSC-CMs. The number on top of each bar shows the number of arrhythmic events. ∗p < 0.05; Pearson's Chi-square test. See also Figure S1.
Figure 3
Figure 3
Electrophysiological Properties of iPSC-CMs and AP Response to IKr Blockade (A) Representative current traces of the IKr in Control-, LQT2A422T-, and LQT2A422T-corr-iPSC-CMs. (B) Average current-voltage relationships for peak tail currents in Control-, LQT2A422T-, and LQT2A422T-corr-iPSC-CMs (independent experiments, n = 8, 6, and 5 from independent differentiation experiments, n = 4, 4, and 3, respectively; mean ± SEM; p = 0.029; two-way repeated measures ANOVA). p < 0.05, Fisher's LSD post hoc test for Control versus LQT2A422T; †p < 0.05, LQT2A422T-corr versus LQT2A422T. (C) Representative traces of AP with 1-Hz pacing. (D) APD50 and APD90 in Control-, LQT2A422T-, and LQT2A422T-corr-iPSC-CMs (independent experiments, n = 6, 9, and 11, from independent differentiation experiments, n = 5, 6, and 5, respectively; mean ± SEM; p = 0.010 and p = 0.025 for APD50 and APD90, respectively; one-way ANOVA). p < 0.05, Fisher's LSD post hoc test for APD50 and APD90. (E) Representative AP traces changed by IKr blockade. (F) Percentage of APD prolongation after E4031 treatment (%ΔAPD) at 1-Hz pacing in each cell line. %ΔAPD50 and %ΔAPD90 in Control-iPSC-CMs and LQT2A422T-corr-iPSC-CMs versus LQT2A422T-iPSC-CMs (independent experiments, n = 7, 10, and 10 from independent differentiation experiments, n = 5, 5, and 6 in Control-, LQT2A422T-, and LQT2A422T-corr-iPSC-CMs, respectively; mean ± SEM; p = 0.001; two-way repeated measures ANOVA). p < 0.05, Fisher's LSD post hoc test. See also Figure S2. APA, action potential amplitude; MDP, maximum diastolic potential.
Figure 4
Figure 4
Functional Analysis of iPSC-CMs Using MEA Following IKs Blockade (A) Representative traces of FP in Control- and LQT1A344Aspl-iPSC-CMs. (B) FPDc at baseline was longer in LQT1A344Aspl-iPSC-CMs than in Control-iPSC-CMs (independent experiments, n = 45 and 21 from independent differentiation experiments, n = 16 and 6 in Control- and LQT1A344Aspl-iPSC-CMs, respectively; mean ± SEM; p < 0.05; unpaired Student's t test). p < 0.05. (C) Representative traces of the FPD following administration of 10 μmol/L (red), 50 μmol/L (green), and 100 μmol/L (blue) chromanol 293B in Control- and LQT1A344Aspl-iPSC-CMs. (D) Averaged %ΔFPDc in each cell line. %ΔFPDc upon treatment with 100 μmol/L chromanol 293B was significantly smaller in LQT1A344Aspl-iPSC-CMs than in Control-, LQT2A422T-, and LQT3N406K-iPSC-CMs (independent experiments, n = 6, 13, 6, and 9 from independent differentiation experiments, n = 3, 5, 3, and 3 in Control-, LQT1A344Aspl-, LQT2A422T-, and LQT3N406K-iPSC-CMs, respectively; mean ± SEM; p = 0.001; two-way repeated measures ANOVA). p < 0.05; Fisher's LSD post hoc test.
Figure 5
Figure 5
Functional Analysis of iPSC-CMs Using MEA Following INa Blockade (A) Representative traces of FP in LQT3N406K- and LQT3corr-iPSC-CMs (left). FPDc at baseline in LQT3N406K-iPSC-CMs was significantly shortened by gene correction (right) (independent experiments, n = 36 and 29 from independent differentiation experiments, n = 8 and 8 in LQT3N406K- and LQT3corr-iPSC-CMs, respectively; mean ± SEM; p < 0.05; unpaired Student's t test). p < 0.05. (B) Representative traces of the FP following administration of 400 nmol/L (red) TTX in Control-, LQT3N406K-, LQT3corr-, LQT1A344Aspl-, and LQT2A422T-iPSC-CMs (left). The response to treatment with 400 nmol/L TTX was significantly larger in LQT3N406K-iPSC-CMs than in Control-, LQT3corr-, LQT1A344Aspl-, and LQT2A422T-iPSC-CMs (right) (independent experiments, n = 9, 11, 17, 5, and 5 from independent differentiation experiments, n = 4, 5, 4, 3, and 3 in Control-, LQT3N406K-, LQT3corr-, LQT1A344Aspl-, and LQT2A422T-iPSC-CMs, respectively; mean ± SEM; p < 0.001; one-way ANOVA). p < 0.05; Fisher's LSD post hoc test. (C) Averaged %ΔFPDc on 10 μmol/L mexiletine in Control-, LQT3N406K-, and LQT3corr-iPSC-CMs (independent experiments, n = 7, 11, and 12 from independent differentiation experiments, n = 3, 3, and 5, respectively; mean ± SEM; p < 0.001; one-way ANOVA). p < 0.05; Fisher's LSD post hoc test. (D) Comparison of AUCs for baseline FPDc and %ΔFPDc upon specific current blockade for recognizing disease-specific iPSC-CMs. Specific current blockade enhanced the accuracy of recognizing disease-specific iPSC-CMs. See also Table S1.

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References

    1. Asakura K., Hayashi S., Ojima A., Taniguchi T., Miyamoto N., Nakamori C., Nagasawa C., Kitamura T., Osada T., Honda Y. Improvement of acquisition and analysis methods in multi-electrode array experiments with iPS cell-derived cardiomyocytes. J. Pharmacol. Toxicol. Methods. 2015;75:17–26. - PubMed
    1. Bett G.C.L., Morales M.J., Beahm D.L., Duffey M.E., Rasmusson R.L. Ancillary subunits and stimulation frequency determine the potency of chromanol 293B block of the KCNQ1 potassium channel. J. Physiol. 2006;576:755–767. - PMC - PubMed
    1. Bett G.C.L., Kaplan A.D., Lis A., Cimato T.R., Tzanakakis E.S., Zhou Q., Morales M.J., Rasmusson R.L. Electronic “expression” of the inward rectifier in cardiocytes derived from human-induced pluripotent stem cells. Heart Rhythm. 2013;10:1903–1910. - PMC - PubMed
    1. Egashira T., Yuasa S., Fukuda K. Induced pluripotent stem cells in cardiovascular medicine. Stem Cells Int. 2011;2011:348960. - PMC - PubMed
    1. Giudicessi J.R., Ackerman M.J. Genotype- and phenotype-guided management of congenital long QT syndrome. Curr. Probl. Cardiol. 2013;38:417–455. - PMC - PubMed

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