Pathogenic KCNH2-G53S variant in the PAS domain influences the electrophysiological phenotype in long QT syndrome type 2

Figures

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  Figure 1. Characterization of the patient carrying KCNH2 p.G53S (A) pedigree of a family with autosomal dominant genetically elusive long QT syndrome (LQT). Squares and circles indicate male and female family members, respectively. Solid symbols indicate the presence of LQT. (B) Representative electrocardiogram (ECG) from the index case (QTc = 548 ms). (C) Episodes of ventricular fibrillation (VF) captured via implantable cardioverter-defibrillator (ICD) monitoring.

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  Figure 2. Schematic of the KCNH p.G53S mutation within the PAS domain (A) schematic representation of the c.157G>A mutation, located within the exon 2 region of KCNH2, and the location of p.G53S in the PAS domain of the hERG channel. (B) Sanger sequencing of the KCNH2G53S hiPSC line showing that it harbors the heterozygous mutation c.157G>A in the KCNH2 gene.

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  Figure 3. Generation of patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) (A) detailed protocol for differentiating pluripotent stem cells into ventricular cardiomyocytes. (B) qRT-PCR to detect GJA5A, NPPA, GJA1, and NPPB in control and KCNH2G53S hiPSC-CMs. (C) Representative immunostaining for NKX2.5, cardiac troponin I (cTNI), MLC2v, and MLC2a in control and KCNH2G53S hiPSC-CMs. Nuclei are stained with DAPI (blue). Scale bar, 100 µm. (D) Western blotting and quantification of MYL2, MYL7, and cardiac troponin I (cTNI). β-Actin is a loading control. (E) qRT-PCR of KCNH2 mRNA expression in control and KCNH2G53S hiPSC-CMs. (F) Western blotting and quantification of hERG in control and KCNH2G53S hiPSC-CMs. β-Actin is a loading control. (G) Immunofluorescence staining of control and KCNH2G53S hiPSC-CMs. Nuclei are stained with DAPI (blue). Scale bar, 50 µm. Data are the mean ± SD. *P < 0.05, **P < 0.01.

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  Figure 4. RNA sequencing of the transcriptome of KCNH2G53S hiPSC-CMs (A) heatmap of the differentially expressed genes (DEGs) in control and KCNH2G53S hiPSC-CMs (n = 3). (B) Volcano plot with yellow points representing upregulated DEGs and blue points representing downregulated DEGs. Grey points represent non-DEGs. (C) Number of genes representing upregulated DEGs and number of genes representing downregulated DEGs for comparison combinations. (D) GO pathway analysis of the DEGs. The plot shows the magnitude of the expression changes (log2 fold change) vs. the statistical significance (-log10 p-value).

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  Figure 5. Electrophysiological profiles of control and KCNH2G53S hiPSC-CMs (A) representative field potential tracings of control and KCNH2G53S hiPSC-CMs and corrected field potential duration (cFPD) of control and KCNH2G53S hiPSC-CMs recorded using a microelectrode array (MEA). (B) Mean beat period of control and KCNH2G53S hiPSC-CMs. (C) Spike amplitude measurement of control and KCNH2G53S hiPSC-CMs. (D) Conduction velocity map illustrating the propagation of electrical impulses between control and KCNH2G53S hiPSC-CMs. (E) Representative action potential traces of control and KCNH2G53S hiPSC-CMs. (F) Comparison of action potential duration at 90% repolarization (APD90) and (G) corrected action potential duration (cAPD90) of control and KCNH2G53S hiPSC-CMs. (H) Corrected action potential duration (cAPD90) of control and KCNH2G53S hiPSC-CMs treated with isoproterenol (100 nM) and propranolol (200 nM). Data are the mean ± SD. *P < 0.05, **P < 0.01.

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  Figure 6. Ca2+ homeostasis analysis in control and KCNH2G53S hiPSC-CMs (A) representative confocal line scans (upper) and normalized Ca2+ transients (lower), (B) normalized Ca2+ transient amplitude (ΔF/F0), and (C) normalized calcium wave duration from control and KCNH2-G53S cardiomyocytes. Data are the mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001.