A Preclinical Study on Brugada Syndrome with a CACNB2 Variant Using Human Cardiomyocytes from Induced Pluripotent Stem Cells

Figures

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  Figure 1. Clinical characteristics of the BrS patient. (A) The pedigree of the BrS patient’s family. The patient recruited for this study is marked by the arrow. He has had sudden cardiac death. (B) The electrocardiogram (ECG) from the BrS patient shows a classic BrS–ECG pattern after infusion of ajmaline. (C) Electrocardiogram of the ICD showing ventricular fibrillation terminated with an appropriate ICD shock after stopping the bisoprolol treatment.

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  Figure 2. Genetic correction of BrS-hiPSCs by CRISPR/Cas9 genome editing. (A) Corrected hiPSCs were generated with a CRISPR guide RNA targeting the CACNB2 exon 4 and a single-stranded oligonucleotide (ssODN) for homology-directed repair. (B) Confirmation of genetic correction, assessed by Sanger sequencing of genomic DNA. (C) Patients’ and CRISPR-corrected hiPSC lines exhibited a typical human stem cell-like morphology. Scale bar: 100 μm. (D) Immunofluorescence staining for key pluripotency markers OCT4, NANOG and TRA1–60 in patients’ and corrected hiPSC lines. Nuclei were counter-stained with DAPI. Scale bar: 100 μm. (E) Purity of patient-specific and CRISPR-corrected hiPSC lines was evaluated by the flow cytometry analysis of pluripotency markers OCT4 and TRA1–60. Gray dots represent the negative controls.

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  Figure 3. Reduction of CACNB2 protein expression level in hiPSC-CMs from the BrS-patient. Western blot and immunostaining analyses were performed to examine the protein levels of CACNB2 in hiPSC-CMs from healthy donor (Healthy), the patient (BrS) and isogenic control cell line (Corrected). (A) Representative immunostaining images. (B) Statistical analyses of fluorescence intensity from immunostained cells. (C) Representative examples of Western blots of cell lysates from healthy donor cell line (healthy), the patient (BrS) and the CRISPR/Cas9-corrected (Corrected) hiPSC-CMs. (D) Statistical analyses of relative expression levels of SCN5A and CACNB2 from Western blot experiments. Data were shown as mean ± standard deviation (SD). Numbers given represent the number of cells (B) or experiments (D). * p < 0.05 versus healthy analyzed by one-way analysis of variance (ANOVA) with Holm–Sidak post-test.

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  Figure 4. Reduction of L-type calcium channel currents in hiPSC-CMs from the BrS patient. Peak L-type calcium (ICa-L) and sodium channel (INa) currents were analyzed in hiPSC-CMs from healthy donors (Healthy), the BrS patient (BrS) and variant-corrected cells (Isogenic). (A) Representative traces of ICa-L in hiPSC-CMs from healthy donors (Healthy), the BrS patient (BrS) and variant-corrected cells (Isogenic). (B) I-V curves of ICa-L from healthy donors (Healthy), the BrS patient (BrS) and variant-corrected cells (Isogenic). (C) Median values of peak ICa-L at −10 mV from healthy donors (Healthy), the BrS patient (BrS) and variant-corrected cells (Isogenic). (D) Activation curves of ICa-L in hiPSC-CMs from each cell line. (E) Inactivation curves of ICa-L in hiPSC-CMs from each cell line. (F) Recovery curves of ICa-L in hiPSC-CMs from each cell line. (G) Median values of the potential at 50% activation (V0.5) of ICa-L in hiPSC-CMs from each cell line. (H) Median values of the potential at 50% inactivation (V0.5) of ICa-L in hiPSC-CMs from each cell line. (I) Median values of the time constants (Tau) of recovery from the inactivation of ICa-L in hiPSC-CMs from each cell line. Numbers given in…

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  Figure 5. Increased beating variability and arrhythmic events in BrS-hiPSC-CMs. Spontaneous calcium transients were recorded in spontaneously beating hiPSC-CMs from the BrS patient, the healthy donor (healthy) and the CRISPR-corrected cells (isogenic). The beating variability (standard deviation (SD) of cell beating intervals) and the occurrence of arrhythmic events (irregular or triggered beats or EAD-like events) were compared among the three cell groups. (A–C) Representative traces of calcium transients in cells from each line. Arrhythmic events are marked by arrows. (D) Median values of standard deviation (SD) of cell beating intervals. (E) Percentage of cells showing arrhythmic events. The numbers given represent number of cells showing arrhythmic events versus total cell number. The numbers given represent number of cells. * p < 0.05 versus healthy according to the one-way ANOVA (D) or Fisher’s exact test (E).

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  Figure 6. Effects of beta-blocker on the beating variability and arrhythmic events in BrS-hiPSC-CMs. Spontaneous calcium transients were recorded in regularly beating hiPSC-CMs from the BrS patient and the isogenic control(isogenic) in the absence (baseline) and presence of 30 nM, 300 nM and 3000 nM bisoprolol (Biso). The beating variability (standard deviation (SD) of cell beating intervals) and the occurrence of arrhythmic events (irregular or triggered beats or EAD-like events) were compared between with and without drug application. (A) Representative trace of BrS (at baseline) and after the administration of different concentrations of bisoprolol. (B) Mean values of standard deviation (SD) of cell beating intervals. (C) Percentage of cells showing arrhythmic events. * p < 0.05 versus isogenic according to Fisher’s exact test (C) or versus baseline according to t-test (B).

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  Figure 7. Effects of quinidine on the beating variability and arrhythmic events in BrS-hiPSC-CMs. (A) Representative traces of calcium transient before and after administration of 10 µM quinidine in BrS-hiPSC-CMs. Only cells with arrhythmic events were used. (B) Median values of standard deviation (SD) of cell beating intervals. (C) Percentage of cells showing arrhythmic events. * p < 0.05 using Fisher’s exact test (C) or versus baseline according to t-test (B).