A PAS-targeting hERG1 activator reduces arrhythmic events in Jervell and Lange-Nielsen syndrome patient-derived hiPSC-CMs

Figures

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  Figure 1. Identification and generation of a potentially novel JLN patient-specific pluripotent stem cell line. (A) Pedigree of the proband with JLN syndrome. (B) Schematic depicting the relative location of the W188X variant with a KCNQ1 subunit. (C) ECG tracings recorded from the proband before (left, QTc: 440 ms) and after (right, QTc: 540 ms) an exercise tolerance test. (D) df19.911 and JLN hiPSC immunolabeled for the pluripotency marker nanog (magenta) and DAPI (blue) to delineate the nuclei. Scale bar: 20 µM.

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  Figure 2. Validation of JLN-derived hiPSC-CMs. (A) Sample max intensity images of df19.9.11 (Control, top) and JLN (bottom) hiPSC-CMs depicting KCNQ1 (magenta), phalloidin (green), and DAPI (blue). Mean fluorescence is quantified (right). (B) Sample max intensity images of df19.911 (top) and JLN (bottom) hiPSC-CMs depicting hERG1 (magenta), phalloidin (green), and DAPI (blue). Mean fluorescence intensity quantified (right). (C) Sample IKs traces recorded from df19.9.11 (top) and JLN (bottom) hiPSC-CMs. Steady-state IKs density plotted as a function of prepulse potential for df19.911 (green) and JLN (magenta) hiPSC-CMs. P values were determined by unpaired, 2-tailed Student’s t test, or ordinary 2-way ANOVA (mixed methods) with multiple comparisons and Šidák post hoc test. **P < 0.01, ****P < 0.0001. Data are presented as mean ± SEM. Scale bar: 20 μm.

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  Figure 3. JLN hiPSC-CMs display prolonged APD90. (A) Representative traces of spontaneous action potentials recorded from df19.911 (green) and JLN (purple) hiPSC-CMs. (B–D) APD90 (B), resting membrane potential (C), and spontaneous firing frequency (D) recorded from df19.911 and JLN hiPSC-CMs. P values were determined by unpaired, 2-tailed Student’s t test. RMP, resting membrane potential. *P < 0.05. Data are presented as mean ± SEM.

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  Figure 4. scFv2.10 transduction accelerates gating in HEK293 cells stably expressing hERG1a. (A) Representative deactivation traces from GFP-transduced (green) or scFv2.10-transduced (purple) HEK 293 cells. Pulse protocol shown in inset. (B) Fast and slow time constants of deactivation measured at –110 mV. Data are presented as mean ± SEM. P values were determined by unpaired, 2-tailed Student’s t test. *P < 0.05. Data are presented as mean ± SEM.

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  Figure 5. scFv2.10 transduction selectively increases IKr density in JLN hiPSC-CMs. (A) Representative IKr traces from JLN hiPSC-CMs transduced with GFP or scFv2.10 elicited by the pulse protocol shown in the inset. (B) Steady-state IKr recorded from GFP-transduced (green circles) and scFv2.10-transduced (magenta squares) hiPSC-CMs plotted as a function of prepulse potential. (C) Peak tail IKr recorded from GFP-transduced (green circles) and scFv2.10-transduced (magenta squares) hiPSC-CMs plotted as a function of prepulse potential. (D) Fast and slow deactivation time constants recorded from JLN hiPSC-CMs at +20 mV. (E) Representative ICa traces from JLN hiPSC-CMs transduced with GFP or scFv2.10. (F) ICa plotted as a function of prepulse potential. P values were determined by unpaired, 2-tailed Student’s t test, or ordinary 2-way ANOVA (mixed methods) with multiple comparisons and Šidák post hoc test. *P < 0.05. Steady-state and Peak tail IKr n values (N = 2) are 7 and 10 for GFP and scFv2.10 transduced hiPSC-CMs, respectively. ICa n value (N = 2) is 10 for GFP and 9 for scFv2.10 transduced hiPSC-CMs. Data are presented as mean ± SEM.

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  Figure 6. scFv2.10 transduction shifts hERG1 subunit abundance. (A and B) Sample max intensity images of JLN hiPSC-CMs depicting hERG1a/1b (magenta), phalloidin (green), and DAPI (blue). scFv2.10 transduction significantly upregulated hERG1a (A) and downregulated hERG1b (B) compared with GFP controls. (C and D) Sample max intensity images of df19.9.11 hiPSC-CMs depicting hERG1a/1b (magenta), phalloidin (green), and DAPI (blue). scFv2.10 transduction of df19.9.11 hiPSC-CMs did not affect hERG1a immunofluorescence (C) but downregulated hERG1b immunofluorescence (D) compared with GFP controls. P values were determined by unpaired, 2-tailed Student’s t test. *P < 0.05, ***P < 0.001. Scale bar: 20 μm. Data are presented as median ± 95% CI.

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  Figure 7. scFv2.10 expression reduces markers of proarrhythmia in JLN hiPSC-CMs. (A) Representative AP recordings from JLN hiPSC-CMs transduced with GFP (green) or scFv2.10 (purple). Left, no-arrhythmia (No EAD); center, phase 2 EADs; right, phase 3 EADs. (B and C) APD90 and AP beat-to-beat variability calculated from AP recordings as shown in A. P values were determined by unpaired, 2-tailed Student’s t test. Data are presented as mean ± SEM. (D) Distribution of GFP-transduced and scFv2.10-transduced JLN hiPSC-CMs generating APs with EADS (Phase 2, blue; Phase 3, purple) or without EADs (green). The 2 groups were compared using a χ2 contingency test. *P < 0.05.