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

Abstract

The hERG1 potassium channel conducts the cardiac repolarizing current, IKr. hERG1 has emerged as a therapeutic target for cardiac diseases marked by prolonged action potential duration (APD). Unfortunately, many hERG1 activators display off-target and proarrhythmic effects that limit their therapeutic potential. A Per-Arnt-Sim (PAS) domain in the hERG1 N-terminus reduces IKr by slowing channel activation and promoting inactivation. Disrupting PAS activity increases IKr and shortens APD in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We thus hypothesized that the hERG1 PAS domain could represent a therapeutic target to reduce arrhythmogenic potential in a long QT syndrome (LQTS) background. To test this, we measured the antiarrhythmic capacity of a PAS-disabling single-chain variable fragment antibody, scFv2.10, in a hiPSC-CM line derived from a patient with Jervell and Lange Nielsen (JLN) syndrome. JLN is a severe form of LQTS caused by autosomal recessive mutations in KCNQ1. The patient in this study carried compound heterozygous mutations in KCNQ1. Corresponding JLN hiPSC-CMs displayed prolonged APD and early afterdepolarizations (EADs). Disrupting PAS with scFv2.10 increased IKr, shortened APD, and reduced the incidence of EADs. These data demonstrate that the hERG1 PAS domain could serve as a therapeutic target to treat disorders of cardiac electrical dysfunction.

Keywords: Arrhythmias; Cardiology; Ion channels; Potassium channels.

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.

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 I_Ks traces recorded from df19.9.11 (top) and JLN (bottom) hiPSC-CMs. Steady-state I_Ks 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.

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.

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.

Figure 5. scFv2.10 transduction selectively increases I_Kr density in JLN hiPSC-CMs.

(A) Representative I_Kr traces from JLN hiPSC-CMs transduced with GFP or scFv2.10 elicited by the pulse protocol shown in the inset. (B) Steady-state I_Kr recorded from GFP-transduced (green circles) and scFv2.10-transduced (magenta squares) hiPSC-CMs plotted as a function of prepulse potential. (C) Peak tail I_Kr 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 I_Ca traces from JLN hiPSC-CMs transduced with GFP or scFv2.10. (F) I_Ca 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 I_Kr n values (N = 2) are 7 and 10 for GFP and scFv2.10 transduced hiPSC-CMs, respectively. I_Ca n value (N = 2) is 10 for GFP and 9 for scFv2.10 transduced hiPSC-CMs. Data are presented as mean ± SEM.

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.

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.