shRNAs Targeting a Common KCNQ1 Variant Could Alleviate Long-QT1 Disease Severity by Inhibiting a Mutant Allele

Abstract

Long-QT syndrome type 1 (LQT1) is caused by mutations in KCNQ1. Patients heterozygous for such a mutation co-assemble both mutant and wild-type KCNQ1-encoded subunits into tetrameric Kv7.1 potassium channels. Here, we investigated whether allele-specific inhibition of mutant KCNQ1 by targeting a common variant can shift the balance towards increased incorporation of the wild-type allele to alleviate the disease in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs). We identified the single nucleotide polymorphisms (SNP) rs1057128 (G/A) in KCNQ1, with a heterozygosity of 27% in the European population. Next, we determined allele-specificity of short-hairpin RNAs (shRNAs) targeting either allele of this SNP in hiPSC-CMs that carry an LQT1 mutation. Our shRNAs downregulated 60% of the A allele and 40% of the G allele without affecting the non-targeted allele. Suppression of the mutant KCNQ1 allele by 60% decreased the occurrence of arrhythmic events in hiPSC-CMs measured by a voltage-sensitive reporter, while suppression of the wild-type allele increased the occurrence of arrhythmic events. Furthermore, computer simulations based on another LQT1 mutation revealed that 60% suppression of the mutant KCNQ1 allele shortens the prolonged action potential in an adult cardiomyocyte model. We conclude that allele-specific inhibition of a mutant KCNQ1 allele by targeting a common variant may alleviate the disease. This novel approach avoids the need to design shRNAs to target every single mutation and opens up the exciting possibility of treating multiple LQT1-causing mutations with only two shRNAs.

Keywords: RNA interference; arrhythmia; gene therapy; hiPSC-cardiomyocytes; long-QT syndrome type 1.

Figure 1

Allele-specific downregulation of KCNQ1 expression in hiPSC-CMs by shRNAs targeting SNP rs1057128: (a) Top, schematic representation of the shRNAs targeting the A allele of rs1057128 on the mutant KCNQ1 allele with the mismatch positions indicated in red. Middle, allele-specific relative mRNA expression of the wild-type and mutant allele and total KCNQ1 in hiPSC-CMs of Line 1 (n = 6). Bottom, allele-specific relative mRNA expression of the wild-type and mutant allele and total KCNQ1 in hiPSC-CMs of Line 2 (n = 6); (b) Top, schematic representation of the shRNAs targeting the G allele of SNP rs1057128 on the wild-type KCNQ1 allele with the mismatch positions indicated in red. Middle, allele-specific relative mRNA expression of the wild-type and mutant allele and total KCNQ1 in hiPSC-CMs of Line 1 (n = 12). Bottom, allele-specific relative mRNA expression of the wild-type and mutant allele and total KCNQ1 in hiPSC-CMs of Line 2 (n = 6). * p < 0.05; ** p < 0.025; *** p < 0.001 compared to shSCR negative control shRNA; error bars indicate SEM.

Figure 2

Allelic imbalance induced by allele-specific shRNAs: (a) Top, schematic representation of the shRNAs targeting the mutant KCNQ1 allele in hiPSC-CMs. Bottom, allelic expression of wild-type and mutant KCNQ1 allele presented as % of total KCNQ1 expression for Line 1 in red (left; n = 6) and Line 2 in blue (right; n = 6); (b) Top, schematic representation of the shRNAs targeting the wild-type KCNQ1 allele. Bottom, allelic expression of wild-type and mutant KCNQ1 allele presented as % of total KCNQ1 expression for Line 1 in red (left; n = 12) and Line 2 in blue (right; n = 6). * p < 0.05; ** p < 0.025; *** p < 0.001 compared to allelic expression in the shSCR negative control shRNA; error bars indicate SEM.

Figure 3

Action potential duration is affected by shifts in allelic balance: (a) Schematic representation of the SNP and mutation in KCNQ1; (b,c) Left, typical recordings of optical action potentials derived from ArcLight fluorescence changes in hiPSC-CMs from Line 1 (b) or Line 2 (c) treated with either negative control shSCR, shA18 targeting the mutant KCNQ1 allele or shG11 targeting the wild-type KCNQ1 allele stimulated at 1 Hz. Right, action potential duration at 20, 50, or 80% of repolarization (APD₂₀, APD₅₀, and APD_80, respectively) of optical action potentials of hiPSC-CMs from Line 1 in red (b) or Line 2 in blue (c). * p < 0.05; *** p < 0.001 compared to shSCR negative control treated hiPSC-CMs; error bars indicate SEM.

Figure 4

The occurrence of arrhythmic events is affected by allele-specific downregulation of the mutant or wild-type KCNQ1 allele. (a) Typical examples of ArcLight traces showing the fluorescence changes over time of hiPSC-CMs with arrhythmic events; (b) Percentage of cells with arrhythmic events in hiPSC-CMs of Line 1 in red (top; shSCR: n = 28, 2 with events; shA18: n = 19, no events; shG11: n = 34, 19 with events) and hiPSC-CMs of Line 2 in blue (bottom; shSCR: n = 43, 5 with events; shA18: n = 47, 4 with events; shG11: n = 41, 21 with events).

Figure 5

Computer simulation of allele-specific mutant KCNQ1 inhibition in an adult human cardiomyocyte: (a) Fraction of slow delayed rectifier potassium current (I_Ks) channels with 0-4 wild-type (WT) subunits in case of equal expression of wild-type and mutant KCNQ1 subunits (WT 1.0/mutant 1.0) and with a 60% suppression of mutant KCNQ1 subunits (WT 1.0/mutant 0.4) assuming random co-assembly of subunits into tetrameric channels; (b) Effects of changes in I_Ks on action potentials of the epicardial (EPI), midmyocardial (MID), and endocardial (ENDO) versions of the human ventricular cell model. Membrane potential (V_m; top) and associated I_Ks (bottom) at 1 Hz stimulation that result from simulations with 100% wild-type KCNQ1 expression (WT), with an equal heterozygous expression of wild-type and E160K mutant KCNQ1 subunits (WT/E160K), and with a 60% suppression of E160K mutant KCNQ1 subunits, assuming that all channels with mutant subunits contribute equally to mutant I_Ks (‘Suppression 1’) or that only channels with one mutant subunit contribute to mutant I_Ks (‘Suppression 2’). Note that the red and orange lines largely overlap; (c) Values of action potential duration at 90% of repolarization (APD₉₀) in each of the simulation settings.