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

Figures

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  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.

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  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.

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  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 APD80, 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.

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  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).

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  Figure 5. Computer simulation of allele-specific mutant KCNQ1 inhibition in an adult human cardiomyocyte: (a) Fraction of slow delayed rectifier potassium current (IKs) 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 IKs on action potentials of the epicardial (EPI), midmyocardial (MID), and endocardial (ENDO) versions of the human ventricular cell model. Membrane potential (Vm; top) and associated IKs (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 IKs (‘Suppression 1’) or that only channels with one mutant subunit contribute to mutant IKs (‘Suppression 2’). Note that the red and orange lines largely overlap; (c) Values of action potential duration at 90% of…