Drug evaluation in cardiomyocytes derived from human induced pluripotent stem cells carrying a long QT syndrome type 2 mutation

Figures

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  Figure 1. Clinical and genetic profile of the LQT2 family. (A) Electrocardiogram of the LQT2 patient, showing prolonged QTc interval of up to 571 ms (lead V5) and arrhythmic episode recorded during telemetry. Electrocardiograms of patient's father (PAT; lead V5) and mother (MAT; lead V5) are also presented. (B) Schematic diagram showing location of the KCNH2 gene on chromosome 7q36.1 and position of autosomal dominant point mutation in exon 7, at base 1681 of the coding sequence (G1681A). This missense mutation results in substitution of the hydrophobic amino acid alanine (Ala), at position 561 of the protein, by the hydrophilic amino acid threonine (Thr; p.Ala561Thr). Results of genomic DNA sequencing in fibroblasts derived from hESCs (HF) and blood samples taken in hospital from LQT2 patient, mother, and father. (C) Schematic representation of the Ikr potassium channel showing that KCNH2 p.Ala561Thr mutation is located in the S5 transmembrane domain (black dot with red halo), which is involved in the pore-forming region of the channel.

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  Figure 2. Derivation and characterization of LQT2 fibroblasts and hiPSCs. (A) (i) Processing a 4 mm skin biopsy isolated from the LQT2 patient. (ii) Fibroblast outgrowths from digested skin sample, (iii) monolayer culture of LQT2 fibroblasts (LQT2–Fib), (iv) hiPSC colony generated from LQT2–Fib following lentiviral transduction with OCT4, NANOG, SOX2, and LIN28, (v) LQT2–hiPSC colony at passage 1, (vi) monolayer culture of LQT2–hiPSCs at passage 15, grown in feeder-free conditions on Matrigel. Scale bars represent 100 µm. (B) Genomic sequencing in LQT2–hiPSCs showing maintenance of the KCNH2 G1681A mutation. (C) Karyogram of LQT2–hiPSCs showing a normal 46XX karyotype, at passage 15. (D) RT–PCR analysis of OCT4, NANOG, SOX2, and LIN28 expression from endogenous (E) and lentiviral (L) loci in LQT2–Fib, LQT2–hiPSCs, LQT2-hiPSC embryoid bodies. (E) Flow cytometry and (F) immunostaining for differentiation marker, SSEA1, and pluripotency markers, TRA-1-81 and SSEA4, in LQT2–hiPSCs. (G) Immunostaining in LQT2-iPSC embryoid bodies for the germ layer markers β-III-tubulin (ectoderm), α-fetoprotein (AFP; endoderm), and α-actinin (mesoderm). Scale bars in (E) and (F) represent 65 µm. (H) DNA…

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  Figure 3. Characterization of cardiac myocytes. (A) Immunostaining for cardiac troponin I (TrpI) and α-actinin in myocytes derived from hESCs, HF–hiPSC, LQT2–hiPSC, and MAT–hiPSC, showing characteristic cardiac muscle striations. (B) Image of an LQT2–hiPSC beating cluster mounted onto a multi-electrode array for electrophysiology analysis, and graph showing prolongation in QT interval and field potential duration in LQT2–hiPSC beaters relative to controls. (C) Image of a single LQT2–hiPSC beating cell undergoing patch-clamp analysis, and action potential curves representing formation of ventricular, atrial, and pacemaker myocytes. (D) Schematic diagram of a multi-electrode array trace, showing how results were analysed to calculate duration of the QT interval and field potential duration. (E) Schematic diagram of a patch-clamp trace, showing how results were analysed to calculate duration of the action potential and the action potential duration at 50 and 90% repolarization (APD50 and APD90, respectively). To determine the type of cardiac myocyte analysed, APD90/50 values <1.4 designated ventricular cells, 1.4–1.7 designated pacemaker cells, and >1.7 designated atrial cells. Scale bars…

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  Figure 4. Electrophysiology analysis of cardiomyocytes. (A) Action potential curves showing prolongation in the action potential duration of ventricular LQT2-hiPSC myocytes relative to controls. (B) Action potential curves showing prolonged action potential duration in atrial LQT2–hiPSC myocytes relative to controls. (C) Box plots and (D) histograms demonstrating the distribution of action potential duration, and action potential duration measured at 50 and 90% repolarization (APD50 and APD90, respectively) in ventricular, atrial, and pacemaker myocytes derived from hESCs and hiPSCs. Histogram results are represented as mean ± standard deviation.

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  Figure 5. Sensitivity of cardiomyocytes to β-adrenoreceptor agonists and antagonists. (A) Multi-electrode array and (B) patch-clamp analysis showing abbreviation of the field potential duration and action potential duration with isoprenaline treatment (blue traces) relative to the spontaneous beating curves (black traces), as well as prolongation of the field potential duration and action potential duration upon subsequent addition of the β-blockers, nadolol (orange traces) or propranolol (green traces). Curves are representative of both control and LQT2–hiPSC myocytes. (C) Graph showing increased sensitivity of LQT2–hiPSC myocytes to isoprenaline treatment relative to controls, and reversal of isoprenaline effects by β-blockers in patient and control cells. (D–E) (i) Averaged and (ii) raw action potential curves of LQT2–hiPSC myocytes showing isoprenaline-induced arrhythmogenesis (blue traces) and attenuation of this phenotype by nadolol (D, yellow trace) or propranolol (E, green trace).

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  Figure 6. Sensitivity of cardiomyocytes to potassium channel blockers and openers. (A) Multi-electrode array and (B) patch-clamp analysis showing elongation of the field potential duration and action potential duration with E4031 treatment (purple traces) relative to the spontaneous beating curves (black traces). Curves are representative of both control and LQT2–hiPSC myocytes. (C) Action potential curves of LQT2–hiPSC myocytes showing E4031-induced arrhythmogenesis. (D) Multi-electrode array and (E and F) patch-clamp analysis showing shortening of the field potential and action potential durations with nicorandil (red traces) and PD-118057 (pink traces) treatment. (G) Action potential curves showing spontaneously occurring early after depolarizations (black trace) abolished by nicorandil treatment (red trace). (H) Action potential traces of sequential drug treatment with isoprenaline, nadolol and PD-118057, resulting in the generation of early after depolarizations due to excessive action potential duration shortening (pink trace). The phenotype was then attenuated with nadolol treatment (yellow trace). (D–F) Curves of both control and LQT2–hiPSC myocytes.