Identification and functional assessment of a KCNH2 compound heterozygosity in a patient with presumed idiopathic ventricular fibrillation ascertains the diagnosis of long QT syndrome type 2

Figures

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  Figure 1. Clinical and genetic characteristics. (A) Sequence analysis of the revealed variants A228V and S1021Qfs*98. (B) Pedigree of the proband’s family; A228V in blue, S1021Qfs*98 in cyan. (C) ECG of the proband at rest (up) and at the 4th minute of recovery after the exercise test (down); 20 mm/mV, 50 mm/s. ECG, electrocardiogram.

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  Figure 2. Impact of S1021Qfs*98- and A228V-hERG variants on channel activation. (A) Scheme of the stimulation protocol (left upper panel) and representative traces in all cell groups, namely WT, S1021Qfs*98, A228V, and S1021Qfs*98/A228V cells (n = 15, 14, 13, and 14, respectively). No current was detected in non-transfected cells (n = 19). (B) Average voltage dependence of tail current activation; **P < 0.01 for S1021Qfs*98 vs. WT, and # and ##P < 0.05 and 0.01, respectively, for S1021Qfs*98/A228V vs. WT (Kruskal–Wallis test with Dunn’s post-test; only significance vs. WT is shown, for others, see Table 1). (C) Voltage dependence of steady-state activation. (D) Voltage dependence of the time constant of activation (τact); n = 15, 9, 13, and 14 cells for WT, S1021Qfs*98, A228V, and S1021Qfs*98/A228V, respectively. WT, wild type.

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  Figure 3. hERG channel deactivation was not significantly changed by S1021Qfs*98 and A228V variants. (A) Scheme of the stimulation protocol. (B) A representative recording in WT channels; inset: a detail view of the key part of the protocol (marked by the dark red rectangle). (C) Voltage dependence of the time constant of deactivation (τdeact) in WT, S1021Qfs*98, A228V, and S1021Qfs*98/A228V channels (n = 15, 15, 12, and 9, respectively). WT, wild type.

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  Figure 4. Time course of inactivation of Kv11.1 channels. (A) Scheme of experimental protocol; inset: a detailed view of the key part of the protocol (marked by the dark red rectangle). (B) Representative traces in WT, S1021Qfs*98, A228V, and S1021Qfs*98/A228V channels (n = 14, 10, 17, and 11, respectively). (C) Voltage dependence of the time constant of inactivation (τinact); for significances, see Table 1. WT, wild type.

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  Figure 5. Voltage dependence of steady-state inactivation and recovery from inactivation of Kv11.1 channels. (A) Scheme of the stimulation protocol; inset: a detailed view of the key part of the protocol (marked by the dark red rectangle). (B) Representative traces in WT, S1021Qfs*98, A228V, and S1021Qfs*98/A228V channels. (C) Voltage dependence of steady-state inactivation (n = 14, 10, 19, and 17 in WT, S1021Qfs*98, A228V, and S1021Qfs*98/A228V channels, respectively). (D) Time constant of recovery from inactivation τrec (n = 14, 10, 19, and 15 in WT, S1021Qfs*98, A228V, and S1021Qfs*98/A228V channels, respectively); ***P < 0.001 for S1021Qfs*98 vs. WT; €€P < 0.01 for A228V vs. WT; #P < 0.05 and ###P < 0.001 for S1021Qfs*98/A228V vs. WT (Kruskal–Wallis test with Dunn’s post-test; only significances vs. WT are shown, for others, see Table 1). WT, wild type.

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  Figure 6. Western blot analysis of Kv11.1 expression after transfection with plasmids for expression of the wild-type (WT) Kv11.1 protein or Kv11.1 protein with the A228V and S1021Qfs*98 variants. (A) Representative western blot (for raw original data from three biological replicates, see Supplementary material online, Figure S3) and average Kv11.1/α-tubulin values (mean ± IQR); *Statistical significance between WT and S1021Qfs*98 variants at P < 0.05 (Kruskal–Wallis test with Dunn’s post-test). (B) A substantially diminished Kv11.1 expression was detected in both S1021Qfs*98 and S1021Qfs*98/A228V (ratio 1:1) channels.

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  Figure 7. Effects of the IKr reduction induced by S1021Qfs*98/A228V channels (by 69%) on action potential (AP) and cytosolic Ca²⁺ transients (CaT) in a human midmyocardial cell model. (A) AP (top) and CaT (bottom) at 1 Hz steady-state stimulation in the control model (grey) and in the model with reduced IKr (red), showing slowed repolarization and prolonged AP duration. (B) Response of the models to an acute reduction of [K⁺]ₑ from 5.4 to 3.0 mM at 1 Hz stimulation. The model with reduced IKr persistently generated early afterdepolarizations (EADs) from the second AP, accompanied by irregularities in CaT, whereas the control model remained stable. (C) Model behaviour at β-adrenergic stimulation induced by 1 µM isoproterenol (2.5 Hz). After 43 s of regular activity, the model with reduced IKr started to generate persistent EADs.