Long-QT founder variant T309I-Kv7.1 with dominant negative pattern may predispose delayed afterdepolarizations under β-adrenergic stimulation

Figures

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  Figure 1. Clinical and genetic characterization of the T309I variant. (A) QTc interval length in the heterozygous T309I carriers (T309I +) and their unaffected relatives (T309I-), both at rest and in the 4th minute of exercise test recovery; ***statistical significance at P < 0.001; the graph was prepared using the software GraphPad Prism, version 6.05. (B) Scheme of T309I-Kv7.1 subunit (upper panel) and birthplaces of the oldest T309I carriers in every family (lower panel; the map was generated using the software QGIS, version 3.10, with data downloaded from https://www.arcdata.cz/produkty/geograficka-data/arccr-500). In the 2 families located in the central part of the country, some earlier ancestors from the eastern part could be identified. Nevertheless, none of them has been living at the time of the study and biological material was not available for molecular analysis. Thus, we can only speculate, whether these ancestors coming from the east really carried the variant, even if it is truly anticipated. (C) Family pedigrees; affected individuals in red, probands indicated with arrows. (D) Haplotype analysis of the KCNQ1 gene and surrounding regions (analysed STR markers shown in…

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  Figure 2. Biophysical impact of T309I mutation. (A) Representative records in wild type (WT), T309I, and WT/T309I channels (KCNE1 subunit cotransfected); arrows point to the tail current. (B) Average voltage dependence of tail current activation (n = 20, 9, and 16 in WT, T309I, and WT/T309I channels, respectively); the half-maximal activation voltage V1/2 = 8.3 ± 2.2 mV in WT channels and 15.6 ± 2.1 mV in WT/T309I channels; P < 0.05). (C) Activation experimental protocol; the dashed lines—steps used for stimulation only in T309I channels. (D) Representative trace from a non-transfected CHO cell. (E) Average voltage dependence of steady-state activation. (F) Average voltage dependence of the time constant of activation (τact) and time constant of deactivation (τdeact; n = 11 and 9 in WT and WT/T309I channels, respectively); inset: deactivation experimental protocol; *, ** and ***statistical significance between WT and WT/T309I channels at P < 0.05, 0.01 and 0.001, respectively. The stimulation frequency was 0.08 Hz. All graphs were prepared using the software Origin, version 8.5.1.

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  Figure 3. Accumulation of WT and WT/T309I currents (KCNE1 subunit cotransfected). (A) Average relative rate-dependent accumulation of the currents at interpulse intervals between 75 and 800 ms (n = 10 and 8 in WT and WT/T309I channels, respectively); inset: experimental protocol applied after a 30-s pause; i.i.—interpulse interval. (B) Average absolute currents at the 1st and 100th pulse of the accumulation protocol with the interpulse interval of 100 ms. (C) Experimental protocol (the stimulation frequency 0.1 Hz), and representative traces of WT and WT/T309I currents under simulated β-adrenergic stimulation (cAMP and OA in the pipette solution); red trace—time 0, control (no cAMP/OA stimulation); green trace—time 120 s, steady-state β-adrenergic stimulation; black traces—time 10 to 110 s in 10-s intervals, development of β-adrenergic response. (D) Time course of the relative increase of the current in presence of cAMP and OA (+ cAMP + OA) in WT and WT/T309I channels (n = 14 and 8; n = 9 in WT without cAMP and OA). (E) Relative steady-state increase of the current in + cAMP + OA; *, ** and ***statistical significance at P < 0.05, 0.01 and 0.001, respectively. All graphs were prepared…

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  Figure 4. Subcellular localization of WT- and T309I-Kv7.1 subunits expressed in CHO cells (KCNE1 subunit cotransfected). (A) Confocal microscopic images of representative cells expressing WT-GFP (i.e. tagged with GFP at the C-terminal end), T309I-GFP, or both WT-GFP and T309I-GFP subunits (upper panels), and respective relative fluorescence intensity profiles (lower panels) under the blue lines in the upper panels; scale bar 25 µm. (B) The resulting maximal relative fluorescence intensity at the cell membrane (M1 and M2) and its mean value in the cytosol (C) in all investigated cells (n = 20, 20 and 12 in WT-GFP, T309I-GFP and WT-GFP/T309I-GFP subunits, respectively; cells from 4 to 7 transfections used in each variant); ** and ***statistical significance of the difference in fluorescence intensity among M1, M2 and C in the respective group of cells at P < 0.01 and 0.001, respectively. (C,E) Subcellular localization of the T309I-GFP subunits, either expressed alone (C) or co-expressed with non-tagged WT (no GFP; E); the cell membrane was stained with red fluorescent dye. Representative cells (upper panels) and respective relative fluorescence intensity profiles (lower panels) under the…

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  Figure 5. Impact of WT/T309I dysfunctional channels on action potential (AP) configuration in a human ventricular cell model. (A) APs (upper panels) and IKs (lower panels) at steady-state stimulation at the cycle length (CL) 1000 ms in control conditions and during β-adrenergic stimulation; APD90—AP duration at 90%-repolarization. (B) APs (upper panels) and IKs (lower panels) at extrastimulation within the vulnerable period of AP in the WT/T309I model during β-adrenergic stimulation—CL suddenly shortened from 1000 to 268 ms. (C) Intracellular Ca2+ concentration at the end of stimulation cycle ([Ca2+]i,end; left upper panel), APs (right upper and middle panels) and intracellular Ca2+ transients ([Ca2+]i; lower panel) after change of CL from 1000 to 500 ms and contemporary start of β-adrenergic stimulation (for sequence of changes, note the time axes in all graphs); arrows—stimuli at CL 500 ms, *delayed afterdepolarization (DAD) or AP activated by DAD. All graphs were prepared using the software Origin, version 8.5.1.