Gene- and variant-specific efficacy of serum/glucocorticoid-regulated kinase 1 inhibition in long QT syndrome types 1 and 2

Figures

• 

  Figure 1. Effect of SGK1-Inh on LQT1 hiPSC-CMs. (A) Dot plot comparing normalized cFPD after 3 h of incubation with DMSO or 0.3, 1, 3, or 10 µM of SGK1-Inh in hiPSC-CMs carrying the KCNQ1-p.A341V variant. N = 9, at baseline from four independent differentiations of three KCNQ1-p.A341V hiPSC lines. (B) Dot plot comparing raw cFPD data after 3 h of incubation with DMSO or 0.3, 1, 3, or 10 µM of SGK1-Inh in hiPSC-CMs carrying the KCNQ1-p.A341V variant. (C) Representative corrected FP traces after 3 h of incubation with DMSO or either 300 nM or 3 µM of SGK1-Inh in hiPSC-CMs carrying the KCNQ1-p.A341V variant. (D) Dot plot comparing normalized cFPD after 3 h of incubation with DMSO or 0.3, 1, 3, or 10 µM of SGK1-Inh in hiPSC-CMs with the KCNQ1-p.R594Q variant. N = 12, at baseline from three independent differentiations. (E) Dot plot comparing raw cFPD data after 3 h of incubation with DMSO or 0.3, 1, 3, or 10 µM of SGK1-Inh in hiPSC-CMs with the KCNQ1-p.R594Q variant. (F) Representative corrected FP traces after 3 h of incubation with DMSO or either 300 nM or 3 µM of SGK1-Inh in hiPSC-CMs with the KCNQ1-p.R594Q variant. *P < 0.05 vs. DMSO. All samples are compared to the respective DMSO.…

• 

  Figure 2. Effect of SGK1-Inh on LQT2 hiPSC-CMs. (A) Dot plot comparing normalized cFPD after 3 h of incubation with DMSO or 0.3, 1, 3, or 10 µM of SGK1-Inh in hiPSC-CMs with the KCNH2-p.A561V variant. N ≥ 12, from three independent differentiations. (B) Dot plot comparing raw cFPD data after 3 h of incubation with DMSO or 0.3, 1, 3, or 10 µM of SGK1-Inh in hiPSC-CMs with the KCNH2-p.A561V variant. (C) Representative corrected FP traces after 3 h of incubation with DMSO or either 300 nM or 3 µM of SGK1-Inh in hiPSC-CMs with the KCNH2-p.A561V variant. (D) Dot plot comparing normalized cFPD after 3 h of incubation with DMSO or 0.3, 1, 3, or 10 µM of SGK1-Inh in hiPSC-CMs with the KCNH2-IVS9-28A/G variant. N ≥ 12 at baseline from three independent differentiations. (E) Dot plot comparing raw cFPD data after 3 h of incubation with DMSO or 0.3, 1, 3, or 10 µM of SGK1-Inh in hiPSC-CMs with the KCNH2-IVS9-28A/G variant. (F) Representative corrected FP traces after 3 h of incubation with DMSO or either 300 nM or 3 µM of SGK1-Inh in hiPSC-CMs with the KCNH2-IVS9-28A/G variant. *P < 0.05 vs. DMSO. All samples are compared to the respective DMSO. Comparison with DMSO was calculated for the whole time…

• 

  Figure 3. Effects of SGK1-Inh on AP characteristics in isolated ventricular rabbit CMs in whole-cell configuration at RT. (A–C) Representative AP traces triggered at 1 Hz in WT (A), LQT1 (B), and LQT2 (C) isolated rabbit CMs treated with DMSO (vehicle) and after 2–6 h of incubation at RT with SGK1-Inh (3 µM). (D–F) Average values for APA, RMP, dV/dtmax, and APD90 with DMSO and after SGK1-Inh incubation (3 µM) in WT (five rabbits), LQT1 (two rabbits), and LQT2 (one rabbit). Results are expressed as mean ± SEM, and every dot represents an individual value derived from one CM (n). N, number of animals. ***P < 0.001. Unpaired Student’s t-test. AP, action potential; APA, AP amplitude; APD90, action potential duration at 90% repolarization; CMs, cardiomyocytes; dV/dtmax, maximal AP upstroke velocity; ns, not significant; RMP, resting membrane potential; RT, room temperature; SEM, standard error of the mean; SGK1-Inh, serum/glucocorticoid-regulated kinase 1 inhibitor; WT, wild type.

• 

  Figure 4. Effects of SGK1-Inh on AP characteristics in isolated ventricular rabbit CMs in perforated patch configuration at 37°C. (A and B) Representative AP traces elicited at 1 Hz WT (A) and LQT2 (B) isolated rabbit ventricular CMs treated with DMSO (vehicle) and after 2–6 h of incubation at 37°C with SGK1-Inh (3 µM). (C and D) Average values for APA, RMP, dV/dtmax, and APD90 with DMSO and after SGK1-Inh incubation (3 µM) in WT (three rabbits) and LQT2 (three rabbits). Results are expressed as mean ± SEM, and every dot represents an individual value derived from one CM (n). N, number of animals. **P < 0.005. Unpaired Student’s t-test. AP, action potential; APA, AP amplitude; APD90, action potential duration at 90% repolarization; CMs, cardiomyocytes; dV/dtmax, maximal AP upstroke velocity; ns, not significant; RMP, resting membrane potential; SEM, standard error of the mean; SGK1-Inh, serum/glucocorticoid-regulated kinase 1 inhibitor; WT, wild type.

• 

  Figure 5. Normalization of APD in isolated LQT2 CMs to the WT level after treatment with SGK1-Inh. Action potentials were recorded either at RT in whole-cell patch configuration (A and C) or at 37°C (body temperature) in perforated patch configuration (B and D). (A and B) Representative AP traces triggered at 1 Hz in WT and LQT2 isolated rabbit CMs incubated with DMSO (vehicle) and LQT2 isolated rabbit CMs after 2–6 h of incubation with SGK1-Inh (3 µM). (C and D) Average values for APD90 in WT and LQT2 CMs treated with DMSO and in LQT2 CMs after SGK1-Inh incubation (3 µM) from Figures 3 and 4 are plotted to evaluate whether SGK1-Inh can restore APD to normal WT levels. Results are expressed as mean ± SEM, and every dot represents an individual value derived from one CM (n). N, number of animals. ** P < 0.01; *** P < 0.001; ****P < 0.0001. One-way ANOVA followed by Tukey’s test for post hoc analysis. AP, action potential; APD, action potential duration; APD90, APD at 90% repolarization; CMs, cardiomyocytes; ns, not significant; RT, room temperature; SEM, standard error of the mean; SGK1-Inh, serum/glucocorticoid-regulated kinase 1 inhibitor; WT, wild type.

• 

  Figure 6. Action potential duration shortening in LQT2 hiPSC-CCS carrying the KCNH2-p.A614V variant. (A) Representative optical AP traces from a single pixel within LQT2 hiPSC-CCS after 4–6 h incubation at 37°C with either DMSO as a vehicle or 3 µM SGK1-Inh. (B) Representative colour-coded APD pixel maps showing the average APD80 within each pixel as calculated for at least eight consecutive beats. (C) Summary of mean APD80 values. (D) Percentage of change in APD80 as calculated for each hiPSC-CCS exposed to DMSO as a vehicle (n = 8 from N = 3 differentiations) or either 300 nM or 3 µM (n = 10 from N = 3 differentiations each) of SGK1-Inh. Results are expressed as mean ± SEM, and every dot represents an individual value derived from one hiPSC-CCS. Data are derived from N = 3 independent differentiations with n ≥ 8 as stated. *P < 0.05, ****P < 0.0001. Statistical analysis was carried out using one-way repeated-measures ANOVA followed by Tukey’s test for post hoc analysis. AP, action potential; APD, action potential duration; APD80, APD at 80% repolarization; hiPSC-CCS, human-induced pluripotent stem cell-derived cardiac cell sheets; ns, not significant; SEM, standard error of the mean;…