Identification of Na+/K+-ATPase inhibition-independent proarrhythmic ionic mechanisms of cardiac glycosides

Figures

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  Figure 1. Structural formula of the 4 CGs adopted in the current study.

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  Figure 2. Effects of CGs on I Ca,L, I Na and I Kr in heterologous expression cell lines. (A) Effects of BF, CBG, Oua and PEA on I Ca,L recorded from Cav1.2-CHO cells. (B) Effects of BF, CBG, Oua and PEA on I Kr (peak tail current) recorded from hERG-HEK293 cells. (C) Effects of BF, CBG, Oua and PEA on I Na recorded from Nav1.5-HEK293 cells. Shown on the left are the % inhibitions plotted in bar-graphs. Shown on the right are representative traces showing the effects of BF on I Ca,L, I Na and I Kr recorded by Patchliner®, the automated patch-clamping system. *p < 0.05, † p < 0.01, ‡ p < 0.001, vs. baseline of each CG (One-Way ANOVA). †(red) p < 0.01, ‡(red) p < 0.001, vs. BF/CBG at same concentrations (Two-Way repeated measures ANOVA). Data are presented as mean ± SEM. (D) Effects of BF on I Ca,L recorded in Cav1.2-CHO cells with Ca2+ depleted in the extracellular solution (recorded by conventional voltage-clamping). (Left) Representative traces of I Ca,L recorded in a Cav1.2-CHO cell at baseline (Ca2+-free extracellular conditions achieved by using BaCl2 to replace CaCl2) and post 10 µM BF treatments. (Right) I-V curves showing the voltage-dependent I Ca,L current densities (pA/pF). *p <…

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  Figure 3. NKA inhibition-independent effects of BF on I Ca,L in Cav1.2-CHO and hESC-CMs. (A) Effects of BF and CBG on I Ca,L recorded in Cav1.2-CHO cells pre-incubated with 10 µM of Oua and with extracellular KCl replaced by CsCl to further suppress the NKA activity. Shown on the left and middle respectively are representative traces showing the effects of BF and CBG on I Ca,L recorded by Patchliner®. Shown on the right is the % inhibition plotted in bar-graph. ‡ p < 0.001, vs. baseline (One-Way ANOVA). Data are presented as mean ± SEM. (B) Effect of BF on I Ca,L in hESC-CMs with NKA fully blocked by Oua (recorded by conventional voltage-clamping). (a) Representative traces of the voltage-gated I Ca,L recorded from a hESC-CM at baseline (with 10 µM Oua incubation for 10 minutes), with subsequently treated with 1.0 and 10 µM of BF for 5 minutes, and finally with Bay K8644 (1 µM) treatment. (b) I-V curves showing the voltage-dependent I Ca,L current densities (pA/pF). *p < 0.05, † p < 0.01, vs. baseline (One-Way ANOVA); *(green) p < 0.05, †(green) p < 0.01, vs. 1.0 µM BF (One-Way ANOVA). n = 7. (c) Comparison of the I Ca,L current densities at peak (I Ca,L peak) and during decay (I Ca,L…

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  Figure 4. Effect of BF on [Ca2+]i transients in hESC-CMs. Spontaneous [Ca2+]i transients were recorded in Fluo-8 loaded hESC-CMs pre-treated with 10 µM Oua for 5 minutes followed by 10 µM BF. (A) Representative traces of [Ca2+]i transients recorded in a cell at baseline (with Oua) and with subsequent exposure to BF. The relative [Ca2+]i transients intensity is plotted as fluorescence ratio (F1/F0). (B) Bar-graphs show [Ca2+]i transient amplitude, time to peak and duration (CaD) at 50% and 80% recovery (CaD50 and CaD80, respectively). * p < 0.05, † p < 0.01, ‡ p < 0.001, vs. baseline. n = 7.

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  Figure 5. Effect of CGs on APs in hESC-CMs. Effects of CGs on APs were recorded in single hESC-CMs. (Aa) Representative AP waveforms recorded in hESC-CMs exposed to 1 µM of BF, CBG, Oua and PEA for 1–2 minutes. (Ab) Representative AP waveforms recorded in hESC-CMs treated with BF (10 µM) for different time points. (Ac) Bar-graphs show APD90, APD30, APA and MDP measured in hESC-CMs 2 minutes post application of BF, CBG, Oua and PEA. * p < 0.05, † p < 0.01, vs. baseline (two-tailed paired Student t-tests). Data are presented as mean ± SEM. (B) Representative superimposed AP waveforms recorded in hESC-CMs exposed to 0.1% DMSO (as vehicle control) and BF (10 nM) for 24 hours. n = 7.

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  Figure 6. Effect of CGs on FPs in hESC-CMs. Effects of BF, CBG, Oua and PEA on cardiac FPs were recorded in clusters of hESC-CMs. (A) % changes of the FPDc over baseline. (B) % changes of the heart rate over baseline. (C) Representative FP traces show the effects of BF. All recordings were started after stabilization for 5 minutes. *p < 0.05, vs. baseline (One-Way ANOVA). *(red) p < 0.05, †(red) p < 0.01, ‡(red) p < 0.001, vs. BF (Two-Way ANOVA). Data are presented as mean ± SEM.

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  Figure 7. Effect of CGs on the APs of Zebrafish larva. Effects of BF, CBG, Oua and PEA (all at 100 µM) on the APs, controlled by digoxin, were analysed in day-3 Zebrafish larva. (A, B and C) Bar-graphs show the effects of various CGs on heart rates, APD and APDc, respectively. (D) Representative waveforms of APs recorded. The dashed lines indicate the representative APDs. *p < 0.05, ‡ p < 0.001, vs. control; ‡ p < 0.001, vs. BF or CBG (One-Way ANOVA). Data are presented as mean ± SD.

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  Figure 8. Simulated effects of reduced I NaK, I Ca,L and I Kr on ventricular APs. ORd model was adopted in A~E. (A) Simulated effect of increased [Na+]i on APs. (B) Simulated effect of reduced I NaK alone on APs. (C) Simulated effect of reduced I Ca,L alone on APs. (D) Simulated effects of I NaK (0%) in combination with reduced I Ca,L on APs. (E) Simulated effect of reduced I Kr on APs. (F) The effect of reduced I Ca,L on APs simulated by the LRd model.