Selective Activation of Tetrodotoxin-Sensitive Sodium Channels by AaH-II Drives Arrhythmogenic Late Na+ Current in the Heart

Figures

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  Figure 1. AaH-II* sensitivity of hNav channels. A, Representative current traces elicited at a test potential of 0 mV for various hNav channels stably expressed in HEK293 cells (all except hNav1.7) or CHO cells (hNav1.7) before and after application of 0.1, 0.3, 1, or 3.3 nM AaH-II*. n.d.: not determined. Scale bars: all 0.5 nA and 10 ms. The appearance of INaL also results in a peak current increase, as the activation process is no longer hidden by fast inactivation. B, Normalized average dose-response curves assessing the potency of AaH-II* on various hNav isoforms. IC50 values were calculated from single (hNav1.1, hNav1.2, hNav1.4, hNav1.6, and hNav1.7) or double (hNav1.3 and hNav1.5) sigmoid fits of the data and provided in Table 1. C, Maximal AUC, comprising both peak and INaL increases, measured at the highest effective AaH-II* concentration for each hNav isoform. Mean ± SEM values are shown.

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  Figure 2. Lack of stimulated Na+ entry through hNav1.5 at a low AaH-II* concentration. A, Top panel, voltage protocol used to simulate an action potential. Lower panels, actual representative Na+ currents through hNav1.5 channels in control condition, 0.1 nM AaH-II* or 10 nM AaH-II* before and after application of 10 μM TTX. B, Persistent inward current traces during the descending ramp after subtraction of the current before by the current after 10 μM TTX for all three conditions. C, Quantification of the subtracted current density in all three conditions.

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  Figure 3. AaH-II* stimulates TTX-S Nav channels in human iPS-derived and rat ventricular cardiomyocytes. A, qRT-PCR experiments quantifying the relative gene expression of seven hNav channels in right ventricle cardiomyocytes derived from iPS cells. B, Left panel: representative Na+ currents from human iPS-derived cardiomyocytes before and after application of 0.1 nM AaH-II* (first) and 100 nM TTX + 0.1 nM AaH-II* (second). Scale bars: 5 ms/1 nA. Right panel: average normalized AUC illustrating INaL increase by AaH-II* and decrease by 100 nM TTX from TTX-S Nav channels. C, as in B but using 10 nM AaH-II* that produces INaL from both TTX-S and Nav1.5 channels. 30 μM TTX inhibits all Nav isoforms. Scale bars: 5 ms/1 nA. *, P < 0.05; **, P < 0.01; ***, P < 0.001. D, Representative Na+ currents from adult rat cardiomyocytes (holding potential −120 mV, test potential −20 mV) before and after application of 0.1 nM AaH-II* (first) and 100 nM TTX + 0.1 nM AaH-II* (second). Scale bars: 200 ms/2 nA and inset 5 ms/2 pA. E, Quantification of all parameters: peak current amplitude, AUC, and INaL on n = 6 cardiomyocytes. F, G, Same as D and E, but using 10 nM AaH-II* to act on the entire Nav population…

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  Figure 4. Selective activation of TTX-S Nav channels by AaH-II* prolongs the APD of human iPS-derived cardiomyocytes. A, Representative APs of a right ventricle cardiomyocyte in control condition, after 0.1 nM AaH-II* application, and the further addition of 100 nM TTX to block TTX-S Nav channels. B, Quantification of various AP parameters (n = 10). **, P < 0.01.

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  Figure 5. Effect of AaH-II* on calcium release from adult rat ventricular cardiomyocytes. A, Calcium transients in control condition and after addition of 0.1, 1, or 3 nM AaH-II*. SCR events are shown by arrows. Scale bars: 1 F/F0; 2 s. B, Percentage of cells presenting SCR events in various AaH-II* concentration conditions. C, Number of SCR events per min of recording as a function of AaH-II* concentration. D, Quantification of the properties of elicited calcium transients in control condition and after application of 0.1, 1, or 3 nM AaH-II*. E, Calcium transients in control condition and after perfusion of 100 nM TTX. Scale bars: 1 F/F0, 2 s. F, Quantification of amplitude, time to peak, and tau decay of calcium transients in control condition and after perfusion of 100 nM TTX. G, Representative calcium transients recorded in control condition and after application of 100 nM TTX. Scale bars: 1 F/F0; 2 s. H, Percentage of cells displaying SCR in various pharmacological conditions. I, Number of SCR per min for cells that displayed SCR. J, Properties of the calcium transients in the double pharmacological treatment condition (100 nM TTX + 0.1 nM AaH-II*).

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  Figure 6. Arrhythmogenic effects of AaH-II* in isolated rat heart models. A, Representative MEA recordings in control conditions and after perfusion of AaH-II* at various concentrations without and with pre-treatment with 100 nM TTX. Scale bar: 250 ms. B, Quantification of ventricular interbeat intervals (IBI) in various pharmacological conditions. *, P < 0.05, ***, P < 0.001. C, Representative ventricular local field potentials at 0.1 and 1 nM AaH-II* without and with 100 nM TTX pre-treatment. D, Quantification of the percentage of increase in repolarization area in various pharmacological conditions. E, Quantification of arrhythmogenic hearts in various pharmacological conditions. F, Representative MEA recordings and phase maps highlighting the arrhythmogenic impact of AaH-II* and 100 nM TTX pre-treatment on the directionality of the electrical propagation. The white arrow indicates this directionality. Scale bar: 100 ms. G, Activation maps in various pharmacological conditions. H, Quantification of activation time in various pharmacological conditions.

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  Figure 7. In vivo pro-arrhythmogenic effects of AaH-II*. A, Circulating plasmatic concentrations of AaH-II* at three injected doses in rats. B, Representative ECG recordings in control conditions or at three different doses of AaH-II*. Scale bar: 200 ms. C, Quantification of ECG parameters at the three doses of AaH-II*. *, P < 0.05. ***, P < 0.001. CV RR: coefficient of variation of RR. D, Representative recordings of A-V blocks and VT/VF at 15 and 30 μg/kg AaH-II*. E, Percentage of arrhythmic rats and associated phenotypes. F, Latency to first arrhythmia as a function of AaH-II* dose injected. G, Effects of AaH-II on ECG parameters in rats pre-treated with 27 μg/kg TTX. Scale bar = 200 ms. H, Percentage of arrhythmic rats after TTX pre-treatment. I, Latency to first arrhythmia at various AaH-II* doses after TTX pre-treatment. J, Various ECG parameters for three AaH-II* doses in rats pre-treated with 27 μg/kg TTX.