Furutani K , Tsumoto K , Chen IS , Handa K , Yamakawa Y , Sack JT , Kurachi Y .

R01 HL128537 NHLBI NIH HHS , U01 HL126273 NHLBI NIH HHS

	Figure 1. Cardiac APs induce hERG facilitation with nifekalant. (A and B) Representative cell currents from hERG channels in Xenopus oocytes evoked by a test pulse from holding potential of –90 to –50 mV before and after AP stimulation (1 Hz, 20×, AP waveform is the same as Fig. 2 H) and a step pulse (+60 mV for 4 s) in the presence of 30 µM nifekalant at room temperature. (C) AP–facilitation relation. The fraction of facilitation induced by repeating APs is normalized to the fraction induced by the +60 mV conditioning step pulse. Experimental data are means ± SEM (n = 8–15). The curve fit indicates exponential increase in the facilitation fraction by AP stimulation (facilitation = 1.02 − 0.70 ⋅ exp[−(#APs)/τ), τ = 5.45 ± 0.02]. (D) Requirement of nifekalant in the induction of facilitation. Effects of AP stimulation (1 Hz, 20×, AP waveform) on the increase in the hERG current were tested in the absence and presence of 30 µM nifekalant in the Xenopus oocytes (n = 7).
	Figure 2. Experimental and simulated macroscopic hERG/IKr currents as modified by nifekalant. (A–C) The macroscopic hERG/IKr currents in response to voltage-clamp pulses from −80 to +60 mV in 10-mV increments from a holding potential of −80 mV at room temperature; representative traces (A), the relationship between membrane voltage and the steady-state current amplitude and membrane voltage (B), and the tail current amplitude (activation curve; C). Experimental data are means ± SEM (n = 11). (D–G) Simulated effects of nifekalant on hERG/IKr currents at room temperature condition. The model assumes two populations of channels, with or without facilitation effect by nifekalant (100 nM). The V1/2 of activation for the facilitated fraction of channel was approximately −31 mV, almost 26 mV negative to that of control channel (see also Materials and methods). (D and E) The macroscopic hERG/IKr currents in response to voltage-clamp pulses from −80 to −30 mV (D) or +30 mV (E) at room temperature. (F and G) The relationship between the tail current amplitude and membrane voltage before (F) and after (G) the induction of facilitation effect. Experimental data are means ± SEM (n = 5). (H) The macroscopic hERG/IKr currents in response to cardiac AP at 37°C. In D–H, black, red, and green solid lines indicate with control, block with facilitation, and conventional block (block without facilitation), respectively. Under the condition in the block with facilitation, IKr comprises two fractions of IKr (see also main text), i.e., facilitated and unfacilitated fractions of IKr. In G right panel, orange and cyan dashed lines represent the facilitated and unfacilitated fractions of IKr, respectively. Experimental data are means ± SEM (n = 6–11).
	Figure 3. Effect of IKr facilitation on cardiac AP. (A–D) Simulated APs in an endocardial ventricular myocyte (A), IKr (B), activation state values for unfacilitated (xr1) and facilitated (xr2) fractions of IKr (C), and IK1 during APs with 100 nM nifekalant (D). Through A to D, black, green, and magenta solid lines indicate with control, block with facilitation, and conventional block (block without facilitation), respectively. In B and C, orange and cyan dashed lines represent the facilitated and unfacilitated fractions of IKr, respectively. Roman numerals above A indicate the phases of the AP in the case of with facilitation.
	Figure 4. Frequency-dependent effect of nifekalant on APD in non–heart failure model. Increases in simulated APD90 from the control condition in normal, non–heart failure model with 100 nM nifekalant (40% IKr block) at each stimulation frequency. Effects of IKr block and facilitation on the APs in normal, non–heart failure model at various simulation frequencies with 100 nM nifekalant.
	Figure 5. Frequency-dependent effect of nifekalant on APD in heart failure model. (A and C) Increases in simulated APD90 from the control condition in heart failure model with 100 nM nifekalant (40% IKr block; A) and 50% IKr block (C) at each stimulation frequency. (B and D) Effects of IKr block and facilitation on the APs in heart failure model at various simulation frequencies with 100 nM nifekalant (40% IKr block; B) and 50% IKr block (D). The small “hook” at the beginning of the AP of 2 Hz is the repolarization phase of the previous one.
	Figure 6. The effect of IKr facilitation on the APD prolongation and EAD development by IKr block. (A–C) The steady-state AP trains with 40% (A), 50% (B), and 55% (C) IKr block in heart failure model with and without facilitation. (D) Effect of IKr block and facilitation on the APD and the development of EADs in heart failure model. Green and magenta lines indicate APD90 of AP (without EAD) for block with facilitation or block without facilitation, respectively. Asterisk, dagger, and double dagger indicate the conditions in A–C, respectively. Sections and pipes indicate the upper limits of IKr block where APs were normally terminated. When EAD was observed, it was classified as either alternated EAD or periodic EAD. In the bottom panel of D, red and deep purple dots indicate APD90 of AP with EAD for block without facilitation, while light and deep green dots indicate APD90 of AP with EAD for block with facilitation. Horizontal bars above the dots indicate alternated EAD, AL, or periodic EAD, EAD; see also main text.
	Figure 7. The preventive effects of IKr facilitation on EAD development in several ventricular AP models. Simulated AP responses in the modified ORd human ventricular myocyte AP models, the modified TNNP human ventricular myocyte AP models, and the modified FRd guinea pig ventricular myocyte AP models of ENDO, MID, and EPI cells. APs in ORd and TNNP models were stimulated at 0.5 Hz, while APs in FRd model were stimulated at 1 Hz. In each panel, the intensity of IKr block is indicated as % Block. Magenta lines indicate AP responses in the IKr block without facilitation, while green lines indicate AP responses in the IKr block with facilitation at the same degree of IKr block as the overlaid magenta line (as an exception, in FRd model of EPI cell, EAD was not observed even by complete IKr block).
	Figure 8. Ionic mechanism of EAD development by IKr block and the influence of IKr facilitation. (A–E) Simulated APs and the changes in the membrane potential (Vm; A and B), L-type Ca2+ channels current, ICaL (C), the net ionic current, Inet (D), and IKr (E) during APs with 55% IKr block in heart failure model. Each simulation of block (left) and facilitation (right) was started from the same initial values. In each panel of B–E, three curves are overlaid. Thin dashed line, thick dashed line, and solid line indicate 6th, 29th, and 31st AP responses after the 55% IKr block, respectively, and these APs are marked with squares in A. In C, an arrowhead indicates the time point of the reactivation of L-type Ca2+ channels, and asterisks indicate the time point of the inward-outward balance of Inet in 31st AP response in the case without facilitation (magenta solid line; see also main text). In E, the increase in the IKr current in the case with facilitation is highlighted by two horizontal dashed lines and an arrow.
	Figure 9. The antiarrhythmic effect of IKr facilitation. Simulation of block and facilitation effect on the reexcitation by the second stimulation (S2) in the ORd model of nonfailing ENDO cell with the prolonged APD90 by 500 ms. AP responses to the S2 stimuli applied at the time indicated.
	Figure 10. Effect of IKr facilitation on the safety window of nifekalant. The changes in the membrane potential (Vm; upper) and IKr (lower) in the presence of various concentrations of nifekalant in normal, non–heart failure model. The therapeutic dose is set as the concentration that prolongs APD90 by 500 ms and effectively suppresses the ectopic excitation as shown in Fig. 9. 2.74× indicates 2.74 times higher drug concentration compared with its therapeutic dose (1×). Green and magenta lines indicate IKr block with and without facilitation, respectively. Roman numerals at the bottom indicate the phases of the AP with facilitation.
	Figure 11. Rabbit cardiac myocyte APs are more stable in nifekalant than dofetilide. AP responses in the isolated rabbit ventricular myocytes were stimulated by minimal current injection at 0.5 Hz in whole-cell current clamp mode at 37°C. (A and B) After recording of the control responses (black lines), cells were treated with either 1 µM dofetilide (pink lines) or 10 µM nifekalant (green line). (A) Representative AP responses without (left) or with (right) EAD in 1 µM dofetilide. Of 19 cells treated with 1 µM dofetilide, five cells showed EAD responses (26%). 14 cells showed the prolongation of APD in 1 µM dofetilide but did not show EAD responses. (B) Representative AP responses in 10 µM nifekalant. All cells treated with 10 µM nifekalant showed prolongation of APD upon 1 µM dofetilide but did not show EAD responses. (C and D) Prolongation of APD and beat-to-beat instability by 1 µM dofetilide and 10 µM nifekalant. Only the data from the cells showing AP responses without EAD were included in this analysis (14 cells of 1 µM dofetilide-treated group; 19 cells of 10 µM nifekalant-treated group). 30 consecutive APs responses in control and under the treatment of either 1 µM dofetilide or 10 µM nifekalant were recorded, and APD90 of the i+1th action potential (APD90i+1) was plotted against APD90 of the one action potential before (ith; APD90i). APD90s were prolonged by treatment with 1 µM dofetilide (C) or 10 µM nifekalant (D) compared with control. 1 µM dofetilide prolonged APD90 longer (P = 4.26 × 10−13, Student’s t test) than 10 µM nifekalant. The control APD90s were identical between dofetilide- and nifekalant-treated cells (P = 0.58).

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