LUF7244, an allosteric modulator/activator of Kv11.1 channels, counteracts dofetilide‐induced torsades de pointes arrhythmia in the chronic atrioventricular block dog model

Figures

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  Figure 1. Experimental set‐up for in vivo studies

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  Figure 2. LUF7244 increases steady‐state IKv11.1 in HEK‐hERG cells. (a) Chemical structure of LUF7244 and dofetilide. (b) Docking pose of LUF7244, after 50 ns of molecular dynamics simulation to the cryo‐EM structure of hERG. (c) Representative traces of Kv11.1 current with or without infusion of LUF7244 (0.5, 3, and 10 μM). Stimulation protocol is shown on top panel. (d) I‐V relationship curve of IKv11.1 under baseline conditions and in the presence of LUF7244 (0.5, 3, and 10 μM; n = 14, 13, and 12 cells). (e) I‐V curve of IKIR2.1 currents obtained from KWGF cells under baseline conditions and in the presence of 10‐μM LUF7244 (n = 9 cells). (f) I‐V relationship curve of INav1.5 measured in HEK‐Nav1.5 cells under baseline conditions and 10‐μM LUF7244 treatment (n = 10 cells). Raw current data were normalized by each cell's membrane capacitance for current density, for comparisons. Values are shown as mean ± SEM

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  Figure 3. LUF7244 shortens APD and suppresses APD prolongation in hiPS‐CMCs. (a) AP recordings in control and after 5‐min incubation with the indicated drug or drug combinations (red) at 10 μM LUF7244 and 30 nM dofetilide. Horizontal bar indicates 1 s. (b) The bar graphs represent changes in normalized APD90 observed with each drug, or drug combination tested (n = 5). Values are shown as mean ± SEM. *P < .05, significantly different from the same drug(s) at 0 min. § P < .05, dofetilide + LUF7244 significantly different from dofetilide

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  Figure 4. LUF7244 shortens APD and reduces dofetilide‐induced EAD in isolated canine cardiomyocytes. (a) Representative examples of LUF7244 induced shortening of APD in cardiomyocytes (left panel). Summarized results of APD90 (middle panel) and STV (right panel) after LUF7244 application to SR and CAVB cardiomyocytes . In SR cardiomyocytes , n = 11, 10, 13, and 14 cells were treated with 0.5, 1, 3, and 10 μM of LUF7244, respectively. In CAVB cardiomyocytes , n = 20, 16, 22, and 17. (b) LUF7244 dose dependently suppressed dofetilide‐induced EAD in SR cardiomyocytes (left panel). Summarized results of APD90 (middle panel) and incidence of EADs after dofetilide infusion in SR CMCs (right panel). Data are represented as number of cells without EADs/total number of cells showing EADs upon dofetilide. n = 7, 11, and 7 cells for 1, 3, and 10 μM of LUF7244, respectively. (c) One, 3, and 10 μM of LUF7244 suppressed dofetilide‐induced APD prolongation and EADs in CAVB cardiomyocytes . Summarized results of APD90 (middle panel) and incidence of EADs after dofetilide infusion in CAVB cardiomyocytes (right panel), n = 10, 10, and 11 cells, respectively. APD90, action potential duration (measured at 90%…

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  Figure 5. LUF7244 increases dofetilide‐sensitive IK in isolated cardiomyocytes and has no effect on ICa‐L. (a) Representative IK traces (left panel) measured from canine cardiomyocytes. Summarized IK current density at +60 mV is shown in right panel (n = 7). (b) Representative IK traces (left panel) measured from canine cardiomyocytes. HMR1556 (500 nM) inhibited the IKs component of total IK. The total IK was further blocked after 1‐μM dofetilide was applied. Summarized IK current density at +60 mV after the different treatments is shown in the right panel (n = 7 cells). (c) ICa‐L measured under baseline conditions or in the presence of 10‐μM LUF7244 or 5‐μM nifedipine (n = 6). (d) Rundown measurement of ICa‐L after 0 min, 5 min or 10 min (n = 6). Current inhibition by 5‐μM nifedipine at the end of the rundown experiment. Left panel: representative traces. Right panel: peak ICa‐L density distribution at +10 mV and corresponding I‐V curves (in box) of ICa‐L. Data shown are individual values with means ± SEM. *P < .05, significantly different from total IK, § P < .05, significantly different from LUF7244, & P < .05, significantly different from HMR, # P < .05, significantly different from…

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  Figure 6. Electrophysiological effects of LUF7244 in dogs with sinus rhythm (n = 5). Representative traces of ECG measured from lead II and LV/RV MAP under baseline infusion and at the end of LUF7244 (2.5 mg·kg−1·15 min−1) infusion, respectively. Values of QT, RR, and LV/RV MAPD are presented next to the corresponding traces. Horizontal line corresponds to 1 s

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  Figure 7. LUF7244 counteracts dofetilide‐induced TdP arrhythmia in CAVB dogs (n = 7). (a) Representative example of ECG (lead II and LV/RV MAP) under baseline infusion and at the first TdP induced by dofetilide (0.025 mg·kg−1·5 min−1). (b) The combined infusion of dofetilide and LUF7244 did not evoke TdP arrhythmia. Values of QT, RR, and LV/RV MAPD are presented next to corresponding traces. Horizontal line corresponds to 1 s. Quantification of incidence of single (sEB), multiple (mEB) ectopic beats and torsades de pointes (TdP) arrhythmia (c) and arrhythmia score (d) from both inducibility and prevention experiments. Data shown are means ± SEM, with individual values in (d). *P < .05 significantly different from TdP in inducibility experiment, # P < .05, significantly different from baseline‐2, ### P < .05, significantly different from baseline‐1