Figures
Figure 1. Effects of HCQ on ion currents attributable to hNav1.5 (INa, A), Cav1.2 (ICa,L, B), Kv4.3 (Ito, C ), hERG (IKr, D), KCNQ1/E1 (IKs, E) and Kir2.1 (IK1, F). (A-F) Left panels (i): Typical currents in the presence of 0, 1, 10 and 100 µM HCQ. Insets summarize the voltage clamping protocols. Right panels (ii): Mean concentration-response plots constructed for the maximum magnitude of each current (n cells).
Figure 2. Effects of AZM on ion currents attributable to hNav1.5 (INa, A), Cav1.2 (ICa,L, B), Kv4.3 (Ito, C ), hERG (IKr, D ), KCNQ1/E1 (IKs, E) and Kir2.1 (IK1, F). (A-F) Left panels (i): Typical currents in the presence of 0, 1, 10 and 100 µM HCQ. Insets summarize the voltage clamping protocols. Right panels (ii): Mean concentration-response plots constructed for the maximum magnitude of each current ((n cells).
Figure 3. Effects of HCQ in combination with 10 µM AZM on ionic currents attributable to hNav1.5 (INa, A), Cav1.2 (ICa,L, B), Kv4.3 (Ito, C ), hERG (IKr, D), KCNQ1/E1 (IKs, E) and Kir2.1 (IK1, F). (A-F) Left panels (i): Typical currents in the presence of 0, 1, 10 and 100 µM HCQ with 10 µM AZM. Insets summarize the voltage clamping protocols. Right panels (ii): Mean concentration-response plots constructed for the maximum magnitude of each current (n cells).
Figure 4. Computational docking studies. (A) hERG homotetramer structure (PDB ID: 5va2) with HCQ molecule (space-filling representation) indicating binding site placement. Residues 544-671 used in IFD of hERG chains A, B, C, and D colored red, green, yellow, and blue, respectively. (B) Overlay of IFD HCQ positions. (C) Representative, top-scoring positions of HCQ (ball-and-stick). Y652 and F656 residues in stick representation colored accordingly to their parent chains. (D) 2-dimensional (2D) diagram depicting interaction types between presented HCQ position and hERG. (E) Representative, top-scoring position of AZM, and (F) 2D diagram depicting AZM-hERG interactions. Diagrams were prepared using Non-bond Interactions Monitor tool in Discovery Studio Visualizer (Dassault Systèmes, Vélizy-Villacoublay, France). IFD = Induced Fit Docking.
Figure 5. Multi-electrode array (MEA) mapping and electrocardiographic (ECG) studies in isolated perfused guinea-pig hearts following HCQ and AZM challenge. (A) Schematic summary of stimulation and MEA and ECG recording configuration. (B) Typical left atrial and left ventricular isochronal conduction maps obtained in the presence of 0, 0.01, 0.1, 1.0 and 10 µM HCQ. (C) (i) Successive LV isochronal maps before and following addition of 10 µM HCQ and 10 µM HCQ and 10 µM AZM in combination followed by drug washout. (ii) Quantified conduction velocities through these conditions. (D) ECG studies: (i) ECG records obtained before and following application of 10 µM HCQ, and 10 µM HCQ and 10 µM AZM combined, followed by drug washout. (ii)-(v): corresponding measured (ii) heart rates (HR), (iii) QT and (iv) PR intervals and (v) QRS durations (n hearts) * p<0.05, ** p<0.01, *** p<0.001
Figure 6. Voltage RH237 and Rhod-2 optical mapping in isolated perfused hearts. Comparisons of results before and following challenge by 1 µM and 10 µM HCQ, and 10 µM HCQ combined with either 1 or 10 µM AZM. (A, B) RH237 mapping: (A)(i) Optical mapping and ECG monitoring configuration (ii) Maps of action potential initiation and conduction; (iii) heart rates and (iv) conduction velocities. (B) Action potential time-courses and recoveries: (i) Maps of AP durations at 90% repolarization (APD90). (ii) Representative APs recorded from defined regions of interest. (iii) APD90s averaged over the field of view. (C) Rhod-2 AM (CaD) mapping of ventricular Ca2+ transients (CaT): (i) maps of CaD durations at 90% recovery (CaD90). (ii) Comparison of typical CaT transients averaged over defined regions of interest; (iii) CaD90 magnitudes; (iv) CaD90 dispersions (n hearts). NS not significant, * p<0.05, ** p<0.01, *** p<0.001.
Figure 7. Voltage RH237 optical mapping of re-entry and ventricular arrhythmic tendency in isolated perfused hearts. (A) Recording and programmed stimulation configuration. (B) Optical action potential traces obtained under programmed pacing at 190 ms, 180 ms and 170 ms cycle lengths (CLs), before (control) and following addition of 10 µM HCM, and 10 µM HCM with added 10 µM AZM. (C) Corresponding voltage maps obtained at the numbered colour-coded timepoints (n hearts).
Figure 8. In silico modelling of action potentials before and following HCQ and combined HCQ and AZM challenge. (A) Simulated action potentials of human ventricular cell under control conditions and in the presence of HCQ (10 µM) and HCQ (10 µM) + AZM (1 µM) combined. HCQ and AZM when combined produced a synergistic action in prolonging action potential duration (APD90), as well as reducing the repolarization rate following the end of phase 0 depolarisation. (B) Effects of HCQ and HCQ + AZM combined on APD90 and the maximal upstroke velocity (MUV) of the AP. (C) Illustration of electrophysiological effects and possible mechanisms underlying the pro-arrhythmic effects of applications of HCQ and HCQ and AZM combined at the ion channel, cellular and tissue levels.