AimsWe investigate mechanisms for potential pro-arrhythmic effects of hydroxychloroquine (HCQ) alone, or combined with azithromycin (AZM), in Covid-19 management supplementing the limited available experimental cardiac safety data.MethodsWe integrated patch-clamp studies utilizing In Vitro ProArrhythmia Assay (CiPA) Schema IC50 paradigms, molecular modelling, cardiac multi-electrode array and voltage (RH237) mapping, ECG studies, and Ca2+ (Rhod-2 AM) mapping in isolated Langendorff-perfused guinea-pig hearts with human in-silico ion current modelling.ResultsHCQ blocked IKr and IK1 with IC50s (10±0.6 and 34±5.0 μM) within clinical therapeutic ranges, INa and ICaL at higher IC50s, leaving Ito and IKs unaffected. AZM produced minor inhibition of INa, ICaL, IKs, and IKr,, sparing IK1 and Ito. HCQ+AZM combined inhibited IKr and IK1 with IC50s of 7.7±0.8 μM and 30.4±3.0 μM, sparing INa, ICaL and Ito. Molecular modelling confirmed potential HCQ binding to hERG. HCQ slowed heart rate and ventricular conduction. It prolonged PR, QRS and QT intervals, and caused prolonged, more heterogeneous, action potential durations and intracellular Ca2+ transients. These effects were accentuated with combined HCQ+AZM treatment, which then elicited electrical alternans, re-entrant circuits and wave break. Modelling studies attributed these to integrated HCQ and AZM actions reducing IKr and IK1, thence altering cell Ca2+ homeostasis.ConclusionsCombined HCQ+AZM treatment exerts pro-arrhythmic ventricular events by synergetically inhibiting IKr, IKs with resulting effects on cellular Ca2+ signalling, and action potential propagation and duration. These findings provide an electrophysiological basis for recent FDA cardiac safety guidelines cautioning against combining HCQ/AZM when treating Covid-19.