Cardiotoxic Potential of Hydroxychloroquine, Chloroquine and Azithromycin in Adult Human Primary Cardiomyocytes

Abstract

Substantial efforts have been recently committed to develop coronavirus disease-2019 (COVID-19) medications, and Hydroxychloroquine alone or in combination with Azithromycin has been promoted as a repurposed treatment. Although these drugs may increase cardiac toxicity risk, cardiomyocyte mechanisms underlying this risk remain poorly understood in humans. Therefore, we evaluated the proarrhythmia risk and inotropic effects of these drugs in the cardiomyocyte contractility-based model of the human heart. We found Hydroxychloroquine to have a low proarrhythmia risk, whereas Chloroquine and Azithromycin were associated with high risk. Hydroxychloroquine proarrhythmia risk changed to high with low level of K+, whereas high level of Mg2+ protected against proarrhythmic effect of high Hydroxychloroquine concentrations. Moreover, therapeutic concentration of Hydroxychloroquine caused no enhancement of elevated temperature-induced proarrhythmia. Polytherapy of Hydroxychloroquine plus Azithromycin and sequential application of these drugs were also found to influence proarrhythmia risk categorization. Hydroxychloroquine proarrhythmia risk changed to high when combined with Azithromycin at therapeutic concentration. However, Hydroxychloroquine at therapeutic concentration impacted the cardiac safety profile of Azithromycin and its proarrhythmia risk only at concentrations above therapeutic level. We also report that Hydroxychloroquine and Chloroquine, but not Azithromycin, decreased contractility while exhibiting multi-ion channel block features, and Hydroxychloroquine's contractility effect was abolished by Azithromycin. Thus, this study has the potential to inform clinical studies evaluating repurposed therapies, including those in the COVID-19 context. Additionally, it demonstrates the translational value of the human cardiomyocyte contractility-based model as a key early discovery path to inform decisions on novel therapies for COVID-19, malaria, and inflammatory diseases.

Keywords: COVID-19; adult human primary cardiomyocyte; contractility; hydroxychloroquine; proarrhythmia risk; translation.

Figure 1.

Human proarrhythmic potential of Hydroxychloroquine (HCQ) and Chloroquine (CQ). Mean % incidence in aftercontraction, contraction failure, premature contraction and pause-dependent arrhythmia when adult human primary cardiomyocytes were treated with HCQ (n = 9 cells, donor heart 200417HHA, A), CQ (n = 8 cells, donor heart 200321HHA, B) and HCQ in combination with low K⁺ (2.5 mM KCl, n = 12 cells, donor heart 200425HHA, C) at a pacing frequency of 1 Hz. The light green zone represents therapeutic free plasma concentration. Abbreviation: min., minute.

Figure 2.

Typical human cardiomyocyte contractility transients after exposure to Hydroxychloroquine (HCQ), Chloroquine (CQ), and Azithromycin (AZ). A–F, transients recorded from 3 independent adult human cardiomyocytes in the presence of vehicle control and after exposure to HCQ (3 µM = 10× the tFPC), CQ (1.2 µM = 3× the tFPC), and AZ (10 µM = 7.5× the tFPC), respectively, at a pacing frequency of 1 Hz. Abbreviations: Sarc. length, Sarcomere length; tFPC, therapeutic free plasma concentration.

Figure 3.

Effects of high Mg²⁺, temperature and Azithromycin (AZ) on Hydroxychloroquine (HCQ) proarrhythmic potential. Mean % incidence in aftercontraction, contraction failure, premature contraction and pause-dependent arrhythmia when adult human primary cardiomyocytes were treated with HCQ in combination with high Mg²⁺ (MgCl₂ 3 mM, n = 9 cells, donor heart 200425HHA, A), and subjected to an increase in temperature after exposure to 0.3 µM HCQ (n = 6 cells, donor heart 200601HHB, B), AZ (n = 9 cells, donor heart 200518HHA, C), HCQ in combination with 0.3 µM AZ (0.75X the tFPC, n = 6 cells, donor heart 200518HHA, D) and AZ in combination with 0.3 µM HCQ (1× the tFPC, n = 7 cells, donor heart 200518HHA, E), at a pacing frequency of 1 Hz. The light green zone represents tFPC. Abbreviations: min., minute; Temp., temperature; tFPC, therapeutic free plasma concentration.

Figure 4.

Human inotropic potential of Hydroxychloroquine (HCQ), Chloroquine (CQ), and Azithromycin (AZ). Cumulative concentration-effect curves for sarcomere shortening are shown as a function of concentrations tested (A and C) or multiple of tFPCs (B and D) at a pacing frequency of 1 Hz. Curves were fitted to sarcomere shortening data using SigmaPlot v14.0 and the concentrations inducing 50% decrease (IC50s) in sarcomere shortening were estimated. HCQ (AZ) and AZ (HCQ) represent HCQ curve in the presence of 0.3 µM AZ and AZ curve in the presence of 0.3 µM HCQ, respectively. The 0.01 µM represents the normalized vehicle data for drugs in (A) and (D). Abbreviation: tFPC, therapeutic free plasma concentration.

Figure 5.

Effects of Hydroxychloroquine (HCQ), Chloroquine (CQ), and Azithromycin (AZ) on cardiac ion channels. A, Stability of current recordings over time in the presence of vehicle, 0.33% dimethyl sulfoxide. B–D, concentration-effect curves for HCQ, CQ, AZ and 3 positive controls (E-4031, Tetracaine and Nifedipine) on hERG, Nav1.5, and Cav1.2 currents, respectively. IC50, concentration inducing 50% decrease in the current (n = 2 replicates per concentration-effect curve).