Synergistic Adverse Effects of Azithromycin and Hydroxychloroquine on Human Cardiomyocytes at a Clinically Relevant Treatment Duration

Abstract

Adverse effects of drug combinations and their underlying mechanisms are highly relevant for safety evaluation, but often not fully studied. Hydroxychloroquine (HCQ) and azithromycin (AZM) were used as a combination therapy in the treatment of COVID-19 patients at the beginning of the pandemic, leading to higher complication rates in comparison to respective monotherapies. Here, we used human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to systematically investigate the effects of HCQ, AZM, and their combination on the structure and functionality of cardiomyocytes, and to better understand the underlying mechanisms. Our results demonstrate synergistic adverse effects of AZM and HCQ on electrophysiological and contractile function of iPSC-CMs. HCQ-induced prolongation of field potential duration (FPDc) was gradually increased during 7-day treatment period and was strongly enhanced by combination with AZM, although AZM alone slightly shortened FPDc in iPSC-CMs. Combined treatment with AZM and HCQ leads to higher cardiotoxicity, more severe structural disarrangement, more pronounced contractile dysfunctions, and more elevated conduction velocity, compared to respective monotreatments. Mechanistic insights underlying the synergistic effects of AZM and HCQ on iPSC-CM functionality are provided based on increased cellular accumulation of HCQ and AZM as well as increased Cx43- and Nav1.5-protein levels.

Keywords: azithromycin; cardiomyocytes; conduction velocity; drug interaction; drug testing; field potential duration; human induced pluripotent stem cells; hydroxychloroquine.

Figure 1

Morphological changes and cytotoxicity in iPSC-CMs treated with HCQ and AZM. (A) Representative brightfield images depicting morphology of iPSC-CMs after 7-day treatments with HCQ and AZM in different concentrations. Arrows indicate formation of vacuoles. Scale bar: 100 µm. (B) Cell viability after 7-day drug treatment as determined by measurement of formazan formation in the MTT assay. (C) LDH activity detected in cell supernatants after 7-day drug treatment. (D) Representative brightfield images depicting morphology of iPSC-CMs after 7-day drug treatment and 7-day washout period. Even after washout, iPSC-CMs treated with a combination of high concentrations of AZM and HCQ show severe morphological changes and increased cell death. Scale bar: 100 µm. (E) Cell viability after 7-day drug washout as determined by using the MTT assay. (F) LDH activity detected in supernatants after 7-day drug washout. Data represent technical replicates (points) and means (squares) of each experiment, N = 3–7 independent experiments using iPSC-CMs from 3 healthy donors (iBM76.1, iBM76.3 in green; iWTD2.1, iWTD2.3 in grey, isWT7.22 in pink). Lines and errors show overall mean and SEM. Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, **** p < 0.0001. N.D.—not determined.

Figure 2

HCQ and AZM cause sarcomeric disorganization in iPSC-CMs. (A) Representative images of α-actinin immunostained iPSC-CMs treated with different concentrations of HCQ and AZM for 7 days. (B,C) Analysis of cell areas after 7-day drug treatment (B) and after subsequent 7-day washout (C). A total of n = 160–240 cells (40 per experiment) from 6 (B) or 4 (C) independent experiments per condition were analyzed. Points represent values of single cells and squares the median values of individual experiments. Lines indicate median and 95% CI of the overall population. (D) Representative images of structurally organized and disorganized iPSC-CMs after drug treatment for 7 days. (E) Percentage of structurally organized and disorganized iPSC-CMs after 7-day drug treatment. Mean and SEM of 5 independent experiments (n = 96–272 cells analyzed per condition from each experiment, same number of cells at different conditions analyzed within one experiment) are shown. (F,G) Sarcomere length after 7-day drug treatment (F) and after 7-day washout (G). Mean and SEM of n = 50–60 cells (10 per experiment) from 6 (F) or 5 (G) independent experiments are shown. Data plots in (F), and (G) show technical replicates (dots) and mean values (squares). Colors indicate iPSC-CM differentiations from 3 healthy donors (iBM76.1, iBM76.3 in green; iWTD2.1, iWTD2.3 in grey, isWT7.22 in pink). Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Contractile dysfunctions in iPSC-CMs treated with HCQ and AZM. (A) Representative motion traces observed in iPSC-CMs using vector-based quantification on treatment day 3 (left) and 7 (right). Values represent mean and SEM of motions from aligned contraction-relaxation cycles of a representative video. (B–D) Effects of AZM and HCQ alone as well as their combination on the beating rate (B), contraction time (C), and relaxation time (D) on treatment day 3 (left) and 7 (right). Data represent technical replicates (points, n = 9–54 videos per condition) and means (squares) of each experiment, N = 4–6 independent experiments using iPSC-CMs from 3 healthy donors (iBM76.1, iBM76.3 in green; iWTD2.1, iWTD2.3 in grey, isWT7.22 in pink). Due to the toxic effects of HCQ or AZM at higher concentrations or in combination, fewer videos could be analyzed under these conditions. Lines show overall mean values and SEM. Statistical analysis based on the mean values of the individual experiments using one-way ANOVA and Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 4

Effects of HCQ and AZM on the field potential duration of iPSC-CMs. (A) Representative recordings of extracellular FP in spontaneous beating iPSC-CMs under different treatment conditions. iPSC-CMs treated with 10 µM AZM and 10 µM HCQ in combination stopped beating at day 8 (one day after initiation of washout). (B,C) Effect of AZM (B) or HCQ (C) on the corrected FPD (FPDc, normalized to day 0) during 7-day treatment and subsequent 7-day washout. (D,E) Effects of HCQ (1, 3, and 10 µM) combined with 1 µM AZM (D) or 10 µM AZM (E) on FPDc during 7-day treatment and following 7-day washout. iPSC-CMs derived from four donors were used for MEA recording. For the initial recording (day 0), 10 ≤ n ≤ 13 for all conditions. Spontaneous beating states of iPSC-CMs are listed in Table S1. Two-way ANOVA with Bonferroni post-hoc test was used for statistical evaluation (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 5

Changes in conduction trajectory and augmented CV in iPSC-CMs treated with AZM and HCQ alone and in combination. (A) Representative heatmaps illustrating the conduction trajectories of electrical signals in iPSC-CMs under different conditions during 7-day drug treatment and following 7-day washout. Due to cell death, no signal was captured in cells treated with 10 µM HCQ and 10 µM AZM in combination in the washout period. (B,C) CV of iPSC-CMs treated with AZM (B, *: 10 µM AZM vs. control) and HCQ (C, ^#: 3 µM HCQ vs. control, *: 10 µM HCQ vs. control) for 7 days and following washout for 7 days (normalized to day 0). (D,E) CV of iPSC-CMs treated with 1 µM (D, *: 1 µM AZM + 10 µM HCQ vs. control) and 10 µM AZM (E, *: 10 µM AZM + 1 µM HCQ vs. control, ^#: 10 µM AZM + 3 µM HCQ vs. control, ^§: 10 µM AZM + 10 µM HCQ vs. control) combined with HCQ (1, 3, and 10 µM) during 7-day treatment and following 7-day washout. iPSC-CMs derived from four donors were used for MEA recording. Spontaneous beating states of iPSC-CMs are listed in Table S1. Two-way ANOVA with Bonferroni post-hoc test was used (*^,§,# p < 0.05, **^,§§,## p < 0.01, ***^,§§§,### p < 0.001, and ****^,#### p < 0.0001).

Figure 6

Nav1.5 and Cx43 protein expression in iPSC-CMs treated with HCQ and/or AZM. (A) Two representative Western blots showing the expression of Nav1.5 and Cx43 in iPSC-CMs under different drug treatment conditions. (B) Quantitation of protein expression levels of Nav1.5 in iPSC-CMs under different conditions; N = 6 independent differentiations. (C) Quantitation of protein expression of Cx43 in iPSC-CMs under different drug treatment conditions; N = 6 independent differentiations. (D) Representative images showing immunostaining for Nav1.5 (green) and Cx43 (red) in iPSC-CMs under different drug treatment conditions. Cell nuclei are shown in blue (Hoechst). Statistical evaluation was performed using one-way ANOVA with Tukey’s multiple comparison test (** p < 0.01, and *** p < 0.001).

Figure 7

Accumulation of HCQ and AZM in iPSC-CMs after the 7-day treatment. (A,B) Concentrations of HCQ (A) and AZM (B) in cell lysates from iPSC-CMs after the 7-day treatment with HCQ and AZM at different conditions, determined using mass spectrometry. Data represent mean and SEM of N = 3 independent experiments, performed with iPSC-CMs from 2 healthy donors (iBM76.1, iBM76.3 in green; iWTD2.1 in grey). N.D., below detection limit. Statistical evaluation was performed using one-way ANOVA with Tukey’s multiple comparison test (* p < 0.05).