In vitro effect of hydroxychloroquine on pluripotent stem cells and their cardiomyocytes derivatives

Abstract

Introduction: Hydroxychloroquine (HDQ) is an antimalarial drug that has also shown its effectiveness in autoimmune diseases. Despite having side effects such as retinopathy, neuromyopathy and controversial cardiac toxicity, HDQ has been presented and now intensively studied for the treatment and prevention of coronavirus disease 2019 (COVID-19). Recent works revealed both beneficial and toxic effects during HDQ treatment. The cardiotoxic profile of HDQ remains unclear and identifying risk factors is challenging. Methods: Here, we used well-established cell-cultured to study the cytotoxic effect of HDQ, mouse induced pluripotent stem cells (miPSC) and their cardiomyocytes (CMs) derivatives were exposed to different concentrations of HDQ. Cell colony morphology was assessed by microscopy whereas cell viability was measured by flow cytometry and impedance-based methods. The effect of HDQ on beating activity of mouse and human induced pluripotent stem cell-derived CMs (miPSC-CMs and hiPSC-CMs, respectively) and mouse embryonic stem cell-derived CMs (mESC-CMs) were captured by the xCELLigence RTCA and microelectrode array (MEA) systems. Results and discussion: Our results revealed that 20 µM of HDQ promotes proliferation of stem cells used suggesting that if appropriately monitored, HDQ may have a cardioprotective effect and may also represent a possible candidate for tissue repair. In addition, the field potential signals revealed that higher doses of this medication caused bradycardia that could be reversed with a higher concentration of ß-adrenergic agonist, Isoproterenol (Iso). On the contrary, HDQ caused an increase in the beating rate of hiPSC-CMs, which was further helped upon application of Isoproterenol (Iso) suggesting that HDQ and Iso may also work synergistically. These results indicate that HDQ is potentially toxic at high concentrations and can modulate the beating activity of cardiomyocytes. Moreover, HDQ could have a synergistic inotropic effect with isoproterenol on cardiac cells.

Keywords: cardiotoxicity; differentiation; hydroxychloroquine; proliferation; stem cells.

FIGURE 1

Effect of HDQ on mouse induced pluripotent stem cells (miPSCs). (A) Experimental workflow for culturing, dissociating and seeding cells on E-Plate cardio 96 of xCELLigence RTCA System. (B) Exemplary recordings showing changes in cell index (CI) in the presence of different concentrations of HDQ as compared to control condition. Cells (25,000 cells/well) were seeded and incubated at 37°C with 5% CO₂ 2 h before measurement. Different concentrations of HDQ were generally added 24 h post plating. (C) Peak cell index values before (pre-treatment) and after treatment with different concentrations of HDQ for 12, 24 36 and 72 h. (D) Representative bright field microscopic images of control and 250 µM HDQ-treated cells after 48 h exposure. (E) Representative flow cytometry histogram showing the effect of different concentrations of HDQ on miPSCs after 48 h exposure. Data are presented as the mean ± SEM of three independent experiments. *p < 0.05 significantly different compared with control.

FIGURE 2

Dynamic monitoring the effect of HDQ on mESC-CMs using RTCA cardio system. (A) Experimental workflow used to examine the effect of HDQ on mouse ES cell-derived cardiomyocytes (mESC-CMs) proliferation and beating activity. (B) Representative kinetics showing change in cell index in the absence (control) or presence of increasing concentration of HDQ. (C) Representative images of changes in beating activity of cardiomyocytes at different time points treated or not (control) with different concentrations of HDQ. (D) Average of beating rate (1/minute) of mESC-CMs recorded at indicated time points before- (pre-treatment) and after treatment with different HDQ concentrations at different time points. Data are shown as mean ± SEM of 6 wells each from three independent experiments. *p < 0.05 vs control.

FIGURE 3

Multielectrode arrays recordings of spontaneously beating EBs/cardiac cluster and effect of HDQ. (A,B) Microscopy images of mES cells (mESC) colony (A) and EBs (B) at day 14 of differentiation. (C) Viable and spontaneously beating mESC-derived cardiac cluster attached to MEA chamber. (D) Representative extracellular field potential (FP) of beating cluster of miPSC-CMs recordings from all 60 electrodes of MEA plate.

FIGURE 4

Effect of HDQ and ß-adrenergic receptor agonist on field potential recorded on beating cluster of miPSC-CMs (A) and hiPSC-CMs (B) using MEA system. Representative field potential (a) traces demonstrating effects of ß-adrenergic agonist isoprenaline (ISO) on clusters of miPSC-CMs (A) and hiPSC-CMs (B) pre-treated with HDQ as indicated. Concentration of both compound, ISO and HDQ were added gradually. The exposure time was at least 3 min for each concentration. Statistical analysis of FP frequencies (b) and voltage (c) in MEA recordings of miPSC-CMs (A) and hiPSC-CMs (B). Data are shown as mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.005 vs. control (−/−: absence of drug).

FIGURE 5

Effect of HDQ on field potential of hiPSC-CMs pre-stimulated with increase concentration of ß-adrenergic receptor agonist. (A) Representative field potential traces and frequencies from MEA analysis of pre-stimulated beating hiPS cell-derived CMs (hiPSC-CMs) cluster exposure to different concentrations of HDQ added gradually. Concentration of ISO and HDQ were added gradually for at least 3 min exposure time for each concentration. (B,C) Statistical analysis of FP frequencies (B) and voltage (C) recorded after treatment as indicated. Data are shown as mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.005, *** p < 0.0005 vs. control (−/−: absence of drug).