In vitro safety “clinical trial” of the cardiac liability of drug polytherapy

Figures

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  FIGURE 1. The cardiac microphysiological system. (a) Photograph of a cardiac MPS under fluorescent lighting with fluidic tubing to deliver media and drugs. (b) Brightfield image of a cardiac MPS containing ~ 2,500 human induced pluripotent stem cell‐derived cardiomyocytes. The cell chamber is separated from adjacent feeding channels via a fenestration barrier of 2 μm wide grooves allowing for nutrient diffusion while protecting the tissue in the cell chamber from media flow‐induced shear stress. The anchoring pillars on either side of the cell chamber help keep the heart muscle elongated and provide resistance for contraction. (c) Representative images of the same tissue under GFP fluorescence for calcium transient recordings (top) or FarRed voltage dye staining (bottom). (d) The top graph shows a typical ECG recording from which the clinical QT interval can be determined. We use APD80 as a proxy for QT duration, corresponding to the duration of the action potential at 80% of its repolarization (bottom). The APD (red) and Ca (green) waveforms are timestamped identifying temporal kinetics. APD80, 80% repolarization time; Ca, calcium; ECG, electrocardiogram; GFP, green fluorescent protein;…

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  FIGURE 2. Electrophysiology analysis of chronic exposure to hydroxychloroquine (HCQ), azithromycin (AZM) or their polytherapy. Doses were chosen to closely mimic clinical trial drug prescription used for coronavirus disease 2019 (COVID‐19) treatment: 0.24 μM HCQ and 0.15 μM AZM on day1 followed by 0.12 μM and 0.075 μM AZM on day 2 to day 10. 19 , 20 Polytherapy was the combination of both monotherapy doses (Table S1). Violin plots demonstrate spontaneous beating during the therapy for HCQ (a), AZM (c) or polytherapy (e). Violin plots depicting the change in 80% repolarization time (APD80) during the therapy for HCQ (b), AZM (d) or polytherapy (f). APD80 values were calculated from spontaneous recording, corrected for beat rate using the Frederica correction. 48 In all graphs, the values were normalized to baseline at day 0 for each tissue. Each point corresponds to one heart muscle. The violin plots show the arithmetic median (solid line) and upper and lower quartile (dashed lines) as well as minimum and maximum values (truncation of violin shape). All tissues analyzed were within inclusion criteria of APD80 less than 500 ms at baseline. Statistics run were one‐way analysis of variance…

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  FIGURE 3. Instability and arrhythmic study of chronic exposure to hydroxychloroquine (HCQ), azithromycin (AZM) and their polytherapy. Poincaré plots 36 , 49 were used to visualize rhythm instabilities in different tissues. Where small clusters of daily traces represent minimal arrhythmic risk, and large complex polygons are indicative of rhythm instability and high arrhythmic risk. (a) Representative Poincaré graph of a tissue exposed to HCQ only. Disorganized polygons can be observed at days 5 and 10 indicative of drug‐induced arrhythmia. (b) Poincaré graph of AZM‐treated heart muscle where instabilities were observed starting on day 6. (c) Representative graph of polytherapy instabilities, which show fewer and smaller polygons when compared to monotherapy, indicative of reduced arrhythmic risk. (d–f) Histograms showing weak or no signal (black), CaD80 above 600 ms with (striated) or without (grey) early afterdepolarization (EAD) and arrhythmic event (pink) as percentage of total heart muscles (i.e., MPS). The analysis was performed for HCQ alone (d), AZM alone (e), and for polytherapy (f). Representative calcium transient trace at baseline and after exposure to 9 days of HCQ (g) or 10…

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  FIGURE 4. Proteomics analysis of microphysiological systems effluent during chronic exposure to hydroxychloroquine (HCQ) and azithromycin (AZM) polytherapy. Scatter plot of biomarkers showing significant changes with polytherapy in the chronic study. The effluent media was analyzed on days 2, 4, 6, 8, and 10. In the data postprocessing, we only included experiments with greater than 65% of samples above limit of detection. The limit of detection is set at three standard deviations above negative control values. Statistics run were one‐way analysis of variance (ANOVA) with multiple comparison to one another and Dunnett’s post hoc correction. *p < 0.05; **p < 0.01. TNNI3 (cardiac troponin I), EPO (erythropoietin), HPGDS (hematopoietic prostaglandin D synthase), CA14 (carbonic anhydrase 14), FES (FES proto‐oncogene, tyrosine kinase)