A predictive in vitro risk assessment platform for pro-arrhythmic toxicity

Cardiotoxicity of pharmaceutical drugs, industrial chemicals, and environmental toxicants can be severe, even life threatening, which necessitates a thorough evaluation of the human response to chemical compounds. Predicting risks for arrhythmia and sudden cardiac death accurately is critical for defining safety profiles. Currently available approaches have limitations including a focus on single select ion channels, the use of non-human species in vitro and in vivo, and limited direct physiological translation. We have advanced the robustness and reproducibility of in vitro platforms for assessing pro-arrhythmic cardiotoxicity using human induced pluripotent stem cell-derived cardiomyocytes and human cardiac fibroblasts in 3-dimensional microtissues. Using automated algorithms and statistical analyses of eight comprehensive evaluation metrics of cardiac action potentials, we demonstrate that tissue-engineered human cardiac microtissues respond appropriately to physiological stimuli and effectively differentiate between high-risk and low-risk compounds exhibiting blockade of the hERG channel (E4031 and ranolazine, respectively).

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Fig: Differentiation of hiPSC-CM, formation of cardiac microtissues, and definitions of metrics. (A) Timeline shows cardiomyocyte differentiation from human-induced pluripotent stem cells (hiPSCs) in high density 2D culture. Cardiac directed differentiation was achieved with Wnt activation at day 1 and inhibition at day 3 (see “Methods”). Cardiac phenotype, visually confirmed by beating cells, appeared between days 8 and 12. Cardiomyocytes differentiated from hiPSCs were used for the production of microtissues or were further purified with a lactate-based metabolic selection protocol. Microtissues self-assembled in microwells after 14–28 days of differentiation of hiPSC-CMs (i.e., without or with lactate purification) and addition of human cardiac fibroblasts (hCFs), and they were cultured in the presence of 1 Hz electrical field stimulation (estim). (B) Schematic of three-dimensional (3D) cardiac microtissue generation shows non-adhesive agarose gels with cylindrical recesses with hemispherical bottoms that guide self-assembly. Cardiac microtissues were cultured for 6–8 days with 1 Hz pacing. (C) Phase contrast image shows consistent spherical microtissue formation after 5 days of 3D culture in all 35 microwells. Scale bar, 800 μm. (D) Confocal image shows a representative cardiac tissue with hiPSC-CM (green) and hCF (red) stained with CellTracker dyes. Scale bar, 200 μm. (E) Confocal image shows a representative cardiac troponin I (red), vimentin (green), and DAPI stained cryosection (10 μm thick) of a microtissue fixed after 7 days in 3D culture. Scale bar, 50 μm. (F) Fluorescence image of microtissues at 3.2 × magnification was obtained during optical mapping. Typically, the action potentials (APs) from 4–9 microtissues were recorded simultaneously. (G-I) Schematics of the AP metrics of that were defined (with units) as: (G) “excitability” (%) measured from the percentage of captured APs during 10 s duration of recording with 2 s pacing cycle length, (H) “stimulation time delay” (ms; stim delay) between stimulation pulse and evoked AP upstroke (dF/dtmax), “rise time” (ms) of AP, “AP duration” (ms) to 30%, 50%, and 80% repolarization (APD30, APD50, APD80), “APD to the maximum repolarization rate” (ms; APDMxR) defined as time between AP upstroke and the end of rapid repolarization marked by d2F/dt2max, “APD triangulation” (ms; APDtri) defined as APDMxR—APD50, and (I) occurrence of “early afterdepolarization” (EAD) reported as (%) of microtissues showing EADs.

Further, we show that the environmental endocrine disrupting chemical bisphenol-A (BPA) causes acute and sensitive disruption of human action potentials in the nanomolar range. Thus, this novel human 3D in vitro pro-arrhythmic risk assessment platform addresses critical needs in cardiotoxicity testing for both environmental and pharmaceutical compounds and can be leveraged to establish safe human exposure levels.

Kofron, C.M., Kim, T.Y., Munarin, F. et al. A predictive in vitro risk assessment platform for pro-arrhythmic toxicity using human 3D cardiac microtissues. Sci Rep 11, 10228 (2021). https://doi.org/10.1038/s41598-021-89478-9

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