Arrhythmia Assessment in Heterotypic Human Cardiac Myocyte-Fibroblast Microtissues.

Risk assessment assays for chemically induced arrhythmia are critical, but significant limitations exist with current cardiotoxicity testing, including a focus on single select ion channels, the use of non-human species in vitro and in vivo, and limited direct physiological translation. To be predictive of actual adverse clinical arrhythmic risk, arrhythmia assessment models for chemicals and drugs should be fit-for-purpose and suited for evaluating compounds in which the mechanism of action may not be entirely known. Here, we describe methods for efficient and reliable screening for arrhythmogenic cardiotoxicity with a 3D human cardiac microtissue model using purified human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and human cardiac fibroblasts. Applying optical mapping of voltage and calcium-sensitive dyes-an established approach to evaluate cardiac action potentials and calcium transients-to 3D heterotypic cardiac myocyte-fibroblast tissues allows for the generation and functional analysis of a large number of individual microtissues to provide greater throughput and high statistical power in analyses. Hundreds of microtissues in standard cell culture plates can be produced with low variability beat-to-beat, microtissue-to-microtissue, and across hiPSC-cardiomyocyte differentiation batches, reducing the number of microtissues required per condition for predictive outputs. The platform described here can be used as a sensitive, efficient, and predictive preclinical model validated for the purpose of assessing human pro-arrhythmic risk.

Human Atrial Cardiac Microtissues for Chamber-Specific Arrhythmic Risk Assessment.

INTRODUCTION: Although atrial fibrillation is the most prevalent disorder of electrical conduction, the mechanisms behind atrial arrhythmias remain elusive. To address this challenge, we developed a robust in vitro model of 3D atrial microtissue from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and evaluated chamber-specific chemical responses experimentally and computationally. METHODS: We differentiated atrial and ventricular cardiomyocytes (aCMs/vCMs) from GCaMP6f-expressing hiPSCs and assessed spontaneous AP activity using fluorescence imaging. Self-assembling 3D microtissues were formed with lactate purified CMs and 5% human cardiac fibroblasts and electrically stimulated for one week before high resolution action potential (AP) optical mapping. AP responses to the atrial-specific potassium repolarizing current I Kur-blocker 4-Aminopyridine (4-AP) and funny current I f-blocker Ivabradine were characterized within their therapeutic window. Finally, we expanded upon a published hiPSC-CM computational model by incorporating the atrial-specific I Kur current, modifying ion channel conductances to match the AP waveforms of our microtissues, and employing the updated model to reinforce our experimental findings. RESULTS: High purity CMs (> 75% cTnT+) demonstrated subtype specification by MLC2v expression. Spontaneous beating rates significantly decreased following 3D microtissue formation, with atrial microtissues characterized by their faster spontaneous beating rate, slower AP rise time, and shorter AP duration (APD) compared to ventricular microtissues. We measured atrial-specific responses, including dose-dependent APD prolongation with 4-AP treatment and dose-dependent reduction in spontaneous activity post-Ivabradine treatment. CONCLUSION: The presented in vitro platform for screening atrial-specific responses is both robust and sensitive, with high throughput, enabling studies focused at elucidating the mechanisms underlying atrial arrhythmias. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00703-x.

A predictive in vitro risk assessment platform for pro-arrhythmic toxicity using human 3D cardiac microtissues.

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). 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.

Monte Carlo simulation of 3D mapping of cardiac electrical activity with spinning slit confocal optics.

Optical techniques used to map transmembrane potential and intracellular Ca2+ activities of intact hearts are restricted to the surface and cannot resolve activity in deeper layers due to the lack of depth resolution. The recent development of spinning slit confocal optics offers advantages of depth resolution as well as high-speed confocal imaging which are necessary for millisecond-scale, depth-resolved mapping of membrane potential and/or intracellular Ca2+ concentration. Here, we show simulated confocal optics derived from confocal slits on a high-speed spinning disk using Monte Carlo method with a numerical heart tissue model and find that depth-resolved optical mapping is feasible down to around 800 microm below the surface using 670-nm excitation light. The numerical model shows that (1) a minimum slit separation, which is found to be a function of depth of the focal plane and the numerical aperture of the objective lens, for minimum background noise exists and (2) narrower slit widths result in slightly greater depth resolution but has a negative impact in significantly lower overall fluorescence intensity. An experimental test of this optics has been performed by imaging two overlapping layers of fluorescent beads and the result confirms the expected depth resolution in non-scattering medium. These results will be able to serve as a benchmark on how a 3D-imaging system can be expected to perform and what kind of theoretical depth-resolution can be expected from it.