(43) Reproductive Toxicity Assessment of Anti-Arrhythmia Drugs Using Stem Cell-Based Embryo Models

Courtney Kehaulani Kurashima¹; Yusuke Marikawa¹

1. Department of Anatomy, Biochemistry and Physiology, University of Hawaiʻi John A. Burns School of Medicine, Honolulu, USA

Abstract Text:

Introduction: Birth defects are the leading cause of infant mortality, affecting about 1 in every 33 babies in the United States. Teratogens are harmful environmental factors that can cause birth defects or miscarriages. Traditionally, teratogens are identified through human epidemiological studies and in vivo animal tests. However, these methods have limitations particularly in elucidating the teratogenic mechanisms during embryonic development. In vitro embryo models made from pluripotent stem cells offer an alternative approach, as they are more amenable to experimental interrogations into molecular mechanism underlying teratogenic effects. This project focuses on the teratogenic effects of amiodarone and dronedarone, medications commonly used to treat arrhythmia. Both drugs are known as teratogens, although the mechanisms by which they affect embryonic development remain unclear. Here, we aim to investigate the teratogenic mechanisms of these drugs using in vitro embryo models derived from mouse and human pluripotent stem cells that recapitulate gastrulation, the key process of embryonic patterning. Methods: P19C5 mouse embryonal carcinoma stem cells and H9 human embryonic stem cells were used to generate 3D aggregates that exhibit growth and axial elongation. These in vitro embryo models were treated with various concentrations of amiodarone and dronedarone, taking into account their therapeutic plasma concentration ranges (Cmax of 2 µM for amiodarone and 3 µM for dronedarone). Adverse effects of the drugs were assessed by analyzing morphology and gene expression profiles. ImageJ software was used to measure morphometric parameters (area, circularity, and aspect ratio) of individual aggregates. Quantitative RT-PCR was performed to evaluate the expression levels of genes integral to embryonic development, such as those involved in pluripotency maintenance, germ layer formation, and axial patterning. Results: Morphological analyses showed a dose-dependent effect of amiodarone and dronedarone treatment in the mouse model. Amiodarone at 4 µM and dronedarone at 3 µM significantly impaired the growth and elongation of aggregates. Expression of key developmental genes, such as Hoxa1 (anterior-posterior axial patterning), Foxc2 (mesodermal patterning), and Meox1 (somite segmentation), were significantly affected at 4 µM and 3 µM for amiodarone and dronedarone. In the human model, several essential embryonic genes, such as ALDH1A2 (retinoic acid synthesis), PAX3 (somite and neural patterning), and TCF15 (paraxial mesoderm patterning), were altered at 2 µM and above of amiodarone. In contrast, dronedarone affected embryonic gene expression at concentrations as low as 0.25 µM. Conclusion: Amiodarone and dronedarone are structurally similar, with dronedarone lacking the iodide moiety and containing an additional methyl sulfonyl group. While amiodarone is a widely used anti-arrhythmic drug, its prolonged use is associated with serious cardiac and thyroid side effects. Dronedarone is considered to have lesser side effects in comparison. However, our data indicates that both drugs exert dose-dependent adverse effects on mouse and human embryo models, with dronedarone having significant effects at lower concentrations compared to amiodarone, particularly in the human model. Our data suggests that these anti-arrhythmia drugs can potentially disrupt embryonic development by affecting mesoderm and neural patterning. In vitro embryo models can be used as an alternative to the traditional approaches used today. This study provides a foundation for further research into the teratogenic mechanisms of these anti-arrhythmia drugs.