IntroductionTyrosine kinase inhibitors (TKIs) are known to induce cardiotoxicity and mitochondrial dysfunction. TKIs can cross the placenta, posing high risks for congenital heart defects. Mitochondria play important roles in the regulation of cardiac differentiation. Previous studies showed that mitochondrial dysfunction is correlated with a decrease of GATA4 expression and impairment of cardiac development. However, the relationship between GATA4 and mitochondrial function during cardiac development upon TKI-exposure remains poorly documented.HypothesisLow-dose exposure to TKIs alters GATA4-mediated regulatory network and metabolic remodeling during early cardiac development, leading to dysfunctions in differentiating cardiomyocytes (CMs).MethodsWe treated human stem cells with several TKI drugs (imatinib, sunitinib, and vandetanib at sublethal levels) during CM differentiation, and profiled the transcriptome and chromatin accessibility landscape in cardiac progenitors and CMs. The GATA4-mediated regulatory network was profiled using ChIP-seq. The functional influence of the early exposure to TKIs in differentiating CMs was evaluated, including Ca-handling and contractility, integrity of sarcomere structure, and mitochondrial respiration.ResultsDevelopmental exposure to TKIs induced impairment of mitochondrial respiration, disarrangement of sarcomere structure, increased calcineurin levels, and significant alterations in contractility and Ca-handling properties of differentiating CMs. Integrated genomic analyses revealed that TKI-exposure altered GATA4-mediated regulatory network, which was highly correlated with mitochondrial functions. Using a gain-of-function approach with CRISPR-activation, we observed that increases in GATA4 expression restored cardiac functions and mitochondrial respiration despite of TKI exposure.ConclusionsWe identified a novel crosstalk between GATA4 activity and mitochondrial respiration during CM differentiation. This study provides new insights into the relationship between gene-regulation and mitochondrial functions, and significantly enhances our understanding of sublethal TKI-induced cardiotoxicity during cardiac development.