Identification of genomic biomarkers for anthracycline-induced cardiotoxicity in human iPSC-derived cardiomyocytes: an in vitro repeated exposure toxicity approach for safety assessment

Abstract

The currently available techniques for the safety evaluation of candidate drugs are usually cost-intensive and time-consuming and are often insufficient to predict human relevant cardiotoxicity. The purpose of this study was to develop an in vitro repeated exposure toxicity methodology allowing the identification of predictive genomics biomarkers of functional relevance for drug-induced cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The hiPSC-CMs were incubated with 156 nM doxorubicin, which is a well-characterized cardiotoxicant, for 2 or 6 days followed by washout of the test compound and further incubation in compound-free culture medium until day 14 after the onset of exposure. An xCELLigence Real-Time Cell Analyser was used to monitor doxorubicin-induced cytotoxicity while also monitoring functional alterations of cardiomyocytes by counting of the beating frequency of cardiomyocytes. Unlike single exposure, repeated doxorubicin exposure resulted in long-term arrhythmic beating in hiPSC-CMs accompanied by significant cytotoxicity. Global gene expression changes were studied using microarrays and bioinformatics tools. Analysis of the transcriptomic data revealed early expression signatures of genes involved in formation of sarcomeric structures, regulation of ion homeostasis and induction of apoptosis. Eighty-four significantly deregulated genes related to cardiac functions, stress and apoptosis were validated using real-time PCR. The expression of the 84 genes was further studied by real-time PCR in hiPSC-CMs incubated with daunorubicin and mitoxantrone, further anthracycline family members that are also known to induce cardiotoxicity. A panel of 35 genes was deregulated by all three anthracycline family members and can therefore be expected to predict the cardiotoxicity of compounds acting by similar mechanisms as doxorubicin, daunorubicin or mitoxantrone. The identified gene panel can be applied in the safety assessment of novel drug candidates as well as available therapeutics to identify compounds that may cause cardiotoxicity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-015-1623-5) contains supplementary material, which is available to authorized users.

Chronic drug‐induced effects on contractile motion properties and cardiac biomarkers in human induced pluripotent stem cell‐derived cardiomyocytes

“…Interestingly, arsenic (10 μM) and doxorubicin (1 μM) caused a transient increase in BR, followed by a decrease in BR together with a decrease in BA and CV. A similar increase in BR upon doxorubicin exposure has been reported by others (Chaudhari et al, 2015). This early transient effect is possibly an initial response of hiPS-CMs to compensate for cardiotoxicity in an attempt to sustain their physiological or metabolic function.…”

Doxorubicin induces cardiotoxicity through upregulation of death receptors mediated apoptosis in cardiomyocytes

“…Notably, the extrinsic apoptosis machinery is conserved in cardiomyocytes across animal species. Doxorubicin treatment increased Fas/FasL expression in rat cardiomyocytes274647 as well as human iPS-CMs48. TNFα/TNFR1 was also shown to be elevated in rat cardiomyocytes following doxorubicin treatment, but its relevance to cardiac injury remains elusive.…”

High throughput physiological screening of iPSC-derived cardiomyocytes for drug development

“…The potential exists to correlate drug dosing and plasma concentrations with cardiotoxic and myopathic risk. Recent studies suggest that iPSC-cardiomyocytes can reveal cardiotoxicities manifesting as alterations in cardiomyocyte viability, contractility, intracellular signaling and gene expression [127, 128]. As above, however, there is great need for validation, especially considering that many anti-cancer drugs elicit their cardiac effects by affecting mitochondrial function [129, 130] and that iPSC-cardiomyocytes are metabolically immature [131].…”