title: 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.
authors.affiliation: Institute of Neurophysiology and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Robert-Koch-Str. 39, 50931, Cologne, NRW, Germany.
authors.forename: Umesh
authors.initials: U
authors.lastname: Chaudhari
authors.affiliation: Institute of Neurophysiology and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Robert-Koch-Str. 39, 50931, Cologne, NRW, Germany.
authors.forename: Harshal
authors.initials: H
authors.lastname: Nemade
authors.affiliation: Institute of Neurophysiology and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Robert-Koch-Str. 39, 50931, Cologne, NRW, Germany.
authors.forename: Vilas
authors.initials: V
authors.lastname: Wagh
authors.affiliation: Institute of Neurophysiology and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Robert-Koch-Str. 39, 50931, Cologne, NRW, Germany.
authors.forename: John Antonydas
authors.initials: JA
authors.lastname: Gaspar
authors.affiliation: Biomolecular Medicine, Department of Surgery and Cancer, Imperial College London, London, UK.
authors.forename: James K
authors.initials: JK
authors.lastname: Ellis
authors.affiliation: Institute of Neurophysiology and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Robert-Koch-Str. 39, 50931, Cologne, NRW, Germany.
authors.forename: Sureshkumar Perumal
authors.initials: SP
authors.lastname: Srinivasan
authors.affiliation: Institute of Neurophysiology and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Robert-Koch-Str. 39, 50931, Cologne, NRW, Germany.
authors.forename: Dimitry
authors.initials: D
authors.lastname: Spitkovski
authors.affiliation: Institute of Neurophysiology and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Robert-Koch-Str. 39, 50931, Cologne, NRW, Germany.
authors.forename: Filomain
authors.initials: F
authors.lastname: Nguemo
authors.affiliation: Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy.
authors.forename: Jochem
authors.initials: J
authors.lastname: Louisse
authors.affiliation: Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy.
authors.forename: Susanne
authors.initials: S
authors.lastname: Bremer
authors.affiliation: Institute of Neurophysiology and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Robert-Koch-Str. 39, 50931, Cologne, NRW, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hescheler
authors.affiliation: Biomolecular Medicine, Department of Surgery and Cancer, Imperial College London, London, UK.
authors.forename: Hector C
authors.initials: HC
authors.lastname: Keun
authors.affiliation: Leibniz Research Centre for Working Environment and Human Factors at the Technical University of Dortmund (IfADo), 44139, Dortmund, Germany.
authors.forename: Jan G
authors.initials: JG
authors.lastname: Hengstler
authors.affiliation: Institute of Neurophysiology and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Robert-Koch-Str. 39, 50931, Cologne, NRW, Germany. a.sachinidis@uni-koeln.de.
authors.forename: Agapios
authors.initials: A
authors.lastname: Sachinidis
mesh_terms: D018943:Anthracyclines / Q000009:adverse effects*; D000903:Antibiotics, Antineoplastic / Q000009:adverse effects; D054316:Biomarkers, Pharmacological / Q000378:metabolism; D054715:Cardiotoxins / Q000009:adverse effects*; D002478:Cells, Cultured; D019295:Computational Biology; D003630:Daunorubicin / Q000009:adverse effects; D004317:Doxorubicin / Q000009:adverse effects; D004353:Drug Evaluation, Preclinical; D015507:Drugs, Investigational / Q000009:adverse effects*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology; D008942:Mitoxantrone / Q000009:adverse effects; D058977:Molecular Sequence Annotation; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D059005:Topoisomerase II Inhibitors / Q000009:adverse effects; D036401:Toxicity Tests, Chronic
chemical_list: D018943:Anthracyclines; D000903:Antibiotics, Antineoplastic; D054316:Biomarkers, Pharmacological; D054715:Cardiotoxins; D015507:Drugs, Investigational; D059005:Topoisomerase II Inhibitors; D004317:Doxorubicin; D008942:Mitoxantrone; D003630:Daunorubicin
keywords: Cardiotoxicity; Genomic biomarkers; Heart failure; Human stem cells derived cardiomyocytes; In vitro test system; Safety assessment; Transcriptomics