Modern diagnostic strategies for early recognition of cancer therapeutics-related cardiac dysfunction involve cardiac troponins measurement. Still, the role of other markers of cardiotoxicity is still unclear. The present study was designed to investigate dynamics of response of human cardiomyocytes derived from induced pluripotent stem cells (hiPCS-CMs) to doxorubicin with the special emphasis on their morphological changes in relation to expression and organization of troponins. The hiPCS-CMs were treated with doxorubicin concentrations (1 and 0.3 µM) for 48 h and followed for next up to 6 days. Exposure of hiPCS-CMs to 1 µM doxorubicininduced suppression of both cardiac troponin T (cTnT) and cardiac troponin I (cTnI) gene expression. Conversely, lower 0.3 µM doxorubicin concentration produced no significant changes in the expression of aforementioned genes. However, the intracellular topography, arrangement, and abundance of cardiac troponin proteins markedly changed after both doxorubicin concentrations. In particular, at 48 h of treatment, both cTnT and cTnI bundles started to reorganize, with some of them forming compacted shapes extending outwards and protruding outside the cells. At later intervals (72 h and onwards), the whole troponin network collapsed and became highly disorganized following, to some degree, overall changes in the cellular shape. Moreover, membrane permeability of cardiomyocytes was increased, and intracellular mitochondrial network rearranged and hypofunctional. Together, our results demonstrate complex effects of clinically relevant doxorubicin concentrations on hiPCS-CM cells including changes in cTnT and cTnI, but also in other cellular compartments contributing to the overall cytotoxicity of this class of cytostatics.

Department of Biology Faculty of Medicine in Hradec Kralove Charles University Prague Zborovská 2089 500 03 Hradec Kralove Czech Republic

Department of Physiology Faculty of Medicine in Hradec Kralove Charles University Prague Simkova 870 500 03 Hradec Kralove Czech Republic

Zobrazit více v PubMed

Suter T.M., Ewer M.S. Cancer drugs and the heart: Importance and management. Eur. Heart J. 2013;34:1102–1111. doi: 10.1093/eurheartj/ehs181. PubMed DOI

Simunek T., Sterba M., Popelova O., Adamcova M., Hrdina R., Gersl V. Anthracycline-induced cardiotoxicity: Overview of studies examining the roles of oxidative stress and free cellular iron. Pharmacol. Rep. 2009;61:154–171. doi: 10.1016/S1734-1140(09)70018-0. PubMed DOI

Zhang S., Liu X., Bawa-Khalfe T., Lu S., Lyu Y.L., Liu L.F., Yeh E.T. Identification of the molecular basis of doxorubicin-induced cardiotoxicity. Nat. Med. 2012;18:1639–1642. doi: 10.1038/nm.2919. PubMed DOI

Herman E.H., Lipshultz S.E., Rifai N., Zhang J., Papoian T., Yu Z.X., Takeda K., Ferrans V.J. Use of cardiac troponin T levels as an indicator of doxorubicin-induced cardiotoxicity. Cancer Res. 1998;58:195–197. PubMed

Adamcová M., Geršl V., Hrdina R., Mělka M., Mazurová Y., Vávrová J., Palička V., Kokštein Z. Cardiac troponin T as a marker of myocardial damage caused by antineoplastic drugs in rabbits. J. Cancer Res. Clin. Oncol. 1999;125:268–274. doi: 10.1007/s004320050273. PubMed DOI

Herman E.H., Zhang J., Lipshultz S.E., Rifai N., Chadwick D., Takeda K., Yu Z.X., Ferrans V.J. Correlation between serum levels of cardiac troponin-T and the severity of the chronic cardiomyopathy induced by doxorubicin. J. Clin. Oncol. 1999;17:2237–2243. doi: 10.1200/JCO.1999.17.7.2237. PubMed DOI

Simunek T., Klimtova I., Kaplanova J., Mazurova Y., Adamcova M., Sterba M., Hrdina R., Gersl V. Rabbit model for in vivo study of anthracycline-induced heart failure and for the evaluation of protective agents. Eur. J. Heart Fail. 2004;6:377–387. doi: 10.1016/j.ejheart.2003.05.003. PubMed DOI

Reagan W.J., York M., Berridge B., Schultze E., Walker D., Pettit S. Comparison of cardiac troponin I and T, including the evaluation of an ultrasensitive assay, as indicators of doxorubicin-induced cardiotoxicity. Toxicol Pathol. 2013;41:1146–1158. doi: 10.1177/0192623313482056. PubMed DOI

Cove-Smith L., Woodhouse N., Hargreaves A., Kirk J., Smith S., Price S.A., Galvin M., Betts C.J., Brocklehurst S., Backen A., et al. An integrated characterization of serological, pathological, and functional events in doxorubicin-induced cardiotoxicity. Toxicol. Sci. 2014;140:3–15. doi: 10.1093/toxsci/kfu057. PubMed DOI

Jirkovsky E., Lencova-Popelova O., Hroch M., Adamcova M., Mazurova Y., Vavrova J., Micuda S., Simunek T., Gersl V., Sterba M. Early and delayed cardioprotective intervention with dexrazoxane each show different potential for prevention of chronic anthracycline cardiotoxicity in rabbits. Toxicology. 2013;311:191–204. doi: 10.1016/j.tox.2013.06.012. PubMed DOI

Bures J., Jirkovska A., Sestak V., Jansova H., Karabanovich G., Roh J., Sterba M., Simunek T., Kovarikova P. Investigation of novel dexrazoxane analogue JR-311 shows significant cardioprotective effects through topoisomerase IIbeta but not its iron chelating metabolite. Toxicology. 2017;392:1–10. doi: 10.1016/j.tox.2017.09.012. PubMed DOI

McGowan J.V., Chung R., Maulik A., Piotrowska I., Walker J.M., Yellon D.M. Anthracycline Chemotherapy and Cardiotoxicity. Cardiovasc. Drugs Ther. 2017;31:63–75. doi: 10.1007/s10557-016-6711-0. PubMed DOI PMC

Dolci A., Dominici R., Cardinale D., Sandri M.T., Panteghini M. Biochemical markers for prediction of chemotherapy-induced cardiotoxicity: Systematic review of the literature and recommendations for use. Am. J. Clin. Pathol. 2008;130:688–695. doi: 10.1309/AJCPB66LRIIVMQDR. PubMed DOI

Cardinale D., Cipolla C.M. Chemotherapy-induced cardiotoxicity: Importance of early detection. Expert Rev. Cardiovasc. Ther. 2016;14:1297–1299. doi: 10.1080/14779072.2016.1239528. PubMed DOI

Cardinale D., Sandri M.T., Martinoni A., Tricca A., Civelli M., Lamantia G., Cinieri S., Martinelli G., Cipolla C.M., Fiorentini C. Left ventricular dysfunction predicted by early troponin I release after high dose chemotherapy. J. Am. Coll. Cardiol. 2000;36:517–522. doi: 10.1016/S0735-1097(00)00748-8. PubMed DOI

Cardinale D., Sandri M.T., Colombo A., Colombo N., Boeri M., Lamantia G., Civelli M., Peccatori F., Martinelli G., Fiorentini C., et al. Prognostic value of troponin I in cardiac risk stratification of cancer patients undergoing high-dose chemotherapy. Circulation. 2004;109:2749–2754. doi: 10.1161/01.CIR.0000130926.51766.CC. PubMed DOI

Cardinale D., Salvatici M., Sandri M.T. Role of biomarkers in cardioncology. Clin. Chem. Lab. Med. 2011;49:1937–1948. doi: 10.1515/CCLM.2011.692. PubMed DOI

Curigliano G., Cardinale D., Suter T., Plataniotis G., de Azambuja E., Sandri M.T., Criscitiello C., Goldhirsch A., Cipolla C., Roila F. ESMO Guidelines Working Group. Cardiovascular toxicity induced by chemotherapy, targeted agents and radiotherapy: ESMO clinical practice guidelines. Ann. Oncol. 2012;23:vii155–vii166. doi: 10.1093/annonc/mds293. PubMed DOI

Cardinale D., Biasillo G., Salvatici M., Sandri M.T., Cipolla C.M. Using biomarkers to predict and to prevent cardiotoxicity of cancer therapy. Expert Rev. Mol. Diagn. 2017;17:245–256. doi: 10.1080/14737159.2017.1283219. PubMed DOI

Henriksen P.A. Anthracycline cardiotoxicity: An update on mechanisms, monitoring and prevention. Heart. 2018;104:971–977. doi: 10.1136/heartjnl-2017-312103. PubMed DOI

Adamcova M., Lencova-Popelova O., Jirkovsky E., Mazurova Y., Palicka V., Simko F., Gersl V., Sterba M. Experimental determination of diagnostic window of cardiac troponins in the development of chronic anthracycline cardiotoxicity and estimation of its predictive value. Int. J. Cardiol. 2015;201:358–367. doi: 10.1016/j.ijcard.2015.07.103. PubMed DOI

Abassi Y.A., Xi B., Li N., Ouyang W., Seiler A., Watzele M., Kettenhofen R., Bohlen H., Ehlich A., Kolossov E., et al. Dynamic monitoring of beating periodicity of stem cell-derived cardiomyocytes as a predictive tool for preclinical safety assessment. Br. J. Pharmacol. 2012;165:1424–1441. doi: 10.1111/j.1476-5381.2011.01623.x. PubMed DOI PMC

Pointon A., Abi-Gerges N., Cross M.J., Sidaway J.E. Phenotypic profiling of structural cardiotoxins in vitro reveals dependency on multiple mechanisms of toxicity. Toxicol. Sci. 2013;132:317–326. doi: 10.1093/toxsci/kft005. PubMed DOI

Doherty K.R., Talbert D.R., Trusk P.B., Moran D.M., Shell S.A., Bacus S. Structural and functional screening in human induced-pluripotent stem cell-derived cardiomyocytes accurately identifies cardiotoxicity of multiple drug types. Toxicol. Appl. Pharmacol. 2015;285:51–60. doi: 10.1016/j.taap.2015.03.008. PubMed DOI

Burridge P.W., Li Y.F., Matsa E., Wu H., Ong S.G., Sharma A., Holmstrom A., Chang A.C., Coronado M.J., Ebert A.D., et al. Human induced pluripotent stem cell-derived cardiomyocytes recapitulate the predilection of breast cancer patients to doxorubicin-induced cardiotoxicity. Nat. Med. 2016;22:547–556. doi: 10.1038/nm.4087. PubMed DOI PMC

Holmgren G., Synnergren J., Bogestal Y., Ameen C., Akesson K., Holmgren S., Lindahl A., Sartipy P. Identification of novel biomarkers for doxorubicin-induced toxicity in human cardiomyocytes derived from pluripotent stem cells. Toxicology. 2015;328:102–111. doi: 10.1016/j.tox.2014.12.018. PubMed DOI PMC

Chaudhari U., Nemade H., Gaspar J.A., Hescheler J., Hengstler G., Sachinidis A. MicroRNAs as early toxicity signatures of doxorubicin in human-induced pluripotent stem cell-derived cardiomyocytes. Arch. Toxicol. 2016;90:3087–3098. doi: 10.1007/s00204-016-1668-0. PubMed DOI PMC

Chaudhari U., Nemade H., Wagh V., Gaspar J.A., Ellis J.K., Srinivasan S.P., Spitkovski D., Nguemo F., Louisse J., Bremer S., et al. Identification of genomic biomarkers for anthracycline-induced cardiotoxicity in human iPSC-derived cardiomyocytes: An in vitro repeated exposure toxicity approach for safety assessment. Arch. Toxicol. 2016;90:2763–2777. doi: 10.1007/s00204-015-1623-5. PubMed DOI PMC

Louisse J., Wust R.C.I., Pistollato F., Palosaari T., Barilari M., Macko P., Bremer S., Prieto P. Assessment of acute and chronic toxicity of doxorubicin in human induced pluripotent stem cell-derived cardiomyocytes. Toxicol. In Vitro. 2017;42:182–190. doi: 10.1016/j.tiv.2017.04.023. PubMed DOI

Balis F.M., Holcenberg J.S., Bleyer W.A. Clinical pharmacokinetics of commonly used anticancer drugs. Clin. Pharmacokinet. 1983;8:202–232. doi: 10.2165/00003088-198308030-00002. PubMed DOI

Hasinoff B.B., Patel D., Wu X. Molecular Mechanisms of the Cardiotoxicity of the Proteasomal-Targeted Drugs Bortezomib and Carfilzomib. Cardiovasc. Toxicol. 2017;17:237–250. doi: 10.1007/s12012-016-9378-7. PubMed DOI

Andersson H., Steel D., Asp J., Dahlenborg K., Jonsson M., Jeppsson A., Lindahl A., Kagedal B., Sartipy P., Mandenius C.F. Assaying cardiac biomarkers for toxicity testing using biosensing and cardiomyocytes derived from human embryonic stem cells. J. Biotechnol. 2010;150:175–181. doi: 10.1016/j.jbiotec.2010.06.023. PubMed DOI

Adamcova M., Simunek T., Kaiserova H., Popelova O., Sterba M., Potacova A., Vavrova J., Malakova J., Gersl V. In vitro and in vivo examination of cardiac troponins as biochemical markers of drug-induced cardiotoxicity. Toxicology. 2007;237:218–228. doi: 10.1016/j.tox.2007.05.016. PubMed DOI

White H.D. Pathobiology of troponin elevations: Do elevations occur with myocardial ischemia as well as necrosis? J. Am. Coll. Cardiol. 2011;57:2406–2408. doi: 10.1016/j.jacc.2011.01.029. PubMed DOI

Kopljar I., De Bondt A., Vinken P., Teisman A., Damiano B., Goeminne N., Van den Wyngaert I., Gallacher D.J., Lu H.R. Chronic drug-induced effects on contractile motion properties and cardiac biomarkers in human induced pluripotent stem cell-derived cardiomyocytes. Br. J. Pharmacol. 2017;174:3766–3779. doi: 10.1111/bph.13713. PubMed DOI PMC

Zhang Y.W., Shi J., Li J.Y., Wei L. Cardiomyocyte death in doxorubicin-induced cardiotoxicity. Arch. Immunol. Ther. Exp. 2009;57:435–445. doi: 10.1007/s00005-009-0051-8. PubMed DOI PMC

Koleini N., Kardami E. Autophagy and mitophagy in the context of doxorubicin-induced cardiotoxicity. Oncotarget. 2017;8:46663–46680. doi: 10.18632/oncotarget.16944. PubMed DOI PMC

Zhou S., Starkov A., Froberg M.K., Leino R.L., Wallace K.B. Cumulative and irreversible cardiac mitochondrial dysfunction induced by doxorubicin. Cancer Res. 2001;61:771–777. PubMed

Carvalho F.S., Burgeiro A., Garcia R., Moreno A.J., Carvalho R.A., Oliveira P.J. Doxorubicin-induced cardiotoxicity: From bioenergetic failure and cell death to cardiomyopathy. Med. Res. Rev. 2014;34:106–135. doi: 10.1002/med.21280. PubMed DOI

De Oliveira B.L., Niederer S.A. Biophysical Systems Approach to Identifying the Pathways of Acute and Chronic Doxorubicin Mitochondrial Cardiotoxicity. PLoS Comput. Biol. 2016;12:e1005214. doi: 10.1371/journal.pcbi.1005214. PubMed DOI PMC

Parra V., Eisner V., Chiong M., Criollo A., Moraga F., Garcia A., Hartel S., Jaimovich E., Zorzano A., Hidalgo C., et al. Changes in mitochondrial dynamics during ceramide-induced cardiomyocyte early apoptosis. Cardiovasc. Res. 2008;77:387–397. doi: 10.1093/cvr/cvm029. PubMed DOI

Lebrecht D., Kokkori A., Ketelsen U.P., Setzer B., Walker U.A. Tissue-specific mtDNA lesions and radical-associated mitochondrial dysfunction in human hearts exposed to doxorubicin. J. Pathol. 2005;207:436–444. doi: 10.1002/path.1863. PubMed DOI

Sardao V.A., Oliveira P.J., Holy J., Oliveira C.R., Wallace K.B. Morphological alterations induced by doxorubicin on H9c2 myoblasts: Nuclear, mitochondrial, and cytoskeletal targets. Cell. Biol. Toxicol. 2009;25:227–243. doi: 10.1007/s10565-008-9070-1. PubMed DOI