Elsevier

Toxicology

Volume 237, Issues 1–3, 31 July 2007, Pages 218-228
Toxicology

In vitro and in vivo examination of cardiac troponins as biochemical markers of drug-induced cardiotoxicity

https://doi.org/10.1016/j.tox.2007.05.016Get rights and content

Abstract

Cardiac troponin T (cTnT) and troponin I (cTnI) are becoming acknowledged as useful biochemical markers of drug-induced cardiotoxicity. In this study we examined the release kinetics of cTnT and cTnI using an in vitro model of isolated rat neonatal ventricular cardiomyocytes (NVCM, 72 h treatment with 0.1–3 μM of daunorubicin) and compared it with data from a rabbit model of chronic anthracycline-induced cardiomyopathy in vivo (3 mg/kg of daunorubicin weekly, 10 weeks). In cell-culture media, the cTnI and cTnT concentrations were concentration- and time-dependently increasing in response to daunorubicin exposure and were negatively exponentially related to cardiomyocyte viability. With 3 μM daunorubicin, the relative increase of AUC of cTnT and cTnI was 2.4- and 5.3-fold higher than the increase of LDH activity, respectively. In rabbits, the daunorubicin-induced cardiomyopathy was associated with progressive increase of both cTnT and cTnI. Although the correlation between cTnT and cTnI cumulative release (AUCs) was found (R = 0.81; P < 0.01) and both cardiac troponins corresponded well with the echocardiographically-assessed systolic dysfunction (R = 0.83 and 0.81 for cTnT and cTnI, respectively; P < 0.001), the first significant increase in cTnI levels was observed earlier (at a cumulative daunorubicin dose of 200 mg/m2) than with cTnT (350 mg/m2). In conclusion, our study has confirmed cTnT and cTnI as very sensitive and specific markers of anthracycline-induced cardiotoxicity. The troponins can become not only the bridge between the clinical and experimental studies of drug-induced cardiotoxicity but also the linkage between the preclinical experiments in vitro and in vivo.

Introduction

Cardiomyocyte injury and associated cell membrane disruption results in the release of intracellular proteins that can be measured in the peripheral circulation. Biochemical assays for detection of myocardial injury were first reported in the 1950s (Karmen et al., 1954). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), myoglobin, creatine kinase (CK), and its isoenzyme MB have all been used to detect myocardial necrosis. However, the expression of these proteins in tissue other than cardiac myocytes begets their common limitations. In 1980s, the attention of cardiologists gradually shifted from the metabolic proteins to the myofibrillar proteins of the myocardium and consequently to the determination of cardiac troponins’ plasma levels. Troponin is a thin-filament-associated complex regulating the formation of actin–myosin cross-bridges during the contraction–relaxation process in the striated muscles, which consists of three different subunits: troponin T (TnT—37 kDa), troponin C (TnC—18 kDa) and troponin I (TnI—24 kDa). Unlike TnC, both TnT and TnI exist in three different isoforms with a unique structure: for the slow-twitch skeletal muscle, fast-twitch skeletal muscle and cardiac muscle, respectively. The cardiac tissue specificity of troponin T and I overcomes the fundamental limitation of the old biomarkers of myocardial necrosis.

Recently, cardiac troponins T and I (cTnT and cTnI) are becoming recognized not only as useful criterion for stratification of patients with acute coronary syndrome (Panteghini, 2002) but also as powerful biomarkers of drug-induced cardiotoxicity. The utility of troponins in this field has been highlighted by a report of The Expert Working Group on Biomarkers of Drug-Induced Cardiac Toxicity of the Nonclinical Subcommittee of the Advisory Committee for Pharmaceutical Science, which is one of the expert panels of the US Food and Drug Administration (FDA) (Expert Working Group, 2002). The main advantages of troponins as biomarkers of drug-induced myocardial injury are: high specificity, high sensitivity, wide diagnostic window, robustness of the assay and bridge between preclinical and clinical studies (Holt, 1998, Wu, 1998, Sparano et al., 2000, Sparano et al., 2002, Herman and Ferrans, 2001, Herman et al., 2003, Wallace et al., 2004, O’Brien, 2006a, O’Brien et al., 2006b). From the clinical point of view, the most important indications for cardiac troponins use for evaluation of drug-induced cardiotoxicity seem to be monitoring of therapy employing β-sympathomimetic and antineoplastic drugs, mainly anthracyclines alone or in a combination with other cytostatic agents (Adamcova et al., 2005a, Adamcova et al., 2005b).

Anthracycline anticancer agents (e.g. doxorubicin, daunorubicin, epirubicin) are notorious for the risk of severe cardiomyopathy and heart failure development (Hrdina et al., 2000, Minotti et al., 2004; Wallace et al., 2004, Lipshultz, 2006, Jones et al., 2006, Berthiaume and Wallace, 2007) and can thus serve as model cardiotoxic drugs. The chronic nature of anthracycline-induced cardiotoxicity requires sensitive and reliable markers of cardiac damage that would allow its early detection, ideally before the first signs of symptomatic heart failure occur.

The aim of this study was to examine and compare the cTnT and cTnI release kinetics in an in vitro primary cardiomyocyte model and to compare it with plasma troponin levels obtained from a validated model of chronic anthracycline-induced heart failure in vivo.

Section snippets

Anthracycline-induced cardiotoxicity in vivo

All the experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication, 1996) and under the supervision of the Ethical Committee of the Faculty of Medicine in Hradec Králové. All manipulations were performed under ketamine anaesthesia (50 mg/kg, i.m.).

The chronic anthracycline cardiomyopathy was induced in Chinchilla male rabbits in a previously validated schedule (daunorubicin 3 mg/kg, i.v.,

Anthracycline-induced cardiotoxicity in vivo

In control rabbits, no premature deaths were observed and the animal well-being was evidenced by 33% body weight increase (from 3.50 ± 0.03 kg at week 1 to 4.64 ± 0.13 kg at week 11; P < 0.05, paired t-test). Repeated 10-week administration of daunorubicin in the weekly dose of 3 mg/kg (i.e. approximately 50 mg/m2 of body surface) resulted in 18% mortality (2 animals out of 11 died in weeks 10 and 11, respectively) and in this group virtually no body weight gain was observed (3.45 ± 0.07 and 3.47 ± 0.23 kg

Discussion

Risk of cardiotoxicity is the most serious drawback of anthracycline antineoplastic drugs. However, these drugs remain among the most powerful and widely used anticancer agents (Lipshultz, 2006). Early, accurate and preferably non-invasive detection of cardiomyocyte injury is therefore of crucial importance. The use of cTnT as a biomarker of doxorubicin cardiotoxicity in animal studies was first reported by Seino et al., in spontaneously hypertensive rats (Seino et al., 1993). Numerous

Acknowledgements

The authors thank Mr. Marcel Bíma and Mrs. Ludmila Latýnová for their skilful technical assistance during the whole study as well as Dr. Eva Čermáková for her help with the statistical analysis. This study was supported by a Research Project of the Czech Ministry of Education, Youth and Sports (MSM 0021620820) and a Grant from the Czech Science Foundation (GAČR 305/05/P156).

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    These authors contributed equally.

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