Dexrazoxane-afforded protection against chronic anthracycline cardiotoxicity in vivo: effective rescue of cardiomyocytes from apoptotic cell death

O Popelová, M Sterba, P Hasková, T Simůnek, M Hroch, I Guncová, P Nachtigal, M Adamcová, V Gersl, Y Mazurová, O Popelová, M Sterba, P Hasková, T Simůnek, M Hroch, I Guncová, P Nachtigal, M Adamcová, V Gersl, Y Mazurová

Abstract

Background: Dexrazoxane (DEX, ICRF-187) is the only clinically approved cardioprotectant against anthracycline cardiotoxicity. It has been traditionally postulated to undergo hydrolysis to iron-chelating agent ADR-925 and to prevent anthracycline-induced oxidative stress, progressive cardiomyocyte degeneration and subsequent non-programmed cell death. However, the additional capability of DEX to protect cardiomyocytes from apoptosis has remained unsubstantiated under clinically relevant in vivo conditions.

Methods: Chronic anthracycline cardiotoxicity was induced in rabbits by repeated daunorubicin (DAU) administrations (3 mg kg(-1) weekly for 10 weeks). Cardiomyocyte apoptosis was evaluated using TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling) assay and activities of caspases 3/7, 8, 9 and 12. Lipoperoxidation was assayed using HPLC determination of myocardial malondialdehyde and 4-hydroxynonenal immunodetection.

Results: Dexrazoxane (60 mg kg(-1)) co-treatment was capable of overcoming DAU-induced mortality, left ventricular dysfunction, profound structural damage of the myocardium and release of cardiac troponin T and I to circulation. Moreover, for the first time, it has been shown that DEX affords significant and nearly complete cardioprotection against anthracycline-induced apoptosis in vivo and effectively suppresses the complex apoptotic signalling triggered by DAU. In individual animals, the severity of apoptotic parameters significantly correlated with cardiac function. However, this effective cardioprotection occurred without a significant decrease in anthracycline-induced lipoperoxidation.

Conclusion: This study identifies inhibition of apoptosis as an important target for effective cardioprotection against chronic anthracycline cardiotoxicity and suggests that lipoperoxidation-independent mechanisms are involved in the cardioprotective action of DEX.

Figures

Figure 1
Figure 1
(A) Echocardiographically determined left ventricular (LV) fractional shortening (FS) during the time course of experiment. (B) Invasively determined index of LV contractility (index dP/dtmax) at the end of the experiment. Statistical significance in comparison with ‘*’ the initial values within each group (paired t-test, P<0.05), ‘c’ control, ‘d’ daunorubicin and ‘x’ DEX+DAU groups (ANOVA, P<0.05).
Figure 2
Figure 2
(A) Plasma concentrations of cardiac troponin T (cTnT) and (B) troponin I (cTnI) in individual animals at the end of the experiment.
Figure 3
Figure 3
(A–C) Histological examination of the left ventricular (LV) myocardium. Masson's blue trichrome; bar=20 μm. In the control group (A), normal structure of the myocardium was observed. In the DAU group (B), foci of cardiomyocytes showing profound degeneration (asterisks) were found. Marked morphological changes comprise cytoplasmic vacuolisation, cell swelling and loss of myofibrils resulting in cell death with subsequent development of interstitial fibrosis (arrow). The LV myocardium of DEX co-treated animals (C) showed largely preserved normal morphology with only subtle changes; signs of cardiomyocyte degeneration were scarce (asterisk).
Figure 4
Figure 4
Terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) assay. (A–C) Representative samples of left ventricular (LV) myocardium labelled with TUNEL in the control (A), DAU (B) and DEX+DAU (C) groups. The arrows indicate examples of TUNEL-positive nuclei; bar=20 μm. (D) Quantitative analysis of TUNEL assay. Statistical significance (ANOVA, P<0.05) in comparison with ‘d’ daunorubicin group. (E) Scatterplot of the LV fractional shortening vs number of TUNEL-positive nuclei per square millimetre and results of the Spearman's correlation analyses.
Figure 5
Figure 5
(A–D) Caspase activities in the left ventricular (LV) myocardium: caspase 3/7 (A), caspase 8 (B), caspase 9 (C) and caspase 12 (D). Statistical significances (ANOVA, P<0.05) in comparison with ‘d’ daunorubicin group.
Figure 6
Figure 6
(A–D) Scatterplots of the cardiac function (LV fractional shortening (FS)) vs activity of individual caspases as determined by Spearman's correlation analyses: caspases 3/7 (A), caspase 8 (B), caspase 9 (C) and caspase 12 (D). LU, luminescence units; FU, fluorescence units.
Figure 7
Figure 7
(A) Total malondialdehyde (MDA, marker of lipoperoxidation) levels in LV myocardium. Statistical significances (ANOVA, P<0.05) in comparison with ‘c’ control group. (B–E) Scatterplots of MDA levels in the left ventricular myocardium vs activity of individual caspases and results of the Spearman's correlation analyses: caspases 3/7 (B), caspase 8 (C), caspase 9 (D) and caspase 12 (E). (F) Scatterplot of myocardial MDA levels vs LV fractional shortening (FS) and results of the Spearman's correlation analyses. LU, luminescence units; FU, fluorescence units.
Figure 8
Figure 8
Representative samples of left ventricular (LV) myocardium showing 4-hydroxynonenal staining in the control (A), DAU (B) and DEX+DAU (C) groups.
Figure 9
Figure 9
(A) Horizontal Hierarchical Tree Plot. The analysis comprised LV functional parameters (FS and dP/dtmax), the plasma levels of cardiac troponins T and I, markers of apoptosis (TUNEL positivity and activities of caspases) and lipoperoxidation (nine variables) in all experimental groups. The analysis resulted in two main clusters. The first cluster covers exclusively the individuals from the control (C) and DEX co-treated group (DD), whereas the second cluster contains only the animals from the daunorubicin group (D), where almost all animals appeared in one cluster. (B) Principal Component Analysis (PCA) scatterplots. This analysis of the same variables also revealed two distinctly separated clusters comprising the DEX+DAU and control groups in the first, with all animals from the DAU group in the second cluster. Treatment groups: control (▵), DAU (▴) and DEX+DAU (□).

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