Yvona Mazurová

Troponin as a marker of myocardiac damage in drug-induced cardiotoxicity

Cardiac troponins T and I (cTnT and cTnI) are becoming the serum biomarkers of choice for monitoring potential drug-induced myocardial injury in both clinical and preclinical studies. The utility of cardiac troponins has been mainly demonstrated following the administration of antineoplastic drugs and β-sympathomimetics, although the routine use of these markers in the monitoring in patients who received anthracyclines therapy is far from settled. Unlike the previous markers, which suffered from numerous shortages, the main advantages of cardiac troponins are their high specificity and sensitivity, wide diagnostic window and the possibility to use commercially available assays in clinical settings as well as in a broad range of laboratory animals. Nevertheless, in spite of vigorous research in this area, a number of questions are still unanswered and these are discussed in this review. The main problems seem to be the lack of standardisation of variety of troponin immunoassays, the assessment of suitable cutoff for drug-induced cardiotoxicity and determination of critical diagnostic window related to the optimal tim-ing of sample collection, which may be drug-dependent.

Proteomic insights into chronic anthracycline cardiotoxicity.

Chronic anthracycline cardiotoxicity is a feared complication of cancer chemotherapy. However, despite several decades of primarily hypothesis-driven research, the molecular basis of this phenomenon remains poorly understood. The aim of this study was to obtain integrative molecular insights into chronic anthracycline cardiotoxicity and the resulting heart failure. Cardiotoxicity was induced in rabbits (daunorubicin 3mg/kg, weekly, 10weeks) and changes in the left ventricular proteome were analyzed by 2D-DIGE. The protein spots with significant changes (p<0.01, >1.5-fold) were identified using MALDI-TOF/TOF. Key data were corroborated by immunohistochemistry, qRT-PCR and enzyme activity determination and compared with functional, morphological and biochemical data. The most important alterations were found in mitochondria - especially in proteins crucial for oxidative phosphorylation, energy channeling, antioxidant defense and mitochondrial stress. Furthermore, the intermediate filament desmin, which interacts with mitochondria, was determined to be distinctly up-regulated and disorganized in its expression pattern. Interestingly, the latter changes reflected the intensity of toxic damage in whole hearts as well as in individual cells. In addition, a marked drop in myosin light chain isoforms, activation of proteolytic machinery (including the proteasome system), increased abundance of chaperones and proteins involved in chaperone-mediated autophagy, membrane repair as well as apoptosis were found. In addition, dramatic changes in proteins of basement membrane and extracellular matrix were documented. In conclusion, for the first time, the complex proteomic signature of chronic anthracycline cardiotoxicity was revealed which enhances our understanding of the basis for this phenomenon and it may enhance efforts in targeting its reduction.

Rabbit model for in vivo study of anthracycline‐induced heart failure and for the evaluation of protective agents

Cardiac toxicity associated with chronic administration of anthracycline (ANT) antibiotics represents a serious complication of their use in anticancer chemotherapy, but can also serve as a useful experimental model of cardiomyopathy and congestive heart failure.

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

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.

Cardiac troponin T as a marker of myocardial damage caused by antineoplastic drugs in rabbits

Abstract Anthracycline derivatives are among the most effective antineoplastic drugs but their therapeutic use is limited by their adverse effects. The cardiac side-effects of antineoplastic drugs were investigated in rabbits in vivo from the viewpoint of release of cardiac troponin T (cTnT) measured by Elecsys Troponin T STAT immunoassay (Boehringer Mannheim, Germany). No increase in cTnT was found following administration of a single dose of daunorubicin (3 mg/kg i.v., n = 4). During development of daunorubicin-induced cardiomyopathy (daunorubicin 3 mg/kg i.v., once a week; maximum nine administrations, n = 7), the levels of cTnT were within the physiological range (i.e. cTnT < 0.1 μg/1) at the beginning of the experiment and before and after the 5th administration, but the pathological values of cTnT after the 8th administration in 43% animals (0.22 ± 0.08 μg/l) correlated with their premature death. In the control group, the levels of cTnT were always lower than 0.1 μg/l during the experiment. Following administration of a new antineoplastic drug – Oracin {6-[2-(2-hydroxyethyl) aminoethyl]-5,11-dioxo-5,6-dihydro-11H-indeno [1,2-c]-isoquinoline hydrochloride, 10 mg/kg i.v., once weekly, ten administrations, n = 7}, there was no increase in cTnT levels. These findings correlated with the PEP: LVET index, histological examination and no animal succumbing to premature death. It is possible to conclude that cTnT is a useful marker for the prediction of experimentally induced anthracycline cardiomyopathy and for the evaluation of cardiotoxic (and, possibly, cardioprotective) effects of new drugs in rabbits.

Comparative study of chronic toxic effects of daunorubicin and doxorubicin in rabbits

This study compares the chronic toxicity of two anthracyclines–daunorubicin and doxorubicin, commonly used for induction of anthracycline cardiomyopathy in the rabbit model. Such a comparative study has not been published until now. Both drugs were administered intravenously to male Chinchilla rabbits in doses at 3 mg/ kg (50 mg/m2) once weekly for 10 weeks. Selected biochemical, haematological and cardiovascular parameters and body weights were regularly monitored; additionally, a histological evaluation of heart, kidney and liver was performed at the end of the experiment. In the daunorubicin group, there were marked signs of the progressive development of heart failure, like the significant increases of the pre-ejection period/left ventricular ejection time index values (up to 134%)–and histological changes within the myocardium were also observed. On the other hand, the 10-week doxorubicin administration did not cause these changes that are typical for heart injury. Haematotoxicity, manifested particularly by aplastic anaemia, was apparent in both the experimental groups. Significant body weight loss (by 45.2%) and high premature mortality (100% versus 36.4%) reflected a greater general toxicity, especially nephrotoxicity of doxorubicin in comparison with daunorubicin. Further studies are necessary to find a possible explanation for these findings.

Deferiprone Does Not Protect against Chronic Anthracycline Cardiotoxicity in Vivo

Anthracycline cardiotoxicity ranks among the most severe complications of cancer chemotherapy. Although its pathogenesis is only incompletely understood, “reactive oxygen species (ROS) and iron” hypothesis has gained the widest acceptance. Besides dexrazoxane, novel oral iron chelator deferiprone has been recently reported to afford significant cardioprotection in both in vitro and ex vivo conditions. Therefore, the aim of this study was to assess whether deferiprone 1) has any effect on the anticancer action of daunorubicin and 2) whether it can overcome or significantly reduce the chronic anthracycline cardiotoxicity in the in vivo rabbit model (daunorubicin, 3 mg/kg i.v., weekly for 10 weeks). First, using the leukemic cell line, deferiprone (1–300 μM) was shown not to blunt the antiproliferative effect of daunorubicin. Instead, in clinically relevant concentrations (>10 μM), deferiprone augmented the antiproliferative action of daunorubicin. However, deferiprone (10 or 50 mg/kg administered p.o. before each daunorubicin dose) failed to afford significant protection against daunorubicin-induced mortality, left ventricular lipoperoxidation, cardiac dysfunction, and morphological cardiac deteriorations, as well as an increase in plasma cardiac troponin T. Hence, this first in vivo study changes the current view on deferiprone as a potential cardioprotectant against anthracycline cardiotoxicity. In addition, these results, together with our previous findings, further suggest that the role of iron and its chelation in anthracycline cardiotoxicity is not as trivial as originally believed and/or other mechanisms unrelated to iron-catalyzed ROS production are involved.

Cardioprotective effects of a novel iron chelator - pyridoxal 2- chlorobenzoyl hydrazone - in the rabbit model of daunorubicin-induced cardiotoxicity.

Iron chelation is the only pharmacological intervention against anthracycline cardiotoxicity whose effectiveness has been well documented both experimentally and clinically. In this study, we aimed to assess whether pyridoxal 2-chlorobenzoyl hydrazone (o-108, a strong iron chelator) can provide effective protection against daunorubicin (DAU)-induced chronic cardiotoxicity in rabbits. First, using the HL-60 leukemic cell line, it was shown that o-108 has no potential to blunt the antiproliferative efficacy of DAU. Instead, o-108 itself moderately inhibited cell proliferation. In vivo, chronic DAU treatment (3 mg/kg weekly for 10 weeks) induced mortality (33%), left ventricular (LV) dysfunction, a troponin T rise, and typical morphological LV damage. In contrast, all animals treated with 10 mg/kg o-108 before DAU survived without a significant drop in the LV ejection fraction (63.2 ± 0.5 versus 59.2 ± 1.0%, beginning versus end, not significant), and their cardiac contractility (dP/dtmax) was significantly higher than in the DAU-only group (1131 ± 125 versus 783 ± 53 kPa/s, p < 0.05), which corresponded with histologically assessed lower extent and intensity of myocardial damage. Although higher o-108 dose (25 mg/kg) was well tolerated when administered alone, in combination with DAU it led to rather paradoxical and mostly negative results regarding both cardioprotection and overall mortality. In conclusion, we show that shielding of free intracellular iron using a potent lipophilic iron chelator is able to offer a meaningful protection against chronic anthracycline cardiotoxicity. However, this approach lost its potential with the higher chelator dose, which suggests that iron might play more complex role in the pathogenesis of this disease than previously assumed.

Proliferation and Differentiation of Adult Endogenous Neural Stem Cells in Response to Neurodegenerative Process within the Striatum

The ongoing process of neurogenesis in the adult mammalian forebrain suggests the possible capacity for limited self-repair after brain injury. Previously, we have demonstrated that in an animal model of Huntington’s disease the neurodegenerative process initiates immediate intensive cell proliferation and differentiation resulting in characteristic enlargement of the subependymal zone (SEZ) of lateral brain ventricles. Now, our interest is focused on the architecture of the neurogenic niche of the SEZ in the identical model, particularly on characteristic features of astrocyte-like cells which are considered to be not only niche cells but also neural stem cells. Our findings prove higher activation of the lateral part of the SEZ (L-SEZ) adjacent to the degenerated striatum compared with the rostral part of the SEZ (R-SEZ). In the activated L-SEZ, niche cells which ensheathe clusters of neural progenitors are of immature astrocytic phenotype because of nestin and vimentin expression (except the expression of glial fibrillary acidic protein). However, the coexpression of all three filaments is not always found. Intermediate filaments also enable us to distinguish the basic shape of astrocytic cells within the SEZ, majority of which resemble protoplasmic rather than fibrillary astrocytes. Furthermore, our results show a wide plasticity of these astrocyte-like cells in immediate response to an extensive pathological process in the brain. These observations are consistent with the fact that adult stem cells undergo different processes in an already mature environment, and therefore can exhibit some specific characteristics unlike the embryonic or fetal neural stem cells.

Lack of cardiotoxicity of a new antineoplastic agent, a synthetic derivative of indenoisochinoline: comparison with daunorubicin in rabbits

Abstract The effect of repeated i.v. administration (once weekly, 12 administrations) of a new antineoplastic agent, Oracin (6-[2-(2-hydroxyethyl)aminoethyl]5,11-dioxo-5,6-dihydro-11H-indeno [1,2-c]-isochinoline hydrochloride, 10 mg/kg) and daunorubicin (3 mg/ kg) were investigated in rabbits in vivo. The criterion of occurrence of cardiotoxicity was compared with a control group of animals. Noninvasive polygraphic records were used to evaluate the function of the heart. The morphological changes of the heart were evaluated after the death of animals. There were no significant changes found in the ratio of the pre-ejection period/left ventricular ejection time (PEP : LVET ratio) after administration of Oracin (values between 0.3080 and 0.3310) or in the control group (values between 0.3425 and 0.3885). The administration of daunorubicin induced a significant, progressive increase in the PEP : LVET ratio (0.3775–0.9473), which was significantly different both from the Oracin-treated and the control group of animals. Histological examination of the hearts from the control group revealed normal structure of the myocardium including minute changes (dispersed cardiomyocytes with intensively eosinophilic cytoplasm, and several single cells with degenerated myofibrils) similar to the normal changes in muscle tissue. A very similar scenario was found in the Oracin group with the exception of one case where a slightly higher number of degenerated and necrotic cells was occurring. Administration of daunorubicin resulted in severe dispersed damage of the myocardium (myocytolysis with subsequent interstitial fibrosis), the changes being markedly different from those of the Oracin treatment and the control group. On the basis of our results it is possible to conclude that the administration of Oracin (10 mg/kg i.v.) did not induce signs of cardiotoxicity in rabbits in vivo.