RESUMEN
Anthracyclines, such as doxorubicin (adriamycin), daunorubicin, or epirubicin, rank among the most effective agents in classical anticancer chemotherapy. However, cardiotoxicity remains the main limitation of their clinical use. Topoisomerase IIß has recently been identified as a plausible target of anthracyclines in cardiomyocytes. We examined the putative topoisomerase IIß selective agent XK469 as a potential cardioprotective and designed several new analogs. In our experiments, XK469 inhibited both topoisomerase isoforms (α and ß) and did not induce topoisomerase II covalent complexes in isolated cardiomyocytes and HL-60, but induced proteasomal degradation of topoisomerase II in these cell types. The cardioprotective potential of XK469 was studied on rat neonatal cardiomyocytes, where dexrazoxane (ICRF-187), the only clinically approved cardioprotective, was effective. Initially, XK469 prevented daunorubicin-induced toxicity and p53 phosphorylation in cardiomyocytes. However, it only partially prevented the phosphorylation of H2AX and did not affect DNA damage measured by Comet Assay. It also did not compromise the daunorubicin antiproliferative effect in HL-60 leukemic cells. When administered to rabbits to evaluate its cardioprotective potential in vivo, XK469 failed to prevent the daunorubicin-induced cardiac toxicity in either acute or chronic settings. In the following in vitro analysis, we found that prolonged and continuous exposure of rat neonatal cardiomyocytes to XK469 led to significant toxicity. In conclusion, this study provides important evidence on the effects of XK469 and its combination with daunorubicin in clinically relevant doses in cardiomyocytes. Despite its promising characteristics, long-term treatments and in vivo experiments have not confirmed its cardioprotective potential.
Asunto(s)
Antraciclinas , Quinoxalinas , Inhibidores de Topoisomerasa II , Ratas , Animales , Conejos , Inhibidores de Topoisomerasa II/toxicidad , Inhibidores de Topoisomerasa II/uso terapéutico , Antraciclinas/toxicidad , Antraciclinas/uso terapéutico , Cardiotoxicidad , Daunorrubicina/toxicidad , Daunorrubicina/uso terapéutico , Doxorrubicina/toxicidad , Antibióticos Antineoplásicos/toxicidad , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/uso terapéutico , Daño del ADNRESUMEN
Background: Anthracycline cardiotoxicity is a well-known complication of cancer treatment, and miRNAs have emerged as a key driver in the pathogenesis of cardiovascular diseases. This study aimed to investigate the expression of miRNAs in the myocardium in early and late stages of chronic anthracycline induced cardiotoxicity to determine whether this expression is associated with the severity of cardiac damage. Method: Cardiotoxicity was induced in rabbits via daunorubicin administration (daunorubicin, 3 mg/kg/week; for five and 10 weeks), while the control group received saline solution. Myocardial miRNA expression was first screened using TaqMan Advanced miRNA microfluidic card assays, after which 32 miRNAs were selected for targeted analysis using qRT-PCR. Results: The first subclinical signs of cardiotoxicity (significant increase in plasma cardiac troponin T) were observed after 5 weeks of daunorubicin treatment. At this time point, 10 miRNAs (including members of the miRNA-34 and 21 families) showed significant upregulation relative to the control group, with the most intense change observed for miRNA-1298-5p (29-fold change, p < 0.01). After 10 weeks of daunorubicin treatment, when a further rise in cTnT was accompanied by significant left ventricle systolic dysfunction, only miR-504-5p was significantly (p < 0.01) downregulated, whereas 10 miRNAs were significantly upregulated relative to the control group; at this time-point, the most intense change was observed for miR-34a-5p (76-fold change). Strong correlations were found between the expression of multiple miRNAs (including miR-34 and mir-21 family and miR-1298-5p) and quantitative indices of toxic damage in both the early and late phases of cardiotoxicity development. Furthermore, plasma levels of miR-34a-5p were strongly correlated with the myocardial expression of this miRNA. Conclusion: To the best of our knowledge, this is the first study that describes alterations in miRNA expression in the myocardium during the transition from subclinical, ANT-induced cardiotoxicity to an overt cardiotoxic phenotype; we also revealed how these changes in miRNA expression are strongly correlated with quantitative markers of cardiotoxicity.
RESUMEN
Angiotensin-converting enzyme inhibitors (ACEis) have been used to treat anthracycline (ANT)-induced cardiac dysfunction, and they appear beneficial for secondary prevention in high-risk patients. However, it remains unclear whether they truly prevent ANT-induced cardiac damage and provide long-lasting cardioprotection. The present study aimed to examine the cardioprotective effects of perindopril on chronic ANT cardiotoxicity in a rabbit model previously validated with the cardioprotective agent dexrazoxane (DEX) with focus on post-treatment follow-up (FU). Chronic cardiotoxicity was induced by daunorubicin (DAU; 3 mg/kg/week for 10 weeks). Perindopril (0.05 mg/kg/day) was administered before and throughout chronic DAU treatment. After the completion of treatment, significant benefits were observed in perindopril co-treated animals, particularly full prevention of DAU-induced mortality and prevention or significant reductions in cardiac dysfunction, plasma cardiac troponin T (cTnT) levels, morphological damage, and most of the myocardial molecular alterations. However, these benefits significantly waned during 3 weeks of drug-free FU, which was not salvageable by administering a higher perindopril dose. In the longer (10-week) FU period, further worsening of left ventricular function and morphological damage occurred together with heart failure (HF)-related mortality. Continued perindopril treatment in the FU period did not reverse this trend but prevented HF-related mortality and reduced the severity of the progression of cardiac damage. These findings contrasted with the robust long-lasting protection observed previously for DEX in the same model. Hence, in the present study, perindopril provided only temporary control of ANT cardiotoxicity development, which may be associated with the lack of effects on ANT-induced and topoisomerase II ß (TOP2B)-dependent DNA damage responses in the heart.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Cardiotoxicidad/prevención & control , Daunorrubicina/efectos adversos , Perindopril/uso terapéutico , Animales , Antibióticos Antineoplásicos , Cardiotoxicidad/tratamiento farmacológico , Cardiopatías/inducido químicamente , Cardiopatías/prevención & control , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/mortalidad , Masculino , Conejos , Troponina T/sangreRESUMEN
BACKGROUND: Anthracycline-induced heart failure has been traditionally attributed to direct iron-catalyzed oxidative damage. Dexrazoxane (DEX)-the only drug approved for its prevention-has been believed to protect the heart via its iron-chelating metabolite ADR-925. However, direct evidence is lacking, and recently proposed TOP2B (topoisomerase II beta) hypothesis challenged the original concept. METHODS: Pharmacokinetically guided study of the cardioprotective effects of clinically used DEX and its chelating metabolite ADR-925 (administered exogenously) was performed together with mechanistic experiments. The cardiotoxicity was induced by daunorubicin in neonatal ventricular cardiomyocytes in vitro and in a chronic rabbit model in vivo (n=50). RESULTS: Intracellular concentrations of ADR-925 in neonatal ventricular cardiomyocytes and rabbit hearts after treatment with exogenous ADR-925 were similar or exceeded those observed after treatment with the parent DEX. However, ADR-925 did not protect neonatal ventricular cardiomyocytes against anthracycline toxicity, whereas DEX exhibited significant protective effects (10-100 µmol/L; P<0.001). Unlike DEX, ADR-925 also had no significant impact on daunorubicin-induced mortality, blood congestion, and biochemical and functional markers of cardiac dysfunction in vivo (eg, end point left ventricular fractional shortening was 32.3±14.7%, 33.5±4.8%, 42.7±1.0%, and 41.5±1.1% for the daunorubicin, ADR-925 [120 mg/kg]+daunorubicin, DEX [60 mg/kg]+daunorubicin, and control groups, respectively; P<0.05). DEX, but not ADR-925, inhibited and depleted TOP2B and prevented daunorubicin-induced genotoxic damage. TOP2B dependency of the cardioprotective effects was probed and supported by experiments with diastereomers of a new DEX derivative. CONCLUSIONS: This study strongly supports a new mechanistic paradigm that attributes clinically effective cardioprotection against anthracycline cardiotoxicity to interactions with TOP2B but not metal chelation and protection against direct oxidative damage.
Asunto(s)
Antraciclinas/farmacología , Cardiotoxicidad/prevención & control , Dexrazoxano/farmacología , Insuficiencia Cardíaca/tratamiento farmacológico , Inhibidores de Topoisomerasa II/metabolismo , Antraciclinas/efectos adversos , Antibióticos Antineoplásicos/efectos adversos , Antibióticos Antineoplásicos/farmacología , Cardiotoxicidad/tratamiento farmacológico , Cardiotoxicidad/metabolismo , ADN-Topoisomerasas de Tipo II/efectos adversos , ADN-Topoisomerasas de Tipo II/metabolismo , Daunorrubicina/metabolismo , Daunorrubicina/farmacología , Dexrazoxano/efectos adversos , Cardiopatías/tratamiento farmacológico , Humanos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Estrés Oxidativo/efectos de los fármacosRESUMEN
The anthracycline (ANT) anticancer drugs such as doxorubicin or daunorubicin (DAU) can cause serious myocardial injury and chronic cardiac dysfunction in cancer survivors. A bisdioxopiperazine agent dexrazoxane (DEX) has been developed as a cardioprotective drug to prevent these adverse events, but it is uncertain whether it is the best representative of the class. The present study used a rabbit model of chronic ANT cardiotoxicity to examine another bisdioxopiperazine compound called GK-667 (meso-(butane-2,3-diylbis(2,6-dioxopiperazine-4,1-diyl))bis(methylene)-bis(2-aminoacetate) hydrochloride), a water-soluble prodrug of ICRF-193 (meso-4,4'-(butan-2,3-diyl)bis(piperazine-2,6-dione)), as a potential cardioprotectant. The cardiotoxicity was induced by DAU (3 mg/kg, intravenously, weekly, 10 weeks), and GK-667 (1 or 5 mg/kg, intravenously) was administered before each DAU dose. The treatment with GK-667 was well tolerated and provided full protection against DAU-induced mortality and left ventricular (LV) dysfunction (determined by echocardiography and LV catheterization). Markers of cardiac damage/dysfunction revealed minor cardiac damage in the group co-treated with GK-667 in the lower dose, whereas almost full protection was achieved with the higher dose. This was associated with similar prevention of DAU-induced dysregulation of redox and calcium homeostasis proteins. GK-667 dose-dependently prevented tumor suppressor p53 (p53)-mediated DNA damage response in the LV myocardium not only in the chronic experiment but also after single DAU administration. These effects appear essential for cardioprotection, presumably because of the topoisomerase IIß (TOP2B) inhibition provided by its active metabolite ICRF-193. In addition, GK-667 administration did not alter the plasma pharmacokinetics of DAU and its main metabolite daunorubicinol (DAUol) in rabbits in vivo. Hence, GK-667 merits further investigation as a promising drug candidate for cardioprotection against chronic ANT cardiotoxicity.
Asunto(s)
Cardiomiopatías/prevención & control , Daño del ADN , Dicetopiperazinas/farmacología , Miocitos Cardíacos/efectos de los fármacos , Profármacos/farmacología , Inhibidores de Topoisomerasa II/farmacología , Disfunción Ventricular Izquierda/prevención & control , Función Ventricular Izquierda/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacos , Animales , Cardiomiopatías/inducido químicamente , Cardiomiopatías/metabolismo , Cardiomiopatías/fisiopatología , Cardiotoxicidad , Enfermedad Crónica , Daunorrubicina , Modelos Animales de Enfermedad , Fibrosis , Células HL-60 , Humanos , Masculino , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Conejos , Proteína p53 Supresora de Tumor/metabolismo , Disfunción Ventricular Izquierda/metabolismo , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
The bisdioxopiperazine topoisomerase IIß inhibitor ICRF-193 has been previously identified as a more potent analog of dexrazoxane (ICRF-187), a drug used in clinical practice against anthracycline cardiotoxicity. However, the poor aqueous solubility of ICRF-193 has precluded its further in vivo development as a cardioprotective agent. To overcome this issue, water-soluble prodrugs of ICRF-193 were prepared, their abilities to release ICRF-193 were investigated using a novel UHPLC-MS/MS assay, and their cytoprotective effects against anthracycline cardiotoxicity were tested in vitro in neonatal ventricular cardiomyocytes (NVCMs). Based on the obtained results, the bis(2-aminoacetoxymethyl)-type prodrug GK-667 was selected for advanced investigations due to its straightforward synthesis, sufficient solubility, low cytotoxicity and favorable ICRF-193 release. Upon administration of GK-667 to NVCMs, the released ICRF-193 penetrated well into the cells, reached sufficient intracellular concentrations and provided effective cytoprotection against anthracycline toxicity. The pharmacokinetics of the prodrug, ICRF-193 and its rings-opened metabolite was estimated in vivo after administration of GK-667 to rabbits. The plasma concentrations of ICRF-193 reached were found to be adequate to achieve cardioprotective effects in vivo. Hence, GK-667 was demonstrated to be a pharmaceutically acceptable prodrug of ICRF-193 and a promising drug candidate for further evaluation as a potential cardioprotectant against chronic anthracycline toxicity.
Asunto(s)
Antraciclinas/efectos adversos , Cardiotónicos/farmacología , Cardiotoxicidad/tratamiento farmacológico , ADN-Topoisomerasas de Tipo II/metabolismo , Dicetopiperazinas/farmacología , Piperazina/farmacología , Inhibidores de Topoisomerasa II/farmacología , Animales , Cardiotónicos/química , Cardiotoxicidad/metabolismo , Dexrazoxano/química , Dexrazoxano/farmacología , Dicetopiperazinas/química , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Piperazina/química , Profármacos/química , Profármacos/farmacología , Conejos , Razoxano/química , Razoxano/farmacología , Inhibidores de Topoisomerasa II/química , Agua/químicaRESUMEN
Bisdioxopiperazine agent dexrazoxane (ICRF-187) has been the only effective and approved drug for prevention of chronic anthracycline cardiotoxicity. However, the structure-activity relationships (SARs) of its cardioprotective effects remain obscure owing to limited investigation of its derivatives/analogs and uncertainties about its mechanism of action. To fill these knowledge gaps, we tested the hypothesis that dexrazoxane derivatives exert cardioprotection via metal chelation and/or modulation of topoisomerase IIß (Top2B) activity in chronic anthracycline cardiotoxicity. Dexrazoxane was alkylated in positions that should not interfere with the metal-chelating mechanism of cardioprotective action; that is, on dioxopiperazine imides or directly on the dioxopiperazine ring. The protective effects of these agents were assessed in vitro in neonatal cardiomyocytes. All studied modifications of dexrazoxane molecule, including simple methylation, were found to abolish the cardioprotective effects. Because this challenged the prevailing mechanistic concept and previously reported data, the two closest derivatives [(±)-4,4'-(propane-1,2-diyl)bis(1-methylpiperazine-2,6-dione) and 4-(2-(3,5-dioxopiperazin-1-yl)ethyl)-3-methylpiperazine-2,6-dione] were thoroughly scrutinized in vivo using a rabbit model of chronic anthracycline cardiotoxicity. In contrast to dexrazoxane, both compounds failed to protect the heart, as demonstrated by mortality, cardiac dysfunction, and myocardial damage parameters, although the pharmacokinetics and metal-chelating properties of their metabolites were comparable to those of dexrazoxane. The loss of cardiac protection was shown to correlate with their abated potential to inhibit and deplete Top2B both in vitro and in vivo. These findings suggest a very tight SAR between bisdioxopiperazine derivatives and their cardioprotective effects and support Top2B as a pivotal upstream druggable target for effective cardioprotection against anthracycline cardiotoxicity. SIGNIFICANCE STATEMENT: This study has revealed the previously unexpected tight structure-activity relationships of cardioprotective effects in derivatives of dexrazoxane, which is the only drug approved for the prevention of cardiomyopathy and heart failure induced by anthracycline anticancer drugs. The data presented in this study also strongly argue against the importance of metal-chelating mechanisms for the induction of this effect and support the viability of topoisomerase IIß as an upstream druggable target for effective and clinically translatable cardioprotection.
Asunto(s)
Antraciclinas/efectos adversos , Cardiotoxicidad/tratamiento farmacológico , ADN-Topoisomerasas de Tipo II/metabolismo , Dexrazoxano/farmacología , Corazón/efectos de los fármacos , Sustancias Protectoras/farmacología , Inhibidores de Topoisomerasa II/farmacología , Animales , Cardiomiopatías/tratamiento farmacológico , Cardiomiopatías/metabolismo , Línea Celular Tumoral , Células HL-60 , Humanos , Masculino , Modelos Animales , Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Conejos , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
Although proteasome inhibitors (PIs) are modern targeted anticancer drugs, they have been associated with a certain risk of cardiotoxicity and heart failure (HF). Recently, PIs have been combined with anthracyclines (ANTs) to further boost their anticancer efficacy. However, this raised concerns regarding cardiac safety, which were further supported by several in vitro studies on immature cardiomyocytes. In the present study, we investigated the toxicity of clinically used PIs alone (bortezomib (BTZ), carfilzomib (CFZ)) as well as their combinations with an ANT (daunorubicin (DAU)) in both neonatal and adult ventricular cardiomyocytes (NVCMs and AVCMs) and in a chronic rabbit model of DAU-induced HF. Using NVCMs, we found significant cytotoxicity of both PIs around their maximum plasma concentration (cmax) as well as significant augmentation of DAU cytotoxicity. In AVCMs, BTZ did not induce significant cytotoxicity in therapeutic concentrations, whereas the toxicity of CFZ was significant and more profound. Importantly, neither PI significantly augmented the cardiotoxicity of DAU despite even more profound proteasome-inhibitory activity in AVCMs compared with NVCMs. Furthermore, in young adult rabbits, no significant augmentation of chronic ANT cardiotoxicity was noted with respect to any functional, morphological, biochemical or molecular parameter under study, despite significant inhibition of myocardial proteasome activity. Our experimental data show that combination of PIs with ANTs is not accompanied by an exaggerated risk of cardiotoxicity and HF in young adult animal cardiomyocytes and hearts.
Asunto(s)
Antraciclinas/toxicidad , Antineoplásicos/toxicidad , Cardiotoxicidad/etiología , Inhibidores de Proteasoma/toxicidad , Animales , Antraciclinas/administración & dosificación , Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidad , Bortezomib/administración & dosificación , Bortezomib/toxicidad , Daunorrubicina/administración & dosificación , Daunorrubicina/toxicidad , Relación Dosis-Respuesta a Droga , Masculino , Miocitos Cardíacos/efectos de los fármacos , Oligopéptidos/administración & dosificación , Oligopéptidos/toxicidad , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/administración & dosificación , Conejos , Ratas , Ratas WistarRESUMEN
Chronic anthracycline (ANT) cardiotoxicity is a serious complication of cancer chemotherapy. Molsidomine, a NO-releasing drug, has been found cardioprotective in different models of I/R injury and recently in acute high-dose ANT cardiotoxicity. Hence, we examined whether its cardioprotective effects are translatable to chronic ANT cardiotoxicity settings without induction of nitrosative stress and interference with antiproliferative action of ANTs. The effects of molsidomine (0.025 and 0.5mg/kg, i.v.) were studied on the well-established model of chronic ANT cardiotoxicity in rabbits (daunorubicin/DAU/3mg/kg/week for 10 weeks). Molsidomine was unable to significantly attenuate mortality, development of heart failure and morphological damage induced by DAU. Molsidomine did not alter DAU-induced myocardial lipoperoxidation, MnSOD down-regulation, up-regulation of HO-1, IL-6, and molecular markers of cardiac remodeling. Although molsidomine increased 3-nitrotyrosine in the myocardium, this event had no impact on cardiotoxicity development. Using H9c2 myoblasts and isolated cardiomyocytes, it was found that SIN-1 (an active metabolite of molsidomine) induces significant protection against ANT toxicity, but only at high concentrations. In leukemic HL-60 cells, SIN-1 initially augmented ANT cytotoxicity (in low and clinically achievable concentrations), but it protected these cells against ANT in the high concentrations. UHPLC-MS/MS investigation demonstrated that the loss of ANT cytotoxicity after co-incubation of the cells in vitro with high concentrations of SIN-1 is caused by unexpected chemical depletion of DAU molecule. The present study demonstrates that cardioprotective effects of molsidomine are not translatable to clinically relevant chronic form of ANT cardiotoxicity. The augmentation of antineoplastic effects of ANT in low (nM) concentrations may deserve further study.
Asunto(s)
Antraciclinas/toxicidad , Antibióticos Antineoplásicos/toxicidad , Cardiotónicos/farmacología , Cardiopatías/inducido químicamente , Cardiopatías/prevención & control , Molsidomina/farmacología , Donantes de Óxido Nítrico/farmacología , Animales , Cardiotoxicidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Enfermedad Crónica , Daunorrubicina/toxicidad , Doxorrubicina/toxicidad , Insuficiencia Cardíaca/prevención & control , Peroxidación de Lípido/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Conejos , Especies Reactivas de Oxígeno/metabolismo , Remodelación Ventricular/efectos de los fármacosRESUMEN
Dexrazoxane (DEX) is a clinically available cardioprotectant that reduces the toxicity induced by anthracycline (ANT) anticancer drugs; however, DEX is seldom used and its action is poorly understood. Inorganic nitrate/nitrite has shown promising results in myocardial ischemia-reperfusion injury and recently in acute high-dose ANT cardiotoxicity. However, the utility of this approach for overcoming clinically more relevant chronic forms of cardiotoxicity remains elusive. Hence, in this study, the protective potential of inorganic nitrate and nitrite against chronic ANT cardiotoxicity was investigated, and the results were compared to those using DEX. Chronic cardiotoxicity was induced in rabbits with daunorubicin (DAU). Sodium nitrate (1g/L) was administered daily in drinking water, while sodium nitrite (0.15 or 5mg/kg) or DEX (60mg/kg) was administered parenterally before each DAU dose. Although oral nitrate induced a marked increase in plasma NOx, it showed no improvement in DAU-induced mortality, myocardial damage or heart failure. Instead, the higher nitrite dose reduced the incidence of end-stage cardiotoxicity, prevented related premature deaths and significantly ameliorated several molecular and cellular perturbations induced by DAU, particularly those concerning mitochondria. The latter result was also confirmed in vitro. Nevertheless, inorganic nitrite failed to prevent DAU-induced cardiac dysfunction and molecular remodeling in vivo and failed to overcome the cytotoxicity of DAU to cardiomyocytes in vitro. In contrast, DEX completely prevented all of the investigated molecular, cellular and functional perturbations that were induced by DAU. Our data suggest that the difference in cardioprotective efficacy between DEX and inorganic nitrite may be related to their different abilities to address a recently proposed upstream target for ANT in the heart - topoisomerase IIß.
Asunto(s)
Cardiotónicos/farmacología , Cardiotoxicidad/prevención & control , Dexrazoxano/farmacología , Nitratos/farmacología , Nitrito de Sodio/farmacología , Animales , Antibióticos Antineoplásicos/efectos adversos , Cardiotoxicidad/metabolismo , Cardiotoxicidad/patología , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Daunorrubicina/efectos adversos , Esquema de Medicación , Infusiones Intravenosas , Masculino , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , ConejosRESUMEN
Due to their cardiac origin, H9c2 cells rank among the most popular cell lines in current cardiovascular research, yet molecular phenotype remains elusive. Hence, in this study we used proteomic approach to describe molecular phenotype of H9c2 cells in their undifferentiated (i.e., most frequently used) state, and its functional response to cardiotoxic drug doxorubicin. Of 1671 proteins identified by iTRAQ IEF/LC-MSMS analysis, only 12 proteins were characteristic for striated muscle cells and none was cardiac phenotype-specific. Targeted LC-SRM and western blot analyses confirmed that undifferentiated H9c2 cells are phenotypically considerably different to both primary neonatal cardiomyocytes and adult myocardium. These cells lack proteins essential for formation of striated muscle myofibrils or they express only minor amounts thereof. They also fail to express many proteins important for metabolism of muscle cells. The challenge with clinically relevant concentrations of doxorubicin did not induce a proteomic signature that has been previously noted in primary cardiomyocytes or adult hearts. Instead, several alterations previously described in other cells of mesodermal origin, such as fibroblasts, were observed (e.g., severe down-regulation of collagen synthesis pathway). In conclusion, the molecular phenotype of H9c2 cells resembles very immature myogenic cells with skeletal muscle commitment upon differentiation and thus, translatability of findings obtained in these cells deserves caution.
Asunto(s)
Doxorrubicina/toxicidad , Miocardio/citología , Proteoma/análisis , Animales , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Miocardio/metabolismo , Fenotipo , Ratas , Relación Estructura-ActividadRESUMEN
BACKGROUND: Cardiac troponins (cTns) seem to be more sensitive for the detection of anthracycline cardiotoxicity than the currently recommended method of monitoring LV systolic function. However, the optimal timing of blood sampling remains unknown. Hence, the aims of the present study were to determine the precise diagnostic window for cTns during the development of chronic anthracycline cardiotoxicity and to evaluate their predictive value. METHODS: Cardiotoxicity was induced in rabbits with daunorubicin (3mg/kg, weekly, for 8 weeks). Blood samples were collected 2-168 h after the 1st, 5th and 8th drug administrations, and concentrations of cTns were determined using highly sensitive assays: hs cTnT (Roche) and hs cTnI (Abbott). RESULTS: The plasma levels of cTns progressively increased with the rising number of chemotherapy cycles. While only a mild non-significant increase in both cTn levels occurred after the first daunorubicin dose, a significant rise was observed after the 5th and 8th administrations. Two hours after these administrations, a significant increase occurred with a peak between 4-6h and a decline until 24h. Discrete cTn release continued even after cessation of the therapy. While greater variability of cTn levels was observed around the peak concentrations, the values did not correspond well with the severity of LV systolic dysfunction. Unlike AMI in cardiotoxicity, cTn elevations may be better associated with cumulative dose and concentrations at steady state than cmax. CONCLUSIONS: To the best of our knowledge, this is the first study to precisely describe the diagnostic window and predictive value of cTns in anthracycline cardiotoxicity.
Asunto(s)
Antraciclinas/toxicidad , Cardiotoxicidad/sangre , Troponina I/sangre , Troponina T/sangre , Animales , Antibióticos Antineoplásicos/toxicidad , Biomarcadores/sangre , Biomarcadores/metabolismo , Cardiomiopatías/sangre , Cardiomiopatías/inducido químicamente , Cardiotoxicidad/diagnóstico por imagen , Daunorrubicina/toxicidad , Modelos Animales de Enfermedad , Ecocardiografía , Corazón/efectos de los fármacos , Corazón/fisiología , Masculino , Valor Predictivo de las Pruebas , Conejos , Análisis de Regresión , Sístole/efectos de los fármacos , Sístole/fisiologíaRESUMEN
Chronic anthracycline cardiotoxicity is a serious clinical issue with well characterized functional and histopathological hallmarks. However, molecular determinants of the toxic damage and associated myocardial remodeling remain to be established. Furthermore, details on the different propensity of the left and right ventricle (LV and RV, respectively) to the cardiotoxicity development are unknown. Hence, the aim of the investigation was to study molecular changes associated with remodeling of the LV and RV in chronic anthracycline cardiotoxicity and post-treatment follow up. The cardiotoxicity was induced in rabbits with daunorubicin (3 mg/kg/week for 10 weeks) and animals were sacrificed either at the end of the treatment or after an additional 10 weeks. Daunorubicin induced severe and irreversible cardiotoxicity associated with LV dysfunction and typical morphological alterations, whereas the myocardium of the RV showed only mild changes. Both ventricles also showed different expression of ANP after daunorubicin treatment. Daunorubicin impaired the expression of several sarcomeric proteins in the LV, which was not the case of the RV. In particular, a significant drop was found in titin and thick filament proteins at both mRNA and protein level and this might be connected with persistent LV down-regulation of GATA-4. In addition, the LV was more affected by treatment-induced perturbations in calcium handling proteins. LV cardiomyocytes showed marked up-regulation of desmin after the treatment and vimentin was mainly induced in LV fibroblasts, whereas only weaker changes were observed in the RV. Remodeling of extracellular matrix was almost exclusively found in the LV with particular induction of collagen I and IV. Hence, the present study describes profound molecular remodeling of myocytes, non-myocyte cells and extracellular matrix in response to chronic anthracycline treatment with marked asymmetry between LV and RV.
Asunto(s)
Antraciclinas/toxicidad , Miocardio/metabolismo , Remodelación Ventricular/fisiología , Animales , Western Blotting , Daunorrubicina/farmacología , Ecocardiografía , Inmunohistoquímica , Filamentos Intermedios/metabolismo , Masculino , Conejos , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/metabolismo , Troponina T/metabolismo , Remodelación Ventricular/efectos de los fármacosRESUMEN
Despite incomplete understanding to its mechanism of action, dexrazoxane (DEX) is still the only clearly effective cardioprotectant against chronic anthracycline (ANT) cardiotoxicity. However, its clinical use is currently restricted to patients exceeding significant ANT cumulative dose (300mg/m(2)), although each ANT cycle may induce certain potentially irreversible myocardial damage. Therefore, the aim of this study was to compare early and delayed DEX intervention against chronic ANT cardiotoxicity and study the molecular events involved. The cardiotoxicity was induced in rabbits with daunorubicin (DAU; 3mg/kg/week for 10 weeks); DEX (60mg/kg) was administered either before the 1st or 7th DAU dose (i.e. after ≈300mg/m(2) cumulative dose). While both DEX administration schedules prevented DAU-induced premature deaths and severe congestive heart failure, only the early intervention completely prevented the left ventricular dysfunction, myocardial morphological changes and mitochondrial damage. Further molecular analyses did not support the assumption that DEX cardioprotection is based and directly proportional to protection from DAU-induced oxidative damage and/or deletions in mtDNA. Nevertheless, DAU induced significant up-regulation of heme oxygenase 1 pathway while heme synthesis was inversely regulated and both changes were schedule-of-administration preventable by DEX. Early and delayed DEX interventions also differed in ability to prevent DAU-induced down-regulation of expression of mitochondrial proteins encoded by both nuclear and mitochondrial genome. Hence, the present functional, morphological as well as the molecular data highlights the enormous cardioprotective effects of DEX and provides novel insights into the molecular events involved. Furthermore, the data suggests that currently recommended delayed intervention may not be able to take advantage of the full cardioprotective potential of the drug.