Anthracycline-induced Cardiotoxicity: Clinical Analysis And Experimental Intervention In Rat Model

Anthracycline(ANTH) is the most effective antineoplastic agents developed to treat solid tumors and hematologic malignancies.However,ANTH may result in dose-dependent cardiotoxicity.As outcomes of many cancers have improved greatly with the development of modern multi-drug chemotherapy,a major concern for this growing number of survivors is the risks of late effects of treatment. Electrocardiography and echocardiography examination as well as optimal chemotherapy schedule for every individual could probably reduce the cardiotoxicity induced by ANTH.In addition,using new protective drugs is also a promising therapeutic option.Based on oxidation stress and free radical injury hypothesis, attentions are focused on antioxidative drugs and iron chelators including dexrazoxane(DZR),the only cardiac protectant approved by FDA for clinical use. However,DZR could aggravate myelosuppression and people worry if it would interfere with antitumor effect of ANTH.So it is necessary to explore new protective drugs.Thrombopoietin(TPO),as a haematopoietic growth factor,can stimulate proliferation and differentiation of megakaryocyte.Recently,TPO was reported to have antiapoptotic and angiogenesis effects.We hypothesize that TPO may protect against cardiotoxicity induced by doxorubicin(DOX).In this study,we retrospectively analized echocardiogram data of leukemia or lymphoma survivors to find the latent abnormality of heart function.A proper rat model was established and was utilized to investigate the effect of TPO on DOX-induced cardiomyopathy and underlying mechanisms. PartⅠClinical Investigation of Anthracycline-induced CardiotoxicityPurposeThis study aimed to determine whether latent cardiac dysfunction is present in children treated with anthracycline because of leukemia or lymphoma and screen for risk factors of cardiac abnormalities.Patients and MethodsSerial echocardiogram data of 110 pediatric survivors with acute leukemia or lymphoma treated with anthracycline were retrospectively reviewed.Measurements of these patients were compared with those of normal children at the same age. Associations between age at diagnosis,cumulative dose of daunorubicin(DNR),sex, length of follow-up,and deviations from normal values in M-mode echocardiograms were evaluated using multivariate linear regression analysis.ResultsCompared to age-matched normal children,the patient group had thinner left ventricle posterior wall(LVPW),increased left ventricular internal dimension at endsystole (LVID_s) and heart rate(HR),decreased left ventricular internal dimension at end-diastole(LVID_d),fractional shortening(FS),ejection fraction(EF),cardiac output(CO) and ratio of mitral E velocity to mitral A velocity(MEV/MAV). Thirty-five of the patients(32%) had EF below 60%.Survivors who received DNR cumulative doses above 210 mg/m~2 had a 5.6-fold excess risk of reduced EF(95%CI, 1.5 to 31.2) compared with those received less than 210 mg/m~2.DNR cumulative dose was correlated with dilated LVID and reduced FS and EF.ConclusionsThis study suggested that patients received anthracycline therapy had decreased heart function and DNR cumulative dose was an independent risk factor for dilated LVID and reduced FS and EF.Survivors treated with DNR doses above 210 mg/m~2 are at higher risk for reduced EF and dilated left ventricle.These patients need long-term follow-up for signs of cardiomyopathy. PartⅡEstablishment of Doxorubicin-induced Cardiomyopathy Model in Rat and Echocardiographic AssessmentObjectiveTo explore the best way of establishing a rat model of doxorubicin-induced cardiomyopathy and assess the value of transthoracic echocardiography in assessment of heart function in rats.MethodsWe established three models of cardiotoxicity induced by doxorubicin in S-D rats. Rats were injected with DOX at one dose of 20 mg/kg intravenously for an acute intravenous model,2.5mg/kg intravenously weekly for 6 weeks for a chronic intravenous model and 2.5mg/kg intraperitoneally weekly for 6 weeks for a chronic intraperitoneal model.Each model had a control group which received equal volume of saline.Transthoracic echocardiography was performed in rats at baseline and before sacrifice and additionally at 6 weeks after beginning of DOX adminiatration for those two chronic models to measure left ventricular dimensions and calculate ejection fraction.Histopathological evaluation was performed by a blinded investigator after rats were sacrificed at week 11.The frequency and severity of myocardial lesions were assessed semi-quantitatively using a light microscope.ResultsThe body weight gain of DOX-treated rats was inhibited seriously compared to control group.At week 11,more dilated LVID_s in DOX-treated rats was found compared to control rats(0.463±0.047 vs.0.412±0.020 cm),as well as significantly decreased FS and EF(31.7±3.5%and 65.6±5.1%,repectively) in chronic intravenous model.Rats which received one large dose of DOX in acute intravenous model had significantly lower FS and EF(39.3±2.6%and 76.1±2.7%,repectively) at day 5 compared to control or baseline,but higher than rats in chronic intravenous model(p＜0.05).No significant decreased FS and EF(45.7±6.3%and 81.8±6.3%, repectively) was shown in rats which received doxorubicin intraperitoneally and had plenty of ascites.Histological examination demonstrated that myocardial injury of DOX-treated rats in chronic intravenous model was more serious than that in acute model.Similarly,no obvious cardiac lesion was found in chronic intraperitoneal group.Analysis of data revealed a significant negative correlation between cardiomyopathy score and FS and EF measured by echocardiography(r=-0.64 and -0.57;p＜0.005).ConclusionsIntravenous injection with 2.5mg/kg of doxorubicin weekly for 6 weeks up to 15 mg/kg is a reliable method to establish doxorubicin-induced cardiomyopathy animal model.Echocardiography appears to be a valuable non-invasive approach to assess heart function and has significant correlation with histological findings.PartⅢThrombopoietin Protects Against Doxorubicin-induced Cardiomyopathy in Rat ModelObjectiveThis study was designed to explore the protective effect of thrombopoietin(TPO) on doxorubicin-induced cardiomyopathy in rat model and related possible mechanisms.MethodsSixty S-D male rats were utilized in tatal.Rats were injected with DOX 2.5mg/kg intravenously once a week for 6 weeks with or without TPO 10μg/kg intraperitoneal pretreatment three times a week till last dose of doxorubicin.As a positive control group,DZR was administered at a dose ratio of 20:1 intraperitoneally 30 min prior every dose of DOX.Ten rats received saline as a normal control group.Left ventricular dimensions were measured and FS,EF was calculated automatically by transthoracic echocardiography in all surviving rats at baseline and week 6 and before sacrifice.Heart morphology was studied by light and electron microscopy to evaluate the severity of cardiac damage in rats.Western blot analysis was used to measure the phophorylation of Akt and ERK in heart tissue.ResultsDOX treatment caused depression in body weight and blood cell counts.TPO resulted in profound stimulation of blood cell production.Rats which received doxorubicin only had dilated left ventricle,significantly decreased FS and EF,severe cardiac damage including mitochondrial swelling and vacuolization under light and electron microsopy.TPO or DZR pretreatment exerted significant cardioprotective effects in terms of improved fractional shortening,ejection fraction and cardiomyopathy score.We also found that phosphorylation of Akt and ERK levels in heart tissue of DOX-treated rats were significantly inhibited and DZR increased both of them.TPO pretreatment also increased the level of Akt activation.ConclusionsOur results suggested that activation of the Akt and ERK signaling pathways was involved in the course of DOX-induced cardiomyopathy and TPO could exert cardiac protective effects probably through upregulating Akt phosphorylation in a rat model. And upregulation of Akt and ERK phosphorylation may also play an important role in cardioprotective effect of DZR.Summary1.Patients received anthracycline therapy had latent decreased heart function and DNR cumulative dose was an independent risk factor for cardiotoxicity.Those survivors treated with DNR doses above 210 mg/m~2 are at higher risk for reduced EF and dilated left ventricle and need long-term follow-up.2.Intermittent intravenous administration with repeated low doses of DOX could result in cardiomyopathy in rats and mimic clinical chemotherapy situation well. Echocardiography was a valuable approach to assess heart function of rats and had significant correlation with histological findings.3.Activation of the Akt and ERK signaling pathways was involved in the course of DOX-induced cardiomyopathy.TPO could exert cardiac protective effects in rat model probably by upregulating Akt phosphorylation level and related antiapoptotic pathway.Upregulation of Akt and ERK phosphorylation may also play a role in cardioprotective effect of DZR.