Experimental Study On Preventive Cardioprotection Of Erythropoietin Against Doxorubicin-induced Cardiomyopathy

Background:Doxorubicin(DOX)is a highly effective antineoplastic agent for treatment of hematologic malignancies and tumors.However,its clinical application is limited by a dose-related cardiomyopathy.Recent evidence suggests that erythropoietin(EPO)plays an important role not only on haematopoiesis but also on protection of organs.It still remains unclear whether EPO can preventively protect against DOX-induced cardiomyopathy and what may be its potential mechanisms.Objectives:To examine the preventive effect of EPO and further determine its specific mechanisms,in vitro and in vivo investigations were performed in rats with DOX-induced cardiomyopathy.Methods:1.In vitro investigation:isolated neonatal Wistar rat cardiomyocytes were treated with vehicle,DOX with or without EPO,or EPO.Twenty-four hours later, cells were used to determine apoptotic and morphologic analyses.2.In vivo investigation:thirty nine Wistar rats were randomly grouped as the Control group,the DOX group,the DOX+EPO group and the EPO group.After two weeks of drug injections and another two weeks of wash-out period,cardiac function was evaluated by echocardiography and haemodynamics.Rats were then sacrificed for histological analysis,terminal deoxynucleotidyltransferase dUTP nick-end labeling(TUNEL) assay,immunohistochemical detection,reverse transcription-polymerase chain reaction(RT-PCR)and Western blot.All values were compared and statistically analyzed by SPSS 13.0,and P＜0.05 was considered significant.Results:1.in vitro investigation:treatment of cardiomyocytes with DOX resulted in a significant increase of apoptotic index.Cardiomyocyte apoptosis in the DOX+EPO group was considerably inhibited by EPO treatment(apoptotic index 1.54±0.74%vs 8.36±1.21%,P＜0.05 compared to the DOX group).2.In vivo investigation:①The present rat model induced by DOX showed similar changes to those of DCM in human by echocardiographic,hemodynamic and histological analyses.②There was significant difference in cardiac function between the DOX+EPO group and the DOX group.Changes included LVEDD(4.70±0.27mm vs 5.91±0.58mm),LVESD (1.93±0.21mm vs 3.54±0.52mm),EF(86.55±3.21%vs 71.58±3.98%),FS (59.89±2.52%vs 40.35±4.74%),LVSP(109.56±6.22mmHg vs 89.54±4.33mmHg), LVEDP(4.05±0.76mmHg vs 9.17±1.36mmHg),+dP/dt max (6127.56±699.42mmHg/s vs 4197.60±626.32mmHg/s)and -dP/dt max (5608.24±771.40mmHg/s vs 4390.03±397.28mmHg/s).From above,preventive treatment with EPO significantly protected from cardiac dysfunction induced by DOX.③Histological analysis showed myofilament loss and marked atrophic cardiomyocytes,surrounded by increased interstitial fibrosis and some compensatory hypertrophic myocytes in the DOX group,whereas EPO apparently exerted a protective effect in the DOX+EPO group.④A very small amount of apoptotic nuclei were detected to scatter about the tissue section in dot distribution in the control group and the EPO group.DOX treatment in the DOX group increased significantly the number of apoptotic nuclei,some of which appeared in focal distribution under the microscope rather than only in dot distribution associated with the above two groups.⑤Protein expressions of Bcl-2 and Bax were detected by immunohistochemistry and Western blot.Compared to the control group,the myocardial level of Bcl-2 was significantly lower in the DOX group from 0.77±0.09 to 0.41±0.05(P＜0.05),but restored to 0.70±0.09 in the DOX+EPO group.Bax protein expression was increased in the DOX group compared to the control group (0.16±0.01 vs 0.04±0.01,P＜0.05).However,there was no significant difference between the DOX group and the DOX+EPO group.⑥RT-PCR assay revealed that compared to the control group,the expression level of mRNA was decreased for Bcl-2 and increased for Bax in the DOX group,and that compared to the DOX group, the expression level was increased for Bcl-2,but remained similar for Bax in the DOX+EPO group.Conclusions:Our results suggest that EPO exerts preventive cardioprotective effects on DOX-induced cardiomyopathy via anti-apoptotic pathways.The up-regulation of Bcl-2 protein expression may contribute to this.