| Anthracycline,represented by daunorubicin（DNR）,is a widely used broad-spectrum antineoplastic drug in clinic.Cardiac toxicity is the most common and serious adverse reaction in anthracycline chemotherapy,which seriously limits its clinical application.The mechanism of anthracycline induced cardiotoxicity（AIC）is not yet completely understood.Despite that the downstream intracellular calcium signal transduction pathways remain unknown,disrupted calcium homeostasis and impaired calcium handling had been regarded as an important mechanism of AIC by multiple evidence.Calcineurin（CaN）-nuclear factor of activated T cells（NFAT）signaling pathway,activated by increased cytosolic free calcium concentration,[Ca2+]i,is a major Ca2+-dependent gene transcription pathway in cardiomyocytes.To date,the exact activation state of CaN-NFAT signaling pathway and its pathophysiological roles in AIC remain unclear.In this study,AIC model was established by treating H9c2 cell with DNR.The changes of expression and activation of CaN and NFAT subfamily members in mRAN and protein levels were compared.An overall blockage of CaN-NFAT signal pathway was induced by specific pharmacological blockers targeting CaN and NFAT in order to assess its effect on DNR induced cardiomyocyte injury in H9c2 cells.Aim:To investigate the activating state and pathophysiological roles of CaN-NFAT signaling pathway in DNR-induced cardiomyocyte injury in H9c2 cells.Methods:H9c2 rat cardiomyocytes were treated with 1μM DNR for 24h to establish DNR myocardial injury model.（1）RT-PCR and Western blotting were used to detect the changes of mRNA and protein expression of CaN and NFAT.（2）Immunofluorescence assay was used to detect the activation of CaN-NFAT signaling pathway in cardiomyocytes.（3）The survival rate of cardiomyocytes was measured by MTS.Creatine kinase（CK）,Lactate dehydrogenase（LDH）biochemical kit was used to detect the degree of myocardial injury.（5）RT-PCR and immunocytochemistry were used to detect the expression of mRNA and protein in ANP.（6）The ratio of Bax to Bcl-2 and the apoptosis rate of cardiomyocytes were detected by Western blotting and Hoechst33342staining.（7）RT-PCR,2,7-Dichlorodihydrofluorescein diacetate（DCFH-DA）reactive oxygen species（ROS）assay was used to detect the level of oxidative stress in cardiomyocytes.Results:（1）Compared with the control group,the expression of CaNBβ,NFATc1/2/4in mRNA and protein level and the nuclear translocation of NFATc1/2/3/4 in DNR treated of H9c2 rats myocardial cells was significantly increased,suggesting an overall activation of CaN-NFAT signaling pathway.（2）The nuclear translocation of NFATc4was depressed by canonical transient receptor potential（TRPCs）channel inhibitor SKF96365,while receptor-operated calcium channel（ROCC）and store-operated calcium channel（SOCC）agonist 1-oleoyl-2-acetyl-sn-glycerol（OAG）and cyclopainic acid（CPA）further increased it.Those results indicated the involvement of TRPCs induced calcium influx in the activation of NFATc4.（3）The activity of LDH and CK increased and the cell survival rate decreased in DNR treated H9c2 cells,those changes were reversed by specifically inhibition of CaN and NFAT.（4）DNR induced increase of ANP in mRNA and protein levels was blocked by CaN and NFAT specific inhibitors.（5）Increased apoptosis rate in DNR treated H9c2 cells was observed by Hoechst33342staining,accompanying with higher Bax/Bcl-2 ratio as compared with the control group.CaN and NFAT inhibition successfully curbed DNR induced cellular apoptosis.（6）CaN and NFAT inhibitors limited excess ROS generation and NOX2 overexpression in DNR treated H9c2 cells.Conclusion:（1）The expression of CaNBβ,NFATc1/2/4 in mRNA and protein levels and the nuclear translocation of NFATc1/2/3/4 was increased in DNR treated H9c2 cells,revealed the overall activation of CaN-NFAT signaling pathway.（2）Specific inhibitor targeting CaN and NFAT effectively inhibited DNR-induced cardiomyocytes death,cellular apoptosis and injury as well as the overactivation of ROS generation,implying a potential effect of CaN-NFAT signaling pathway on myocardial injury and heart failure via regulation of cardiomyocyte apoptosis and oxidative stress in AIC. |
