Autophagic Degradation Of CTGF Induced By Sunitinib Caused The Cardiotoxicity

Objectives:Sunitinib,a multi-targeted tyrosine kinases inhibitor,is approved for the first-line treatment of metastatic renal cell carcinoma,imatinib-resistant gastrointestinal stromal tumor and pancreatic neuroendocrine tumors.However,a substantial number of sunitinib-treated patients develop cardiac dysfunction and the mechanism remains poorly understood,which lead to no effective strategy to reverse the cardiotoxicity and limite its long-term and safe clinical application.We found that autophagy played a critical role in sunitinib-induced cardiotoxicity,but how it affects the heart and what is the key protein target are supposed to further study.It is well-known that over-activated autophagy in cardiomyoeytes could cause structure damage or dysfunction and even lead to cells death by clearing some necessary proteins.We found that CTGF was largely accumulated in autophagic lysosome after sunitinib treatment through MS assay.In further study,we will confirm the effects with autophagic degradation of CTGF on sunitinib-induced cardiotoxicity and whether Tollip regulates the physiological process as an autophagical adaptor protein.We are aimed to clarify the underlying molecular mechanism of sunitinib-induced cardiotoxicity and contribute to find the potential strategy to reverse this side effect.Methods:1.Effects of autophagy in sunitinib-induced cardiotoxicity.(1)Transmission electron microscope and western blot were applied to detect the autophagy in heart after sunitinib treatment;(2)Transgenic mouse model with cardiomyocyte-specific deletion of Atg7 was established through cre-loxp system.Echcardiography,HE staining and myocardial enzyme assaying were applied to confirm whether sunitinib-induced cardiotoxicity was stimulated by autophagy.2.Effects of down-regulated CTGF in sunitinib-induced cardiotoxicity.(1)MS assessment was used to detect the critical proteins accumulated in autophagic lysosome;(2)Western blot was applied to analyse the CTGF protein level in cardiomyocytes or mouse heart after sunitinib treatment;(3)Silence CTGF by siRNA and then survival rate of cardiomyocytes was detected by SRB;(4)Mouse model with cardiomyocyte-specific over-expression of CTGF was established by AAV9.Echcardiography,HE staining and myocardial enzyme assaying were applied to confirm if over-expressed CTGF in cardiomyocytes could block sunitinib-induced cardiotoxicity.3.Effects of autophagy in down-regulated CTGF induced by sunitinib.(1)The mRNA level of CTGF was detected using Real-Time PCR;(2)Cardiomyocytes were exposed to sunitinib in the presence or absence of CHX and the synthetic protein level of CTGF were measured by western blot analysis;(3)Cardiomyocytes were exposed to sunitinib in the presence or absence of MG 132 or CQ and western blot was used to confirm which protein degradation pathway was corresponding to CTGF;(4)Knock-out of Atg5 or Atg7 in MEF cells or cardiomyocytes and detected the level of CTGF by western blot.4.Effects of autophagic adaptor protein Tollip in regulating autophagic degradation of CTGF induced by sunitinib.(1)Western blot was used to detect the protein level of Tollip;(2)Silence Tollip by siRNA and then CTGF was detected using western blot;(3)Lipofectamine transfection of Flag-CTGF and HA-Tollip plasmids were applied to overexpress CTGF and Tollip in COS7.And Immunofluorescence assay was used to inspect co-location of CTGF and Tollip.Immunoprecipitation assay was used to clarify the interaction between CTGF and Tollip.Results:1.Cardiotoxicity of sunitinib was induced by autophagy.(1)We demonstrated that sunitinib apparently activated autophagy in cardiomyocytes;(2)Cardiomyocyte-specific deletion of Atg7 could block autophagy and reverse sunitinib-induced cardiotoxicity.2.Down-regulation of CTGF stimulated sunitinib-induced cardiotoxicity.(1)We found that CTGF was largely accumulated in autophagic lysosome and down-regulated after sunitinib treatment;(2)Silence of CTGF could reduce the survival rate of cardiomyocytes;(3)Cardiomyocyte-specific over-expression of CTGF could reverse sunitinib-induced cardiotoxicity.3.Sunitinib activated autophagic degradation of CTGF.(1)We confirmed that sunitinib had little influence on the mRNA or protein synthesis level of CTGF;(2)The degradation of CTGF was not occurred through ubiquitin-proteosome pathway;(3)Inhibition of autophagy could block the down-regulation of CTGF induced by sunitinib.3.Tollip regulated autophagic degradation of CTGF induced by sunitinib as an autophagic adaptor.(1)We clarified that sunitinib could up-regulate Tollip;(2)Silence of Tollip inhibited down-regulation of CTGF stimulated by sunitinib;(3)Sunitinib promoted the co-location and interaction between CTGF and Tollip.Conclusions:1.Autophagy stimulated by sunitinib caused the cardiotoxicity.2.The autophagic degradation of CTGF played a key role in sunitinib-induced cardiotoxicity.3.Tollip regulated the autophagic degradation of CTGF induced by sunitinib as an autophagic adaptor.