Mechanism Of Parkin-Mediated Mitophagy Regulates DOX-Induced Cardiotoxicity

Objective Doxorubicin(DOX)is an effective anti-tumor drug,however its clinical usage is limited due to cardiotoxicity.Various doxorubicin-induced mechanisms of irreversible cardiotoxicity have been proposed,including reactive oxygen species(ROS)production,lipid peroxidation and mitochondrial dysfunction.Among these pathological factors,mitochondrial dysfunction is considered to be a key target for doxorubicin-induced cardiotoxicity.Parkin participates in the selective clearance of damaged mitochondria through mitophagy,which plays an important role in maintaining intracellular homeostasis and normal mitochondrial morphology.In addition,there is increasing evidence that Yes-associated protein(YAP)is associated with cardiac development,cell proliferation,and heart disease such as myocardial infarction,but whether it is associated with cardiotoxicity has not been reported,and the target protein of YAP is Parkin.Therefore,this study was designed to determine whether YAP/Parkin-mediated mitophagy regulates doxorubicin-induced cardiotoxicity and explores its underlying mechanisms.Method Six healthy 8 week old male mice and six healthy 8 week old Parkin transgenic male mice produced the model of cardiotoxicity in mice by DOX(model group),six healthy 8 week old male mice and six healthy 8 week old Parkin transgenic male mice were injected with saline as control group.Apoptosis was determined by TUNEL(terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick-end labeling);mitochondrial fission was detected by two-photon inverted microscope and electron microscopy;protein and RNA expression of Parkin and YAP were detected by immunoblotting and real-time quantitative PCR.Level;Chromatin immunoprecipitation(ChIP)assay confirmed that endogenous YAP interacted with Parkin;cardiac function and myocardial remodeling were assessed by small animal echocardiography and histological staining.Result In the present study,we found that Parkin RNA and protein levels were revealed to be downregulated in mouse hearts and in H9c2 cells upon DOX treatment.Enforced expression of Parkin led to mitophagy activation and attenuated cell apoptosis.In addition,Parkin transgenic mice exhibited DOX-induced mitochondrial fission andapoptosis.Furthermore,Yes-associatd protein(YAP),as a transcription co-activator,regulated the gene expression of Parkin,and in turn Parkin protected against cardiotoxicity induced by DOX treatment.Conclusion In the present study used a doxorubicin-induced cardiotoxicity model produced by cardiomyocyte H9c2 and Parkin transgenic mice,and the use of adenovirus or plasmid overexpression and knockdown of the target gene to detect protein levels,RNA levels,mitochondrial fission,and mitophagy.Chromatin immunoprecipitation was used to detect the interaction between YAP and Parkin.It was concluded that YAP-targeted Parkin regulates doxorubicin-induced cardiotoxicity by enhancing mitophagy and inhibiting apoptosis.Our results revealed a novel apoptotic pathway involving YAP-Parkin axis and provided a potential protective mechanism against DOX-induced cardiotoxicity during cancer therapy.The following conclusions were drawn:(1)DOX induces mitochondrial fission and mitophagy in cardiomyocytes.(2)Parkin suppresses DOX–induced mitochondrial fission and apoptosis by by enhancing mitophagy in cells.(3)Parkin represses DOX-induced cardiotoxicity in mice and DOX-induced cardiotoxicity promotes deleterious myocardial remodeling.(4)YAP as a transcription co-activator,regulated the gene expression of Parkin,and in turn Parkin protected against DOX-induced cardiotoxicity.