Chromatin Remodeling Factor Brg1 Protects Doxorubicin-induced Cardiotoxicity By Improving Cardiac DNA Damage Repair

Doxorubicin has been widely used as clinical chemotherapeutic agent to cure cancer.The Doxorubicin-induced cardiotoxicity limites the the patients receiving maximum and uninterrupted treatments.People endevour to find out the mechanisms of the doxorubicin-induced cardiotoxicity,mechanisms like the DNA damage,genetic transcriptional regulation,mithchondrial dysfunction and ROS production have been proved invloving the initiation of the doxorubicin-induced cardiotoxiciy.However,the molecular mechanisms of the doxorubicin-induced cardiotoxicity still unclear.The chromatin remodeling complexes change the structure of the nuclearsome by its ATP-dependent translocases,leading to regulates the DNA replicates,transcription,DNA damage response and recombination.We use the intravanous Doxorubicin-induced mouse cardiotoxicity model to elucidate the molecular mechanisms of the Brgl regulates the Doxorubicin-induced cardiotoxicity.Part OneModel of Doxorubicin-induced cardiotoxicityObjective:To explore cardiac function,DNA double strand breaksand telomere function after doxorubicin administration.Doxorubicin administrated by tail-vein intravenous injection to create the doxorubicin-induced cardiotoxicity mouse model.Methods:8-10 weeks old mouse were used to intravenouse injecte doxorubicin,21 mouse were used,body weight between 22-24 grams.All mice separate into 4 groups:WT+NS,WT+Dox 5mg/kg,WT+Dox 10 mg/kg,WT+Dox 20mg/kg.Echocardiograph used to analysis the cardiac function,mitral valve flow was used to analysis the cardiac diastolic function,EF and FS were used analysis the cardiac systolic function,DHE staining was used to detect the creation of the ROS.7 mouse,body weight between 22-24 grams,were used to monitor the y-H2AX dynamic changes after doxorubicin administration.Mouse were seperated into 2 groups.WT+NS and WT+Dox 5mg/kg.Mouse were sacrificed 10 mins,20 mins,30 mins,60 mins,90 mins and 120 mins after doxorubicin administration.WB and IF were used to analysis the y-H2AX changes.7 mouse,body weight between 22-24 grams,were used to monitor the y-H2AX dynamic changes after doxorubicin administration.Mouse were seperated into 2 groups.WT+NS and WT+Dox 5mg/kg.Mouse were sacrificed 2 hours,4 hour,8 hours,16 hours,24 hours and 48 hours after doxorubicin administration.WB and IF were used to analysis the y-H2AX changes.Results:Our results found that E/A ratio inverted 2 hours after doxorubicin 20 mg/kg treatment,which indicating that the doxorubicin induced cardiac diastolic dyfunction.2 weeks after doxorubicin treatment,doxorubicin induced progressive cardiac systolic function decline.Doxorubicin chronic treatment induced cardiac dysfunction with doxorubicin accumulated administration.The DNA double strand break indicator,y-H2AX,shows the dynamic changes after acute doxorubicin treatment,the persitent y-H2AX foci formation after 2 weeks of doxorubicin treamtnet and at the chronic doxorubicin treatment.The persistent y-H2AX signal co-localize with telomere signal forms the telomere,which indicating telomere dysfunction induced foci formation.Cardiac necrosis was found after chronic doxorubicin treatment.Conclusion:Doxorubicin-induced cardiotoxicity mouse model can be successfully builted with tail-vein doxorubicin injection.Identified the progressive cardiac function decline.Telomere dysfunction and necrosis were found in cardiomyocytes after doxorubicin treatment.The dosage of 10 mg/kg i.v.and chronic multiple 5mg/kg doxorubicin treatment were fixed for further experimental.Part twoBrgl improve the doxorubicin-induced mouse cardiac toxicityObjective:To explore the impaction of Brg1 to doxorubicin-induced cardiotoxicity,gene editing technics was used to create the Brgl cardiac specific over expression mouse model and Brgl cardiac specific knock out mouse model.Observe the cardiac function,cardiomyocytes function,DNA damage repair ability and cardiomyocytes necrosis changes within differenct genotypic mouse model challenge with the doxorubicin.Methods:8-10 weeks old WT,Brg1 OE and Brg1 KO mice were used to induce cardiotoxicity mouse model.Cardiac function,DNA damage repair,cardiac necrosis were analysed at acute and chronic doxorubicin induced cardiotoxicity mouse model.Body weight and heart weight were recorded,hearts were fixed in paraformamide.IF was proceeded to analysis the y-H2AX foci,western blots was used to observe the dynamic changes and expression of y-H2AX in cardiomyocytes after doxorubicin treatment.EBD staining to observe the cardiac necosis.Results:Brgl attenuate the doxorubicin-induced cardiotoxicity by initiate y-H2AX induction facilitating the cardiac DNA damage repair.Brg1 protects heart from doxorubicin induced EF and FS decline.Brg1 protects heart from doxorubicin induced cardiac necosis and EF and FS decline.Brgl cardiac specific knock out shows the opposite effects.Conclusion:Brgl protects heart from doxorubicin induced cardiotoxicity by attenuating the cardiac dysfunction,cardiac necrosis,cardiac DNA damage.Part three Brgl attenuate the doxorubicin-induced cardiac telomere dysfunctionObjective:To explore the difference of cardiac telomere function between WT,Brgl OE and Brgl KO mice after doxorubicin treatment.Gene editing technics was used to create the Brgl OE and Brgl KO mouse model.Methods:Doxorubicin was administrated on 8-10 weeks old WT,Brg1 OE and Brg1 KO mice.Hearts were harvested,fixed by paraformamide and proceeded with Q-FISH.Results:TIFs were analysised.Brg1 decrease the TIFs numbers in Brg1 OE doxorubicin group.TIFs increase in Brgl KO doxorubicin group and the telomeric volume increased,at the same time,the telomere fusion also increased.Conclusion:Brgl protect heart from doxorubicin-induced cardioxtoxicity by attenuating the doxorubicin-induced cardiac telomere dysfunction.