Investigation Of The Role Of ECSIT And Its Localization On Mitochondria In Myocardial Ischemia/reperfusion Injury

BackgroundExcessive activation of the innate immune contributes to the development of myocardial ischemia/reperfusion injury;and Toll-like receptors mediated signaling pathway plays an important role in innate immune activation and inflammatory responses.ECSIT(an evolutionarily conserved signaling intermediate in Toll pathways)is an important intermediate in the Toll/IL-1 signal transduction pathway.Recent studies have found that,in macrophages cells,TLR1/2/4 induces mitochondria production of reactive oxygen species(ROS)via the interaction of ECSIT with mitochondrial respiratory chain complex.Furthermore,mitochondrial reactive oxygen species burst resulting mitochondrial dysfunction is another pathological mechanisms of myocardial ischemia/reperfusion injury.However,the link between mitochondrial function and immune response mediated by TLRs post myocardial ischemia/reperfusion injury is still unclear.Therefore,whether ECSIT relates to myocardial ischemia/reperfusion injury relevance,involves in the regulation of mitochondrial function and immune response post myocardial ischemia/reperfusion injury,and whether the role of ECSIT in myocardial ischemia/reperfusion injury is dependent on its localization on mitochondria have not been reported.Objective1.To observe the role of ECSIT in the development of myocardial I/R injury.2.To illuminate the role and molecular mechanism of mitochondrial localization of ECSIT in cardiomyocytes post-H/R injury.3.Clarify the localization of ECSIT on mitochondria involved in regulation of myocardial I/R injury.Method1.Animal modelsWe establish myocardial ischemia/reperfusion injury model by ligation left anterior descending coronary artery(LAD)for 45min and followed by 24hr reperfusion.To clarify the relevance between ECSIT and the development of myocardial I/R injury in mice,8-9 weeks C57/BL6 male mice were randomly divided into two groups,including sham and I/R.Monitor the change of ECSIT in total protein and mitochondrial protein and the interaction between ECSIT and NDUFAF1 post-myocardial I/R injury.To clarify the role of ECSIT in myocardial ischemia/reperfusion injury in mice,we injected adenovirus overexpression ECSIT,which mostly located on mitochondria,by intramyocardial injection one week before LAD ligation.8-9 weeks C57/BL6 male mice were randomly divided into four groups,including sham,I/R,I/R+AdvGFP and I/R+AdvECSIT.Monitor the role of overexpression ECSIT in cardiac function and cell apoptosis post-myocardial I/R injury.To clarify the role of ECSIT localization on mitochondria in myocardial ischemia/reperfusion injury in mice,we construct transgenic mice overexpressing ECSIT targeting on mitochondria.We add OTC-leader(N-terminal 32 amino acids of ornithine transcarbamylase)on N-terminal site of ECSIT to make sure that ECSIT is targeting mitochondria.8-9 weeks male transgenic mice or wild type mice were randomly divided into four groups,including WT Sham,WT I/R,TG Sham and TG I/R.Monitor the role of ECSIT localization on mitochondria in cardiac function and cell apoptosis post-myocardial I/R injury.2.Cell modelsMitochondrial dysfunction and myocytes apoptosis are major pathological changes in myocardial ischemia/reperfusion injury,we use primary cardiomyocytes cultured from 1-3 days SD neonatal rats and H9C2 cell line.To establish the hypoxia/reoxygenation model,we use sugar-free medium and put cells in hypoxic incubator for 1 hour hypoxia and then convent to normal medium and normoxic incubator for 4 hours.To clarify the relevance between ECSIT and hypoxia/reoxygenation(H/R)injury in myocytes,The myocytes divided into Control group and H1/R4 group.Monitor the change of ECSIT in total protein and mitochondrial protein and the interaction between ECSIT and NDUFAF1 after H/R injury.To clarify the role of ECSIT localization on mitochondrial in H/R injury in myocytes,we transfect cell with adenovirus overexpression ECSIT(AdvECSIT)or deletion mutant ECSIT(Adv△ECSIT,which is without N-terminal 48 amino acids)48 hours before H/R.The N-termlnal 48 amino acids of ECSIT is essential for ECSIT located on mitochondria,the deletion mutant of ECSIT cannot located on mitochondria.The myocytes divided into five group,including:Control,H1/R4,H1/R4+AdvGFP,H1/R4+AdvECSIT and H1/R4+Adv△ECSIT.Monitor the role of ECSIT localization on mitochondria in mitochondrial structure and function and cell apoptosis post H/R injury.3.Observation targetsTo evaluate myocardial infarct size via Evans Blue-TTC staining.To detect cardiac function by echocardiography.To detect myocardial mitochondrial structure via transmission electron microscope.To explore the molecular mechanisms of myocardial ischemia/reperfiusion injury,we use co-immunoprecipitation to observe the interaction between ECSIT and TRAF6、NDUFAF1.To evaluate the activation of apoptosis signaling pathway,we use Western blot to calculate the level of Cleaved-Caspase3.To estimate myocytes injury though MTT assay and PI,Hoechrst33342 double staining.To detect the production of reactive oxygen species in myocytes by DHE and Mito Sox staining.To evaluate myocytes mitochondrial structure,transmission electron microscope was performed.To detect the localization of the ECSIT that we constructed adenovirus overexpressed,we use immunofluorescence staining and Western blot.And we use mitochondrial oxygen consumption assay to detect mitochondrial function.We detect the level of apoptosis by Caspase 3/7 activity assay.To further evaluate the activation of apoptosis signaling pathway,we use Western blot to calculate the level of Caspase9 and Cleaved-Caspase3.Results1.The change of ECSIT on mitochondria was decreased after myocardial I/R1.After ligation LAD 45min following 24hr reperfusion,Evans Blue-TTC staining showed the ratio of ischemia area/risk area(IA/RA)was 48.6±3.25%;the echo data showed ejection fraction(EF%)and fractional shortening(FS%)were decreased by 36.94%and 43.12%after I/R injury,respectively.Furthermore,we observed that mitochondria were swelling and ultrastructural were disrupted by transmission electron microscope after myocardial I/R injury.These data imply that myocardial I/R injury increases the infract size and induces cardiac dysfunction and mitochondria ultrastructural disruption.2.Compared with sham group,after myocardial I/R injury,the expression of total ECSIT did not change in myocardium,but the expression of ECSIT in mitochondrial protein was significantly decreased.The proteinase K sensitivity experiment showed that the reduction of ECSIT in mitochondria was mostly on the inner membrane of mitochondria.In addition,compared to the sham group,myocardial I/R injury induced TRAF6 recruitment to the outer mitochondrial membrane,increased the interaction between TRAF6 and ECSIT and the ubiquitination of ECSIT,and decreased the interaction of ECSIT and NDUFAF1 which participates in assembling mitochondrial respiratory chain complex.These data suggest that the expression of ECSIT on mitochondria is related to myocardial I/R injury,and its mechanism may be associated with the interaction between ECSIT and TRAF6/NDUFAF1.3.Evans Blue-TTC staining showed intramyocardial injection adenovirus packaging ECSIT the ratio of ischemia area/risk area(IA/RA)was increased by 38.9%(49.26±2.97%vs.30.52±1.31%)compared with I/R group.Compared to I/R group,the echo data showed ejection fraction(EF%)and fractional shortening(FS%)were increased by 21.8%(43.49±2.29%vs.52.98±2.14%)and 39.0%(19.95±1.56%vs.27.73±0.94%)while infected with AdvECSIT,respectively.Intramyoeardial inj ection adenovirus packaging ECSIT persevered eardiac injury and suppressed apoptosis pathway activation indueed by I/R injury,showing ECSIT play an role in the development of myocardial I/R injury.4.Compared to I/R group,intramyocardial injection adenovirus packaging ECSIT enhanced the interaction of ECSIT and NDUFAF1 induced by I/R injury,showing ECSIT may though located on mitochondria play an role in the development of myocardial I/R injury.5.We constructed mitochondria targeting overexpression ECSIT(1ECSIT)transgenic mice to detect the role of mitochondria localization ECSIT in myocardial I/R injury in vivo.The mECSIT vector plasmid entirely located on mitochondria by immunofluorescence staining.Compared to wild type(WT)mice,TTC staining showed in transgenic(TG)mice group,the ratio of ischemia area/risk area(IA/RA)post I/R injury was reduced by 40.5%(49.87±2.17%vs.29.67±1.73%).Compared to WT I/R group,the echo data showed TG mice post-I/R ejection fraction(EF%)and fractional shortening(FS%)were increased by 23.9%(39.63±2.49%vs.49.09±1.77%)and 29.7%(18.54±1.45%vs.24.04±1.05%),respectively.Ⅱ.Mitochondrial targeting overexpression of ECSIT protects cardiomyocytes from hypoxia/reoxygenation injury and its mechanism1.After H/R injury,the ultrastructural of mitochondria were disrupted and production of ROS in cardiac myocytes was increased.These data imply that H/R injury indueed mitochondria dysfunetion in cardiac myocytes.2.Compared with the control group,in HI/R1 group the expression of EC SIT in neither the total protein nor the mitochondrial protein was significant changed;but in H1/R4 group,the expression of ECSIT in mitochondria were signifieantly decreased which in total protein remained unchanged,and the interaction of ECSIT and NDUFAF1 was decreased,showing that the reduction of ECSIT on mitochondrial are related to H/R injury in cardiac myocytes.3.Transfection overexpression ECSIT adenovirus and adenovirus overexpression deletion mutant ECSIT,which deleted N-terminal 48 anuno acids of ECIST into myocytes.AdvECSIT significantly increased the expression of ECSIT in mitochondrial protein and cell immunology influences staining showed the overexpression ECSIT adenovirus mostly located on mitochondria.These data imply that the adenovirus we construct worked well and it is a mitochondrial targeting overexpression ECSIT adenovirus.4.Compared to H/R group,the myocytes were transfected with adenovirus mitochondrial targeting overexpression ECSIT preserved mitochondria structure and mitochondrial dysfunction induced by H/R injury.Western blot data and Caspase3 activity assay showed that overexpression ECSIT inhibited myocytes apoptosis induced by H/R injury.These data imply that ECSIT plays a role in mitochondrial morphology and function,and cell apoptosis post H/R injury.5.Without N-terminal 48 amino acids,ECSIT cannot play a similar role as full-length ECSIT.ECSIT localization on mitochondria play an improtant role in cell death and excessive production of reactive oxygen species induced by H/R injury.These data indicate that ECSIT protection cardiac myocytes from H/R injury is dependent on its mitochondrial localization.6.Overexpression ECSIT by adenovirus in myocytes inhibited the reduced interaction between ECSIT and NDUFAF1 induced by H/R injury.These data imply that the mechanism of overexpression ECSIT protects myocytes from H/R injury may relate to its mitochondrial localization and interaction with NDUFAF1.However,compared to the H/R+AdvECS IT group,the deletion mutant EC SIT cannot increased the interaction between NDUFAF1 and ECSIT on mitochondria,showing ECSIT protection H/R injury may rely on its interaction with NDUFAFI.ConclusionIntegrated the in vivo experiments and in vitro experiments,suggesting myocardial I/R signifieantly increased ubiquitination of ECSIT and decreased the level of ECSIT localized to the inner mitochondrial membrane in the myocardium.In addition,we found that myocardial I/R significantly caused a decrease in the interaction between ECSIT and complex I assembly chaperone NDUFAF1 in the mitochondrial of I/R myocardium.In contrast,increased expression of ECSIT ameliorated hypoxia/reoxygenation(H/R)-induced cardiomyocytes injury.Collectively,our data demonstrated that ECSIT is required for correct complex I assembly and/or stability and for mitochondrial function.These studies will generate novel data for understanding the signaling molecular sensor that is critical for myocardial I/R injury.It may be possible to apply this find in a practical fashion to identify new and novel therapeutic approaches to prevent or manage myocardial I/R inJury.