The Role Of Pellino1 SUMO Modification In The Regulation Of Mycardial Apoptosis In Ischemia-reperfusion Injury

Background Acute myocardial infarction(acute myocardial infarction,AMI)is one of the most dangerous type of coronary heart disease.It is thee most effective treatment strategy to restore coronary blood flow in time after mycardial infarction.However,mycardial ischemia-reperfusion(ischemia/reperfusion,I/R)injury is one of the main restricting factors that hinder its optimal efficacy.When myocardial I/Rdamages occurs,the myocardial tissue metabolic function is disturbed,resulting in serious cell damage and even apoptosis.There are many mechanisms involved in regulating apoptosis in the body.The study shows that natural immunity plays a key role in the apoptosis of I/R damaged myocardium.The TLR(Toll like receptor,TLR)is one of the most important modes of the natural immune response,and its mediated signaling pathway is involned in the development of apoptosis.Pellino1 is an important regulatory protein in the TLRs signaling pathway,and plays an important role in TLRs regulating intracelluar signal transduction mediated by TLRs.It is known that Pellino1 can undergo phosphorylation,ubiquitination and SUMO modification,and the post-translational modification of Pellino1 is closely related to its E3 ubiquitin ligase activity.However,what is the effect of Pellino1 SUMO modification on its function,and whether it is possible to participate in the development of cardiomyocyte apoptosis by regulating intracellular signal transduction mediated by TLRs is not clear.In this study,the effect of Pellino1 SUMO modification on myocardial cell apoptosis induced by I/R damage in myocardium was investigated through the inhibition of SUMO modification of the mutant Pellino1,and the mechanism of Pellino1 SUMO modification was discussed.Objective To clarify the effect of Pellino1 SUMO modification on myocardial I/R induced cardiomyocyte apoptosis,the mechanism was preliminarily clarified whether it was closely related to the intracellular signal transduction mediated by TLRs by Pellino1 SUMO modification.Methods 1.Animal models in vivo We establish myocardial ischemia/reperfusion injury model by ligation heart left anterior descending coronary artery(LAD)for 45 min and followed by 72 h reperfusion.To determine the correlation between Pellino1 SUMO modification and mycardial I/R injury,we selected the male adult C57/BL6 mice aged eight weeks randomly divided into: sham and I/R.In order to study the role of Pellino1 SUMO modification in cardiomyocyte apoptosis induced by mycardium I/R injury.We select eight weeks old male C57BL6 mice and injeced the SUMO modification locus mutation Pellino1 packed by adenovirus(mutant 5 SUMO modified locus of Pellino1)at the edge of the left auricle mycardial,to make its mycardial overexpressed Pellino1 cannot occur SUMO modification.Simultaneously injected control adenovirus GFP,After ten days,sham or I/R surgery were randomly subjected: sham group(sham),myocardial I/R group(I/R),myocardoal I/R+GFP adenovirus group(I/R + Adv-GFP)and mutant Pellino1 adenovirus group(I/R + Adv-Peli1-5M). 2.Cell models in vitro Primary neonatal rat cardiomyocytes(NRVMs)were cultured in vitro.NRVMs were isolated from hearts of 1-to3-day-old Sprague Dawley rat pups,To eatablished the hypoxia/reoxygenation(H/R)damage model:cells were transferred to a hypoxic conditioned incubator for 1h of hypoxia,then transferred to a normoxic incubator for 4h.To analyse the role of SUMOylation of Pellino1 in cardiomyocyte apoptosis induced by H/R injury,we transfect cell with adenovirus packed mutant Pellino1(Adv-Peli1-5M).The experiment was divded into four groups: control group(con),hyoxia/reoxygenation(H/R),transfected with GFP adenovirus and hyoxia/reoxygenation(Adv-GFP+H/R),transfected with Adv-Peli1-5M adenovirus and hyoxia/reoxygenation(Adv-Peli1-5M +H/R).The effect and mechanism of Pellino1 SUMO modification on cell function and cardiomyocyte apoptosis after H/R injury were investigated.Observation targets Western blot and immunoprecipitation test confirmed the correlation between Pellino1 SUMO modification and mycardial I/R injury,the relation between E3 enzyme activity of Pellino1 and mycardial I/R injury was also detected.To evaluate cardiac function via echocardiography,including the left ventricular systolic function,the index with ejection fraction(EF%)and shortened fraction(FS%);The size of mycardial infarction area was evaluated by Evans Blue-TTC double staining.TUNEL staining were used to assessed the apoptotic cell,Western blot was used to test the cleaved Caspase9,cleaved Caspase3,BCL-2 and BAX protein expression levels in the myocardial tissue cells and in vitro cultured myocardial cells.Results ?.Regulation of Pellino1 SUMO modification in Myocardial tissue on Myocardial Ischemia/Reperfusion Injury 1.After ligation LAD 45 min following 3d reperfusion,the expression level of Pellino1 protein in mycardial tissue was increased,whether the level of SUMOylation of Pellino1 was significantly reduced,indicating that myocardial I/R injury may be associated with SUMO modification of Pellino1.The activity of Pellino1 E3 ubiquitin ligase was drastically increased,which suggested that mycardial I/R injury may be related to the E3 ubiquitin ligase activity of Pellino1.2.The cardiac function of mice was decreased after operation,and Evans Blue-TTC double staining showed the apparent white infarction.Compared to injection control adenovirus GFP group,the infract area was significantly increased in which transfection of SUMO modified mutant Pellino1,the cardiac function showed that EF% and FS% were decreased significantly.Which prompt that mutation of Pellino1 SUMOytion can increase cardiac dysfunction in mycardiac I/R injury.3.Compared with the control group,the number of apoptotic cell in the mycardial tisssue showed by TUNEL staining was siginificantly increased in the local transfected mutant Pellino1 mice.It showed that inhibition of Pellino1 SUMO modification increased the apoptosis induced by mycardial I/R.4.After ligation LAD 45 min following 3d reperfusion,increase the expression of pro-apoptotic protein BAX,cleaved-Caspase9 and cleaved-Caspase3,and decrease the expression of pro-survival protein BCL-2,after transfection mutant Pellino1 inhibition of SUMO modification,further increase the above change,which further increase in promoting apoptosis proteins,reduce the pro-survival protein.5.Compared to the control adenovirus GFP group,intramyocardial injection adenvirus packaging mutant Pellino1 future increase the activity of E3 ubiquitin ligase of Pellino1,further to increase its downstream c-IAP2 ubiquitin modification.It suggested that inhibition of Pellino1 SUMO modification aggravates I/R damage in mice,which may be related to the enhancement of Pellino1 E3 ubiquitin ligase activity and the ubiquitination of c-IAP2.??.The molecular mechanism of Pellino1 SUMO modification regulating on H/R injury 1.Adv-Peli1-5M adenvirus was constructed and transfected to cardiomyocytes.The mutant Pellino1 could effectively reduce the Pellino1 SUMO modification.2.The number of apoptotic cell increased induced by H/R,while transfection Adv-Peli1-5M,obvious mutiply the apoptosis,the specific performance is as followers: increase the expression of pro-apoptotic protein BAX,cleaved-caspase9 and cleaved-caspase3,and decrease the expression of pro-survival proteins BCL-2,TUNEL staining show the same tendency.3.The activity of E3 ubiquitin ligase of Pellino1 increased after H/R,resulting in an increase in the ubiquitination of c-IAP2 and Lys48 ubiquitination of TRAF3,Inhibition of Pellino1 SUMO modification further enhance the activity of its E3 ubiquitin ligase,leading to promoting the degradation of TRAF3.4.When delivered the Adv-Peli1-5M to mutuant the SUMO conjugation sites in cardiomyocyte following H/R,which triggered the H/R-induced phosphorylation of p38,ERK1/2 and JNK,suggest accelerated the activation of MAPK signaling.Conclusion Above all,Pellino1 SUMO modification may participate in the myocardial apoptosis induced by myocardial I/R,the mechanism may be related to the reduce the SUMOylation of Pellino1,consequencely increase the activity of E3 ubiquitin ligase of Pellino1,resulting in activate the c-IAP2/TRAF3/MAPK signaling activation.The results of this paper can help to elucidate the pathophysiological mechanism of myocardial I/R injury and provide an novel view for clinical prevention of myocardial I/R injury.