Conditional Down-regulation Of HSP60 In Heart Ameliorates Septic Cardiomyopathy Via Complete Induction Of Autophagy

Research background:Sepsis is a systemic inflammatory response syndrome which is caused by infection and the release of a variety of pro-inflammatory factors can lead to the failure of multiple organs.Cardiac dysfunction,characterized by decreased left ventricular fraction shortening and ejection fraction,is a common complication of sepsis and also a major cause of high mortality and high medical cost for sepsis.Strengthening myocardial protection is an important means to resisit the cardiac dysfunction caused by sepsis.However,there are few studies on how to improve the sepsis-induced myocardial injury.At present,researchers have detected the activation of autophagy in sepsis-induced cardiac dysfunction,however,autophagic flow is inhibited with the progress of sepsis.Recent studies have shown that complete induction of autophagy is essential for cardioprotection in sepsis.HSP60 is an important member of the heat shock protein family and has been shown to be closely related to the development of cardiovascular disease,autoimmune system diseases,etc.Under normal conditions,HSP60 is an important chaperone protein that is mainly distributed in mitochondria,and it also is a key component for the mitochondrial matrix protein folding and repair system.However,the expression of HS60 is increased significantly or transferred from the cytoplasm to the cell membrane and released outside the cell in the stress state.Wheeler DS compared the serum from 63 children with sepsis,10 non-sepsis,and 24 healthy children and found that serum HSP60 levels in children with sepsis were much higher than those in normal healthy children and non-sepsis patients.That means HSP60 may play an important role in the development of sepsis.Our previous study also found that the expression of HSP60 in myocardium was significantly increased after sepsis.The further researches need to elucidate the role of HSP60 in sepsis-induced cardiac dysfunction and whether the intervention against HSP60 could play a protective role in cardiac functionObjective:The purpose of this study was to elucidate the role of HSP60 in sepsis-induced cardiac dysfunction and whether the intervention against HSP60 could play a protective role in cardiac function.Methods:Both the primary isolated SD neonatal rat cardiomyocytes,C57/BL6 male mice and conditional down-regulation HSP60 mice were used as subjects.Western Blot,ELISA,adenovirus interference.PCR,qPCR and small animal ultrasound were used to study the effect of HSP60 on the sepsis-induced cardiac dysfunction.Results:1.The expression of HSP60 and autophagy markers LC3,p62 during sepsisIn vitro,neonatal rat cardiomyocytes were stimulated by LPS at different time points,the expression of HSP60 increased gradually with the prolonging of stimulating time(F=23.090,P<0.05).LC3 peaked at 6 h and then began to decline,but the protein expression level was still higher than the baseline level(F=35.000,P<0.05).p62 began to accumulate in 6h(F=3.830,P<0.05).In vivo,adult male mice were administered by cecal ligation and puncture(CLP)at different time points,the expression of HSP60 increased gradually with the prolongation of sepsis development time(F=5.501,P<0.05).LC3 began to increase at 6 h after CLP and continued to increase to 24 h after CLP(F=28.027,P<0.05),which is the longest observation time point.p62 began to increase at 18 h and continued until 24 h after CLP(F=71.563,P<0.05).Compared with the basal level,the content of HSP60 in the cells culture medium and mice serum all increased during sepsis(F=17.531,P<0.05 and F=53.724,P<0.05,respectively).2.HSP60-/+mice were established by intraperitoneal injecting tamoxifen to Cre(+)HSP60(+)mice,the expression of HSP60 in the heart was different between the HSP60+/+mice and HSP60-/+mice(F=49.606,P<0.05),and the expression of HSP60 in liver had no statistics significance(F=3.378,P>0.05).3.Effects of conditional down-regulation HSP60 in heart on cardiac functionAt basal level,there was no difference of cardiac function between HSP60+/+and HSP60-/+mice(P>0.05).Cardiac function of HSP60+/+ mice was decreased 12 h after CLP compared with 0 h(P<0.05),but the cardiac function of HSP60-/+mice declined until 24 h after CLP(P<0.05).4.The expression of HSP60 and autophagy markers LC3 and p62 in HSP60+/+mice were significantly increased at 36 h after sepsis(P<0.05);however,there was no significant change in HSP60 and p62,and LC3 was significantly increased at 36 h after sepsis in HSP60-/+ group(P<0.05).No significant accumulation of p62 in HSP60-/+group indicating that conditional down-regulation HSP60 in heart could promote the formation of a complete autophagic flow.Conclusions:1.Sepsis mediated the increase of HSP60 and autophagy.2.The incomplete activation of autophagy was detected during sepsis3.Conditional down-regulation of HSP60 in heart ameliorates septic cardiomyopathy via complete induction of autophagy.