| Background and objectiveAcute myocardial infarction(MI)is the main cause of death in patients with coronary heart disease.Timely reperfusion to restore coronary blood flow is the most effective way to revive surviving ischemic cardiomyocytes and reduce the mortality caused by MI.However,it also causes fatal cell injury,clled ischemia/reperfusion(I/R)injury.I/R injury attenuates the beneficial effect of reperfusion therapy in patients with MI.Limiting I/R injury improves the outcomes of patients with acute MI.Pharmacological postconditioning is easier to perform and has shown potential in both experimental and clinical setting,therefore has better prospect of clinical application.Impaired autophagic flux contributes to cardiomyocyte death in I/R injury.Restoring the impaired autophagic flux by using agents may be a promising strategy that alleviates myocardial I/R injury.In recent years,it is found that calreticulin(CRT)involved in regulating diverse and important biological processes.We also found that CRT is involved in cardioprotection of ischemia postconditioning.The present study aimed to investigate the effects of CRT postconditioning on the myocardial I/R injury in rat,and evaluate the effect of exogenous CRT postconditioning on impaired autophagic flux induced by hypoxia/reoxygenation(H/R)injury in caltured H9c2 cells.Methods1.A rat myocardial I/R injury model was established:The left anterior descending coronary artery was ligated resulting in myocardial ischemia for 40min,followed by reperfusion for 24h.CRT postconditioning was performed by an intraperitoneal injection(CRT 0.5 mg/kg)5 minutes before reperfusion.2.The cardiac function,serum myocardial injury markers,and myocardial infarction range were determined,and the morphological changes of myocardial tissue were observed.3.The H/R injury models of neonatal rat cardiomyocyte and H9c2 cell were established.CRT postconditioning was performed by adding 25pg/ml CRT to the medium at the onset of reoxygenation.4.The cell viability and lactate dehydrogenase(LDH)leakage of neonatal rat cardiomyocyte was measured.The cell death rate,LDH leakage,intracellular reactive oxygen species(ROS)and malondialdehyde(MDA)of H9c2 were assessed.5.Autophagic flux was monitored by mRFP-GFP-LC3 adenovirus infection.The number of autophagosomes and autolysosomes in cells were determined by counting the fluorescence dots.6.Western blot assay was used to determine the expression of autophagy-related proteins:Beclinl,LC3,LAMP-2 and p62.7.Statistical analysis:GraphPad Prism 6.0 software was used for statistical analysis.One way ANOVA was used to determine the differences between groups,and LSD method was used for comparison between groups.P<0.05 was considered statistically significant.Results1.CRT postconditioning improved the cardiac function and myocardial morphological structure,reduced the content of serum myocardial injury markers,and improved the infarct size.2.CRT postconditioning attenuated the decrease of cell viability and LDH leakage from the neonatal rat cardiomyocyte subjected to H/R.CRT postconditioning reduced the cell death rate,LDH leakage and the degree of oxidative stress in H9c2 cells subjected to H/R.3.CRT postconditioning reduced autophagosomes accumulation in H9c2 cells stujected to H/R.CRT postconditioning suppressed H/R-induced excessive formation of autophagosomes,as shown by a decrease of autophagosomes,the ratio of LC3-?/LC3-?,and expression of LC3-? and Beclinl.Meanwhile,it also improved H/R-induced impaired autophagosomes clearance,as shown by an increase of autolysosomes and LAMP-2 expression,and a decrease of p62 expression.Conclusion1.CRT postconditioning reduces myocardial ischemia/reperfusion injury in vivo.2.CRT postconditioning protected cultured cardiomyocytes in vitro against H/R injury.3.CRT postconditioning inhibited the excessive formation of autophagosomes,promoted the clearance of autophagosomes,and resorted the autophagic flux,consequently reduced the H/R injury in H9c2 cells. |
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