Autophagic Flux And Its Association With P62 Mediated Degradation Of Ubiquitinated Proteins Involve In Exercise Preconditioning-induced Cardioprotection

Objective:Exercise preconditioning has a strong myocardial protective effect and has received much attention.The mechanism of myocardial protective effect may be related to myocardial autophagy.Autophagy is a physiological process in which cells degrade and utilize their own cytoplasm,which plays an important role in maintaining the intracellular energy balance and protein homeostasis.Our group also conducted some studies on myocardial autophagy in early days.Interestingly,cardiac autophagy was up-regulated in both exhaustive exercise induced myocardial injury and EP-induced cardioprotection.As is well-known that autophagy is a double-edged sword.Excessive autophagy and impaired clearance of autophagosomes are important factors that aggravate cell injury or even apoptosis.What's the role does autophagy play in exhaustive exercise and exercise preconditioning are still under investigation.Accordingly,in this dissertation,we will reveal the true face of autophagy in exhaustive exercise-induced myocardial injury and EP-induced cardioprotection through the investigation of autophagic flux in myocardium.In addition,endoplasmic reticulum stress induces the accumulation of ubiquitination during exhausted exercise,which might aggravate myocardial injury.p62 promotes proteins aggregation and deliver them for autophagic degradation via both LC3-interacting region?LIR?and PB1 domains.However,studies on the degradation of ubiquitination by selective autophagy through the mediation of p62 during exercise have not been reported.In this study,the interaction of p62 and LC 3 was studied to explore weather ubiquitination could be degraded by selective autophagy through activation of p62 in EP-induced cardioprotective effects against exhaustive exercise.Methods:120 male Sprague-Dawley rats?SPF grade?were randomly divided into 6 groups,namely,C group,control group;EE group,exhausted exercise group,the rats performed a high-intensity treadmill exercise until exhaustion with a speed of 25³0m/min,slope is 0°;EEP group,early exercise preconditioning group,rats undergoing high-intensity intermittent treadmill exercise for 80 minutes,which cantains 4 period of exercise?30 m/min?and rest each last for 10 minutes,to established EP model;the LEP group,the late exercise preconditioning group,the exercise program were the same as those in the EEP group;the EEP+EE group,the early exercise preconditioning+exhaustive exercise group,the rats established an EP model followed by exhaustive exercise after 30 min;LEP+EE group,late exercise preconditioning+exhaustive exercise group,the rats establish EP model followed by,exhaustive exercise after 24 h.Rats in group C were sacrificed after being placed on the treadmill for 3 hours.LEP rats were sacrificed after the EP model was established for 24 hours,the other rats were sacrificed after 30 minutes of exercise.To evaluate EP-induced cardioprotective effects,plasma cTnI,H-FABP and NT-proBNP were measured by chemiluminescence immunoassay and enzyme-linked immunosorbent assay?ELISA?.The level of myocardial ischemia and hypoxia was evaluated by HBFP staining.Transmission electron microscopy was used to observe the ultrastructural changes of myocardial cells.Using TUNEL staining to analysis myocardial apoptosis and the levels of apoptosis-related proteins Bax and Bcl-2 were detected by and Western blot.The expression of BNP in myocardium was detected by immunoblotting and immunohistochemistry.To evaluate the autophagic flux in myocardium,the expression levels of Beclin 1,Bcl-2,LC3,LAMP 2 and Cathepsin D in myocardium were detected by immunoblotting and immunofluorescence.Beclin1 and Bcl-2 were detected by immunofluorescence double labeling to evaluate autophagy initiation.The level of co-localization of-2 and LC 3 with LAMP 2 was used to analyze the level of autolysosome in myocardium.The expression of myocardial ubiquitylated protein and p62 was detected by immunoblotting and immunofluorescence staining.The level of co-localization of ubiquitinated proteins with p62 and p62 with LC 3 were examined using immunofluorescence double labeling to evaluate the effect of p62 on ubiquitinated proteins and the level of p62-mediated selective autophagy of ubiquitinated proteins.All measures were expressed as mean±standard error.One-way ANOVA was used for comparison between groups.The difference was considered statistically significant at p<0.05.The changes and mechanisms of autophagic flux and p62-mediated selective autophagy of ubiquitinated proteins in the protection of exercise preconditioning were analyzed via the alteration of the above indicators.Results1^st part:Myocardial damage and protection results.1)Exhausted exercise significantly increased plasma cTnI and H-FABP levels and myocardial ischemia and hypoxia,while early and late exercise preconditioning significantly reduced the up-regulation of plasma cTnI and H-FABP levels caused by exhaustive exercise.The degree of ischemia and hypoxia.2)Myofibers and mitochondrial damage were observed in the EE group,while early and late exercise preconditioning improved the ultrastructural damage of the myocardium induced by exhaustive exercise.Myocardial tissue apoptosis results.3)In the EE,EEP+EE,and LEP+EE groups,positive TUNEL-positive nuclei were found in the interstitial cells of the myocardium,while positive TUNEL staining was not observed in the cardiomyocytes.In addition,in these three groups,apoptotic protein Bax increased significantly,but there was no significant change in the ratio of Bcl-2 and Bax to Bcl-2.4)Exhausted exercise significantly increased plasma NT-proBNP levels,while early and late exercise preconditioning significantly reduced the up-regulation of plasma NT-proBNP levels caused by exhausted exercise.In addition,the expression of BNP increased significantly in EE,EEP+EE,and LEP+EE groups.2^nd part:Alterations of autophagic flux in myocardial.1)In the EE,EEP+EE and LEP+EE groups,the expression of Beclin 1 in myocardium was significantly increased,but the ratio of Bcl-2 and Beclin 1 to Bcl-2 was not significantly changed.2)The co-localization levels of Beclin 1 and Bcl-2 in myocardium were significantly decreased in EEP,LEP,EE,EEP+EE,and LEP+EE groups.Compared with EE group,the co-localization levels of Beclin 1 and Bcl-2 was increased in EEP+EE and LEP+EE groups 3)In the EE,EEP+EE,and LEP+EE groups,the ratio of LC3 II to LC3 II and LC3 I was significantly increased,but there was no significant change in the expression of LC3 I.4)In the EEP+EE and LEP+EE groups,the expression of LAMP 2 in the myocardium was significantly increased,whereas it was decreased in the EE group.5)In the EEP+EE and LEP+EE groups,the co-localization of cardiac myocardium LC 3 and LAMP 2 was significantly increased,while it was decreased in the EE group.6)In the EE,EEP+EE,and LEP+EE groups,the expression of cardiomyocytes Cathepsin D in the immature and intermediate types was significantly increased,while no significant changes were observed in the mature type.3^rd part:Changes of p62 mediates in ubiquitinated protein selective autophagy in myocardium.1)The expression of ubiquitinated proteins in the myocardium of EE and EEP+EE groups was significantly increased;compared with EE group,the ubiquitinated proteins in the LEP+EE group had a tendency to increase.2)The expression of p62 in EE and EEP+EE myocardium was significantly increased.Compared with EE group,the expression of p62 in EEP+EE group had a decreasing trend,while the expression of p62 in LEP+EE group was significantly decreased.3)EE,EEP+EE and LEP+EE groups showed a significant increase in the co-localization level of ubiquitinated protein and p62 in myocardium;compared with EE group,the co-localization levels of ubiquitinated protein and p62 in EEP+EE and LEP+EE groups was significantly decreased.4)The co-localization of p62 and LC3 in myocardium in EE and EEP+EE groups was significantly increased.Compared with EE group,the colocalization level of p62 and LC3 in EEP+EE group had a decreasing trend,while that in LEP+EE group was decreased significantly.ConclusionExhaustive exercise can cause myocardial ischemia,hypoxia and injury,but it does not cause cardiomyocyte apoptosis.Early and late EP can reduce myocardial damage caused by exhausted exercise and lead to compensatory increase of myocardial BNP levels,but failed to prevent myocardial interstitial cell from apoptosis caused by exhaustive exercise.Restriction of autophagic flux might lead to the accumulation of p62 and its ubiquitinated proteins aggregates,which might aggravate the exhaustive exercise-induced myocardial injury.Restoration of autophagic flux was observed in early and late exercise preconditioning-induced myocardial protection,which might promote the clearance of p62-mediated degradation of ubiquitinated proteins and bring out cardioprotective effects.