Autophagic Flux And Myocardial Damage Is Mediated By Changed Lysosomal V-ATPase Activity Following Burns Under Plain And Plateau Environment

Background and PurposeBurn shock caused by severe burns and hemorrhagic shock caused by severe trauma can lead to severe myocardial hypoxia damage.A large number of clinical practice showed that,compared with the plains,the hypoxia damage of heart and other important organs caused by burn shock and traumatic hemorrhagic shock was significantly increased in plateau hypoxia environment.As a circulatory organ,severe heart damage will further reduce the body oxygen supply,aggravate the injury of other tissues and organs,increase the mortality and overall mortality in severe burn and traumatic shock[1,2].Therefore,to explore the mechanism of hypoxic organ damage,especially heart injury,under the conditions of plateau hypoxia is of great significance.The mechanism and regulation of hypoxic myocardial injury under plateau hypoxia condition is not clear.Previous studies have found that hypoxia and other stress can damage the process of autophagy(autophagic flux),and further promote the amplification of apoptosis and necrosis and other pathological damage[3].Under physiological conditions,the unblocked autophagic flux can provide energy for the tissue and help the cells detoxify,to ensure the smooth completion of life activities such as metabolism of cell.Thus,under stress or hypoxia stress conditions,a complete autophagy process is essential for cells to maintain normal physiological function.Many pathological factors can cause cell autophagic damage,and thus lead to cell death and dysfunction[4,5].Previous studies have found that burns in plain anoxic enviroment can cause increase of myocardial autophagosomes,which is an important factor in myocardial injury and dysfunction.However,the critical role of hypoxic myocardial autophagy damage in the plateau environment has not been reported.In this study,we will focus on hypoxic myocardial autophagic damage in the hypoxic environment in plain and the plateau.We speculated the decrease of lysosomal V-ATPase activity caused by severe burn is the key molecular mechanism of hypoxic myocardial autophagic flux and myocardial damage.To confirm this hypothesis,we established a severe burn model in plain and simulated plateau hypoxic environment.Myocardial lysosomal V-ATPase,autophagic flux and indices of myocardium were determined to demonstrate speculation in order to provide theoretical basis for the prevention and treatment of hypoxic myocardial damage in the hypoxic environment.Materials and Methods1.Establishment of severe burn Model in plain normoxic and plateau hypoxic environment.SD rats were used for this study.Ten rats were treated with 88.8 ? water bath for 35 s to simulate a 40%TBSA scald in plain normoxic environment.After pretreatment in 5000 m plateau for 12 h,ten rats were inflicted with 40% TBSA of third degree burn on the back with 88.8 ? water bath for 35 s under 3000 m plateau.Then the rats were transferred to 5000 m plateau for 24 h,and then samples were collected for pathomorphological observations.2.Assay of myocardial damage,myocardial autophagic flux and lysosomal V-ATPase activityThe serum myocardial enayme spectrum and myocardic troponin T(cTnT)were tested by rate method and electrochemiluminescence immunoassay.The expression of myocardium autophagy-related proteins LC3 and P62 was determined by WB.Lysosomal V-ATPase activity was measured by enzyme coupling assay.3.Measurement of V-ATPase,autophagic flux and myocardial damage after intervention of V-ATPase activityAfter using V-ATPase activator(ATP)and inhibitor(baflomycin),the myocardial enzyme spectrum and cTnT were tested by rate method and electrochemiluminescence immunoassay.The expression of myocardium autophagy-related proteins LC3 and P62 were determined by WB.Enzyme coupling assay was used to detect lysosomal V-ATPase activity.4.Measurement of myocardial damage and autophagic flux in rats in plateau hypoxia environment.After the burned rats were observed in the plateau 5000 m environment for 24 h,blood was collected and the serum separated.The serum myocardial enayme spectrum and myocardic troponin T(cTnT)were tested by rate method and electrochemiluminescence immunoassay.Protein from myocardium was extracted to detect the expression of myocardium autophagy-related proteins LC3 and P62 by WB.Result1.Establishment of severe burn model in plain normoxic and plateau hypoxic environmentDisorganization of the scalded skin tissues were observed.The basement membrane was ruptured and loss of continuity.Edema of papillary layer of dermis,fragmentation of collagen fibers in reticular layer,destruction of the hair follicle,edema and necrosis of subcutaneous tissue,and fragmentation of the muscle fibers were found,indicating that a typical fullthickness scald model was established successfully.2.Changes of myocardial damage,autophagic flux and V-ATPase activity after severe burn in normal oxygen environmentLevels of cTnT,AST,LDH,?-HBDH,CK,CK-MB in the burn group(4.3±0.38 ?g/L,1112.0±51.6 IU/L?2174.0±201.3 IU/L,833.1±52.8 IU/L,1304.0±184.3 IU/L,1004.0±54.0 IU/L)were significantly increased as compared with those of normal control group(2.2±0.4 ?g/L,100.7±5.1 IU/L,484.3±76.5 IU/L,183.9±24.3 IU/L,340.5±41.9 IU/L,485.9±45.6 IU/L)(P values were less than 0.05).The expression of LC3 and P62 in myocardium of burned rats was significantly increased as compared with the normal control group.The activity of V-ATPase in the burned rats was 2.44 ± 0.33 ?mol /protein mg /h,which was significantly lower than the normal control group(6.12 ± 0.22 ?mol / mg protein /h),(P <0.05).3.The changes of V-ATPase activity,autophagic flux and myocardial damage after regulating the V-ATPase activity in burn rats under plain normoxic environmentThe activity of V-ATPase in the normal control group was 4.15 ± 0.47 ?mol / mg protein /h,and the activity of V-ATPase decreased to 1.93±0.56 ?mol / mg protein /h after treatment with V-ATPase activity inhibitor(baflomycin)(P <0.05).The activity of V-ATPase in burn rats treated with ATP was increased significantly(P <0.05).The autophagic flux was impaired in the rats of normal control treated with baflomycin,and the degree of autophagic flux damage in burn rats treated with ATP was alleviated as compared with the rats in the burn control group.Levels of cTnT,AST,LDH,?-HBDH,CK and CK-MB in normal control rats treated with bafilomycin were significantly higher than those in the non-treated control group(P values were less than 0.05).However,the levels of cTnT,AST,LDH,?-HBDH,CK and CK-MB in burn rats treated with ATP were significantly lower than those in the non-treated burn group(P values were less than 0.05).4.Effects of severe burns on myocardial damage and myocardial autophagy under plateau hypoxic environmentThe serum levels of cTnT,AST,LDH,?-HBDH,CK and CK-MB in the plateau control group were were significantly higher than those in plain normal control.The serum levels of cTnT,AST,LDH,?-HBDH,CK and CK-MB in the plateau burn group were also significantly higher than those in plain burn rats,indicating more severe myocardial damage.The expression of autophagy-related proteins LC3 and P62 in the normal plain control,the plateau control,the plain burn and the plateau burn groups increased successsively,indicating that the damage of myocardial autophagic flux and the myocardium were more serious on rat in plateau burns than that in plain burn.Conclusion1.We successfully established a full-thickness scald model in rats under simulated 5000 m plateau environment.2.Obvious damage of myocardium and autophagy disorders were found in rats 24 h following severe burns in plain normoxic environment.The autophagy disorders caused by inhibition of the myocardial lysosomal V-ATPase activity after burns may be one of the important mechanisms of myocardial damage.3.The rats underwent myocardial damage and autophagic flux damage both in the plateau hypoxic and in plain normoxic environment.However,myocardial and autophagic flux damage were more serious in the plateau burn than that in plain burn.