The Effects Of HuR Shuttle Impairment In Impaired Autophagy Flux In Hypoxic Cardiomyocytes And Its Mechanism

Background: Under acute tableland hypoxia,severe burns,traumatic shock,etc.,autophagosomes accumulate due to hypoxia,thus amplifying myocardial damage.Acute hypoxia plays a central role in autophagy flux impairment.Autophagy is a key process in maintaining cellular metabolic balance under physiological and pathological conditions.In autophagy,lysosomes need to maintain an acidic intracavitary environment(p H 4.5-5.0)to ensure the normal functions of acid hydrolases.Lysosomal acidification is critical to the degradation of autophagosomes and the maintenance of autophagy.However,when cardiomyocytes suffer from acute hypoxic injury due to various environments,lysosomal acidification is impaired,and the intracavitary acidification is weakened,which hinders the autophagy process,resulting in the accumulation of autophagosomes which cannot be degraded.The lysosomal acidification disorder becomes an important cause of autophagy flux impairment.Therefore,studying the mechanism and the regulatory factors of lysosomal acidification will provide a therapeutic basis for improving lysosomal acidification of hypoxic myocardium.Human antigen R(Hu-Antigen R,HuR)is an RNA binding protein that has been deeply and extensively studied.In normal states,HuR is mainly located in the nucleus and inactive.In certain pathological environments,HuR will bind a variety of m RNAs in the nucleus and "escort" m RNA to the cytoplasm,during which HuR maintains the stability of the m RNA.In previous studies,the enrichment of HuR in the cytoplasm is used as the criterion for the activation of its function.Our previous study found that a variety of autophagy activation states were accompanied by HuR enrichment in cytoplasm,suggesting that HuR may be involved in autophagy.Studies have shown that HuR can maintain the stability of m RNA of a partial unit of vacuolar H+ ATPase(v-ATPase).V-ATPase,which drives the cytoplasmic H+ into lysosomes by utilizing the energy generated by hydrolysis of ATP,is a key molecule for the acidification and degrading function of lysosomes.However,the relationship between HuR and lysosomal acidification has rarely been reported.We hypothesize that HuR is involved in maintaining lysosomal acidification during cardiomyocyte autophagy.In this study,we constructed the mice 30%TBSA third-degree scald model and a cardiomyocytes hypoxia model to study autophagy,lysosomal acidification and HuR changes.By artificially regulating HuR,we explored its role in lysosomal acidification during autophagy,and provided a theoretical basis for improving lysosomal acidification disorders in pathological environments such as acute hypoxia.Methods Adult C57BL/6 mice were used to establish the model of 30% TBSA third-degree scald.The autophagy level and HuR nucleoplasm distribution were detected by western blotting.And primary C57BL/6 neonatal mouse cardiomyocytes were used to establish the hypoxia model.The autophagy activation model was established using sugar-free medium,serving as positive control.HuR levels as well as changes of lysosomal acidification were observed.The WB assay was used to detect the changes of HuR protein in whole cell total protein,cytoplasmic protein and nuclear protein of cardiomyocytes.At the same time,the protein distribution of HuR was detected by immunofluorescence staining.After that,cells were treated with HuR-small interfering RNA(HuR-si RNA,200 n M)or Cyclosporin A(Cs A,5.0?g/m L)to change the expression of HuR.By detecting changes of lysosomal acidification level,cell viability as well as autophagy,a regulatory effect of HuR on lysosomal acidification during autophagy was determined.Furthermore,after silencing or activating HuR,the expression of ATP6V1E1 protein and m RNA was detected by RT-q PCR and WB.Results: In mice scald models,myocardial Hif-1? protein increased,suggesting hypoxic myocardium.Expressions of autophagy marker ATG5 were significantly increased.At the same time,the level of LC3 as well as p62 increased,suggesting the impairment of autophagy flux.More over,HuR shuttle cannot be identified.And the same results were viewed in hypoxia model.In hypoxia models,Cs A,the activator of HuR shuttle,improved the lysosomal acidification as well as cathepsin D levels,suggesting the positive roles of HuR in lysosomal acidification.On the contrary,HuR-si RNA was used to decrease HuR expression,and the level of lysosomal acidification and cathepsin D decreased,suggesting that HuR can regulate lysosomal acidification.Further more,HuR knockdown decreased the viability of cardiomyocytes,suggesting its positive role in maintaining cardiomyocytes viability.Further detection of the m RNA and protein levels of ATP6V1E1 revealed that HuR is involved in the regulation of the molecular stability of v-ATPase on lysosomes.Conclusions: After severe scald,the myocardial cells were injured by hypoxia,and the autophagy level was significantly increased.However,HuR shuttle was restricted,and lysosomal acidification was blocked,resulting in inhibition of autophagosomes degradation.Upregulation of HuR shuttle by activator can significantly improve the level of lysosomal acidification.It was found that HuR can stabilize the molecular stability of ATP6V1E1 m RNAs,thereby maintaining the protein levels of ATP6V1E1.These results suggest that HuR is involved in sustained expression of v-ATPase after autophagy activation,which further promotes myocardial autophagy and lysosomal acidification.Improving the lysosomal acidification by regulating HuR in patients suffering from burn may become a new target for the treatment of myocardial autophagy flux injury.