DJ-1 Participates In The Regulation Of Protective Autophagy In Hypoxic H9c2 Cardiomyocytes In A C106-dependent Manner

Objective:The study aims to determine whether DJ-1 participates in the regulation of protective autophagy in hypoxic H9c2 cardiomyocytes in a C106-dependent manner.Methods:1.The H9c2 cardiomyocytes were pretreated with or without 5 m M autophagy inhibitor 3-methyladenine(3-MA)for 12 h followed by hypoxia for 3 h.To observe the expression level of DJ-1 protein and the changes of autophagy in hypoxia-treated H9c2 cells,the following indexes were detected.(1)Western blot was used to detect the expression of DJ-1,p62,and LC3;(2)Transmission Electron Microscope was used to detect the number of autophagosomes;(3)The changes of autophagy flow traced by m RFP-GFP-LC3 were detected by laser confocal;(4)CCK-8 kit was used to detect the cell viability;(5)LDH Cytotoxicity Assay Kit was used to detect LDH release.2.The H9c2 and H9c2/sh DJ-1 cells were subjected to hypoxia for 3 h.To explore the effect of DJ-1 down-regulation on the protective autophagy in hypoxic H9c2 cardiomyocytes,the following indexes were detected.(1)Western blot was used to detect the expression of DJ-1,p62,and LC3;(2)Transmission Electron Microscope was used to detect the number of autophagosomes;(3)The changes of autophagy flow traced by m RFP-GFP-LC3 were detected by laser confocal;(4)CCK-8 kit was used to detect the cell viability;(5)LDH Cytotoxicity Assay Kit was used to detect LDH release.3.The H9c2 and H9c2/DJ-1 cells were pretreated with or without 5 m M 3-MA for 12 h followed by hypoxia for 3 h.To explore the effect of DJ-1 up-regulation on the protective autophagy in hypoxic H9c2 cardiomyocytes,the following indexes were detected.(1)Western blot was used to detect the expression of DJ-1,p62,and LC3;(2)Transmission Electron Microscope was used to detect the number of autophagosomes;(3)The changes of autophagy flow traced by m RFP-GFP-LC3 were detected by laser confocal;(4)CCK-8 kit was used to detect the cell viability;(5)LDH Cytotoxicity Assay Kit was used to detect LDH release.4.The H9c2/DJ-1 and H9c2/DJ-1(C106S)cells were subjected to hypoxia for 3h.To explore the effect of C106 mutation of DJ-1 protein on the protective autophagy in hypoxic H9c2 cardiomyocytes,the following indexes were detected.(1)Western blot was used to detect the expression of DJ-1,p62,and LC3;(2)Transmission Electron Microscope was used to detect the number of autophagosomes;(3)The changes of autophagy flow traced by m RFP-GFP-LC3 were detected by laser confocal;(4)CCK-8 kit was used to detect the cell viability;(5)LDH Cytotoxicity Assay Kit was used to detect LDH release.Results:1.Compared with the control group,the expression level of DJ-1 protein in hypoxic H9c2 cells was up-regulated,along with increased LC3II/LC3 I,decreased p62,increased autophagosome number,and increased autophagy flow and simultaneously the cell viability decreased and the LDH release increased.However,the expression level of DJ-1 protein had no significant change in hypoxic H9c2 cells pretreated with 3-MA,but the LC3II/LC3 I decreased,the expression level of p62 increased,the number of autophagosomes decreased and the autophagy flow decreased and simultaneously the cell viability further decreased and the LDH release further increased.These results suggested that hypoxia can up-regulate the expression of DJ-1 protein and induce protective autophagy in H9c2 cardiomyocytes,indicating that the protective autophagy induced by hypoxia in H9c2 cardiomyocytes may be related to the expression of DJ-1 protein.2.Compared with the hypoxic H9c2 cells,the expression level of DJ-1 protein in hypoxic H9c2/sh DJ-1 cells was down-regulated,along with decreased LC3II/LC3 I,increased p62,decreased autophagosome number and decreased autophagy flow and simultaneously the cell viability decreased and the LDH release increased.These results suggested that DJ-1 down-regulation could inhibit protective autophagy in hypoxic H9c2 cardiomyocytes and aggravate cell hypoxia injury.It was also reversely proved that DJ-1 protein mediated protective autophagy in hypoxic H9c2 cardiomyocytes.3.Compared with the hypoxic H9c2 cells,the expression level of DJ-1 protein in hypoxic H9c2/DJ-1 cells was up-regulated significantly,along with increased LC3II/LC3 I,decreased p62,increased autophagosome number and increased autophagy flow and simultaneously the cell viability decreased and the LDH release increased.Interestingly,the above effects induced by DJ-1 up-regulation were reversed by pretreatment with the autophagy inhibitor 3-MA.These results indicated that DJ-1 up-regulation promoted protective autophagy in hypoxic H9c2 cardiomyocytes and alleviated cell hypoxic injury.At the same time,it further positively proved that DJ-1 protein was involved in the regulation of protective autophagy in hypoxic H9c2 cardiomyocytes.4.Compared with the hypoxic H9c2/DJ-1 cells,the expression of DJ-1 protein had no significance in hypoxic H9c2/DJ-1(C106S)cells,however,the LC3II/LC3 I decreased,the expression level of p62 increased,the number of autophagosomes decreased and the autophagy flow decreased and simultaneously the cell viability further decreased and the LDH release further increased.These results indicated that the C106 mutation of DJ-1 could cancel DJ-1-mediated protective autophagy in hypoxic H9c2 cardiomyocytes,in other words,DJ-1 participates in the regulation of protective autophagy in hypoxic H9c2 cardiomyocytes in a C106-dependent manner.Conclusion:In H9c2 cardiomyocytes,hypoxia can induce protective cell autophagy;DJ-1participates in the regulation of protective autophagy induced by hypoxia in a C106-dependent manner.