DJ-1 Mediates Detrimental Autophagy Of H9C2 Cardiomyocytes Under Anoxia/reoxygenation In A C106 Oxidation-state-dependent Manner

Objective:Taking the C106 oxidative modification of DJ-1 protein as the entry point,this study discussed whether DJ-1 protein participates in the detrimental autophagy induced by anoxia/reoxygenation（A/R）in a C106 oxidation-state-dependent manner from the level of H9c2 cardiomyocytes.Methods:1.H9c2 cardiomyocytes were pretreated with 5 m M autophagy inhibitor 3-Methyladenine（3-MA）or 50μM autophagy agonist Rapamycin（Rapa）for 12 h,and the A/R injury model was then established by anoxia for 3 h and reoxygenation for 2 h.To verified whether A/R caused detrimental autophagy and C106 oxidation of DJ-1protein in H9c2 cardiomyocytes,the following indexes were detected:（1）Flow cytometry was applied to detect the change of reactive oxygen species（ROS）in cells;（2）Western Blot and immunoprecipitation（IP）were used to detect the expression level of oxidized-DJ-1（ox-DJ-1）,P62,and LC3 in cells;（3）CCK-8 was used to measure the cell viability and spectrophotometry was used to measure the release of intracellular lactate dehydrogenase（LDH）.2.H9c2 cardiomyocytes were pretreated with oxidant H₂O₂ of 100,200,and 400μM for 4 h respectively to promote the C106 oxidation of DJ-1 protein.To explore whether DJ-1 protein oxidized to ox-DJ-1 through C106 site could induce the detrimental autophagy of H9c2 cells,the following indexes were detected:（1）Flow cytometry was applied to detect the change of ROS in cells;（2）Western Blot and IP were used to detect the expression level of ox-DJ-1,P62,and LC3 in cells;（3）The changes of autophagy flow traced by m RFP-GFP-LC3 were detected by laser confocal;（4）CCK-8 was used to measure the cell viability and spectrophotometry was used to measure the intracellular LDH release.3.H9c2 cardiomyocytes were pretreated with 500μM antioxidant N-acetylcysteine（NAC）for 12 h to inhibit the C106 oxidation of DJ-1 protein,and the A/R injury model was then established.To further explore whether down-regulating the expression of ox-DJ-1 can inhibit the detrimental autophagy induced by A/R in H9c2 cells,the following indexes were detected:（1）Flow cytometry was applied to detect the change of ROS in cells;（2）Western Blot and IP were used to detect the expression level of ox-DJ-1,P62,and LC3 in cells;（3）The changes of intracellular autophagosomes were observed by transmission electron microscope;（4）The changes of autophagy flow traced by m RFP-GFP-LC3 were detected by laser confocal;（5）CCK-8 was used to measure the cell viability and spectrophotometry was used to measure the intracellular LDH release.4.The H9c2/DJ-1 and H9c2/DJ-1（C106S）cells were pretreated with 400μM H₂O₂ for 4 h,and the A/R injury model was then established.To observe whether the up-regulation of ox-DJ-1 pretreated by H₂O₂in H9c2/DJ-1 cells promoted A/R-induced detrimental autophagy,and whether the reduction of ox-DJ-1 caused by the C106mutation of DJ-1 protein in H9c2/DJ-1（C106S）cells inhibited the above effects,the following indexes were detected:（1）Flow cytometry was applied to detect the change of ROS in cells;（2）Western Blot and IP were used to detect the expression level of ox-DJ-1,P62,and LC3 in cells;（3）The changes of intracellular autophagosomes were observed by transmission electron microscope;（4）The changes of autophagy flow traced by m RFP-GFP-LC3 were detected by laser confocal;（5）CCK-8 was used to measure the cell viability and spectrophotometry was used to measure the intracellular LDH release.5.H9c2 cardiomyocytes were pretreated with 5μM specific C106 oxidation inhibitor Compoud-23（Comp-23）for 12 h,and the A/R injury model was then established.To further clarified the role of C106 oxidation of DJ-1 protein in A/R-induced detrimental autophagy,the following indexes were detected:（1）Flow cytometry was applied to detect the change of ROS in cells;（2）Western Blot and IP were used to detect the expression level of ox-DJ-1,P62,and LC3 in cells;（3）The changes of autophagy flow traced by m RFP-GFP-LC3 were detected by laser confocal;（4）CCK-8 was used to measure the cell viability and spectrophotometry was used to measure the intracellular LDH release.Results:1.Compared with the Control group,the autophagy level of H9c2 cardiomyocytes increased significantly after A/R,which displayed an increase in LC3-II/LC3-I and a decrease in P62,at the same time,the cell viability decreased and the LDH release increased.However,after the autophagy level under A/R was inhibited by the autophagy inhibitor 3-MA,the cell viability increased and LDH release decreased remarkably.On the contrary,after the autophagy level under A/R was further increased by the autophagy agonist Rapa,the cell viability further decreased and the LDH release further increased.These results suggested that A/R could induce the detrimental autophagy in H9c2 cardiomyocytes.In addition,the level of ROS and the expression of ox-DJ-1 in H9c2 cardiomyocytes were significantly increased after A/R,suggesting that the detrimental autophagy in H9c2 cardiomyocytes under A/R may be related to the DJ-1 oxidized at C106.2.After pretreatment of H9c2 cells with different concentrations（100,200,400μM）of H₂O₂,with the increase of intracellular ROS level,the expression level of ox-DJ-1 was up-regulated in a concentration-dependent manner,while the expression level of P62 was down-regulated,the ratio of LC3-II/LC3-I was up-regulated,the autophagy flow was enhanced,and the cell viability was decreased and the LDH release was increased in a concentration-dependent manner.Besides,the above effects caused by the up-regulation of ox-DJ-1 pretreated by H₂O₂were consistent with those of A/R group.The above results suggested that the DJ-1 oxidized at C106 could be involved in the detrimental autophagy of H9c2 cells under A/R.3.Compared with the A/R group,the ROS level in A/R-treated H9c2 cells decreased significantly after pretreated with the antioxidant NAC,and the expression level of ox-DJ-1 decreased,along with the increase of P62,the decrease of LC3-II/LC3-I,the decline of intracellular autophagy flow and the reduction of autophagosome number.At the same time,the cell viability increased and the LDH release decreased.These results suggested that inhibiting the C106 oxidation of DJ-1 protein and decreasing the expression level of ox-DJ-1 can alleviate the detrimental autophagy in H9c2 cardiomyocytes under A/R.4.H₂O₂ pretreatment before A/R in H9c2/DJ-1 cells could markedly increase the level of ox-DJ-1 and promote the detrimental autophagy induced by A/R,which was manifested as increased LC3-II/LC3-I,decreased P62 protein level,enhanced autophagy flow,and increased autophagosome number.In addition,the cell viability decreased and LDH release increased.However,H9c2/DJ-1（C106S）cells pretreated with H₂O₂ before A/R showed no significant change in A/R-induced detrimental autophagy because there was no significant change in ox-DJ-1 expression due to C106mutation.These results suggested that promoting the C106 oxidation of DJ-1 protein and up-regulating the expression of ox-DJ-1 can promote A/R-induced detrimental autophagy in H9c2 cells,while C106 mutation can inhibit this promoting effect.5.H9c2 cells pretreated with the specific C106 oxidation inhibitor Comp-23 could significantly reduce the expression level of ox-DJ-1,along with increased P62,decreased LC3-II/LC3-I,decreased intracellular autophagy flow,increased cell viability and decreased LDH release.The above results further suggested that DJ-1mediated the detrimental autophagy of H9c2 cardiomyocytes under A/R in a C106oxidation-state-dependent manner.Conclusion:In this study,it was confirmed that A/R could promote the C106 oxidation of DJ-1 protein and induce detrimental autophagy in H9c2 cells,that is,DJ-1 mediated the detrimental autophagy of H9c2 cardiomyocytes under A/R was in a C106 oxidation-state-dependent manner.