Molecular Mechanism Underlying Hypoxic Preconditioning-promoted Mitochondrial Localization Of DJ-1 In Hypoxia/reoxygenation H9c2 Cells

Objective:This study aims to investigate the underlying molecular mechanism by which hypoxic preconditioning?HPC?promotes the mitochondrial translocation of DJ-1 in hypoxia/reoxygenation?H/R?H9c2 cells from the perspective of the interaction of DJ-1 and Grp75.Methods:1.Three candidate RNA interference sequences and a negative control sequence were designed according to the GeneBank gene sequence?NM₀01100658.2?of rat Grp75 and were constructed into lentiviral vector?LV-shGrp75-A,B,C and nagtive control?.The constructed Grp75 shRNA and nagtive control shRNA lentiviral vectors were respectively infected into H9c2 cardiomyocytes,and their efficacies in extinguishing Grp75 expression were evaluated by Real-time PCR and Western Blot.The RNAi lentivirus testified being most effective was used for subsequent experiments.2.To observe whether the interaction between DJ-1 and Grp75 was influenced by HPC in H9c2 cells subjected to H/R,the cellular model of HPC from rat heart-derived H9c2 cells was induced 24 h prior to H/R.The interaction of Grp75 with DJ-1 was assessed by Co-immunoprecipitation.3.In order to observe whether HPC promotes the mitochondrial translocation of DJ-1 in H9c2 cells subjected to H/R by increasing the interaction between DJ-1 and Grp75,the H9c2 cells were infected by LV-shGrp75 lentiviral vectors for 72 h and then subjected to HPC 24 h prior to H/R or transfected with pFlag-DJ-1 for 48 h followed by H/R.Subsequently,the whole-cell lysates and cytosolic and mitochondrial fractions were prepared,and mitochondrial,cytosolic,and total DJ-1 levels were examined by Western blot.Moreover,the mitochondrial localization of DJ-1 was further detected by Immunofluorescence technique.4.To examine the effects of interfering Grp75 protein on DJ-1-mediated the protection of mitochondrial complex I,inhibition of mitochondrial ROS generation,and maintenance of mitochondrial membrane potential by HPC in H9c2 cells subjected to H/R,the H9c2 cells were infected with LV-shGrp75 lentiviral vectors for 72 h and then subjected to HPC 24 h prior to H/R or transfected with pFlag-DJ-1 for 48 h followed by H/R.Subsequently,the activities of mitochondrial complex I was assessed by spectrophotometry.The mitochondria-derived superoxide production was detected by MitoSOX Red probe.The mitochondrial membrane potential was analyzed by JC-1 staining.5.To further analyze the influence of Grp75 knockdown on DJ-1-mediated the delayed cytoprotection of HPC against oxidative stress induced by H/R,the H9c2 cells were infected with LV-shGrp75 lentiviral vectors for 72 h and then subjected to HPC 24 h prior to H/R or transfected with pFlag-DJ-1 for 48 h followed by H/R.Subsequently,the cellular damage was monitored by measuring cell viability and lactate dehydrogenase?LDH?release,and oxidative stress was analyzed by quantifying the activities of antioxidant enzymes?SOD,CAT and GPx?and Malondialdehyde?MDA?content.Cell viability was detected by cell counting kit-8?CCK8?method.LDH,MDA,and the activities of antioxidant enzymes?SOD,CAT and GPx?were detected by a colorimetric method.Results:1.Three recombinant lentiviral vectors of shRNA targeting the Grp75 gene?LV-shGrp75-A,B,C?were successfully constructed and could effectively reduce the expression of Grp75 gene in H9c2 cells.The silencing effect of LV-shGrp75-A on the expression of Grp75 was most remarkable and its inhibition ratio to the expression of Grp75 mRNA and protein were respectively 77% and 74% by Real-time PCR and Western Blot analysis,which can be selected for follow-up research.2.HPC significantly up-regulated the expression of DJ-1 and further enhanced the interaction between DJ-1 and Grp75 in H9c2 cells subjected to H/R.3.HPC obviously promoted DJ-1 translocation from cytoplasm to mitochondria in H9c2 cells subjected to H/R,which was mimicked by DJ-1 overexpression induced by pFlag-DJ-1 transefection.Importantly,downregulating Grp75 expression by LV-shGrp75 could inhibit the increase of mitochondrial translocation of DJ-1 by HPC and pFlag-DJ-1 transefection.4.HPC significantly attenuated H/R-induced mitochondrial complex I suppression,mitochondrial reactive oxygen species burst,and loss of mitochondrial inner membrane potential in H9c2 cells,which was mimicked by DJ-1 overexpression induced by pFlag-DJ-1 transefection.More intriguingly,the above beneficial effects induced by HPC and pFlag-DJ-1 transefection were blocked by Grp75 knockdown.5.HPC significantly attenuated H/R-induced viability loss,LDH release,suppression of antioxidant enzymes?SOD,CAT and GPx?activities,and elevation of MDA content,which was also mimicked by DJ-1 overexpression induced by pFlag-DJ-1 transefection.Likewise,the aforementioned effects induced by HPC and pFlag-DJ-1 transefection were antagonized by Grp75 knockdown with LV-shGrp75.Conclusion:Increasing the interaction between DJ-1 and Grp75 is a crucial mechanism by which HPC promotes the mitochondrial translocation of DJ-1 in H9c2 cells,thereby preserving mitochondrial complex I activity,inhibiting mitochondrial ROS generation,and subsequently providing a delayed cardioprotection against oxidative stress by H/R.