The Regulatory Effect Of Zinc Transporter Zip7 (SLC39A7) On Mitophagy And The Underlying Molecular Mechanism

Objective 1.To test whether the up-regulation of Zip7 expression during reperfusion increases mitochondrial ROS generation by inhibiting mitophagy in cardiac cells.2.To explore the molecular mechanism underlying the Zip7 regulation of mitophagy.Methods The changes of Zip7 expression were measured in isolated rat hearts subjected to ischemia followed by reperfusion and in cardiac H9c2 cells experiencing hypoxia/reoxygenation.Zip7 knockout heterozygous mice were generated and their embryonic tissues were collected to detect protein expression of TOM20,a marker protein of mitophagy.A stable H9c2 cell line in which Zip7 genes were knocked out was constructed.Protein expression level of TOM20,PINK1,and Parkin were detected by Western blotting.Zip7 siRNA was transiently transfected into H9c2 cells to test its effect on TOM20 protein expression.Mitosox red fluorescent probe was used to detect the changes of the mitochondrial superoxide.Changes of the mitochondrial membrane potential(??m)were detected using JC-1 fluorescent probe.The intracellular localization of PINK1 and Parkin was determined using the cellular Immunofluorescence technique.Zinpyr-1 was used to detect intracellular free zinc.Differential genes related to autophagy and mitophagy in H9c2 cells lacking Zip7 genes were detected and screened by RNA-Seq.Results 1.QPCR and Western blotting data showed that Zip7 m RNA and protein expression was increased in isolate rat hearts subjected to ischemia/reperfusion.In addition,Zip7 m RNA and protein expression was upregulated in H9c2 cells subjected to hypoxia and reoxygenation,which was inhibited by the selective Ca2+ chelators BAPTA-AM and EGTA,suggesting that Ca2+ overload may lead to the increase of Zip7 expression in the setting of hypoxia/reoxygenation.2.Western blotting analysis revealed that the expression of Zip7 was up-regulated in H9c2 cells treated with CaCl2 under the physiological conditions.3.Western blotting data showed that the transfection of Zip7 siRNA decreased TOM20 expression compared to the negative control.Similarly,the expression of TOM20 protein was decreased in Zip7 heterozygous knockout mouse embryonic tissues as well as in the Zip7 gene knockout H9c2 cells.In contrast,Zip7 overexpression enhanced TOM20 protein expression.These data suggest that Zip7 knockout may promote mitophagy,and vise versa.In addition,western blotting data showed that LC3?/?was increased but P62 was decreased in Zip7 heterozygous knockout mouse embryonic tissues as well as in the Zip7 gene knockout H9c2 cells line.The GFP-LC3 fluorescence intensity was increased in the Zip7 gene knockout H9c2 cells.In contrast,Zip7 overexpression enhanced P62 protein expression but decreased LC3 ?/?.These data suggest that Zip7 knockout may promote autophagy,and vise versa.4.Western blotting data showed that the expression of TOM20 was significantly decreased at reoxygenation in Zip7 knockout H9c2 cells,indicating that Zip7 may suppress mitophagy upon reperfusion.5.Studies using Mitosox red fluorescence revealed that under physiological conditions,mitochondrial ROS were reduced in H9c2 cells lacking Zip7 genes,whereas Zip7 overexpression increased mitochondrial ROS.Importantly,knockout of Zip7 reduced mitochondrial ROS in cells subjected to hypoxia/reoxygenation.6.Experiments with the immunofluorescence and Western blotting showed that Zip7 was located in mitochondria.7.Confocal imaging results showed that mitochondrial free Zn2+ was significantly increased after Zip7 knockout.8.Studies adopting flow cytometry and confocal imaging showed that compared to wild-type H9c2 cells,the mitochondrial membrane potential was reduced(depolarization)in Zip7 knockout cells.In contrast,compared to the empty vector group,Zip7 overexpression increased the mitochondrial membrane potential(hyperpolarization)in H9c2 cells,suggesting that Zip7 regulates mitophagy by altering mitochondrial membrane potential.9.Western blotting data showed that compared to wild-type H9c2 cells,Parkin expression was increased both in the cytoplasm and in mitochondria of Zip7 knockout cells,indicating that Zip7 knockout induces mitophagy presumably through the PINK1/Parkin pathway.10.Immunofluorescence data showed that compared to wild-type H9c2 cells,the co-localization of PINK1 and Parkin in mitochondria was increased after Zip7 knockout,indicating that Zip7 knockout lead to an increase of PINK1 and Parkin in mitochondria by altering mitochondrial membrane potential.11.Quantitative PCR data showed that compared to the Zip7 gene knockout group,the TOM20 m RNA level was significantly increased after Parkin siRNA transfection,suggesting that Parkin knockdown inhibits mitophagy induced by Zip7 gene knockout,confirming the above observation that Zip7 inhibits mitophagy through the PINK1/Parkin pathway.Conclusions 1.Zip7 is upregulated in the setting of myocardial ischemia/reperfusion or hypoxia/reoxygenation.2.Zip7 overexpression results in an increase in mitochondrial ROS generation leading to cardiac injury by inhibiting mitophagy.3.Zip7 regulates mitophagy through the PINK1/Parkin pathway.