Roles Of STAT3 In Zinc-Induced Cardioprotection Against Ischemia/Reperfusion Injury

ObjectiveTo test whether zinc improves mitochondrial respiratory function and prevents mitochondrial ROS generation at reperfusion by phosphorylating mitochondrial STAT3 at Ser⁷2727 and to explore the underlying mitochondrial molecular mechanism.MethodsIsolated rats hearts and cardiac H9c2 cells were subjected to ischemia/reperfusion or hypoxia/reocygenation.Western blotting analysis was used to determine the expression levels of STAT3,ERK,Tubulin,COX IV and phosphorylation of STAT3,ERK.Immunofluorescence staining was used to determine the co-localization of STAT3 and mitochondria.The high-resolution respirometry（Oxygraph-2k）was used to determien the respiratory capacity and oxidative phosphorylation in isolated mitochondria.Adenosine-5’-triphosphate（ATP）content and Citrate Synthase Activity were measured using the ATP determination kit and Citrate synthase activity assay kit according to the manufacturer’s instructions.Mitochondrial fluorescence probes including JC-1,DCFH-DA,and Mitosox Red were used to determine mitochondrial membrane potential（△Ψm）,reactive oxygen species（ROS）,and mitochondrial superoxide respectively.mRNA expressions of the mitochondria-coded genes including ND1,ND5,ND6 were detected with Real-Time quantitative PCR using the CFX96 Real-Time System.The mitochondrial succinate dehydrogenase（SDH）activity was determined using a SDH activity colorimetric assay kit.Results1.Under normoxic conditions,ZnCl₂ increased the co-localization of phopsphorylated-STAT3 at Ser⁷²⁷（green）and mitochondria（red）.Western blotting analysis showed that ZnCl₂ phosphorylated STAT3 at Ser⁷²⁷ and translocated it into mitochondria in cultured H9c2 cells and in isolated rat hearts.ZnCl₂-induced STAT3phosphorylation was reversed by the treatment with the selective zinc chelator TPEN.2.TPEN reduced myocardial STAT3 phosphorylation(Ser⁷²⁷)under normoxic conditions,establishing the positive correlation between the Zn²⁺level and STAT3phosphorylation at Ser⁷²⁷.However,STAT3 phosphorylation at Ser⁷²⁷ was not altered by TPEN in isolated mitochondria.3.Western blotting analysis showed that in the settings of I/R and H/R,ZnCl₂ also increased STAT3 phosphorylation at Ser⁷²⁷.In addition,ZnCl₂ enhanced both the mitochondrial phospho-STAT3 and the mitochondrial total STAT3 at reperfusion and reoxygenation.These effects of zinc were blocked by TPEN,pointing to that zinc phosphorylated STAT3 and translocated phospho-STAT3 into the mitochondria at reperfusion or reoxygenation.4.Western blotting analysis showed that ZnCl₂ increased ERK as well as STAT3phosphorylation both in the normoxic hearts and in the hearts subjected to ischemia/reperfusion.However,these effects were abolished by the MEK inhibitor PD98059.These data suggest that ERK mediates the action of zinc on STAT3phosphorylation.5.ZnCl₂ can improve the mitochondrial respiratory function under normoxic conditions and in the setting of I/R.In support,ZnCl₂ increased the myocardial ATP level and the citrate synthase activity.Moreover,zinc could improve the mitochondria oxidative phosphorylation through phosphorylated STAT3.These data indicate that zinc prevents ischemia/reperfusion injury by improving mitochondrial metabolic capacity via STAT3 phosphorylation at Ser⁷²⁷.6.H/R markedly increased both DCF and MitoSOX Red fluorescence,which was prevented by ZnCl₂.However,ZnCl₂ failed to reduce MitoSOX Red fluorescence in cells transfected with the STAT3 mutant STAT3S727A,indicating that serine residue-phosphorylated mitochondrial STAT3 are involved in the protective effect of ZnCl₂ on ROS generation in the setting of H/R.7.ZnCl₂ prevented dissipation of mitochondrial membrane potential（ΔΨm）at reoxygenation.However,this effect of ZnCl₂ disappeared in cells transfected with STAT3S727A,suggesting that serine residue-phosphorylated STAT3 mediates zinc-induced cardioprotection against hypoxia/reoxygenation injury.8.ZnCl₂ increased the mRNA level of ND6 through STAT3 phosphorylation at Ser⁷²⁷ in the hearts subjected to ischemia/reperfusion,implying that zinc improves oxidative phosphorylation presumably by preventing the damage to mtDNAs that encode the electron transport chain（ETC）subunits.9.ZnCl₂ suppressed SDH activity via phospho-STAT3 at Ser⁷²⁷,which may lead to the prevention of mitochondrial ROS generation upon reoxygenation.Conclusions1.Zinc increases myocardial STAT3 phosphorylation at Ser⁷2727 and translocates it into the mitochondria through the ERK signaling pathway.2.Zinc improves mitochondrial oxidative phosphorylation and prevents mitochondrial ROS generation at reperfusion by phosphorylating STAT3 at Ser⁷²⁷.3.Zinc improves mitochondrial oxidative phosphorylation by preventing the damage of mitochondrial ND6 gene through phospho-STAT3 at Ser⁷²⁷.4.Zinc alleviates mitochondrial ROS generation upon reperfusion by suppressing SDH activity via phospho-STAT3 at Ser⁷²⁷.