Therole Of The Zinc Transporter Zip2(SLC39A2) In Myocardial Ischemia/Reperfusion Injury

Objective 1.To test if Zip2 expression is upregulated at reperfusion and whether Zip2upregulation is caused by cardiac zinc loss.2.To clarify the exact role of the zinc transporter Zip2 in myocardial ischemia/reperfusion injury.3.To determine whether zinc loss induces the Zip2 upregulation by activating STAT3during reperfusion.Methods In vivo mouse hearts were subjected to 30 min ischemia followed 2 h of reperfusion,whereas cardiac H9c2 cells experienced hypoxia/reoxygenation.Cardiac zinc concentration was measured by inductively coupled plasma optical emission spectrometer?ICPOES?.Cardiac Zip2 mRNA expression was detected by real-time quantitative PCR?RT-PCR?,Western blotting analysis was used to determine expression levels of Zip2,phospho-STAT3?Tyr705?,STAT3 and tubulin.Cell viability was determined by a CCK-8 assay kit.Serum concentration of the enzymes including LDH,cTnI,and CK-MB were determined using ELISA kits.Myocardial infarct size was determined by the Evans Blue and TTC double staining.Results 1.Studies using ICPOES showed that cardiac zinc levels were decreased during reperfusion,indicating that reperfusion induces zinc loss.2.RT-PCR and Western blotting analysis showed that Zip2 mRNA and protein expression was increased at reperfusion,an effect that was blocked by exogenous ZnCl₂ given at the beginning of reperfusion,indicating that the up-regulation of Zip2 expression during reperfusion was caused by cardiac zinc loss.3.Myocardial infarct size of the Zip2-knockout?KO?mice was larger than that of the wild-type mice.In support,serum LDH,cTnI,and CK-MB levels were also significantly higher in the KO mice than those in the wild-type mice,indicating that the knockout of Zip2 gene worsened myocardial ischemia/reperfusion injury.In contrast,the delivery of adeno-associated virus?AAR?carrying Zip2-overexpressing plasmid transfection of Zip2 with adeno-associated virus?AAV?reduced infarct size as well as the serum levels of LDH,cTnI,and CK-MB,indicating that Zip2overexpression significantly alleviated ischemia/reperfusion injury.4.In H9c2 cells subjected to hypoxia/reoxygenation,Zip2 siRNA significantly reduced cell viability compared to the NC group,indicating that Zip2 knockdown significantly aggravated hypoxia/reoxygenation injury.However,ZnCl₂ reversed the cellular injury caused by Zip2 siRNA by increasing cell viability.On the contrary,the Zip2 ovexpression significantly increased cell viability,indicating that Zip2 overexpression can alleviate hypoxia/reoxygenation injury.Moreover,the zinc chelator TPEN diminished the protection induced by Zip2 overexpression.These data suggest that Zip2 exerts its myocardial protection by maintaining the cellular zinc homeostasis.5.Compared to the sham group,the phosphorylated level of STAT3?Tyr705?was significantly increased in I/R group,which was blocked by ZnCl₂ given at the beginning of reperfusion,suggesting that the STAT3 activation is caused by zinc loss at reperfusion.6.Western blotting analysis and RT-PCR data showed that stattic,an inhibitor of STAT3,reduced Zip2 expression as well as STAT3 phosphorylation during reperfusion,pointing to that STAT3 serves as an upstream signal to regulate Zip2expression at reperfusion.7.Compared to the vector group,STAT3?Tyr705?phosphorylation and Zip2expression were significantly increased by the transfection of STAT3 but not by.the STAT3 Y705F,further confirming the above observation that as an upstream signal,STAT3 regulates Zip2 expression.8.Infarct size as well as the serum LDH,cTn I,and CK-MB activities were reduced in the STAT3 AAV group compared to the Vector group,an effect that was reversed by STAT3 Y705F transfection.In addition,Zip2-KO mice transfected with STAT3AAV failed to reduce infarct size as well as the serum LDH,cTnI and CK-MB activities.These data suggest that Zip2 knockout inhibits the protection induced by STAT3 overexpression,and that STAT3 exerts its myocardial protection via Zip2.Conclusions 1.Cardiac zinc loss at reperfusion triggers Zip2 upregulation.2.Zip2 exerts its myocardial protection by maintaining the zinc homeostasis during reperfusion and the Zip2 up-regulation at reperfusion may serve as an important intrinsic protective mechanism by which the heart is resistant to ischemia/reperfusion injury.3.Cardiac zinc loss up-regulates Zip2 expression by activating STAT3 during reperfusion,and STAT3 exerts its cardioprotective effect through Zip2.