The Regulatory Effect Of The Zinc Transporter Zip2 (SLC39A2) On Mitochondrial Respiration At Reperfusion In Mouse Hearts

Objective 1.To investigate the regulatory effect of the zinc transporter Zip2(SLC39A2)on mitochondrial respiration upon reperfusion.2.To explore the molecular mechanism of the regulation of mitochondrial respiration by the zinc transporter Zip2.Methods The Zip2 knockout homozygous mice were generated,and an in vivo mouse myocardial ischemia/reperfusion model was established.Mice were transfected with adeno-associated virus(AAV)in vivo.Western blotting analysis was used to determine the expression levels of STAT3,phospho-STAT3(Ser727),phosphoERK,ERK and Tubulin.Cardiac zinc concentration was measured by inductively coupled plasma optical emission spectrometer(ICPOES).The high-resolution respirometry(Oxygraph-2k)was used to determine the mitochondrial respiratory function and oxidative phosphorylation.Results 1.Compared to the sham group,zinc concentration in reperfused myocardium was decreased in wild-type mice,which was further reduced in the Zip2 KO group,indicating that Zip2 knockout reduced cardiac zinc concentration at reperfusion.This result imply that Zip2 may contribute to maintenance of the zinc homeostasis during reperfusion.2.Studies using high-resolution respirometry(Oxygraph-2k)showed that compared to the wild-type mice,both the mitochondrial respiratory control rate(RCR)and oxidative phosphorylation were decreased in the Zip2 KO group,which was further worsened by ischemia/reperfusion,indicating that Zip2 knockout inhibits the cardiac mitochondrial respiratory function.Zip2 may be involved in maintaining mitochondrial respiration upon reperfusion.3.Western blotting analysis showed that compared to the WT I/R group,expression levels of phospho-STAT3(Ser727),phospho-ERK were significantly decreased in Zip2 KO I/R group,suggesting that Zip2 may regulate mitochondrial respiration through the ERK-STAT3(Ser727)pathway.4.The further studies using high-resolution respirometry(Oxygraph-2k)showed that STAT3 overexpression(STAT3 WT group)significantly improved the mitochondrial respiratory function,while the STAT3 dominant negative mutant(STAT3 S727A)markedly inhibited mitochondrial respiratory function.In addition,compared with the Zip2 KO-Vector,mitochondrial respiration was significantly improved at reperfusion in the Zip2 KO STAT3 group,suggesting that the impairment of mitochondrial function by Zip2 KO was reversed by STAT3 overexpression.These results suggest that Zip2 regulates mitochondrial respiration through phosphorylation of STAT3(Ser727)during myocardial ischemia/reperfusion,which may accout for Zip2’s cardioprotection against ischemia/reperfusion injury.Conclusions 1.Zip2 knockout reduced cardiac zinc concentration at reperfusion in mouse hearts.2.Zip2 knockout inhibited the mitochondrial respiratory function in mouse hearts,suggesting that Zip2 contributes to the maintenance of mitochondrial respiration upon reperfusion.3.Zip2 knockout inhibited the phosphorylation of ERK and STAT3(Ser727)at reperfusion,Zip2 may regulate mitochondrial respiration through the ERK-STAT3(Ser727)signaling pathway.4.STAT3 overexpression improved the mitochondrial respiratory function at reperfusion and reversed the impairment of mitochondrial respiration by Zip2 KO,suggesting that Zip2 regulates mitochondrial respiration through phosphorylation of STAT3(Ser727)during myocardial ischemia/reperfusion.