Roles Of Mitochondrial Src Tyrosine Kinase And The Zinc Transporter Zip2 In Cardioprotection Against Ischemia/reperfusion Injury

Part I The role of mitochondrial Src tyrosine kinase in ischemic preconditioning induced cardioprotection in isolated rat heartsObjectives While ischemic preconditioning(IPC) and other cardioprotective interventions have been proposed to protect the heart from ischemia/reperfusion(I/R)injury by inhibiting mitochondrial complex I activity upon reperfusion, the exact mechanism underlying the modulation of complex I activity remains elusive. This study was aimed to test the hypothesis that IPC induced cardioprotection against I/R injury by modulating complex I activation at reperfusion via activating mitochondrial Src tyrosine kinase.Methods To establish the models of I/R injury, male Wistar rats were anesthetized and removed rapidly. Then, hearts were mounted on a Langendorff apparatus and subjected to 30 min regional ischemia followed by 2 h of reperfusion. IPC were induced by 3 cycles of 5 min ischemia and 5 min reperfusion before the index ischemia. Triphenyltetrazolium chloride(TTC) was used to test infarct size. To deter mine the activity of mitochondrial Src tyrosine kinase, we detected the phosphorylation of this kinase at Tyr416 by western blotting. To test mitochondrial complex I activity, we used complex I enzymatic activity microplate assay kit.Immunoprecipitation technique was used to exam whether mitochondrial Src tyrosine kinase interacts with complex I.Results Compared to the control group, myocardial infarct size was significantly reduced by IPC which was inhibited by selective Src tyrosine kinase inhibitor PP2(1μM). Western blotting analysis of mitochondrial Src phosphorylation at Tyr416 showed that I/R injury dramatically reduced mitochondrial Src phosphorylation at 30 min after the onset of ischemia(I30), and 10 min of reperfusion(R10). IPC increased mitochondrial Src phosphorylation upon reperfusion which was inhibited by the tyrosine kinase inhibitor genistein. In support, IPC’s anti-infarct effect was inhibited by the selective Src tyrosine kinase inhibitor PP2. Complex I activity wassignificantly increased upon reperfusion which was prevented by IPC in a Src tyrosine kinase dependent manner. Further experiments showed that Src and phosphor-Src were found in complex I. IPC increased protein tyrosine phosphorylation of UDUFS3 subunit of complex I,suggestting that the UDUFS3 subunit protein tyrosine phosphorylation may account for the inhibitory action of IPC on complex I.Conclutions Mitochondrial Src tyrosine kinase accounts for the inhibitory action of IPC on complex I, and thereby plays a role in the cardioprotective effect of IPC.Part II The critical role of the zinc transporter Zip2(SLC39A2) in hypoxia/reoxygenation injuryObjectives Although exogenous zinc has been demonstrated to be cardioprotective,the exact role of endogenous zinc and its homeostasis during cardiac ischemia/reperfusion injury remain elusive. We aimed to test whether Zip2, an important zinc importer that increases cytosolic free zinc levels, plays a role in hypoxia/reoxygenation(H/R) injury.Methods To induce H/R injury, Rat heart tissue-derived H9c2 cardiac myoblast cells cultured in a 24-well plate filled with the hypoxia medium were exposed to hypoxia(1% O2) by placing the plate in a humidified hypoxia glove box for 21 h. Then the hypoxia medium was replaced by the normal DMEM and cells were cultured in an incubator under normoxic conditions(room air with 5% CO2) for 4 h. Zip2, STAT3 m RNA expressions were evaluated by quantitative RT-PCR.Cells with >80%subconfluency were transfected with Zip2 plasmid or Zip2-si RNA or Stat3-si RNA using transfection reagents(Santa Cruz) or Lipofactamine(Invitrogen). All experiments were done 48 h after transfection. Intracellular free Zn2+was detected with Fluo Zin-3. The cell viability was assessed by propidium iodide fluorometry using a fluorescence reader.Results Intracellular free zinc levels were decreased in cardiac cells experienced 21 h of hypoxia(1% O2) followed by 4 h of reoxygenation(66.4 ± 4.7%, P<0.05),indicating that H/R causes zinc loss in cardiac cells. Evaluation of zinc transporters by RT-PCR assay revealed that the m RNA expression of Zip2 was significantly upregulated at reoxygenation(5 folds). These results suggest Zip2’s attempt to homeostatically compensate for zinc loss caused by H/R. To examine the role of Zip2 during H/R, we altered Zip2 levels using genetic tools and tested their impacts on cell viability at reoxygenation. H/R reduced cell viability to 73.7 ± 1.7% of the baseline(control)(P<0.05). Zip2 plasmid transfection increased cell viability(83.5 ± 2.9%,P<0.05) compared to the control, whereas Zip2 si RNA dramatically decreased cell viability(57.7 ± 2.3%, P<0.05) compared to the control, implying that Zip2 is acytoprotective, rescuing cells from H/R injury. The upregulation of Zip2 expression was reversed by stattic, an inhibitor of signal transducers and activators of transcription 3(STAT3), indicating that STAT3 may mediate Zip2 upregulation at reoxygenation. In support, both H/R and the selective zinc chelator N,N,N,N’-tetrakis-(2-pyridylmethyl) ethylenedia mine(TPEN) increased STAT3 phosphorylation at Tyr705, suggesting that H/R-induced zinc loss may account for STAT3 activation. Furthermore, stattic reduced cell viability after H/R.Conclusion Zip2 is upregulated upon reoxygenation via STAT3 and this upregulation may serve as an important intrinsic mechanism by which cells are resistant to H/R injury.