The Role Of Zn²⁺ In ER Stress Inhibition-induced Cardioprotection Against Ischemia/reperfusion Injury

Objective To investigate the role of Zn2+in ER stress inhibition-inducedcardioprotection against ischemia/reperfusion injury.Methods Rat heart tissue-derived H9c2cells were exposed to H2O2to induceendoplasmic reticulum stress. First we examined if H2O2could increase GRP78andGRP94expressions with western blot. We then detected intracellular free Zn2+concenctrations and mitochondrial membrane potential (Ψm) with laser scanningconfocal microscopy. Third, we determind the effects of TUDCA and TPEN on GRP78expressions at rat hearts subjecterd to ischemia followed by reperfusion. Lastly, weobserved the morphological changes of myocardial tissue with HE staining and TEM.Results1) H9c2cells were exposed to different doses of ZnCl2(1,5, and10μM),Compared to normal group (82.2±3.2%), zinc significantly increased the Newport GreenDCF fluorescence intensity (135.1±11.6%,141.7±8.5%, and157.1±20.7%respectively, P＜0.05), indicating that exogenous ZnCl2can increase intracellular freeZn2+.2) Compared to normal group (82.2±3.2%), the Newport Green DCF fluorescenceintensity was significantly increased (112.7±5.4%) in cells treated with TUDCA for10min, pointing to that TUDCA can also increase intracellular free zinc.3) Compared tocontrol group (100.0±12.8%、100.0±7.9%), treatment of cells with H2O2(800μM) for20min enhanced the expressions of the ER stress protein GRP78and GRP94(296.2±41.0%and150.6±13.5%, respectively), suggesting that H2O2can induce ER stress.4)Cells treated with H2O2showed a significant decrease (40.6±7.4%) in TMREfluorescence compared to the normal group (92.6±0.1%), indicating that oxidative stresscan induce the mPTP opening. In contrast, TUDCA (30μM) prevented the loss of TMREfluorescence (77.8±6.8%), suggesting that inhibition of ERS can prevent the mPTPopening. This effect of TUDCA was blocked by the chelation of free zinc with TPEN (37.7±13.0%) indicating that Zn2+may mediate the cardioprotective effect of TUDCA on bytargeting the mPTP.5) Western blot analysis showed that GRP78expression was notaltered at10min of ereperfusion in perfused rat hearts. In contrast, GRP78expression wasmarkedly increased30and60min after the onset of reperfusion (191.3±25.3%and139.0±7.3%, respectively), suggesting that ischemia/reperfusion causes ER stress. Heartstreated with TUDCA showed a significant reduction of GRP78expression (105.2±8.1% and101.2±6.2%), and effect that waws reversed by TPEN (183.2±22.0%and131.2±5.4%), implying that Zn2+may mediate the effect of TUDCA on ERS.6) Theimmunofluorescence study showed that compared with the sham group, the greenfluorescence intensity of GRP78was markedly increased in the I/R group, which wawsinhibited by TUDCA, pointing to that TUDCA inhibited I/R-induced increase in theexpression of GRP78. Furtehr experiments revealed that TPEN reversed the effect ofTUDCA on GRP78expression implying that Zn2+may mediate the effect of TUDCA onER stress.7) Studies with HE staining showed that myocardial fibers were disarranged andruptured, and the gaps were widened in the I/R group compare to the sham group, whichwas prevented by TUDCA. However, theeffect of TUDCA was again inhibited by TPEN,suggesting that zinc mediates the effect of TUDCA.8) Studies with TEM revealed that10min after the onset reperfusion, the mitochondrial structure was comparatively complete,some mitochondria swelling, mitochondrial crista clearly visible, only crista gap widened,ER expanded partly.30min after the onset reperfusion, filaments was dissolved, damagedor even disappear, Z line is not clear, mitochondrial swelling, mitochondrial doublemembrane fused even rupture, cristae broken, dissolved and disappeared, mitochondriaoccur vacuolar degeneration, ER expanded, part of ER degranulation. Treated withTUDCA, the structure of mitochondria were comparatively intact, partial mitochondrialstill has swelling, but mitochondrial cristae intact and clear, the ER expanded, suggestingthat TUDCA could protect reperfusion heart. But TPEN reversed the effect of TUDCA oncardioprotection, futher suggesting that Zn2+mediated the effect of TUDCA oncardioprotection.Conclusions These data suggest that increased intracellular free Zn2+accounts for thecardioprotective effect of ER stress inhibition on reperfusion injury through prevention ofthe mPTP opening.