Inositol-requiring Enzyme 1 Attenuates Myocardial Ischemia Injury by Restoring the Blocked Autophagy Flux in Mice
Objectives:To explore the impact of inositol-requiring enzyme 1(IRE1)signaling pathway on autophagy flux and cardiac function in mice with myocardial ischemia/reperfusion(I/R)injury.Methods:H9c2 cells were divided into control group,IRE1 group,oxygen glucose deprivation/reoxygenation(OGD/R)group,OGD/R+IRE1 group,chloroquine group,IRE1+chloroquine group,OGD/R+IRE1+chloroquine group,OGD/R+chloroquine group,OGD group,OGD+chloroquine group,OGD+IRE1+RNAi X-box binding protein 1(si-XBP1)group and OGD+IRE1+XBP1 overexpression(XBP1-OE)group.Autophagy flux of cells in each group was evaluated by autophagy double-labeled adenovirus(Adv-RFP-GFP-LC3).The nuclear translocation of X-box binding protein 1(XBP1)was analyzed by immunofluorescence and western blot.Adult male C57BL/6 J mice were randomly divided into sham operation group,I/R group,IRE1 group and I/R+IRE1 group(n=8 each).Cardiac function was evaluated by echocardiography.Quantitative western blot analysis was used to detect protein expression of autophagy-related molecules.Results:(1)Compared with OGD/R group,the expression level of IRE1 protein was significantly upregulated(P<0.001),and the expressions of microtubule-associated proteins light chain 3B(LC3Ⅱ)and sequestosome 1(p62)proteins were significantly decreased in IRE1+OGD/R group(all P<0.05).Compared with OGD/R+chloroquine group,the expression of LC3Ⅱ and p62 protein was significantly increased in OGD/R+IRE1+chloroquine group(all P<0.05).Compared with control group,the fluorescence intensity ratio of IRE1 nucleus/cytoplasm was significantly increased in OGD/R group(P<0.001),and further increased in IRE1+OGD/R group(P<0.001).The level of XBP1 in the nuclear protein was significantly higher in IRE1+OGD/R group than in OGD/R group(P<0.01).Compared with OGD/R+IRE1 group,the yellow punctures was significantly decreased in OGD/R+IRE1+si-XBP1 group(P<0.01),and significantly increased in OGD/R+IRE1+XBP1-OE group(P<0.05).(2)Compared with Sham group,the left ventricular ejection fraction(LVEF)and fractional shortening(FS)were significantly decreased in I/R group(both P<0.05),LVEF and FS reduction could be partly reversed in I/R+IRE1 group.Compared with Sham group,the number of autophagic vacuoles and the expressions of IRE1,LC3Ⅱ and p62 were significantly increased in I/R group(P<0.05).The number of autophagic vacuoles and the expression of p62 were significantly downregulated(both P<0.05),and the expressions of IRE1 and LC3Ⅱ in myocardial tissue were further increased in I/R+IRE1 group as compared to the I/R group(all P<0.05).Conclusions:IRE1 restores the blocked autophagy flux induced by OGD/R and I/R by promoting the nuclear translocation of XBP1,and the recovery of autophagy flux is associated with cardiac function improvement post I/R injury in mice.
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