Nrg1β Released in Remote Ischemic Preconditioning Improves Myocardial Perfusion and Decreases Ischemia/Reperfusion Injury via ErbB2-Mediated Rescue of Endothelial Nitric Oxide Synthase and Abrogation of Trx2 Autophagy

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  Figure 1.. Superoxide-induced Nrg1 (neuregulin 1) release by the microvascular endothelial bed of the hindlimb acts as remote ischemic preconditioning (RIPC)-factor and protects the myocardium from ischemia/reperfusion (I/R)-induced injury. A, Mice were either subjected to sham or three cycles of 5 min hindlimb ischemia followed by 5 min reperfusion. Thirty minutes after the last I/R cycle, serum Nrg1 levels were determined by ELISA. Mean±SEM was plotted as a bar graph (n=5). *P<0.01 vs sham. B, Formaldehyde fixed paraffine embedded gastrocnemius muscle sections from sham or RIPC-treated mice were immunostained with anti-Nrg1 and anti-CD31 antibodies along with isolectin GS-IB4, scale bar=100 mm. C, Nrg1 specific fluorescent signals were quantitated, and mean±SEM fluorescent values were plotted as a bar graph (n=6). *P<0.01 vs sham (Student t test). D, To confirm the source of Nrg1, gastrocnemius muscle sections were hybridized with mouse Nrg1 Stellaris fluorescent in situ hybridization (FISH) Probe set labeled with CAL Fluor Red 590 Dye. Capillaries in the sections were stained with isolectin GS-IB4 conjugated with Alexa Fluor 488. Scale bar=25 mm. E, Mean fluorescence values from RNA FISH…

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  Figure 2.. Remote ischemic preconditioning (RIPC) improved cardiac perfusion via Nrg1 (neuregulin 1)-dependent eNOS (endothelial nitric oxide synthase) activation. A, Mice were subjected to sham or myocardial ischemia/reperfusion (I/R) or RIPC+I/R surgery, and at the end of the reperfusion period, the nontargeted contrast agent was injected to mice via femoral vein while collecting B-mode/contrast mode images of the heart from long-axis view using Vevo 3100. To block the function of circulating Nrg1 in mice, neutralizing anti-Nrg1 antibodies (150 µg/kg) was administered to mice before RIPC and then subjected to RIPC+I/R injury. B, Contrast intensities in the left ventricle (LV) anterior wall downstream to the ligation site were quantitated and plotted over time. Loss of peak height and the rightward shift of peak indicates hypoperfusion. C, From the rate of contrast agent intensity change, the perfusion index was calculated. Percentage of perfusion was calculated based on the perfusion index assuming its level in the sham group is 100% and blotted as a bar graph (n=5) and shown as a heat map over the anterior LV in A. Values are means±SEM (n=5 mice). *P<0.01 vs sham, **P<0.01 vs I/R,…

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  Figure 3.. Only preischemic and not postischemic Nrg1β (neuregulin 1) administration protects myocardium due to loss of endothelial-ErbB2 (Erb-B2 receptor tyrosine kinase 2) during ischemia. A, To determine the protective function of Nrg1 during ischemia or reperfusion, recombinant Nrg1β (4 µg/kg) was injected into mice before ischemia (preischemia) or after ischemia (postischemia) but before reperfusion. After ischemia/reperfusion (I/R), the heart was isolated and perfused with annexin-V-Fe complex. The infarcted tissue was excised, and tissue bound annexin-V was quantified by electron spin resonance (EPR). B, The absolute spin count was calculated from the EPR spectra of paramagnetic-Fe bound to annexin-V and plotted as a bar graph. Values are means±SD (n=5 mice). *P<0.01 vs sham; **P<0.01 vs I/R, †P<0.01 vs Nrg1 (preischemic) plus IR (ANOVA). C, Mice were subjected to sham, I/R, remote ischemic preconditioning (RIPC) or RIPC+I/R, and the infarcted tissue region was excised. Protein extract was prepared from the excised tissue and analyzed by Western blotting for ErbB2 and ErbB4 levels. D, Sections of sham and I/R mouse heart were subjected to immunofluorescence staining using anti-ErbB2…

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  Figure 4.. Remote ischemic preconditioning (RIPC) protects ErbB2 (Erb-B2 receptor tyrosine kinase 2) from ischemia/reperfusion (I/R)-mediated degradation and the loss of ErbB2 results in Trx2 (thioredoxin 2) autophagy and apoptosis. A, Mice were subjected to a sham procedure, 30 min ischemia, I/R, RIPC or RIPC+I/R, and the infarcted tissue region were excised. Protein extract was prepared from the excised tissue and analyzed by Western blotting for ErbB2, Trx2, catalase, SOD1, SOD2, and total actin. B, ErbB2 and Trx2 levels in the blots were quantified and plotted as a bar graph (n=3). *P<0.01 vs sham; **P<0.01 vs I/R. C, HCAECs were transfected with nontarget (NT) or ErbB2 siRNA (25, 50, 100 nmol/L). After 48 h of transfection, cell lysates were prepared, and an equal amount of protein from each cell lysate was analyzed for Trx1, Trx2, SOD1, SOD2, and catalase levels using their specific antibodies. Blots were reprobed with anti-ErbB2 and anti-β-actin antibodies to determine the depletion of ErbB2 and equal protein loading, respectively. Bar graph shows the level of Trx2. D, HCAECs were transfected with pcDNA3 empty or pcDNA3-ErbB2 plasmids. After 24 h of transfection, cells were exposed…

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  Figure 5.. Endothelial-specific deletion of ErbB2 (Erb-B2 receptor tyrosine kinase 2) results in loss of remote ischemic preconditioning (RIPC)-mediated protection of cardiac perfusion. A, To generate a mouse model of endothelial-specific inducible deletion of ErbB2, we crossed ErbB2 floxed mice with VECad-Cre-ERT2 mice. To test the endothelial-specific deletion of ErbB2, we isolated endothelial cells from WT and ErbB2fl/fl:VECad-Cre-ERT2 mice and treated with tamoxifen (30 µg/mL) for 48 h. After the treatment, cells were lysed, and an equal amount of protein from cell lysates was analyzed for ErbB2 levels by Western blotting using anti-ErbB2 antibodies. Blots were reprobed with anti-Cre, anti-CD31, and anti-β-actin antibodies. B, To induce deletion of ErbB2 in endothelial cells, ErbB2fl/fl:VECad-Cre-ERT2 mice were injected with three doses of tamoxifen (75 mg/kg). After the induced deletion, ErbB2fl/fl:VECad-Cre-ERT2 and nontarget (NT) mice were subjected to sham or myocardial ischemia/reperfusion (I/R) or RIPC+I/R surgery. At the end of the reperfusion period, the nontargeted contrast agent was injected into mice via the left femoral vein while collecting B-mode/contrast mode images of…

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  Figure 6.. Endothelial-ErbB2 (Erb-B2 receptor tyrosine kinase 2) directly associates with Src and activate eNOS (endothelial nitric oxide synthase). A, Human coronary artery endothelial cells (HCAECs) were treated with preconditioned medium (PM) for the indicated period, and cell lysates were prepared. An equal amount of protein from cell lysates was immunoprecipitated using the anti-ErbB2 antibody. The immunoprecipitates were analyzed by Western blotting for tyrosine phosphorylation of ErbB2, level of associated Src, ErbB4, and ErbB2 using their specific antibodies. B, HCAECs were treated with CM for 1 or 4 h, and proximity ligation assay (PLA) was performed using anti-ErbB2 and anti-Src antibodies. Scale bar=20 mm. C, PLA signals were counted and plotted as a bar graph (n=5). *P<0.01 vs control (ANOVA). D, To determine the role of Nrg1 (neuregulin 1) released by HMVEC in PM, PM was pretreated with neutralizing anti-Nrg1 and then incubated with HCAECs for 1 h. At the end of treatment, cell lysates were prepared and analyzed for ErbB2 tyrosine phosphorylation and its interaction with Src immunoprecipitation followed by Western blotting. E, HCAECs were incubated with PM or Nrg1…

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  Figure 7.. Endothelial ErbB2 (Erb-B2 receptor tyrosine kinase 2) dimerizes with ErbB4, recruits Src and mediates Nrg1 (neuregulin 1)-dependent eNOS (endothelial nitric oxide synthase) activation during ischemic preconditioning. A, HCAECs were treated with preconditioned medium (PM) for the indicated period, and cell lysates were prepared. An equal amount of protein from cell lysates was immunoprecipitated using anti-ErbB4 antibody, and the immunoprecipitates were analyzed by Western blotting for tyrosine phosphorylation of ErbB4 and associated ErbB2 levels using their specific antibodies. B, To study the in vivo interaction of ErbB2 and ErbB4, HCAECs were treated with PM for 1 or 4 h, and proximity ligation assay (PLA) was performed using anti-ErbB2 and anti-ErbB4 antibodies. Scale bar=20 mm. C, Green foci-proximity signals of ErbB2 and ErbB2 association were counted and plotted as a bar graph. *P<0.01 vs control (ANOVA). D–F, HCAECs were incubated with PM or Nrg1 neutralized-PM for 1 h, and either cell lysates were prepared and analyzed for ErbB4 tyrosine phosphorylation and its interaction with ErbB2 by Western blotting as described in A or subjected to PLA (E) using anti-ErbB2 and…