Abstract
OBJECTIVE: Remote ischemic preconditioning (RIPC) is an intervention process where the application of multiple cycles of short ischemia/reperfusion (I/R) in a remote vascular bed provides protection against I/R injury. However, the identity of the specific RIPC factor and the mechanism by which RIPC alleviates I/R injury remains unclear. Here, we have investigated the identity and the mechanism by which the RIPC factor provides protection. APPROACH AND RESULTS: Using fluorescent in situ hybridization and immunofluorescence, we found that RIPC induces Nrg1β expression in the endothelial cells, which is secreted into the serum. Whereas, RIPC protected against myocardial apoptosis and infarction, treatment with neutralizing-Nrg1 antibodies abolished the protective effect of RIPC. Further, increased superoxide anion generated in RIPC is required for Nrg1 expression. Improved myocardial perfusion and nitric oxide production were achieved by RIPC as determined by contrast echocardiography and electron spin resonance. However, treatment with neutralizing-Nrg1β antibody abrogated these effects, suggesting Nrg1β is a RIPC factor. ErbB2 (Erb-B2 receptor tyrosine kinase 2) is not expressed in the adult murine cardiomyocytes, but expressed in the endothelial cells of heart which is degraded in I/R. RIPC-induced Nrg1β interacts with endothelial ErbB2 and thereby prevents its degradation. Mitochondrial Trx2 (thioredoxin) is degraded in I/R, but rescue of ErbB2 by Nrg1β prevents Trx-2 degradation that decreased myocardial apoptosis in I/R. CONCLUSIONS: Nrg1β is a RIPC factor that interacts with endothelial ErbB2 and prevents its degradation, which in turn prevents Trx2 degradation due to phosphorylation and inactivation of ATG5 (autophagy-related 5) by ErbB2. Nrg1β also restored loss of eNOS (endothelial nitric oxide synthase) function in I/R via its interaction with Src.
Highlights
Nrg1β is a Remote ischemic preconditioning (RIPC) factor that interacts with endothelial ErbB2 and prevents its degradation, which in turn prevents Trx[2] degradation due to phosphorylation and inactivation of ATG5 by ErbB2
Since Nrgs are secreted by endothelial cells in response to I/R of the heart,[13] we determined whether RIPC would induce Nrg1β expression in a remote vascular bed that is subjected to short-episodes of I/R
We determined whether microvascular endothelial cells would secrete Nrg1β during short I/R episodes similar to that applied in RIPC
Summary
Recombinant human Nrg1β1 active domain (catalog No 396-HB) and neutralizing anti-Nrg[1] antibody (catalog No AF-396-NA) were bought from R&D Systems. Anti-Nrg[1] (to detect full-length Nrg[1], catalog No 2573), anti-ErbB2 (catalog No 4290), anti-phospho-eNOS (Ser1177; catalog No 9571), anti-phospho-Src (Tyr[416]; catalog No 2101), anti-Ambra[1] (catalog No 24907), and anti-BCLN1 (catalog No 3495) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Antiheregulin antibody (catalog No RB-276-P0), anti-ErbB4 neutralizing antibody (catalog No MA5-13016), and antiErbB4 antibody for proximity ligation assay (PLA; catalog No MA1-861) were purchased from Pierce Biotechnology (Rockford, IL). Anti-ErbB2 antibody (catalog No GTx117479) used for PLA was bought from GeneTex. Anti-Src antibody (catalog No 05-184) and anti-phospho-Tyr antibody (catalog No 05-321) were purchased from Millipore. Construction of Ad-dnSrc and production of antiphosphoeNOS (Tyr 83) antibodies were described previously.[30,31] pcDNA3-ErbB2 plasmid[32] was a gift from Mein-Chie Hung and was obtained through Addgene (plasmid No 16257)
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