Abstract 13357: Tumor Necrosis Factor Associated Factor 2 Mediates Cardiac Protection Following Ischemia Reperfusion Injury in an NF-kB Dependent Manner
Background: Previous studies have shown that tumor necrosis factor signaling is cytoprotective in the heart following ischemia/reperfusion (I/R) injury; however, the mechanism(s) for this effect is not known. We have shown that TNF receptor associated factor 2 (TRAF2), a scaffolding protein downstream from TNF receptor signaling, confers cytoprotective effects following I/R injury, mimicking exogenous TNF. Accordingly, th.
purpose of this study was to determine the mechanisms responsible for the cytoprotective effects of TRAF2.
Methods: As an initial approach to evaluate a potential role of NF-κB, lines of mice with cardiac-restricted overexpression of TRAF2 (MHC-TRAF2) were backcrossed with mice with cardiac-restricted overexpression of a dominant-negative form of IkB (DNIkB), or with p50 knockout mice. Hearts were subjected to ischemia (30 min) reperfusion (60 min) injury ex vivo using a buffer perfused Langendorff system. LV injury was assessed by measuring % recovery of LV developed pressure compared to baseline, creatine kinase (CK) release, and uptake of Evans Blue dye by injured myocytes. Western blot analysis was performed on nuclear and cytoplasmic fractions for NF-κB family members (p65/RelA, p50, p52, RelB, and c-Rel). Oligonucleotide pull-down assays were performed on the nuclear fractions with consensus NF-κB binding site-containing oligos.
Results: Hearts from MHC-TRAF2/MHC-DNIkB mice demonstrated a significant (p<0.05) decrease in % recovery of LV developed pressure after I/R injury as well as a significant increase (p<0.05) in CK release and Evans blue uptake compared to hearts expressing MHC-TRAF2 alone. Hearts from MHC-TRAF2/p50 KO mice also demonstrated a significant decrease in cardioprotection compared to MHC-TRAF2 mouse hearts, albeit to a lesser extent. Hearts from MHC-TRAF2 mice showed a significant increase in nuclear localization of p50 and small increase in p65. Oligonucleotide pull-down assays also demonstrate an increase in p50 (but not p65) binding to consensus NF-κB oligos.
Conclusion: Taken together, these studies suggest for the first time that NF-κB mediated signaling is required for the cytoprotective effects of TRAF2 following I/R injury, likely through p50-mediated signaling.
- © 2012 by American Heart Association, Inc.