Abstract 17562: TRAF2 Mediates Tumor Necrosis Factor Induced Mitochondrial Autophagy
Background: Activation of mitochondrial death pathway contributes to adverse left ventricular remodeling in cardiac-restricted TNF transgenic mice (MHCsTNF). Autophagy, a prosurvival pathway, is essential for removal of ubiquitin-tagged damaged mitochondria. TNF receptor associated factor-2 (TRAF2) is a multifunctional protein with E3-ubiquitin ligase activity that coordinates cytoprotective signaling downstream of both TNF receptors. Therefore, we hypothesized that TRAF2 mediates autophagic removal of TNF-damaged mitochondria.
Methods and Results: MHCsTNF hearts and TNF-treated neonatal rat cardiac myocytes (NRCMs) demonstrate upregulation of TRAF2 protein and increased abundance of autophagosome-bound LC3-II, suggesting activation of autophagy. Adenovirally transduced TRAF2 is sufficient to induce GFP-LC3 labeled autophagosomes (3.0-fold over control, P<0.05) that colocalize with MitoTracker Red, suggesting that mitochondria are substrates of TRAF2-induced autophagy. TNF-treated NRCMs demonstrate increased mitochondrial depolarization as measured with mitochondrial JC-1 dye, and TRAF2 knockdown with adenovirally-transduced shRNA results in increased mitochondrial biomass, as assessed with nonyl acridine orange fluorescence, in TNF-treated NRCMs (by 1.6-fold; P<0.01, vs. nontargeting shRNA control), mimicking the effects of autophagy inhibitor, 3-methyladenine. Additionally, TRAF2 overexpression induces a decline in mitochondrial biomass in TNF-treated NRCMs (by 50±17%, P<0.05, vs LacZ control), indicating that TRAF2 is both necessary and sufficient for the autophagic clearance of TNF-damaged mitochondria. Furthermore, TRAF2 knockdown prevents loss of mitochondrial biomass induced by CCCP, an uncoupler, and results in accumulation of mitochondria in untreated NRCMs, suggesting that TRAF2 may mediate a common pathway for the autophagic removal of damaged mitochondria. Finally, transgenic expression of dominant negative TRAF2 in MHCsTNF mice impairs autophagy with accumulation of autophagosome-bound LC3-II and p62.
Conclusion: TRAF2 is essential for autophagic clearance of TNF-damaged mitochondria, which may contribute to TRAF2-mediated cytoprotective signaling.
- © 2012 by American Heart Association, Inc.