Abstract 12198: TRAF2 Coordinates With PARKIN to Mediate Mitochondrial Autophagy in Cardiomyocytes
Background: Mitochondrial permeabilization provokes cardiomyocyte death, and causes adverse left ventricular remodeling in cardiac-restricted TNF transgenic mice (MHCsTNF). Autophagy, a lysosomal degradative pathway, is essential for removal of ubiquitin-tagged damaged mitochondria. Our prior work suggests that TNF receptor associated factor-2 (TRAF2), a multifunctional protein with E3 ubiquitin ligase activity, facilitates removal of damaged mitochondria. The role of TRAF2 vis-à-vis PARKIN, another E3 ubiquitin ligase essential for mitophagy, is unknown.
Methods and Results: MHCsTNF hearts and TNF-treated neonatal rat cardiac myocytes (NRCMs) demonstrate upregulation of both TRAF2 and PARKIN, with increased evidence for mitochondrial autophagy and reduced mitochondrial mass. To evaluate the role of TRAF2 and PARKIN in autophagic removal of damaged mitochondria, we employed gain-of-function and loss-of-function approaches; and assessed mitochondrial mass with nonyl-acridine orange fluorescence and mitochondrial DNA content; and mitochondrial depolarization by JC-1. Treatment of NRCMs with TNF or an uncoupling agent, CCCP, provokes mitochondrial depolarization with increased colocalization of endogenous TRAF2 with adaptor protein, p62, ubiquitin, and mitochondrial proteins, within LC3-bound autophagosomes. TRAF2 also colocalizes and interacts with PARKIN (by FRET analysis) on damaged mitochondria. Knockdown of endogenous TRAF2 results in increased mitochondrial mass with accumulation of depolarized mitochondria, mimicking the effects of PARKIN knockdown and of treatment with autophagy inhibitor, 3-methyladenine. Exogenous expression of wild type TRAF2, but not of mutants with impaired E3 ubiquitin ligase activity, is sufficient to partially restore removal of depolarized mitochondria in CCCP-treated NRCMs, in the setting of PARKIN knockdown. Analogously, exogenous PARKIN is sufficient to partially overcome TRAF2 deficiency; suggesting overlapping roles for both as E3 ubiquitin ligases in mitochondrial autophagy.
Conclusion: TRAF2 plays an essential role in concert with PARKIN as an E3 ubiquitin ligase for mitophagy, which may contribute to TRAF2-induced cytoprotective signaling.
- © 2013 by American Heart Association, Inc.