Abstract 90: Akt Mediated Mitochondrial Dysfunction Involves Mitochondrial Endothelial Nitric Oxide Synthase Translocation
Pulmonary arterial hypertension (PH) is a fatal disease characterized by uncontrolled pulmonary vascular cell proliferation. Mitochondrial dysfunction (MD) of pulmonary endothelial cells (EC) was shown to be one of the primary events implicated into proliferative, apoptosis resistant cell phenotype. However, the particular molecular mechanisms responsible for MD remain unclear. The development of PH in patients is associated with severe nitrosative stress, leading to post-translational protein modifications. Thus, we have recently found that Akt is susceptible to nitration of tyrosine Y350 residue. We hypothesize that nitration of Akt induces activation of Akt signaling and contributes to the development of MD. Nitrosative stress in EC was initiated by eNOS uncoupler ADMA or peroxynitrite donor SIN-1 and resulted in significant Akt nitration and activation (1.5±0.1 fold control; p=0.007; N=3-4), as well as activation of anti-apoptotic (BAD phosphorylation), and proliferative (mTOR phosphorylation) signaling cascades. Increased Akt signaling induced phosphorylation of eNOS at serine S615 (1.47±0.08 fold control, p=0.005, N=3) and S1177 (0.22±0.04 vs. 0.47±0.07, p=0.029, N=3). Phosphorylation of eNOS resulted in its translocation to mitochondria (3± 0.3 fold control; p=0.003; N=3) which, in turn, significantly decreased basal mitochondrial respiration (oxygen consumption rate, pmol/min: untransfected cells 1022±96 vs. mitochondrial targeted phospho-mimetic eNOS mutants S615D 102±9 and S1177D 117±7, p<0.001, N=4-6), perhaps due to previously reported inhibitory effect of NO on mitochondrial respiratory chain. Finally, we have created an anti-oxidant conjugated “shielding” peptide that, by shielding the Akt nitration site, is capable to prevent Akt activation. Indeed, pre-treatment with shielding peptide (100μg/ml, 30min) completely abolished SIN-1 induced nitration of Akt in EC. We conclude that Akt nitration may contribute to proliferative/apoptosis resistant EC phenotype through pathological activation of Akt signaling and Akt mediated mitochondrial eNOS translocation. Besides, our novel shielding peptide based therapeutic strategy opens new avenues in MD prevention.
Author Disclosures: R. Rafikov: None. O. Rafikova: None. X. Sun: None. S. Aggarwal: None. S.M. Black: None.
This research has received full or partial funding support from the American Heart Association, National Center.
- © 2015 by American Heart Association, Inc.