Research Project
10402122
Project Summary Pulmonary circulation undergoes adaptive processes critical for survival of the newborn at birth. For a successful postnatal transition, several events come to play, including increased pulmonary blood flow and remodeling of muscularized small-medium size pulmonary arteries (PAs). One important mechanism regulating these processes is the activation of adaptive processes that regulate mitochondrial oxidative stress. However, the mechanism is unknown. For the past five years, through the mentored career development award, we have been exploring the molecular processes that maintain the activity of mitochondrial-localized superoxide dismutase (SOD2) appropriate for ROS levels in mitochondria during postnatal transition. Our findings demonstrate that phosphorylation of heat shock protein-70 (hsp70), a major cytosolic molecular chaperone is a critical mechanism regulating mitochondrial oxidative stress during exposure of the fetal lungs to high oxygen environment. We found that elevated ROS induces the activation of protein kinase-B (AKT1), which in turn phosphorylates Hsp70 on Serine-(S631) to promote the import of SOD2 into the mitochondria in response to stress. We also found that when phosphorylated on S631, Hsp70 recruits Obg-like ATPase-1 (OLA1) to Hsp70-SOD2 complexes and the binding of OLA1 drives the mitochondrial SOD2 import by antagonizing CHIP-mediated ubiquitination and proteasomal degradation of Hsp70 and its downstream target, SOD2. Disruption of AKT-mediated phosphorylation of Hsp70S631 increases mitochondrial ROS levels. We also observed that AKT activity is significantly decreased in persistent pulmonary hypertension of the newborn (PPHN) and contributes to impaired vasorelaxation in the disease. However, all these studies were done in vitro, therefore it is important to verify this critical mechanism in vivo. Our working hypothesis is that dynamic phosphorylation of Hsp70S631 by AKT is a novel mechanism promoting postnatal adaptation of pulmonary circulation at birth through mechanisms regulating mitochondrial import of SOD2 and redox balance. To test this hypothesis in an in vivo model, we will test the effect of disrupting OLA1 binding to hsp70 or AKT-mediated phosphorylation of Hsp70 in vivo using Sprague Dawley (SD) rats. We have generated a series of cell-permeable decoy peptide inhibitors of AKT and OLA1. For this, 4 weeks old rats will be treated with GSG (Akt inhibitor), or GLGIV (OLA1 inhibitor) peptide for 4 weeks in normoxic conditions. Echocardiographic measurements of tricuspid regurgitant jets (TR jets) and cardiac function will be performed. We will also quantify mitochondrial ROS levels using mitoSOX staining and HPLC. Vasorelaxation responses of PA in the treated and untreated groups to physiological stimuli in an ex-vivo preparations will be determined. It is anticipated that interruptions of hsp70 interactions with Akt or OLA1 will induce vascular remodeling and PPHN. While CHIP inhibition would increase vasorelaxation response of PAs to stimuli. Thus, increasing AKT activity may be a potential therapeutic option in the treatment of PPHN.
