Abstract 17362: Regulation of Oxidative Stress and Apoptotic Signaling in Cardiac Myocytes by G Protein-Coupled Receptor Kinase-2: The Role of NADPH Oxidases

Abstract

Oxidative stress due to generation of reactive oxygen species (ROS) is an important mechanism of cardiac myocyte death following ischemia-reperfusion (I-R) injury and can lead to heart failure. NADPH oxidases (Nox) are major cellular enzymatic sources for superoxide (O 2 ) radical production and Nox2 and Nox4 are the two predominant isoforms expressed in the heart. The mechanisms by which these Nox isoforms are regulated in the heart after various stresses are unclear. G protein-coupled receptor kinase-2 (GRK2) is a critical regulator of β-adrenergic signaling and cardiac function. We hypothesized that GRK2 may play a role in regulating cardiac myocyte oxidative stress and apoptosis. Cardiac myocytes were exposed to 3 hours of hypoxia (2% O 2 , 5% CO 2 , 93% N 2 ) followed by 3 hours of reoxygenation (5% CO 2 , 95% air). Hypoxia-reoxygenation (H-R) led to significant 3.5-fold upregulation of GRK2 activity as well as ROS production (4.7±0.7 vs 1.1±0.4 nmoles/min/mg protein) and apoptosis measured by caspase-3 activity (6.2±1.5 vs 1.8±0.6 fluorimetric units/mg protein) compared to normoxic controls. Interestingly, 4-fold overexpression of GRK2 by adenoviral infection led to an identical 3-fold increase in ROS production under basal conditions (3.5±1.2 vs 1.3±0.5 nmoles/min/mg protein with null vector controls) which was completely inhibited by pre-treatment with 10μM apocyanin, a Nox inhibitor. Inhibition of GRK2 by adenoviral-mediated expression of βARKct led to a significant reduction in ROS production and caspase 3 activity following H-R which was similar to that observed by knocking down Nox4. Immunoprecipitated Nox4 demonstrated a strong total serine phosphorylation under H-R conditions. Finally, confocal imaging of Nox4 and GRK2 in cardiac myocytes exposed to H-R demonstrated a primarily peri-nuclear co-localization that was absent under control normoxic conditions. These data provide evidence that GRK2 may be an important regulator of oxidative stress via NADPH oxidases in cardiac myocytes. Further investigation of this novel interaction between GRK2 and Nox4 will provide additional insight into the potential role of GRK2 as a regulator of oxidative stress and apoptotic signaling in the heart.