Abstract 18635: G Protein-Coupled Receptor Kinase 2 Regulates Cellular Oxidative Stress and Survival
Cardiac myocyte oxidative stress and apoptosis are considered important mechanisms for the development of heart failure (HF). Chronic HF is characterized by increased levels of circulating catecholamines to augment cardiac output. Long-term stimulation of myocardial β-adrenergic receptors (β-ARs) is known to be deleterious in cardiac myocytes, however, the potential mechanisms underlying increased cell death are unclear. We hypothesize that G protein-coupled receptor kinase-2 (GRK2), a critical regulator of myocardial β-AR signaling and function, plays an important role in mediating cellular oxidative stress and apoptotic cell death in response to β-agonist stimulation. Stimulation of H9c2 cells with a non-selective β-agonist, isoproterenol (ISO) [10-5M] led to a 3-fold increase in oxidative stress/reactive oxygen species (ROS) generation as quantified by Mitosox staining and a significant increase in apoptosis measured by TUNEL and caspase-3 activity compared to PBS-treated controls. There was also a 2.5-fold increase in NADPH oxidase-4 (Nox4) expression but no change in Nox2, the primary NADPH isoforms and major sources of ROS generation in cardiac myocytes. ISO stimulation led to a 3-fold increase in GRK2 activity, similar to the myocardial GRK2 activity present in chronic HF. A 3-fold adenoviral-mediated overexpression of GRK2 led to similar increases in ROS production and apoptosis as seen with ISO stimulation. The increases in oxidative stress by either ISO stimulation or GRK2 overexpression were abolished by pre-treatment with apocynin, a non-specific Nox inhibitor, or siRNA-mediated knockdown of Nox4. Direct activation of adenylyl cyclase with forskolin did not lead to increased ROS production suggesting that β-agonist-induced oxidative stress is likely mediated by activation of GRK2 and increased Nox4 activity and not cAMP/PKA. Adenoviral-mediated expression of the GRK2 inhibitor, βARKct, inhibited Nox4 upregulation, ROS production and apoptosis in response to ISO stimulation. These data demonstrate that GRK2 may play an important role in regulating oxidative stress and apoptosis in the heart and provides another mechanism for the beneficial effects of cardiac-targeted GRK2 inhibition.
- © 2012 by American Heart Association, Inc.