Abstract 17485: Novel Role of NADPH Oxidase Subunit p67phox C-terminus Region in Mediation of α1-Adrenergic Receptor-Induced Cardiac Hypertrophy
We previously reported that α1-adrenergic receptor (α1AR)-induced hypertrophy in adult rat ventricular myocytes (ARVM) was mediated via activation of MEK1/2-ERK1/2 cascade. The hypertrophy was attenuated by NADPH oxidase (NOX) inhibitors and a dominant-negative (DN) mutant (V204A) of NOX subunit p67phox that completely inhibits NOX activity of reactive oxygen species (ROS) generation, suggesting the roles of NOX and ROS in α1AR signaling. We also showed that cardiac-specific overexpression of this V204A mutant attenuated chronic pressure-overload-induced hypertrophy in transgenic mouse heart. In this study, we further tested the potential ROS-independent role of p67phox in α1AR signaling using a truncated mutant (1-235) of p67phox, which lacks the p67phox C-terminus region and is known not affecting NOX activity of ROS generation. Adenoviral constructs containing β-galactosidase (AdLacZ) or p67phox mutant 1-235 (Ad235) were expressed in ARVM, and their effects on α1AR stimulation (1 μM norepinephrine + 2 μM propranolol)-induced hypertrophy and activation of MEK-ERK cascade was studied. Overexpression of Ad235 significantly attenuated α1AR -induced activation of MEK1/2 (58±7% ↓) and ERK1/2 (65±8% ↓) vs. AdLacZ as measured by Western blots. Furthermore, α1AR-induced hypertrophy (48 hr) was also prevented by Ad235 as measured by 3H-leucine incorporation (71±5% ↓) and fetal gene ANF mRNA expression (54±11% ↓) using qRT-PCR. These data confirmed that p67phox mediates α1AR-induced hypertrophy and MEK1/2-ERK1/2 activation, also suggested an interesting notion that the reported non-ROS generating region of p67phox C-terminus may also contribute to α1AR-induced hypertrophic signaling in ARVM. In addition, immunoprecipitation of ARVM lysates with anti-p67phox antibody followed by Western blotting with anti-ERK or anti-MEK antibodies showed the co-localization of p67phox with ERK and MEK kinases, suggesting the direct interaction between MEK-ERK cascade and p67phox in ARVM. Taken together, our data showed that the non-ROS generating region of p67phox C-terminus mediates α1AR-induced hypertrophy in ARVM, suggesting a novel role of p67phox as a signaling molecule in α1AR-induced hypertrophy via a potential ROS-independent mechanism.
- Cardiac hypertrophy
- Reactive oxygen intermediates
- Signal transduction
- Alpha-adrenergic receptor blockers
- © 2011 by American Heart Association, Inc.