Abstract 12981: Reactive Oxygen Species (ROS) Regulate 5’ Amp-activated Protein Kinase (AMPK) Phosphorylation and Decrease Muscle Ring Finger Protein 1 (MuRF1) Expression in Chronic Pressure Overload-induced Cardiac Hypertrophy

We recently reported a transgenic (Tg) mouse model with cardiac-specific overexpression of a dominant-negative (DN) mutant of NADPH oxidase (NOX) subunit p67 phox that inhibits NOX-mediated ROS production in the heart. The transverse aortic constriction (TAC)-induced cardiac hypertrophy in the wild type (WT) mice was significantly attenuated in this DN-p67 Tg mice. AMPK is a metabolic switch regulator and is recently reported to regulate the transcription of an ubiquitin ligase MuRF1, which was shown to involve in cardiac growth and hypertrophy. In order to understand the signaling roles of ROS and AMPK in cardiac hypertrophy, we thus hypothesized that increased cardiac ROS level inhibits AMPK phosphorylation (p), which decreases MuRF1 expression that leads to cardiac growth or hypertrophy. In our studies, TAC or sham surgery was performed in both DN-p67 Tg and WT mice at 12-week of age. Mouse hearts were harvested 4-week post TAC for histological and biochemical analysis. Western blot results show that the expression levels of p-AMPKα and Murf1 decrease 52±10% and 31±9%, respectively, in the WT-TAC hearts compared to that of WT-sham; whereas TAC-induced decrease expression of p-AMPKα and Murf1 in Tg hearts was significantly attenuated compared to that of WT-TAC. The Tg-TAC mice also show significant attenuation of the increase of membrane lipid peroxidation by 4-hydroxynonenal staining in heart tissue sections (1.35±0.1 fold ↑ in Tg-TAC vs. 1.74±0.16 fold ↑ in WT-TAC), indicating less ROS production in the Tg-TAC myocardium compared to that of WT-TAC. To further test our hypothesis in vitro , we used primary cultured adult rat ventricular myocytes (ARVM) subjected to nutrient deprivation (4-hr culture in HBSS buffer), which induces AMPKα phosphorylation (1.7±0.3 fold ↑ vs. in normal culture medium) and also increases MuRF1 expression by Western blotting. When ARVM was co-treated with 0.5 mM H 2 O 2 , the increase of p-AMPKα by nutrient deprivation was abolished, confirming that increase of ROS decreases AMPK activation in ARVM in vitro . In summary, our data show that pressure-overload-induced cardiac oxidative stress inhibits AMPK activation and MuRF1 expression, which may contribute to TAC-induced cardiac hypertrophy.