Abstract 19478: The Protective Effect of Beta3-Adrenergic Signaling Occurs via Differential Phosphorylation of nNOS in Cardiac Myocytes
Rationale: Stimulation of β3-adrenoreceptor (β3-AR) signaling improves left ventricular function via a nNOS/NO-dependent manner in hypertrophied and failing animal hearts. nNOS, which is expressed in cardiac myocytes, is regulated by modification of stimulatory phosphorylation residue Ser1412 and inhibitory residue Ser847. However, how differential phosphorylation of these residues contribute to cardiomyocyte β3-AR-mediated NOS activation and signaling has yet to be explored.
Objective: The aim of this study is to identify the molecular mechanisms involved in β3-AR-dependent regulation of nNOS activation and its downstream signaling in cardiomyocytes in the setting of hypertrophy.
Methods and Results: In neonatal rat ventricular cardiomyocytes isolated from 4 day old pups, 48 hours of ET-1or 72 hours of norepinephrine lead to increased cell size and β3-AR mRNA. In hypertrophied cells, specific β3-AR agonist BRL-37433 induced NOS activity, nNOS phosphorylation at site Ser1412, dephosphorylation of site Ser847, and ROS suppression. BRL-dependent NOS activity and ROS suppression were attenuated by the nNOS inhibitor L-VNIO. NOS activity was also attenuated in phosphomimetic mutants Ser1412A (constitutively dephosphorylated) and Ser847D (constitutively phosphorylated). However, BRL failed to reduce ROS in the presence of Ser847D. In addition, Akt inhibition decreased BRL-induced Ser1412 phosphorylation and NOS activity, while Gi/o blockade altered BRL-regulation of both phosphorylation sites, NOS activity, and ROS reduction. In vivo, BRL administration completely dephosphorylated Ser847 and moderately increased Ser1412 phosphorylation in mice hearts under pressure overload.
Conclusion: Our data suggest that β3-AR regulates myocardial NOS activity and ROS via alterations in nNOS phosphorylation in hypertrophied myocytes and failing hearts. This is the first study to demonstrate a role for nNOS phosphorylation as a key factor in cardiomyocyte and β3-AR signaling. These results contribute significantly to our understanding the protective properties of β3-AR signaling during cardiac sympathetic overdrive, and will ultimately aid in drug discoveries that target molecular mechanisms to treat heart failure.
- © 2012 by American Heart Association, Inc.