Anti-hypertrophic and anti-oxidant effect of beta3-adrenergic stimulation in myocytes requires differential neuronal NOS phosphorylation

Rationale: Stimulation of beta 3-adrenoreceptors (beta 3-AR) blunts contractility and improves chronic left ventricular function in hypertrophied and failing hearts in a neuronal nitric oxide synthase (nNOS) dependent manner. nNOS can be regulated by post-translational modification of stimulatory phosphorylation residue Ser1412 and inhibitory residue Ser847. However, the role of phosphorylation of these residues in cardiomyocytes and beta 3-AR protective signaling has yet to be explored. Objective: We tested the hypothesis that beta 3-AR regulation of myocyte stress requires changes in nNOS activation mediated by differential nNOS phosphorylation. Methods and results: Endothelin (ET-1) or norepinephrine induced hypertrophy in rat neonatal ventricular cardiomyocytes (NRVMs) was accompanied by increased beta 3-AR gene expression. Co-administration of the beta 3-AR agonist BRL-37433 (BRL) reduced cell size and reactive oxygen species (ROS) generation, while augmenting NOS activity. BRL-dependent augmentation of NOS activity and ROS suppression due to NE were blocked by inhibiting nNOS (L-VNIO). BRL augmented nNOS phosphorylation at Ser1412 and dephosphorylation at Ser847. Cells expressing constitutively dephosphorylated Ser1412A or phosphorylated Ser847D nNOS mutants displayed reduced nNOS activity and a lack of BRL modulation. BRL also failed to depress ROS from NE in cells with nNOS-Ser847D. Inhibiting Akt decreased BRL-induced nNOS-Ser1412 phosphorylation and NOS activation, whereas G(1/o) blockade blocked BRL-regulation of both post-translational modifications, preventing enhancement of NOS activity and ROS reduction. BRL resulted in near complete dephosphorylation of Ser847 and a moderate rise in Ser1412 phosphorylation in mouse myocardium exposed to chronic pressure-overload. Conclusion: beta 3-AR regulates myocardial NOS activity and ROS via activation of nNOS involving reciprocal changes in phosphorylation at two regulatory sites. These data identify a novel and potent anti-oxidant and anti-hypertrophic pathway due to nNOS post-translational modification that is coupled to beta 3-AR receptor stimulation. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.