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

• Published in: Journal of molecular and cellular cardiology Vol. 62; pp. 8 - 17
• Main Authors: Watts, Vabren L, Sepulveda, Fernando M, Cingolani, Oscar H, Ho, Alice S, Niu, Xiaolin, Kim, Rosa, Miller, Karen L, Vandegaer, Koenraad, Bedja, Djahida, Gabrielson, Kathleen L, Rameau, Gerald, O'Rourke, Brian, Kass, David A, Barouch, Lili A
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-09-2013
• Subjects: Adrenergic beta-Agonists - pharmacology; Animals; Antioxidants - pharmacology; Beta3-adrenergic receptors; Cardiovascular; Cells, Cultured; Ethanolamines - pharmacology; Heart failure; Hypertrophy; Immunoprecipitation; Male; Mice; Mice, Inbred C57BL; Muscle Cells - drug effects; Muscle Cells - metabolism; Neuronal nitric oxide synthase; Nitric Oxide Synthase Type I - metabolism; Phosphorylation; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, Adrenergic, beta-3 - metabolism; Heart failure; Neuronal nitric oxide synthase; Reactive oxygen species; Beta3-adrenergic receptors; Hypertrophy
• ISSN: 0022-2828; 1095-8584
• DOI: 10.1016/j.yjmcc.2013.04.025

Abstract Rationale Stimulation of β3-adrenoreceptors (β3-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 β3-AR protective signaling has yet to be explored. Objective We tested the hypothesis that β3-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 β3-AR gene expression. Co-administration of the β3-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 Gi/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 β3-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 β3-AR receptor stimulation.