Abstract 92: The Protective Effect of β3-Adrenergic Signaling Occurs via Differential Phosphorylation of nNOS in Cardiac Myocytes

Abstract

Objective: The aim of this study is to identify the mechanisms involved in β3-adrenoreceptor (β3-AR)-dependent cardiac protection via nNOS signaling in cardiomyocytes in the setting of hypertrophy. Background: β3-AR and its downstream signaling pathways are recognized as novel modulators of heart function. Unlike β1- and β2-ARs, β3-ARs are stimulated at high catecholamine concentrations and induce negative inotropic effects, it serves as a “brake” to protect the heart from catecholamine overstimulation. We previously showed that β3-agonism restored left ventricular function, generated nitric oxide, and suppressed reactive oxygen species in mouse myocardium after pressure overload. Interestingly, cardioprotection was lost after acute nNOS inhibition and in nNOS -/- animals. Methods: Neonatal rat ventricular cardiomyocytes (NRVMs) were isolated from 2-4 day old Sprague-Dawley pups. Cells were treated with hypertrophic agents (angiotensin II, endothelin-1, and norepinephrine), the specific β3-AR agonist (BRL-37433),and phosphometic Sindbis viruses for nNOS. Results: Forty-eight hours of ET-1 (100nM) or 72 hours of NE (100μ M) treatment increased cell size and β3-AR mRNA expression vs. untreated cells. In hypertrophied cardiomyocytes, BRL (75nM) reduced cell size and induced NOS activity, nNOS phosphorylation at stimulatory site Ser1412, dephosphorylation of deactivation site Ser847, and ROS suppression. BRL-dependent NOS activity and ROS suppression were both attenuated by the nNOS inhibitor L-VNIO. NOS activity was also attenuated by phosphomemetic mutants Ser1412A (constitutively dephosphorylated) and Ser847D (constitutively phosphorylated). In addition, G i/o and Akt inhibition suppressed BRL-induced nNOS-Ser1412 phosphorylation and NOS activity. Conclusion: Our data suggest that BRL regulates β3-specific myocardial NOS activity via alterations in nNOS phosphorylation in isolated hypertrophied myocytes and failing hearts of murine animals. This is the first study to demonstrate a role for nNOS phosphorylation as a key factor in cardiac myocyte and β3-AR signaling. These results contribute significantly to our understanding of the cardiac protection.