Abstract 17320: Signaling Mechanisms Involved In Beta3-adrenergic Receptor Regulation of Nnos in Isolated Cardiomyocytes

Abstract

Beta3 -adrenergic (beta3-AR) receptors play a pivotal role in modulating cardiac function, though their precise role in the heart remains controversial. We have recently demonstrated that beta3-mediated cardiac protection via negative inotropy is lost in nNOS-/- animals under pressure overload as compared with WT. However, the exact manner in which beta3-AR activation regulates nNOS signaling to stimulate NOS activity at the cardiomyocyte level is unknown. In this study we used a specific beta3-AR agonist, BRL37344 (BRL), to assess the role of beta3-AR in nNOS regulation in hypertrophied isolated neonatal rat ventricular cardiomyocytes (NRVM). To induce hypertrophy we pretreated cells with norepinephrine for 72 hours, which resulted in a 70% increase in cell size and a 25% increase in beta3-AR mRNA expression as compared with non-hypertrophied cells, analyzed by immunocytochemistry and real-time PCR. In hypertrophied cardiomyocytes, BRL administration lead to a time-dependent 5-fold increase in NOS activity, which was significantly suppressed by nNOS specific inhibitor LVNIO. NOS activity was measured by the arginine-to-citrulline conversion assay. BRL also cause a time-dependent 1.5-fold increase in nNOS phosphorylation at positive regulatory site Ser1416, dephosphorylation of negative regulatory site Ser847, and decreased superoxide generation as compared with unstimulated control. In addition BRL induced phosphorylation eNOS-Ser114, which indicates eNOS deactivation. Pretreatment with pertussis toxin (PTX) suppressed BRL-induced nNOS-Ser1416 phosphorylation, nNOS-Ser847 dephosphorylation, and NOS activity, and unaltered superoxide generation, suggesting Gi/o dependency. Taken together, our data suggest that BRL regulates NOS signaling in ventricular cardiomyocytes via phosphorylation regulation of nNOS. To our knowledge this is first study to demonstrate a role for nNOS phosphorylation as a key factor in beta3-AR signaling. These results contribute significantly to our understanding the negative inotropic properties of myocardial beta3-AR at cellular levels during cardiac sympathetic overstimulation, and will ultimately aid in drug discoveries that target the molecular mechanisms associated heart failure.