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

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.

Keywords: Beta3-adrenergic receptors; Heart failure; Hypertrophy; Neuronal nitric oxide synthase; Reactive oxygen species.

Fig. 1

Norepinephrine and ET-1 induced hypertrophy and β3-AR expression in isolated NRVMs. Cells were treated with or without ET-1 (100 nM) for 48 h, and NE (100 mM) for 72 h. A–B.) Cell surface area was determined by phase contrast microscopy. C.) β3-AR mRNA expression was measured by qPCR, and normalized to baseline expression. *p < 0.05 vs. basal; #p < 0.05 vs. respective agonist control in immunostaining experiment; and #p < 0.05 vs. ET-1 in qPCR experiment.

Fig. 2

BRL restores NOS activity and suppresses hypertrophy-induced superoxide generation via a nNOS-dependent manner in hypertrophied cardiomyocytes. Cells were stimulated for 45 min with BRL in the presence or absence of 72 h NE pretreatment, or LVNIO, nNOS inhibitor. Hypertrophied cells were stimulated at indicated time points to measure A.) NOS activity, B.) superoxide generation in the absence or C–D.) presence of LVNIO. NOS activity was measured by 14C arginine to citrulline conversion and superoxide was measured by Lucigenin Assay. Data is presented as mean ± SEM for 3–6 experiments. *p < 0.001 vs. hypertrophied control; #p < 0.05 vs. LVNIO treated.

Fig. 3

BRL alters phosphorylation of nNOS and eNOS in hypertrophied cardiomyocytes. Cells were stimulated for 45 min with BRL in the presence or absence of 72 h NE pretreatment. Cells were stimulated at indicated time points to measure A.) nNOS-Ser1412 and -Ser847, and B.) eNOS-Ser1177 and -Ser114 phosphorylations. Phosphorylation was measured by Immunoblot analysis with phosphospecific antibodies. Data is presented as mean ± SEM for 3–6 experiments. *p < 0.05 vs. basal.

Fig. 4

BRL administration alters nNOS phosphorylation in mice myocardium after pressure overload. C57BL/6J mice underwent 3-weeks of transverse aortic constriction (TAC) in the presence or absence of BRL administration (0.1 mg/kg/day). Left ventricular myocardium was homogenized and probe against phospho-specific antibodies for A.) nNOS-Ser847 and B) Ser1412. Phosphorylation was measured by western blot analysis. Data is presented as mean ± SEM for three experiments. *p < 0.05 vs. SHAM; #p < 0.05 vs. TAC-BRL.

Fig. 5

nNOS phosphomimetic mutants alter BRL-induced NOS activity and superoxide suppression in hypertrophied cardiomyocytes. Hypertrophied cells were infected with Myc-tagged Sindbis viruses encoding an aspartate (D) or alanine (A) site-mutation for Ser847 or Ser1412 for 24 h prior to 45 min BRL treatment. A.) Infection was determined using Immunoblot analysis with antibodies against Myc protein. B.) NOS activity was measured by 14C arginine to citrulline conversion and C.) superoxide was measured by Lucigenin Assay. GFP Sindbis virus was used to define basal levels of transformation. Data is presented as mean ± SEM for 3–6 experiments. *p < 0.05 vs. respective viral control. #p < 0.05 vs. GFP baseline.

Fig. 6

Akt inhibition blocks β3-AR-induced NOS activity and Ser1412 phosphorylation in hypertrophied cardiomyocytes. Cells were stimulated for 45 min with BRL in the presence or absence of Akt-i, Akt inhibitor, after NE pretreatment. Phosphorylation, NOS activity and superoxide were measured by A–B.) immunoblotting, C.) 14C arginine to citrulline conversion, and D.) lucigenin assay, respectively. E.) Left ventricular myocardium was homogenized and immunoprecipitated with anti-nNOS then probed with anti-Akt. Data is presented as mean ± SEM for three experiments. p < 0.05 vs. basal; #p < 0.05 vs. Akt-i + BRL group.

Fig. 7

G_i/o inhibition blocks β3-induced Ser1412 phosphorylation, NOS activity, and superoxide suppression while partially restoring Ser847. Cells were pretreated with NE for 72 h and stimulated for 45 min with BRL in the presence or absence of Pertussis Toxin, G_i/o inhibitor. Phosphorylation was detected with A.) Ser1412 and B) nNOS-Ser847 phospho-specific antibodies. C.) NOS activity and D.) superoxide generation were measured by 14C arginine to citrulline conversion and Lucigenin assay, respectively. Data is presented as mean ± SEM for three experiments. *p < 0.05 vs. basal; #p < 0.05 vs. BRL alone.

Fig. 8

Proposed signal transduction cascade by which β3-AR activation leads to NOS signaling and ROS scavenging in cardiac myocytes.