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

RATIONALE: Stimulation of ß3-adrenoreceptors (ß3-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 ß3-AR protective signaling has yet to be explored. OBJECTIVE: We tested the hypothesis that ß3-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 ß3-AR gene expression. Co-administration of the ß3-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: ß3-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 ß3-AR receptor stimulation.

Beneficial cardiac effects of caloric restriction are lost with age in a murine model of obesity.

Obesity is associated with increased diastolic stiffness and myocardial steatosis and dysfunction. The impact of aging on the protective effects of caloric restriction (CR) is not clear. We studied 2-month (younger) and 6-7-month (older)-old ob/ob mice and age-matched C57BL/6J controls (WT). Ob/ob mice were assigned to diet ad libitum or CR for 4 weeks. We performed echocardiograms, myocardial triglyceride assays, Oil Red O staining, and measured free fatty acids, superoxide, NOS activity, ceramide levels, and Western blots. In younger mice, CR restored diastolic function, reversed myocardial steatosis, and upregulated Akt phosphorylation. None of these changes was observed in the older mice; however, CR decreased oxidative stress and normalized NOS activity in these animals. Interestingly, myocardial steatosis was not associated with increased ceramide, but CR altered the composition of ceramides. In this model of obesity, aging attenuates the benefits of CR on myocardial structure and function.

Cardioprotective effect of beta-3 adrenergic receptor agonism: role of neuronal nitric oxide synthase.

OBJECTIVES: The aim of this study was to determine whether activation of ß3-adrenergic receptor (AR) and downstream signaling of nitric oxide synthase (NOS) isoforms protects the heart from failure and hypertrophy induced by pressure overload. BACKGROUND: ß3-AR and its downstream signaling pathways are recognized as novel modulators of heart function. Unlike ß1- and ß2-ARs, ß3-ARs are stimulated at high catecholamine concentrations and induce negative inotropic effects, serving as a "brake" to protect the heart from catecholamine overstimulation. METHODS: C57BL/6J and neuronal NOS (nNOS) knockout mice were assigned to receive transverse aortic constriction (TAC), BRL37344 (ß3 agonist, BRL 0.1 mg/kg/h), or both. RESULTS: Three weeks of BRL treatment in wild-type mice attenuated left ventricular dilation and systolic dysfunction, and partially reduced cardiac hypertrophy induced by TAC. This effect was associated with increased nitric oxide production and superoxide suppression. TAC decreased endothelial NOS (eNOS) dimerization, indicating eNOS uncoupling, which was not reversed by BRL treatment. However, nNOS protein expression was up-regulated 2-fold by BRL, and the suppressive effect of BRL on superoxide generation was abrogated by acute nNOS inhibition. Furthermore, BRL cardioprotective effects were actually detrimental in nNOS(-/-) mice. CONCLUSIONS: These results are the first to show in vivo cardioprotective effects of ß3-AR-specific agonism in pressure overload hypertrophy and heart failure, and support nNOS as the primary downstream NOS isoform in maintaining NO and reactive oxygen species balance in the failing heart.

Restoring leptin signaling reduces hyperlipidemia and improves vascular stiffness induced by chronic intermittent hypoxia.

Chronic intermittent hypoxia (IH) during sleep can result from obstructive sleep apnea (OSA), a disorder that is particularly prevalent in obesity. OSA is associated with high levels of circulating leptin, cardiovascular dysfunction, and dyslipidemia. Relationships between leptin and cardiovascular function in OSA and chronic IH are poorly understood. We exposed lean wild-type (WT) and obese leptin-deficient ob/ob mice to IH for 4 wk, with and without leptin infusion, and measured cardiovascular indices including aortic vascular stiffness, endothelial function, cardiac myocyte morphology, and contractile properties. At baseline, ob/ob mice had decreased vascular compliance and endothelial function vs. WT mice. We found that 4 wk of IH decreased vascular compliance and endothelial relaxation responses to acetylcholine in both WT and leptin-deficient ob/ob animals. Recombinant leptin infusion in both strains restored IH-induced vascular abnormalities toward normoxic WT levels. Cardiac myocyte morphology and function were unaltered by IH. Serum cholesterol and triglyceride levels were significantly decreased by leptin treatment in IH mice, as was hepatic stearoyl-Coenzyme A desaturase 1 expression. Taken together, these data suggest that restoring normal leptin signaling can reduce vascular stiffness, increase endothelial relaxation, and correct dyslipidemia associated with IH.

Beta 3-adrenoreceptor regulation of nitric oxide in the cardiovascular system.

The presence of a third beta-adrenergic receptor (beta 3-AR) in the cardiovascular system has challenged the classical paradigm of sympathetic regulation by beta1- and beta2-adrenergic receptors. While beta 3-AR's role in the cardiovascular system remains controversial, increasing evidence suggests that it serves as a "brake" in sympathetic overstimulation - it is activated at high catecholamine concentrations, producing a negative inotropic effect that antagonizes beta1- and beta2-AR activity. The anti-adrenergic effects induced by beta 3-AR were initially linked to nitric oxide (NO) release via endothelial NO synthase (eNOS), although more recently it has been shown under some conditions to increase NO production in the cardiovascular system via the other two NOS isoforms, namely inducible NOS (iNOS) and neuronal NOS (nNOS). We summarize recent findings regarding beta 3-AR effects on the cardiovascular system and explore its prospective as a therapeutic target, particularly focusing on its emerging role as an important mediator of NO signaling in the pathogenesis of cardiovascular disorders.

Distinct roles of protease-activated receptors in signal transduction regulation of endothelial nitric oxide synthase.

Protease-activated receptors (PARs), such as PAR1 and PAR2, have been implicated in the regulation of endothelial NO production. We hypothesized that PAR1 and PAR2 distinctly regulate the activity of endothelial NO synthase through the selective phosphorylation of a positive regulatory site, Ser(1179), and a negative regulatory site, Thr(497), in bovine aortic endothelial cells. A selective PAR1 ligand, TFLLR, stimulated the phosphorylation of endothelial NO synthase at Thr(497). It had a minimal effect on Ser(1179) phosphorylation. In contrast, a selective PAR2 ligand, SLIGRL, stimulated the phosphorylation of Ser(1179) with no noticeable effect on Thr(497). Thrombin has been shown to transactivate PAR2 through PAR1. Thus, thrombin, as well as a peptide mimicking the PAR1 tethered ligand, TRAP, stimulated phosphorylation of both sites. Also, thrombin and SLIGRL, but not TFLLR, stimulated cGMP production. A G(q) inhibitor blocked thrombin- and SLIGRL-induced Ser(1179) phosphorylation, whereas it enhanced thrombin-induced Thr(497) phosphorylation. In contrast, a G(12/13) inhibitor blocked thrombin- and TFLLR-induced Thr(497) phosphorylation, whereas it enhanced the Ser(1179) phosphorylation. Although a Rho-kinase inhibitor, Y27632, blocked the Thr(497) phosphorylation, other inhibitors that targeted Rho-kinase failed to block TFLLR-induced Thr(497) phosphorylation. These data suggest that PAR1 and PAR2 distinctly regulate endothelial NO synthase phosphorylation and activity through G(12/13) and G(q), respectively, delineating the novel signaling pathways by which the proteases act on protease-activated receptors to potentially modulate endothelial functions.

Role of protease-activated receptor-1 in endothelial nitric oxide synthase-Thr495 phosphorylation.

Protease activated receptors (PARs) are G protein-coupled receptors that are known to regulate endothelial nitric oxide synthase (eNOS) activity in part by phosphorylating the enzyme at various sites. Ser1177 is a positive regulatory site, which leads to the enhanced production of nitric oxide (NO), a vasodilator of arteries. Thr495 is a negative regulatory site, which inhibits NO production. We have shown that thrombin, a PAR agonist, mediates eNOS-Ser1177 phosphorylation through Gq and a calcium and protein kinase C (PKC)-delta sensitive, but phosphatidylinositol 3-kinase (PI3K)/Akt-independent pathway. However, the mechanism for eNOS-Thr495 phosphorylation by PAR agonists is unknown. We used a specific synthetic PAR-1 activating peptide, TFLLR, and thrombin to assess the role of PAR-1 involvement in the phosphorylation of eNOS-Thr495 in human umbilical vein endothelial cells (HUVECs). Using Western blot analysis and the Griess Reagent assay, we found that both agonists phosphorylated Thr495 in a time- and dose-dependent manner and significantly decreased nitrite production, respectively. Pretreatment of cells with the PAR-1 inhibitor, SCH-79797, resulted in a significant decrease in thrombin- and TFLLR-induced phosphorylation of eNOS-Thr495 and an increase in nitrite production. We further demonstrated that inhibition of Rho with C3 exoenzyme or dominant negative (dn) RhoA, and inhibition of Rho-Kinase (ROCK) with Y-27632 caused a significant decrease in thrombin and TFLLR-induced Thr495 phosphorylation. Blockade of the Rho/ROCK pathway also caused an increase in nitrite production. This suggests that PAR-1 regulates eNOS activity via phosphorylation of eNOS-Thr495, which is dependent upon activation of the Rho/ROCK pathway. These findings will be beneficial in further understanding the signaling pathways that regulate eNOS-induced NO production, which plays an important role in endothelial dysfunction associated with cardiovascular disease.