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.

Deficiency of neuronal nitric oxide synthase increases mortality and cardiac remodeling after myocardial infarction: role of nitroso-redox equilibrium.

BACKGROUND: Neuronal nitric oxide synthase (NOS1) plays key cardiac physiological roles, regulating excitation-contraction coupling and exerting an antioxidant effect that maintains tissue NO-redox equilibrium. After myocardial infarction (MI), NOS1 translocates from the sarcoplasmic reticulum to the cell membrane, where it inhibits beta-adrenergic contractility, an effect previously predicted to have adverse consequences. Counter to this idea, we tested the hypothesis that NOS1 has a protective effect after MI. METHODS AND RESULTS: We studied mortality, cardiac remodeling, and upregulation of oxidative stress pathways after MI in NOS1-deficient (NOS1(-/-)) and wild-type C57BL6 (WT) mice. Compared with WT, NOS1(-/-) mice had greater mortality (hazard ratio, 2.06; P=0.036), worse left ventricular (LV) fractional shortening (19.7+/-1.5% versus 27.2+/-1.5%, P<0.05), higher LV diastolic diameter (5.5+/-0.2 versus 4.9+/-0.1 mm, P<0.05), greater residual cellular width (14.9+/-0.5 versus 12.8+/-0.5 microm, P<0.01), and equivalent beta-adrenergic hyporesponsiveness despite similar MI size. Superoxide production increased after MI in both NOS1(-/-) and WT animals, although NO increased only in WT. NADPH oxidase (P<0.05) activity increased transiently in both groups after MI, but NOS1(-/-) mice had persistent basal and post-MI elevations in xanthine oxidoreductase activity. CONCLUSIONS: Together these findings support a protective role for intact NOS1 activity in the heart after MI, despite a potential contribution to LV dysfunction through beta-adrenergic hyporesponsiveness. NOS1 deficiency contributes to an imbalance between oxidative stress and tissue NO signaling, providing a plausible mechanism for adverse consequences of NOS1 deficiency in states of myocardial injury.

Interleukin 10 knockout frail mice develop cardiac and vascular dysfunction with increased age.

UNLABELLED: Cardiovascular dysfunction is a primary independent predictor of age-related morbidity and mortality. Frailty is associated with activation of inflammatory pathways and fatigue that commonly presents and progresses with age. Interleukin 10 (IL-10), the cytokine synthesis inhibitory factor, is an anti-inflammatory cytokine produced by immune and non-immune cells. Homozygous deletion of IL-10 in mice yields a phenotype that is consistent with human frailty, including age-related increases in serum inflammatory mediators, muscular weakness, higher levels of IGF-1 at midlife, and early mortality. While emerging evidence suggests a role for IL-10 in vascular protection, a clear mechanism has not yet been elucidated. METHODS: In order to evaluate the role of IL-10 in maintenance of vascular function, force tension myography was utilized to access ex-vivo endothelium dependent vasorelaxation in vessels isolated from IL-10 knockout IL-10(tm/tm) and control mice. Pulse wave velocity ((PWV), index of stiffness) of vasculature was measured using ultrasound and blood pressure was measured using the tail cuff method. Echocardiography was used to elucidated structure and functional changes in the heart. RESULTS: Mean arterial pressures were significantly higher in IL-10(tm/tm) mice as compared to C57BL6/wild type (WT) controls. PWV was increased in IL-10(tm/tm) indicating stiffer vasculature. Endothelial intact aortic rings isolated from IL-10(tm/tm) mice demonstrated impaired vasodilation at low acetylcholine doses and vasoconstriction at higher doses whereas vasorelaxation responses were preserved in rings from WT mice. Cyclo-oxygenase (COX-2)/thromboxane A2 inhibitors improved endothelial dependent vasorelaxation and reversed vasoconstriction. Left ventricular end systolic diameter, left ventricular mass, isovolumic relaxation time, fractional shortening and ejection fraction were all significantly different in the aged IL-10(tm/tm) mice compared to WT mice. CONCLUSION: Aged IL-10(tm/tm) mice have stiffer vessels and decreased vascular relaxation due to an increase in eicosanoids, specifically COX-2 activity and resultant thromboxane A2 receptor activation. Our results also suggest that aging IL-10(tm/tm) mice have an increased heart size and impaired cardiac function compared to age-matched WT mice. While further studies will be necessary to determine if this age-related phenotype develops as a result of inflammatory pathway activation or lack of IL-10, it is essential for maintaining the vascular compliance and endothelial function during the aging process. Given that a similar cardiovascular phenotype is present in frail, older adults, these findings further support the utility of the IL-10(tm/tm) mouse as a model of frailty.

Reduced neuronal nitric oxide synthase expression contributes to cardiac oxidative stress and nitroso-redox imbalance in ob/ob mice.

Disruption of leptin signaling in the heart may contribute to obesity-related cardiac disease, as leptin deficient (oblob) mice display cardiac hypertrophy, increased cardiac apoptosis and reduced survival. Since leptin maintains a tonic level of neuronal nitric oxide synthase (NOS1) expression in the brain, we hypothesized that leptin deficiency would decrease NOS1 cardiac expression, in turn activating xanthine oxidoreductase (XOR) and creating nitroso-redox imbalance. We studied 2- to 6-month-old oblob (n=26) and C57Bl/6 controls (n=27). Cardiac NOS1 protein abundance (P<0.01) and mRNA expression (P=0.03) were reduced in oblob (n=10 and 6, respectively), while NOS3 protein abundance and mRNA expression were unaltered. Importantly, cardiac NOS1 protein abundance was restored towards normal in oblob mice after leptin treatment (n=3; P<0.05 vs leptin untreated oblob mice). NO metabolite (nitrite and nitrate) production within the myocardium was also reduced in oblob mice (n=5; P=0.02). Furthermore, oxidative stress was increased in oblob mice as GSH/GSSG ratio was decreased (n=4; P=0.02). Whereas XOR activity measured by Amplex Red fluorescence was increased (n=8; P=0.04), XOR and NADPH oxidase subunits protein abundance were not changed in oblob mice (n=6). Leptin deficiency did not disrupt NOS1 subcellular localization, as NOS1 co-localized with ryanodine receptor but not with caveolin-3. In conclusion, leptin deficiency is linked to decreased cardiac expression of NOS1 and NO production, with a concomitant increase in XOR activity and oxidative stress, resulting in nitroso-redox imbalance. These data offer novel insights into potential mechanisms of myocardial dysfunction in obesity.

Activation of the cardiac ciliary neurotrophic factor receptor reverses left ventricular hypertrophy in leptin-deficient and leptin-resistant obesity.

Disruption of the leptin signaling pathway within the heart causes left ventricular hypertrophy (LVH). Because human obesity is a syndrome of leptin resistance, which is not amenable to leptin treatment, the identification of parallel signal transduction pathways is of potential therapeutic value. Ciliary neurotrophic factor (CNTF), which acts parallel to leptin in the hypothalamus, is not previously recognized to have cardiac activity. We hypothesized that CNTF receptors are present on cardiomyocytes and their activation reverses LVH in both leptin-deficient ob/ob and leptin-resistant db/db mice. The localization of CNTF receptors (CNTFRalpha) to the sarcolemma in C57BL/6, ob/ob and db/db was confirmed in situ with immunohistochemistry, and immunoblotting (60 and 40 kDa) on isolated myocytes. ob/ob mice were randomly assigned to receive s.c. recombinant CNTF (CNTF(Ax15); 0.1 mg x kg(-1) per day; n = 11) calorie-restriction (n = 9), or feeding ad libitum (n = 11). db/db mice were allocated to three similar groups (n = 8, 7, and 8, respectively) plus a leptin group (1 mg x kg(-1) per day; n = 7). Echocardiography showed that CNTF(Ax15) reduced cardiac hypertrophy [posterior wall thickness decreased by 29 +/- 8% (P < 0.01) in ob/ob and by 21 +/- 3% in db/db mice (P < 0.01)], which was consistent with the reduction of myocyte width. Western blotting showed that leptin and CNTF(Ax15) activated Stat3 and ERK1/2 pathway in cultured adult mice cardiomyocytes and cardiac tissue from in ob/ob and db/db mice. Together, these findings support the role of a previously undescribed signaling pathway in obesity-associated cardiac hypertrophy and have therapeutic implications for patients with obesity-related cardiovascular disease and other causes of LVH.