BRL-37344 Research Articles

Chronic β1-adrenergic blockade enhances myocardial β3-adrenergic coupling with nitric oxide-cGMP signaling in a canine model of chronic volume overload: new insight into mechanisms of cardiac benefit with selective β1-blocker therapy.

The β1-adrenergic antagonist metoprolol improves cardiac function in animals and patients with chronic heart failure, isolated mitral regurgitation (MR), and ischemic heart disease, though the molecular mechanisms remain incompletely understood. Metoprolol has been reported to upregulate cardiac expression of β3-adrenergic receptors (β3AR) in animal models. Myocardial β3AR signaling via neuronal nitric oxide synthase (nNOS) activation has recently emerged as a cardioprotective pathway. We tested whether chronic β1-adrenergic blockade with metoprolol enhances myocardial β3AR coupling with nitric oxide-stimulated cyclic guanosine monophosphate (β3AR/NO-cGMP) signaling in the MR-induced, volume-overloaded heart. We compared the expression, distribution, and inducible activation of β3AR/NO-cGMP signaling proteins within myocardial membrane microdomains in dogs (canines) with surgically induced MR, those also treated with metoprolol succinate (MR+βB), and unoperated controls. β3AR mRNA transcripts, normalized to housekeeping gene RPLP1, increased 4.4 × 10(3)- and 3.2 × 10(2)-fold in MR and MR+βB hearts, respectively, compared to Control. Cardiac β3AR expression was increased 1.4- and nearly twofold in MR and MR+βB, respectively, compared to Control. β3AR was detected within caveolae-enriched lipid rafts (Cav3(+)LR) and heavy density, non-lipid raft membrane (NLR) across all groups. However, in vitro selective β3AR stimulation with BRL37344 (BRL) triggered cGMP production within only NLR of MR+βB. BRL induced Ser (1412) phosphorylation of nNOS within NLR of MR+βB, but not Control or MR, consistent with detection of NLR-specific β3AR/NO-cGMP coupling. Treatment with metoprolol prevented MR-associated oxidation of NO biosensor soluble guanylyl cyclase (sGC) within NLR. Metoprolol therapy also prevented MR-induced relocalization of sGCβ1 subunit away from caveolae, suggesting preserved NO-sGC-cGMP signaling, albeit without coupling to β3AR, within MR+βB caveolae. Chronic β1-blockade is associated with myocardial β3AR/NO-cGMP coupling in a microdomain-specific fashion. Our canine study suggests that microdomain-targeted enhancement of myocardial β3AR/NO-cGMP signaling may explain, in part, β1-adrenergic antagonist-mediated preservation of cardiac function in the volume-overloaded heart.

β3-Adrenoreceptor stimulation protects against myocardial infarction injury via eNOS and nNOS activation.

β3-adrenergic receptor (AR) and the downstream signaling, nitric oxide synthase (NOS) isoforms, have been emerged as novel modulators of heart function and even potential therapeutic targets for cardiovascular diseases. However, it is not known whether β3-AR plays cardioprotective effects against myocardial infarction (MI) injury. Therefore, the present study was designed to determine the effects of β3-AR on MI injury and to elucidate the underlying mechanism. MI model was constructed by left anterior descending (LAD) artery ligation. Animals were administrated with β3-AR agonist BRL37344 (BRL) or β3-AR inhibitor SR59230A (SR) respectively at 0.1 mg/kg/hour one day after MI operation. The scar area, cardiac function and the apoptosis of myocardial were assessed by Masson's trichrome stain, echocardiography and TUNEL assay respectively. Western blot analysis was performed to elucidate the expressions of target proteins. β3-AR activation with BRL administration significantly attenuated fibrosis and decreased scar area after MI. Moreover, BRL also preserved heart function, and reduced the apoptosis of cardiomyocyte induced by MI. Furthermore, BRL treatment altered the phosphorylation status of endothelial NOS (eNOS) and increased the expression of neuronal NOS (nNOS). These results suggested that β3-AR stimulation has a substantial effect on recovery of heart function. In addition, the activations of both eNOS and nNOS may be associated with the cardiac protective effects of β3-AR.

The essential role for aromatic cluster in the β3 adrenergic receptor

To explore the function of the conserved aromatic cluster F213(5.47), F308(6.51), and F309(6.52) in human β3 adrenergic receptor (hβ3AR). Point mutation technology was used to produce plasmid mutations of hβ3AR. HEK-293 cells were transiently co-transfected with the hβ3AR (wild-type or mutant) plasmids and luciferase reporter vector pCRE-luc. The expression levels of hβ3AR in the cells were determined by Western blot analysis. The constitutive signalling and the signalling induced by the β3AR selective agonist, BRL (BRL37344), were then evaluated. To further explore the interaction mechanism between BRL and β3AR, a three-dimensional complex model of β3AR and BRL was constructed by homology modelling and molecular docking. For F308(6.51), Ala and Leu substitution significantly decreased the constitutive activities of β3AR to approximately 10% of that for the wild-type receptor. However, both the potency and maximal efficacy were unchanged by Ala substitution. In the F308(6.51)L construct, the EC(50) value manifested as a "right shift" of approximately two orders of magnitude with an increased E(max). Impressively, the molecular pharmacological phenotype was similar to the wild-type receptor for the introduction of Tyr at position 308(6.51), though the EC(50) value increased by approximately five-fold for the mutant. For F309(6.52), the constitutive signalling for both F309(6.52)A and F309(6.52)L constructs were strongly impaired. In the F309(6.52)A construct, BRL-stimulated signalling showed a normal E(max) but reduced potency. Leu substitution of F309(6.52) reduced both the E(max) and potency. When F309(6.52) was mutated to Tyr, the constitutive activity was decreased approximately three-fold, and BRL-stimulated signalling was significantly impaired. Furthermore, the double mutant (F308(6.51)A_F309(6.52)A) caused the total loss of β3AR function. The predicted binding mode between β3AR and BRL revealed that both F308(6.51) and F309(6.52) were in the BRL binding pocket of β3AR, while F213(5.47) and W305(6.48) were distant from the binding site. These results revealed that aromatic residues, especially F308(6.51) and F309(6.52), play essential roles in the function of β3AR. Aromatic residues maintained the receptor in a partially activated state and significantly contributed to ligand binding. The results supported the common hypothesis that the aromatic cluster F[Y]5.47/F[Y]6.52/F[Y]6.51 conserved in class A G protein-coupled receptor (GPCR) plays an important role in the structural stability and activation of GPCRs.

BETA3‐ADRENERGIC RECEPTOR REGULATION OF nNOS SIGNALING IN CARDIOMYOCYTES

β3‐AR plays a pivotal role in modulating cardiac function. We recently showed that β3‐mediated cardioprotection is lost in nNOS−/− animals under pressure overload as compared with WT. However, the manner in which β3‐AR regulates nNOS signaling at the cardiomyocyte level is unknown. Here, we used specific β3‐ AR agonist BRL37344 (BRL) to assess the role of β3‐AR regulation of nNOS in hypertrophied isolated neonatal rat ventricular cardiomyocytes. Hypertrophy was induced by 72‐hour norepinephrine pretreatment, which increased cell size and β3‐AR mRNA as compared with non‐hypertrophied cells. In hypertrophied myocytes, BRL increased NOS activity, nNOS phosphorylation at stimulatory site Ser1416, and dephosphorylation of deactivation site Ser847. NOS activity was attenuated by nNOS inhibitor LVNIO and phosphomemetic mutants Ser1416A and Ser847D. In addition, Gi/o and Akt inhibition suppressed BRL‐induced nNOS‐Ser1416 phosphorylation and NOS activity. Our data suggest that BRL regulates myocardial NOS activity via nNOS phosphorylation. This is first study to demonstrate a role for nNOS phosphorylation as a key factor in β3‐AR signaling. These results contribute significantly to our understanding of the negative inotropic properties of cardiac β3‐AR signaling during sympathetic overstimulation, and will ultimately aid in drug discoveries that target molecular mechanisms associated heart failure.

Abstract P215: β3-Adrenergic Receptor Mediated NOS Signaling in Cardiomyocytes

Beta3 -adrenergic receptors play a pivotal role in modulating cardiac function, though their precise role in the heart remains controversial. We have recently demonstrated alterations in Ca 2+ -dependent NOS isoforms and decreased NOS activity in left ventricular tissue of beta3 -/- mice after pressure overload. However, the exact manner in which beta3-AR signaling regulates these isoforms to stimulate NOS activity at the cardiomyocyte level is not well understood. In this study we used a specific beta3-AR agonist, BRL37344 (BRL), to assess the role of beta3-AR in eNOS and 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, measured by the arginine-to-citrulline conversion assay. beta3-activation also caused a 1.5-fold increase in nNOS phosphorylation at positive regulatory site Ser1416, and dephosphorylation of negative regulatory site Ser847 as compared with unstimulated control. NOS activity and nNOS phosphorylation overlapped in time. 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, suggesting G i/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.

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

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.

E0072 Cardioprotective effect of β3 adrenocepter agonism in pressure overload induced hypertrophy

Objectiveβ3-adrenergic receptors (β3-AR) and its downstream signalling are recognised as novel modulators of heart function. We have recently shown impaired cardiac functional compensation in a model of pressure overload. We...

Up‐Regulation and Functional Effect of Cardiac β3‐Adrenoreceptors in Alcoholic Monkeys

Recent studies link altered cardiac beta-adrenergic receptor (AR) signaling to the pathology of alcoholic cardiomyopathy (ACM). However, the alteration and functional effect of beta(3)-AR activation in ACM are unknown. We tested the hypothesis that chronic alcohol intake causes an up-regulation of cardiac beta(3)-AR, which exacerbates myocyte dysfunction and impairs calcium regulation, thereby directly contributing to the progression of ACM. We compared myocyte beta(3)- and beta(1)-AR expression and myocyte contractile ([Ca(2+)](i)), transient ([Ca(2+)](iT)), and Ca(2+) current (I(Ca,L)) responses to beta- and beta(3)-AR stimulation in myocytes obtained from left ventricle (LV) tissue samples obtained from 10 normal control (C) and 16 monkeys with self-administered alcohol for 12 months prior to necropsy: 6 moderate (M) and 10 heavy (H) drinkers with group average alcohol intakes of 1.5 +/- 0.2 and 3.3 +/- 0.2 g/kg/d, respectively. Compared with control myocytes (C), in alcoholic cardiomyocytes, basal cell contraction (dL/dt(max), -39%, H: 69.8 vs. C: 114.6 microm/s), relaxation (dR/dt(max), -37%, 58.2 vs. 92.9 microm/s), [Ca(2+)](iT) (-34%, 0.23 vs. 0.35), and I(Ca,L) (-25%, 4.8 vs. 6.4pA/pF) were all significantly reduced. Compared with controls, in moderate and heavy drinkers, beta(1)-AR protein levels decreased by 23% and 42%, but beta(3)-AR protein increased by 46% and 85%, respectively. These changes were associated with altered myocyte functional responses to beta-AR agonist, isoproterenol (ISO), and beta(3)-AR agonist, BRL-37344 (BRL). Compared with controls, in alcoholic myocytes, ISO (10(-8) M) produced significantly smaller increases in dL/dt(max) (H: 40% vs. C: 71%), dR/dt(max) (37% vs. 52%), [Ca(2+)](iT) (17% vs. 37%), and I(Ca,L) (17% vs. 27%), but BRL (10(-8) M) produced a significantly greater decrease in dL/dt(max) (H: -23% vs. C: -11%), [Ca(2+)](iT) (-30% vs. -11%), and I(Ca,L) (-28% vs. -17%). Chronic alcohol consumption down-regulates cardiac beta(1)- and up-regulates beta(3)-ARs, contributing to the abnormal response to catecholamines in ACM. The up-regulation of cardiac beta(3)-AR signaling enhances inhibition of LV myocyte contraction and relaxation and exacerbates the dysfunctional [Ca(2+)](i) regulation and, thus, may precede the development of ACM.

Lateral Paracapsular GABAergic Synapses in the Basolateral Amygdala Contribute to the Anxiolytic Effects of β3 Adrenoceptor Activation

Norepinephrine (NE) is known to play an integral role in the neurobiological response to stress. Exposure to stressful stimuli increases NE levels in brain regions that regulate stress and anxiety, like the basolateral amygdala (BLA). NE is thought to increase excitability in these areas through alpha- and beta-adrenoceptors (ARs), leading to increased anxiety. Surprisingly, recent studies have shown that systemic beta 3-AR agonist administration decreases anxiety-like behaviors, suggesting that beta 3-ARs may inhibit excitability in anxiety-related brain regions. Therefore, in this study we integrated electrophysiological and behavioral approaches to test the hypothesis that the anxiolytic effects of beta 3-AR agonists may be mediated by an increase in BLA GABAergic inhibition. We examined the effect of a selective beta 3-AR agonist, BRL37344 (BRL), on GABAergic synapses arising from local circuit interneurons and inhibitory synapses originating from a recently described population of cells called lateral paracapsular (LPCS) interneurons. Surprisingly, BRL selectively enhanced LPCS-evoked inhibitory postsynaptic currents (eIPSCs) with no effect on local GABAergic inhibition. BRL also had no effect on glutamatergic synaptic excitation within the BLA. BRL potentiation of LPCS eIPSCs was blocked by the selective beta 3-AR antagonist, SR59230A, or by intracellular dialysis of Rp-CAMPS (cAMP-dependent protein kinase inhibitor), and this enhancement was not associated with any changes in spontaneous IPSCs or LPCS paired-pulse ratio. BRL also increased the amplitude of unitary LPCS IPSCs (uIPSCs) with no effect on uIPSC failure rate. Finally, bilateral BLA microinjection of BRL reduced anxiety-like behaviors in an open-field assay and the elevated plus-maze. Collectively, these data suggest that beta 3-AR activation selectively enhances LPCS, but not local, BLA GABAergic synapses, and that increases in LPCS-mediated inhibition may contribute to the anxiolytic profile of beta 3-AR agonists.