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There is much evidence supporting the role of β₂-adrenoceptors (β₂AR) in angiogenesis but the mechanisms underlying their effects have not been elucidated. Hence, we studied post-ischaemic angiogenesis in the hindlimb (HL) of β₂AR knock-out mice (β₂AR-/-) in vivo and explored possible molecular mechanisms in vitro.Femoral artery resection (FAR) was performed in wild-type and β₂AR-/- mice and adaptive responses to chronic HL ischaemia were explored; blood flow was measured by ultrasound and perfusion of dyed beads, bone rarefaction, muscle fibrosis and skin thickness were evaluated by immunoflourescence and morphometric analysis. Intrafemoral delivery of an adenovirus encoding the human β₂AR (ADβ₂AR) was used to reinstate β₂ARs in β₂AR-/- mice. Molecular mechanisms were investigated in mouse-derived aortic endothelial cells (EC) in vitro, focusing on NFκB activation and transcriptional activity.Angiogenesis was severely impaired in β₂AR-/- mice subjected to FAR, but was restored by gene therapy with ADβ₂AR. The proangiogenic responses to a variety of stimuli were impaired in β₂AR-/- EC in vitro. Moreover, removal of β₂ARs impaired the activation of NFκB, a transcription factor that promotes angiogenesis; neither isoprenaline (stimulates βARs) nor TNFα induced NFκB activation in β₂AR(-/-) EC. Interestingly, cAMP response element binding protein (CREB), a transcription factor that counter regulates NFκB, was constitutively increased in β₂AR(-/-) ECs. ADβ₂AR administration restored β₂AR membrane density, reduced CREB activity and reinstated the NFκB response to isoprenaline and TNFα.Our results suggest that β₂ARs control angiogenesis through the tight regulation of nuclear transcriptional activity.

Aims Myocardial infarction (MI) is the most common cause of heart failure (HF) worldwide. G protein-coupled receptor kinase 5 (GRK5) is upregulated in failing human myocardium and promotes maladaptive cardiac hypertrophy in animal models. However, the role of GRK5 in ischemic heart disease is still unknown. In this study, we evaluated whether myocardial GRK5 plays a critical role post-MI in mice and included the examination of specific cardiac immune and inflammatory responses. Methods and results Cardiomyocyte-specific GRK5 overexpressing transgenic mice (TgGRK5) and non-transgenic littermate control (NLC) mice as well as cardiomyocyte-specific GRK5 knockout mice (GRK5cKO) and wild type (WT) were subjected to MI and, functional as well as structural changes together with outcomes were studied. TgGRK5 post-MI mice showed decreased cardiac function, augmented left ventricular dimension and decreased survival rate compared to NLC post-MI mice. Cardiac hypertrophy and fibrosis as well as fetal gene expression were increased post-MI in TgGRK5 compared to NLC mice. In TgGRK5 mice, GRK5 elevation produced immuno-regulators that contributed to the elevated and long-lasting leukocyte recruitment into the injured heart and ultimately to chronic cardiac inflammation. We found an increased presence of pro-inflammatory neutrophils and macrophages as well as neutrophils, macrophages and T-lymphocytes at 4-days and 8-weeks respectively post-MI in TgGRK5 hearts. Conversely, GRK5cKO mice were protected from ischemic injury and showed reduced early immune cell recruitment (predominantly monocytes) to the heart, improved contractility and reduced mortality compared to WT post-MI mice. Interestingly, cardiomyocyte-specific GRK2 transgenic mice did not share the same phenotype of TgGRK5 mice and did not have increased cardiac leukocyte migration and cytokine or chemokine production post-MI. Conclusions Our study shows that myocyte GRK5 has a crucial and GRK-selective role on the regulation of leucocyte infiltration into the heart, cardiac function and survival in a murine model of post-ischemic HF, supporting GRK5 inhibition as a therapeutic target for HF., Graphical Abstract

Following injury, leukocytes are released from hematopoietic organs and migrate to the site of damage to regulate tissue inflammation and repair; however, leukocytes lacking beta(2)-adrenergic receptor (beta(2)-AR) expression have marked impairments in these processes. Beta blockade is a common strategy for the treatment of many cardiovascular etiologies; therefore, the objective of our study was to assess the impact of prior beta blacker treatment on baseline leukocyte parameters and their responsiveness to acute injury. In a temporal and beta-adrenergic receptor isoform-dependent manner, chronic beta blocker infusion increased splenic vascular cell adhesion molecule 1 expression and leukocyte accumulation (monocytes/macrophages, mast cells, and neutrophils) and decreased chemokine receptor 2 (CCR2) expression and migration of bone marrow and peripheral blood leukocytes (PBLs) to, as well as infiltration into, the heart following acute cardiac injury. Further, CCR2 expression and migratory responsiveness were significantly reduced in the PBLs of patients receiving beta blocker therapy compared with beta blocker-naive patients. These results highlight the ability of chronic beta blocker treatment to alter baseline leukocyte characteristics that decrease leukocytes' responsiveness to acute injury and suggest that prior beta blockade may act to reduce the severity of innate immune responses.

A vast body of literature has established G protein–coupled receptor kinase 2 (GRK2; family: β-adrenergic receptor kinases [βARKs]) as a key player in the development and progression of heart failure. Inhibition of GRK2 improves cardiac function after injury in numerous animal models. In recent years, discovery of several noncanonical GRK2 targets has expanded our view of this kinase. This article describes the exciting finding that cardiac GRK2 activity can regulate whole-body metabolism. Transgenic mice with cardiac-specific expression of a peptide inhibitor of GRK2 (TgβARKct) display an enhanced obesogenic phenotype when fed a high-fat diet (HFD). In contrast, mice with cardiac-specific overexpression of GRK2 (TgGRK2) show resistance to HFD-induced obesity. White adipose tissue (WAT) mass was significantly enhanced in HFD-fed TgβARKct mice. Furthermore, regulators of adipose differentiation were differentially regulated in WAT from mice with gain or loss of GRK2 function. Using complex metabolomics, we found that cardiac GRK2 signaling altered myocardial branched-chain amino acid (BCAA) and endocannabinoid metabolism. In addition, it modulated circulating BCAA and endocannabinoid metabolite profiles on mice fed an HFD. We also found that one of the BCAA metabolites identified here enhances adipocyte differentiation in vitro. These results suggest that metabolic changes in the heart due to GRK2 signaling on mice fed an HFD control whole-body metabolism.

G protein–coupled receptor kinases (GRKs) phosphorylate activated receptors to promote arrestin binding, decoupling from heterotrimeric G proteins, and internalization. GRK2 and GRK5 are overexpressed in the failing heart and thus have become therapeutic targets. Previously, we discovered two classes of GRK2-selective inhibitors, one stemming from GSK180736A, a Rho-associated coiled-coil containing kinase 1 (ROCK1) inhibitor, the other from paroxetine, a selective serotonin-reuptake inhibitor. These two classes of compounds bind to the GRK2 active site in a similar configuration but contain different hinge-binding “warheads”: indazole and benzodioxole, respectively. We surmised from our prior studies that an indazole would be the stronger hinge binder and would impart increased potency when substituted for benzodioxole in paroxetine derivatives. To test this hypothesis, we synthesized a series of hybrid compounds that allowed us to compare the effects of inhibitors that differ only in the identity of the warhead. The indazole-paroxetine analogs were indeed more potent than their respective benzodioxole derivatives but lost selectivity. To investigate how these two warheads dictate selectivity, we determined the crystal structures of three of the indazole hybrid compounds (CCG224061, CCG257284, and CCG258748) in complex with GRK2–Gβγ. Comparison of these structures with those of analogous benzodioxole-containing complexes confirmed that the indazole-paroxetine hybrids form stronger interactions with the hinge of the kinase but also stabilize a distinct conformation of the kinase domain of GRK2 compared with previous complexes with paroxetine analogs. This conformation is analogous to one that can be assumed by GRK5, at least partially explaining the loss in selectivity.

Background Short-term changes of neurohormones can give important prognostic information in heart failure (HF) patients. In this study, we evaluate whether changes in plasma Norepinephrine (NE) and serum N-terminal pro-brain natriuretic peptide (NT-proBNP) after exercise training predict cardiac mortality in HF patients. Methods and results We enrolled 221 HF patients (mean age 72.5 ± 10.2 year) followed-up for a mean period of 27.64 ± 10.7 months. All pts underwent a 3-month exercise training. Before training, clinical examination, echocardiography, peak VO2 determination, and blood draw for NT-proBNP and NE measurements were performed. Primary end-point was cardiac related mortality. Eighty-six-nine percent of patients were in NYHA class III, mean left ventricular ejection fraction (LVEF) was 32.5 ± 10.4%, and mean peak VO2 was 12.36 ± 1.45 ml/kg/min. At baseline, mean NT-proBNP was 2111.4 ± 1145.6 pg/ml and mean NE was 641.8 ± 215.3 pg/ml. One hundred-one subjects died for cardiac causes. Training was associated with a significant increase of peak VO2 and LVEF, whereas NE, NT-proBNP, and heart rate decreased. Multiple Cox proportional hazards regression analysis was performed using delta% values (post vs pre-training) of LVEF, heart rate, NE, and NT-proBNP along with baseline covariates, revealing delta value of NE as the strongest predictor of cardiac mortality. Noteworthy, training reduced NT-proBNP in both survivor and non-survivor patients, while a lack of reduction of NE was observed in non survivors. Conclusions In our HF population, short-term changes of NE after exercise training independently predicted long-term cardiac mortality.

Background— The sphingosine-1-phosphate receptor 1 (S1PR1) and β 1 -adrenergic receptor (β1AR) are G-protein–coupled receptors expressed in the heart. These 2 receptors have opposing actions on adenylyl cyclase because of differential G-protein coupling. Importantly, both of these receptors can be regulated by the actions of G-protein–coupled receptor kinase-2, which triggers desensitization and downregulation processes. Although classic signaling paradigms suggest that simultaneous activation of β1ARs and S1PR1s in a myocyte would simply result in opposing action on cAMP production, in this report we have uncovered a direct interaction between these 2 receptors, with regulatory involvement of G-protein–coupled receptor kinase-2. Methods and Results— In HEK (human embryonic kidney) 293 cells overexpressing both β1AR and S1PR1, we demonstrated that β1AR downregulation can occur after stimulation with sphingosine-1-phosphate (an S1PR1 agonist), whereas S1PR1 downregulation can be triggered by isoproterenol (a β-adrenergic receptor agonist) treatment. This cross talk between these 2 distinct G-protein–coupled receptors appears to have physiological significance, because they interact and show reciprocal regulation in mouse hearts undergoing chronic β-adrenergic receptor stimulation and in a rat model of postischemic heart failure. Conclusions— We demonstrate that restoration of cardiac plasma membrane levels of S1PR1 produces beneficial effects that counterbalance the deleterious β1AR overstimulation in heart failure.

Cardiac gene transfer is an attractive tool for developing novel heart disease treatments. Adeno-associated viral (AAV) vectors are widely used to mediate transgene expression in animal models and are being evaluated for human gene therapy. However, it is not clear which serotype displays the best cardiac tropism. Therefore, we curried out this study to directly compare AAV serotypes 1-9 heart transduction efficiency after indirect intracoronary injection. AAV-cytomegalovirus immediate early enhancer promoter (CMV)-luciferase serotypes 1-9 were injected in the left ventricular cavity of adult mice, after cross-clamping the ascending aorta and pulmonary artery. An imaging system was used to visualize luciferase expression at 3, 7, 21, 70, and 140 days postinjection. Echocardiography was performed to evaluate cardiac function on day 140. At the end of the study, luciferase enzyme activity and genome copies of the different AAV serotypes were assessed in several tissues and potential AAV immunogenicity was evaluated on heart sections by staining for macrophage and lymphocyte antigens. Among AAV serotypes 1-9, AAV6 showed the best capability of achieving high transduction levels in the myocardium in a tissue-specific manner, whereas the other serotypes had less cardiac transduction and more extracardiac expression, especially in the liver. Importantly, none of the serotypes tested with this marker gene affected cardiac function nor was associated with inflammation.

Background— The incidence of heart failure is ever-growing, and it is urgent to develop improved treatments. An attractive approach is gene therapy; however, the clinical barrier has yet to be broken because of several issues, including the lack of an ideal vector supporting safe and long-term myocardial transgene expression. Methods and Results— Here, we show that the use of a recombinant adeno-associated viral (rAAV6) vector containing a novel cardiac-selective enhancer/promoter element can direct stable cardiac expression of a therapeutic transgene, the calcium (Ca 2+ )-sensing S100A1, in a rat model of heart failure. The chronic heart failure–rescuing properties of myocardial S100A1 expression, the result of improved sarcoplasmic reticulum Ca 2+ handling, included improved contractile function and left ventricular remodeling. Adding to the clinical relevance, long-term S100A1 therapy had unique and additive beneficial effects over β-adrenergic receptor blockade, a current pharmacological heart failure treatment. Conclusions— These findings demonstrate that stable increased expression of S100A1 in the failing heart can be used for long-term reversal of LV dysfunction and remodeling. Thus, long-term, cardiac-targeted rAAV6-S100A1 gene therapy may be of potential clinical utility in human heart failure.

The understanding of molecular mechanisms of complex physiological phenomena, such as cardiac contractile function or blood pressure homeostasis, has generated the need for a new generation of scientists who must be able to conjugate intrinsic biological mechanisms and clinical manifestations of disease. From this body of knowledge new strategies of disease management or therapeutic tools are derived, creating the ground for translational medicine, which provides the bridge from basic science to the medical arena. The investigation of G protein-coupled receptor kinases in the cardiovascular system is one example of the successful transposition of basic science to the field of heart and vascular disorders. This review attempts to assemble the currently available information in this continuing area of research for the class of scientists now referred to as 'translational researchers'. © 2006 Adis Data Information BV. All rights reserved.

The medical treatment of chronic heart failure has undergone a dramatic transition in the past decade. Short-term approaches for altering hemodynamics have given way to long-term, reparative strategies, including beta-adrenergic receptor (betaAR) blockade. This was once viewed as counterintuitive, because acute administration causes myocardial depression. Cardiac myocytes from failing hearts show changes in betaAR signaling and excitation-contraction coupling that can impair cardiac contractility, but the role of these abnormalities in the progression of heart failure is controversial. We therefore tested the impact of different manipulations that increase contractility on the progression of cardiac dysfunction in a mouse model of hypertrophic cardiomyopathy. High-level overexpression of the beta(2)AR caused rapidly progressive cardiac failure in this model. In contrast, phospholamban ablation prevented systolic dysfunction and exercise intolerance, but not hypertrophy, in hypertrophic cardiomyopathy mice. Cardiac expression of a peptide inhibitor of the betaAR kinase 1 not only prevented systolic dysfunction and exercise intolerance but also decreased cardiac remodeling and hypertrophic gene expression. These three manipulations of cardiac contractility had distinct effects on disease progression, suggesting that selective modulation of particular aspects of betaAR signaling or excitation-contraction coupling can provide therapeutic benefit.

The actions of G-protein coupled receptor kinases (GRKs) critically regulate beta-adrenergic receptor (betaAR) signalling. In the cardiovascular system, the betaAR signalling pathway controls important responses of the heart such as the ability to contract (inotropy), the ability to contract faster (chronotropy) and the ability to relax (lusotropy). The observation that the betaAR kinase (betaARK1, also known as GRK2), the most abundant GRK in the heart, is increased in cardiovascular disease associated with impaired cardiac function, suggests that this molecule could have pathophysiological relevance in the setting of heart failure. Technological advances in the genetic engineering of mice have provided a powerful tool to study the physiological implications of altering GRK activity and expression in the heart. Recent studies have demonstrated that betaARK1 plays a key role in not only the regulation of myocardial signalling, but also in cardiac function and development. Importantly, targeting the activity of GRKs, and betaARK1 in particular, appears to represent a novel therapeutic strategy for the treatment of the failing heart. At present, gene therapy modalities are being tested which inhibit the activity of betaARK1 in the heart. This technology makes it possible to test directly whether betaARK1 inhibition in the setting of heart disease will improve the function of the compromised heart. Thus, these genetic approaches or the development of small molecule inhibitors of GRK activity, may lead to novel therapeutic approaches for cardiovascular disease.

In the fetus and neonate, β -adrenoceptor stimulation fails to produce physiological desensitization. The current study explores the mechanisms underlying the response pattern in neonatal rats. Homologous cardiac β -adrenergic desensitization caused by isoproterenol treatment in vivo was demonstrable in adult rats by the immediate (2 h) and specific loss of the ability of isoproterenol, but not glucagon, to stimulate adenylyl cyclase in vitro . Homologous desensitization was absent when the same treatment was given to neonates. By 12 h post-treatment, the adults showed heterologous desensitization (loss of the response to glucagon), an effect which was once again absent in the immature rats. The absence of desensitization in neonates did not reflect a deficiency in the activity or subcellular distribution of β ARK1, the enzyme that initiates the phosphorylation and consequent desensitization of β -adrenoceptors. On the other hand, neonates showed relatively poor receptor-G s transduction as assessed by the GTP-induced shift in receptor ligand binding. Repeated isoproterenol treatment of adult rats led to uncoupling of receptor-G-protein transduction but the same treatment in neonates enhanced transduction. Furthermore, neonatal sympathectomy with 6-OHDA interfered with the ontogenetic rise in β -adrenoceptor-G s interactions. These results indicate that the maintenance of agonist responses in the face of neonatal adrenergic stimulation does not reflect simply an absence of the ability to elicit homologous or heterologous desensitization but rather represents an active regulatory mechanism in which neural input exerts a positive trophic role at the level of G-protein function.

Aims: We investigated whether exercise training could promote angiogenesis and improve blood perfusion and left ventricular (LV) remodelling of the post-myocardial infarction (MI) failing heart. We also explored the contribution of ameliorated beta-adrenergic receptor signalling and function on the overall improvement of cardiac contractility reserve induced by exercise.Methods and results: Adult Wistar male rats were randomly assigned to one of four experimental groups. Sham-operated and post-MI heart failure (HF) rats were housed under sedentary conditions or assigned to 10-weeks of a treadmill exercise protocol. At 4 weeks after MI, sedentary HF rats showed LV eccentric hypertrophy, marked increase of LV diameters associated with severely impaired fractional shortening (14 +/- 5%), increased LV end diastolic pressure (20.9 +/- 2.6 mmHg), and pulmonary congestion. In addition, cardiac contractile responses to adrenergic stimulation were significantly blunted. In trained HF rats, exercise was able to (i) reactivate the cardiac vascular endothelial growth factor pathway with a concurrent enhancement of myocardial angiogenesis, (ii) significantly increase myocardial perfusion and coronary reserve, (iii) reduce cardiac diameters, and (iv) improve LV contractility in response to adrenergic stimulation. This latter finding was also associated with a significant improvement of cardiac beta-adrenergic receptor downregulation and desensitization.Conclusions: Our data indicate that exercise favourably affects angiogenesis and improves LV remodelling and contractility reserve in a rat model of severe chronic HF.

Chronic heart failure (HF) is characterized by sympathetic hyperactivity reflected by increased circulating catecholamines (CAs), which contributes significantly to its morbidity and mortality. Therefore, sympatholytic treatments, that is, treatments that reduce sympathetic hyperactivity, are being pursued currently for the treatment of HF. Secretion of CAs from the adrenal gland, which is a major source of CAs, is regulated by alpha(2)-adrenoceptors (alpha(2)ARs), which inhibit, and by beta-adrenoceptors (betaARs), which enhance CA secretion. All ARs are G-protein-coupled receptors (GPCRs), whose signaling and function are regulated tightly by the family of GPCR kinases (GRKs). Despite the enormous potential of adrenal ARs for the regulation of sympathetic outflow, elucidation of their properties has only begun recently. Here, recent advances regarding the roles of adrenal ARs in the regulation of sympathetic outflow in HF and the regulatory properties of ARs are discussed, along with the potential benefits and challenges of harnessing their function for HF therapy.

Cardiac overstimulation by the sympathetic nervous system (SNS) is a salient characteristic of heart failure, reflected by elevated circulating levels of catecholamines. The success of beta-adrenergic receptor (betaAR) antagonists in heart failure argues for SNS hyperactivity being pathogenic; however, sympatholytic agents targeting alpha2AR-mediated catecholamine inhibition have been unsuccessful. By investigating adrenal adrenergic receptor signaling in heart failure models, we found molecular mechanisms to explain the failure of sympatholytic agents and discovered a new strategy to lower SNS activity. During heart failure, there is substantial alpha2AR dysregulation in the adrenal gland, triggered by increased expression and activity of G protein-coupled receptor kinase 2 (GRK2). Adrenal gland-specific GRK2 inhibition reversed alpha2AR dysregulation in heart failure, resulting in lowered plasma catecholamine levels, improved cardiac betaAR signaling and function, and increased sympatholytic efficacy of a alpha2AR agonist. This is the first demonstration, to our knowledge, of a molecular mechanism for SNS hyperactivity in heart failure, and our study identifies adrenal GRK2 activity as a new sympatholytic target.

The blockade of heptahelical receptor coupling to heterotrimeric G proteins by the expression of peptides derived from G protein Galpha subunits represents a novel means of simultaneously inhibiting signals arising from multiple receptors that share a common G protein pool. Here we examined the mechanism of action and functional consequences of expression of an 83-amino acid polypeptide derived from the carboxyl terminus of Galpha(s) (GsCT). In membranes prepared from GsCT-expressing cells, the peptide blocked high affinity agonist binding to beta(2) adrenergic receptors (AR) and inhibited beta(2)AR-induced [35S]GTPgammaS loading of Galpha(s). GsCT expression inhibited beta(2)AR- and dopamine D(1A) receptor-mediated cAMP production, without affecting the cellular response to cholera toxin or forskolin, indicating that the peptide inhibited receptor-G(s) coupling without impairing G protein or adenylyl cyclase function. [35S]GTPgammaS loading of Galpha(q/11) by alpha(1B)ARs and Galpha(i) by alpha(2A)ARs and G(q/11)- or G(i)-mediated phosphatidylinositol hydrolysis was unaffected, indicating that the inhibitory effects of GsCT were selective for G(s). We next employed the GsCT construct to examine the complex role of G(s) in regulation of the ERK mitogen-activated protein kinase cascade, where activation of the cAMP-dependent protein kinase (PKA) pathway reportedly produces both stimulatory and inhibitory effects on heptahelical receptor-mediated ERK activation. For the beta(2)AR in HEK-293 cells, where PKA activity is required for ERK activation, expression of GsCT caused a net inhibition of ERK activation. In contrast, alpha(2A)AR-mediated ERK activation in COS-7 cells was enhanced by GsCT expression, consistent with the relief of a downstream inhibitory effect of PKA. ERK activation by the G(q/11)-coupled alpha(1B)AR was unaffected by GsCT. These findings suggest that peptide G protein inhibitors can provide insights into the complex interplay between G protein pools in cellular regulation.

A mouse model of hypertrophic cardiomyopathy (HCM) was created by expression of a cardiac α-myosin transgene including the R403Q mutation and a deletion of a segment of the actin-binding domain. HCM mice show early histopathology and hypertrophy, with progressive hypertrophy in females and ventricular dilation in older males. To test the hypothesis that dilated cardiomyopathy (DCM) is part of the pathological spectrum of HCM, we studied chamber morphology, exercise tolerance, hemodynamics, isolated heart function, adrenergic sensitivity, and embryonic gene expression in 8- to 11-mo-old male transgenic animals. Significantly impaired exercise tolerance and both systolic and diastolic dysfunction were seen in vivo. Contraction and relaxation parameters of isolated hearts were also decreased, and lusitropic responsiveness to the β-adrenergic agonist isoproterenol was modestly reduced. Myocardial levels of the G protein-coupled β-adrenergic receptor kinase 1 (β-ARK1) were increased by more than twofold over controls, and total β-ARK1 activity was also significantly elevated. Induction of fetal gene expression was also observed in transgenic hearts. We conclude that transgenic male animals have undergone cardiac decompensation resulting in a DCM phenotype. This supports the idea that HCM and DCM may be part of a pathological continuum rather than independent diseases.

The beta-adrenergic signaling cascade is an important regulator of myocardial function. Significant alterations of this pathway are associated with several cardiovascular diseases, including congestive heart failure (CHF). Included in these alterations is increased activity and expression of G protein-coupled receptor kinases (GRKs), such as the beta-adrenergic receptor kinase (beta ARK1), which phosphorylate and desensitize beta-adrenergic receptors (beta ARs). A body of evidence is accumulating that suggests that GRKs, in particular beta ARK1, are critical determinants of cardiac function under normal conditions and in disease states. Transgenic mice with myocardial-targeted alterations of GRK activity have shown profound changes in the in vivo functional performance of the heart. Included in these studies is the compelling finding that inhibition of beta ARK1 activity or expression significantly enhances cardiac function and potentiates beta AR signaling in failing cardiomyocytes. This article summarizes the advances made in the study of beta ARK1 in the heart and addresses its potential as a novel therapeutic target for CHF.

Proliferation of vascular smooth muscle (VSM) severely affects the outcome of coronary artery bypass and angioplasty procedures, causing the failure of venous grafts or restenosis of the reopened vessel. Investigation into the mechanisms underlying the process of VSM cellular proliferation has provided evidence that intracellular signaling mechanisms triggered by extracellular hormonal factors acting through G protein-coupled receptors, can mediate and sustain this pathological process. Inhibition of common pathways of G protein-coupled receptor signaling has recently proven effective in preventing VSM cellular activation and proliferation. In particular, inhibition of the G beta gamma-mediated mitogen-activated protein (MAP) kinase signaling pathway results in the inhibition of VSM proliferation in vitro. Moreover, use of adenoviral vectors to deliver a peptide inhibitor of G beta gamma signaling in vivo has resulted in inhibition of intimal hyperplasia in experimental models of vein-graft failure and restenosis.

BACKGROUND: Intimal hyperplasia remains the leading cause of vein graft failure. Various external stenting devices have been shown to reduce the development of intimal hyperplasia in vein grafts. Mitogenic and mechanotransduction signals are known to be mediated by G protein-coupled receptors. Therefore in this study we examined the alterations in G protein expression and receptor coupling in vein grafts stented with external tube support. METHODS: Thirty New Zealand White male rabbits had a right carotid interposition bypass graft with use of the ipsilateral jugular vein. Fifteen animals received external support and 15 were controls. In a subset the animals either had removal of the external support or a sham-control neck exploration at 14 days after the initial implantation (n = 5 per group). RESULTS: External support reduced G alpha i3 proteins by 30% in vein grafts without changes in G alpha s by Western blot. Vein grafts with external support were significantly less sensitive to pertussis toxin inactivation than controls were in response to both norepinephrine and serotonin. A 24% decrease in intimal thickness was maintained after withdrawal of the initial external support. CONCLUSIONS: The placement of an external support is associated with alternations in G protein expression and receptor coupling function in vein grafts. The results of this study suggest that the development of vein graft intimal hyperplasia may involve G protein-mediated events.

Abstract —Pressure overload ventricular hypertrophy is accompanied by dysfunctional β-adrenergic receptor signaling due to increased levels of the β-adrenergic receptor kinase-1, which phosphorylates and desensitizes β-adrenergic receptors. In this study, we examined whether increased β-adrenergic receptor kinase 1 expression is associated with myocardial hypertrophy induced by adrenergic stimulation. With use of implanted mini-osmotic pumps, we treated mice with isoproterenol, phenylephrine, or vehicle to distinguish between α 1 - and β-adrenergic stimulation. Both treatments resulted in cardiac hypertrophy, but only isoproterenol induced significant increases in β-adrenergic receptor kinase-1 protein levels and activity. Similarly, in isolated adult rat cardiac myocytes, 24 hours of isoproterenol stimulation resulted in a significant 2.8-fold increase in β-adrenergic receptor kinase-1 protein levels, whereas 24 hours of phenylephrine treatment did not alter β-adrenergic receptor kinase-1 expression. Our results indicate that increased β-adrenergic receptor kinase-1 is not invariably associated with myocardial hypertrophy but apparently is controlled by the state of β-adrenergic receptor activation.

Transgenic mice were generated with cardiac-specific overexpression of the G protein-coupled receptor kinase 3 (GRK3) to explore the in vivo role of this GRK in cardiac function. GRK3 is expressed in the heart along with the beta-adrenergic receptor kinase (beta-ARK1) and GRK5. We have previously demonstrated that myocardial-targeted overexpression in transgenic mice of beta-ARK1 (Koch, W.J., H. A. Rockman, P. Samama, R. A. Hamilton, R. A. Bond, C. A. Milano, and R. J. Lefkowitz. Science 268: 1350-1353, 1995) or GRK5 (Rockman, H.A., D.-J. Choi, N. U. Rahman, S. A. Akhter, R. J. Lefkowitz, and W. J. Koch. Proc. Natl. Acad. Sci. USA 93: 9954-9959, 1996) results in significant attenuation of beta-adrenergic signaling and in vivo cardiac function and selective desensitization of angiotensin (ANG) II-mediated cardiac responses. Surprisingly, myocardial overexpression of GRK3 resulted in normal biochemical signaling through beta-adrenergic receptors (beta-ARs), and in vivo hemodynamic function in response to a beta-AR agonist was indistinguishable from that in nontransgenic controls. Furthermore, in vivo signaling and functional responses to ANG II were unaltered. However, myocardial thrombin signaling, as assessed by p42/p44 mitogen-activated protein (MAP) kinase activation, was significantly attenuated in GRK3 transgenic mouse hearts, indicating a distinct in vivo substrate specificity for GRK3.

BACKGROUND: Vein graft intimal hyperplasia is associated with changes in G protein expression. The carboxyl terminus of the beta-adrenergic receptor kinase-1 (beta ARKCT) is known to inhibit G beta gamma-mediated mitogen-activated signaling pathways. This study examines the effects of adenoviral-mediated beta ARKCT infection on the development of intimal hyperplasia in vein grafts. METHODS: New Zealand White rabbits underwent bypass grafting of the carotid artery with the jugular vein. Vein grafts were infected with adenoviral vectors encoding for beta ARKCT (n = 19), beta-galactosidase (n = 3), or empty viral constructs (n = 12). In control animals, vein grafting was performed without infection (n = 10). RESULTS: The efficacy of beta ARKCT infection in vein grafts was verified by reverse transcriptase-polymerase chain reaction. X-gal staining of beta-galactosidase-infected vein grafts demonstrated the transgene in cells throughout the vessel wall. Adenoviral infection of vein grafts without gene transfer did not alter wall thicknesses or sensitivities to contractile agonists, compared with control grafts. beta ARKCT infection, however, reduced intimal thickness by 36% (P < .001) and medial thickness by 24% (P < .001), compared with empty viral infection. beta ARKCT-infected vein grafts also demonstrated increased sensitivity in response to contractile agonists. CONCLUSIONS: These results show that inhibition of G beta gamma signaling with adenoviral-mediated beta ARKCT in vivo infection effectively modifies the structural and functional hyperplastic abnormalities in vein grafts.

Heart failure is accompanied by severely impaired β-adrenergic receptor (βAR) function, which includes loss of βAR density and functional uncoupling of remaining receptors. An important mechanism for the rapid desensitization of βAR function is agonist-stimulated receptor phosphorylation by the βAR kinase (βARK1), an enzyme known to be elevated in failing human heart tissue. To investigate whether alterations in βAR function contribute to the development of myocardial failure, transgenic mice with cardiac-restricted overexpression of either a peptide inhibitor of βARK1 or the β 2 AR were mated into a genetic model of murine heart failure ( MLP −/− ). In vivo cardiac function was assessed by echocardiography and cardiac catheterization. Both MLP −/− and MLP −/− /β 2 AR mice had enlarged left ventricular (LV) chambers with significantly reduced fractional shortening and mean velocity of circumferential fiber shortening. In contrast, MLP −/− /βARKct mice had normal LV chamber size and function. Basal LV contractility in the MLP −/− /βARKct mice, as measured by LV dP/dtmax, was increased significantly compared with the MLP −/− mice but less than controls. Importantly, heightened βAR desensitization in the MLP −/− mice, measured in vivo (responsiveness to isoproterenol) and in vitro (isoproterenol-stimulated membrane adenylyl cyclase activity), was completely reversed with overexpression of the βARK1 inhibitor. We report here the striking finding that overexpression of this inhibitor prevents the development of cardiomyopathy in this murine model of heart failure. These findings implicate abnormal βAR-G protein coupling in the pathogenesis of the failing heart and point the way toward development of agents to inhibit βARK1 as a novel mode of therapy.