Inhibition Of G Protein-coupled Receptor Kinase 2 Research Articles

Cardiac Progenitor Cells Engineered With βARKct Have Enhanced β-Adrenergic Tolerance

Stem cell survival and retention in myocardium after injury following adoptive transfer is low. Elevated catecholamine levels coinciding with myocardial injury adversely affect cardiac progenitor cell (CPC) survival. The G protein-coupled receptor kinase 2 (GRK2)-derived inhibitory peptide, βARKct, enhance myocyte contractility, survival, and normalize the neurohormonal axis in failing heart, however salutary effects of βARKct on CPC survival and proliferation are unknown. Herein, we investigated whether the protective effects of βARKct expression seen in the failing heart relate to CPCs. Modified CPCs expressing βARKct enhanced AKT/eNOS signaling through protective β2-adrenergic receptors (β2-ARs). In addition, to the actions of βARKct expression on β2- AR signaling, pharmacologic inhibition of GRK2 also increased β2-AR signaling in nonengineered CPCs (lacking βARKct) but had limited effects in βARKct engineered CPCs providing evidence for the strength of the βARKct in inhibiting GRK2 in these cells. Increased proliferation and metabolic activity were observed in βARKct-engineered CPCs following catecholamine stimulation indicating improved adrenergic tolerance. βARKct modification of CPCs increased survival and proliferation following adoptive transfer in an acute myocardial infarction model concomitant with increased expression of β-AR. Thus, βARKct engineering of CPCs promotes survival and proliferation of injected cells following myocardial infarction, which includes improved β-adrenergic tolerance essential for stem cell survival.

SAR study and conformational analysis of a series of novel peptide G protein‐coupled receptor kinase 2 inhibitors

G protein-coupled receptor kinase 2 (GRK2) plays a central role in the cellular transduction network. In particular, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. Thereby, its inhibition offers a potential therapeutic solution to several pathological conditions. In the present study, we performed a SAR study and a NMR conformational analysis of peptides derived from HJ loop of GRK2 and able to selectively inhibit GRK2. From Ala-scan and D-Ala point replacement, we found that Arg residues don't affect the inhibitory properties, while a D-amino acid at position 5 is key to the activity. Conformational analysis identified two β-turns that involve N-terminal residues, followed by a short extended region. These information can help the design of peptides and peptido-mimetics with enhanced GRK2 inhibition properties.

Design, synthesis and efficacy of novel G protein-coupled receptor kinase 2 inhibitors

G protein-coupled receptor kinase 2 (GRK2) is a relevant signaling node of the cellular transduction network, playing major roles in the physiology of various organs/tissues including the heart and blood vessels. Emerging evidence suggests that GRK2 is up regulated in pathological situations such as heart failure, hypertrophy and hypertension, and its inhibition offers a potential therapeutic solution to these diseases. We explored the GRK2 inhibitory activity of a library of cyclic peptides derived from the HJ loop of G protein-coupled receptor kinases 2 (GRK2). The design of these cyclic compounds was based on the conformation of the HJ loop within the X-ray structure of GRK2. One of these compounds, the cyclic peptide 7, inhibited potently and selectively the GRK2 activity, being more active than its linear precursor. In a cellular system, this peptide confirms the beneficial signaling properties of a potent GRK2 inhibitor. Preferred conformations of the most potent analog were investigated by NMR spectroscopy.

Desensitization of G-protein-coupled receptors induces vascular hypocontractility in response to norepinephrine in the mesenteric arteries of cirrhotic patients and rats

The increased beta-arrestin-2 and its combination with G-protein-coupled receptors (GPCRs) lead to GPCRs desensitization. The latter may be responsible for decreased contractile reactivity in the mesenteric arteries of cirrhotic patients and rats. The present study is to investigate the machinery changes of alpha-adrenergic receptors and G proteins and their roles in the contractility of mesenteric arteries of cirrhotic patients and animal models. Patients with cirrhosis due to hepatitis B and cirrhotic rats induced by CCl4 were studied. Mesenteric artery contractility in response to norepinephrine was determined by a vessel perfusion system. The contractile effect of G protein-coupled receptor kinase-2 (GRK-2) inhibitor on the mesenteric artery was evaluated. The protein expression of the alpha1 adrenergic receptor, G proteins, beta-arrestin-2, GRK-2 as well as the activity of Rho associated coiled-coil forming protein kinase-1 (ROCK-1) were measured by Western blot. In addition, the interaction of alpha1 adrenergic receptor with beta-arrestin-2 was assessed by co-immunoprecipitation. The portal vein pressure of cirrhotic patients and rats was significantly higher than that of controls. The dose-response curve to norepinephrine in mesenteric arteriole was shifted to the right, and EC50 was significantly increased in cirrhotic patients and rats. There were no significant differences in the expressions of the alpha1 adrenergic receptor and G proteins in the cirrhotic group compared with the controls. However, the protein expressions of GRK-2 and beta-arrestin-2 were significantly elevated in cirrhotic patients and rats compared with those of the controls. The interaction of the alpha1 adrenergic receptor and beta-arrestin-2 was significantly aggravated. This interaction was significantly reversed by GRK-2 inhibitor. Both the protein expression and activity of ROCK-1 were significantly decreased in the mesenteric artery in patients with cirrhosis compared with those of the controls, and this phenomenon was not shown in the cirrhotic rats. Norepinephrine significantly increased the activity of ROCK-1 in normal rats but not in cirrhotic ones. Norepinephrine significantly increased ROCK-1 activity in cirrhotic rats when GRK-2 inhibitor was used. beta-arrestin-2 expression and its interaction with GPCRs are significantly upregulated in the mesenteric arteries in patients and rats with cirrhosis. These upregulations result in GPCR desensitization, G-protein dysfunction and ROCK inhibition. These may explain the decreased contractility of the mesenteric artery in response to vasoconstrictors.

Inhibition of G-protein-coupled Receptor Kinase 2 (GRK2) Triggers the Growth-promoting Mitogen-activated Protein Kinase (MAPK) Pathway

Inhibition of G-protein-coupled receptor kinase 2 (GRK2) is an emerging treatment option for heart failure. Because GRK2 is also indispensable for growth and development, we analyzed the impact of GRK2 inhibition on cell growth and proliferation. Inhibition of GRK2 by the dominant-negative GRK2-K220R did not affect the proliferation of cultured cells. In contrast, upon xenograft transplantation of cells into immunodeficient mice, the dominant-negative GRK2-K220R or a GRK2-specific peptide inhibitor increased tumor mass. The enhanced tumor growth upon GRK2 inhibition was attributed to the growth-promoting MAPK pathway because dual inhibition of the GRK2 and RAF-MAPK axis by the Raf kinase inhibitor protein (RKIP) did not increase tumor mass. The MAPK cascade contributed to the cardioprotective profile of GRK2 inhibition by preventing cardiomyocyte death, whereas dual inhibition of RAF/MAPK and GRK2 by RKIP induced cardiomyocyte apoptosis, cardiac dysfunction, and signs of heart failure. Thus, cardioprotective signaling induced by GRK2 inhibition is overlapping with tumor growth promotion.

MCARD-Mediated Gene Transfer of GRK2 Inhibitor in Ovine Model of Acute Myocardial Infarction

β-Adrenergic receptor (βAR) dysfunction in acute myocardial infarction (MI) is associated with elevated levels of the G-protein-coupled receptor kinase-2 (GRK2), which plays a key role in heart failure progression. Inhibition of GRK2 via expression of a peptide βARKct transferred by molecular cardiac surgery with recirculating delivery (MCARD) may be a promising intervention. Five sheep underwent scAAV6-mediated MCARD delivery of βARKct, and five received no treatment (control). After a 3-week period, the branch of the circumflex artery (OM1) was ligated. Quantitative PCR data showed intense βARKct expression in the left ventricle (LV). Circumferential fractional shortening was 23.4 ± 7.1 % (baseline) vs. -2.9 ± 5.2 % (p < 0.05) in the control at 10 weeks. In the MCARD-βARKct group, this parameter was close to baseline. The same trend was observed with LV wall thickening. Cardiac index fully recovered in the MCARD-βARKct group. LV end-diastolic volume and LV end-diastolic pressure did not differ in both groups. MCARD-mediated βARKct gene expression results in preservation of regional and global systolic function after acute MI without arresting progressive ventricular remodeling.

Abstract 18646: The Dynamic Relationship Between GRK2 and NOS/NO Critically Determines Cardiac Ischemia/Reperfusion Injury

Background and Aim s: G protein-coupled receptor kinase 2 (GRK2) and endothelial nitric oxide synthase (eNOS) play key roles in cardiac signaling and function. Increased GRK2, seen in injured myocardium, causes increased cell death and injury after ischemia while reciprocally, eNOS/NO exerts protection. A link between these two systems occurs as GRK2 can be regulated via S-nitrosylation (SNO) at Cys340, which inhibits its activity. Alternatively, increased GRK2 activity inhibits NO in the heart. The aim of this study was to determine whether there is a dynamic interaction between cardiac GRK2 and eNOS/NO. Methods: We investigated a direct interaction between GRK2 and eNOS in cultured myocytes and hearts. Cardiac-specific transgenic mice overexpressing GRK2 (Tg-GRK2) and mice with expression of the GRK2 inhibitor βARKct (Tg-βARKct) were used to create hybrid mice with concurrent modulation of eNOS. Finally, we created novel knock-in mice where all endogenous GRK2 was a mutant form (Cys340Ser) resistant to SNO regulation. All mice were characterized basally and subjected to ischemia/reperfusion (I/R) injury and infarct size and cardiac phenotypes determined. Results: 1). Co-immunoprecipitation discovered an interaction between GRK2 and eNOS in cardiac tissue, which was significantly increased in the ischemic heart. 2). TgGRK2/TgeNOS hybrid mice showed significantly reduced infarct size and improved function post I/R compared to Tg-GRK2 mice. Protection offered by βARKct was reversed in mice without eNOS. 3). SNO-GRK2 was increased by isoproterenol and GSNO in WT mice but was not observed in GRK2-C340S mice. 4). Interestingly, GRK2-C340S mice exhibited significantly larger injury post I/R compared to WT mice with decreased cardiac dysfunction. GSNO treatment reduced infarct size in WT mice but had no effects in GRK2-C340S mice. Also, pre-treatment with the NOS inhibitor L-NAME increased infarct size in WT mice but not in GRK2-C340S mice. Conclusions: Our data demonstrates a unique relationship between GRK2 and NO signaling in the heart, which has direct implications for mechanisms of cardiac injury. This includes the demonstration that GRK2 is a nodal pro-death kinase after I/R injury and its SNO is a requirement for NO-mediated cardioprotection.

Abstract 18635: G Protein-Coupled Receptor Kinase 2 Regulates Cellular Oxidative Stress and Survival

Cardiac myocyte oxidative stress and apoptosis are considered important mechanisms for the development of heart failure (HF). Chronic HF is characterized by increased levels of circulating catecholamines to augment cardiac output. Long-term stimulation of myocardial β-adrenergic receptors (β-ARs) is known to be deleterious in cardiac myocytes, however, the potential mechanisms underlying increased cell death are unclear. We hypothesize that G protein-coupled receptor kinase-2 (GRK2), a critical regulator of myocardial β-AR signaling and function, plays an important role in mediating cellular oxidative stress and apoptotic cell death in response to β-agonist stimulation. Stimulation of H9c2 cells with a non-selective β-agonist, isoproterenol (ISO) [10 -5 M] led to a 3-fold increase in oxidative stress/reactive oxygen species (ROS) generation as quantified by Mitosox staining and a significant increase in apoptosis measured by TUNEL and caspase-3 activity compared to PBS-treated controls. There was also a 2.5-fold increase in NADPH oxidase-4 (Nox4) expression but no change in Nox2, the primary NADPH isoforms and major sources of ROS generation in cardiac myocytes. ISO stimulation led to a 3-fold increase in GRK2 activity, similar to the myocardial GRK2 activity present in chronic HF. A 3-fold adenoviral-mediated overexpression of GRK2 led to similar increases in ROS production and apoptosis as seen with ISO stimulation. The increases in oxidative stress by either ISO stimulation or GRK2 overexpression were abolished by pre-treatment with apocynin, a non-specific Nox inhibitor, or siRNA-mediated knockdown of Nox4. Direct activation of adenylyl cyclase with forskolin did not lead to increased ROS production suggesting that β-agonist-induced oxidative stress is likely mediated by activation of GRK2 and increased Nox4 activity and not cAMP/PKA. Adenoviral-mediated expression of the GRK2 inhibitor, βARKct, inhibited Nox4 upregulation, ROS production and apoptosis in response to ISO stimulation. These data demonstrate that GRK2 may play an important role in regulating oxidative stress and apoptosis in the heart and provides another mechanism for the beneficial effects of cardiac-targeted GRK2 inhibition.

Paroxetine Is a Direct Inhibitor of G Protein-Coupled Receptor Kinase 2 and Increases Myocardial Contractility

G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. Herein we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine-Gβγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice with paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.

Inhibitor of G Protein-Coupled Receptor Kinase 2 Normalizes Vascular Endothelial Function in Type 2 Diabetic Mice by Improving β-Arrestin 2 Translocation and Ameliorating Akt/eNOS Signal Dysfunction

In type 2 diabetes, although Akt/endothelial NO synthase (eNOS) activation is known to be negatively regulated by G protein-coupled receptor kinase 2 (GRK2), it is unclear whether the GRK2 inhibitor would have therapeutic effects. Here we examined the hypotensive effect of the GRK2 inhibitor and its efficacy agonist both vascular (aortic) endothelial dysfunction (focusing especially on the Akt/eNOS pathway) and glucose intolerance in two type 2 diabetic models (ob/ob mice and nicotinamide+streptozotocin-induced diabetic mice). Mice were treated with a single injection of the GRK2 inhibitor or vehicle, and the therapeutic effects were compared by examining vascular function and by Western blotting. The GRK2 inhibitor lowered blood pressure in both diabetic models but not in their age-matched controls. The GRK2 inhibitor significantly improved clonidine-induced relaxation only in diabetic (ob/ob and DM) mice, with accompanying attenuations of GRK2 activity and translocation to the plasma membrane. These protective effects of the GRK2 inhibitor may be attributable to the augmented Akt/eNOS pathway activation (as evidenced by increases in Akt phosphorylation at Ser(473) and at Thr(308), and eNOS phosphorylation at Ser(1177)) and to the prevention of the GRK2 translocation and promotion of β-arrestin 2 translocation to the membrane under clonidine stimulation. Moreover, the GRK2 inhibitor significantly improved the glucose intolerance seen in the ob/ob mice. Our work provides the first evidence that in diabetes, the GRK2 inhibitor ameliorates vascular endothelial dysfunction via the Akt/eNOS pathway by inhibiting GRK2 activity and enhancing β-arrestin 2 translocation under clonidine stimulation, thereby contributing to a blood pressure-lowering effect. We propose that the GRK2 inhibitor may be a promising therapeutic agent for cardiovascular complications in type 2 diabetes.

Paeoniflorin Inhibits Proliferation of Fibroblast-Like Synoviocytes through Suppressing G-Protein-Coupled Receptor Kinase 2

Paeoniflorin (Pae) is a monoterpene glucoside and the main component of the total glucosides of paeony (TGP) extracted from the roots of Paeonia lactiflora. Its anti-inflammatory effect is associated with regulating G-protein-coupled receptors (GPCRs) signaling. The aim of this study was to explore the expression change of G-protein-coupled receptor kinase 2 (GRK2) in fibroblast-like synoviocytes (FLS) and the effect of Pae. Pae was obtained and purified from the roots of Paeonia lactiflora. We investigated the expression of GRK2 in synovium during the inflammatory process and assessed the effects of a specific GRK2 inhibitor and Pae on proliferation, cAMP level, and protein kinase A (PKA) activity of FLS in vitro. Additionally, the effect of Pae on GRK2 expression in FLS was detected in vitro. Expression of GRK2 in synovium from CIA rats increased during the inflammatory process. The specific GRK2 inhibitor suppressed proliferation and increased the cAMP level as well as PKA activity of FLS, and Pae had the same effects. Furthermore, Pae decreased GRK2 expression in FLS in vitro. Our results indicate that a chronic inflammatory process in CIA induces upregulation of GRK2 expression in FLS, and Pae can reverse this change, which might be one of the important mechanisms for Pae regulating GPCRs signaling and suppressing the proliferation of FLS in CIA.

AAV6. ARKct cardiac gene therapy ameliorates cardiac function and normalizes the catecholaminergic axis in a clinically relevant large animal heart failure model

G protein-coupled receptor kinase 2 (GRK2), which is markedly upregulated in failing human myocardium, has been implicated as a contributing factor or consequence of heart failure (HF). Importantly, cardiac-specific GRK2 knockout mice have recently proved the pathological nature of GRK2 in HF. Targeted inhibition of GRK2 is possible using a peptide inhibitor known as the βARKct, which has rescued several disparate small animal HF models. This study was designed to evaluate long-term βARKct expression in a clinically relevant large animal HF model, using stable myocardial gene delivery with adeno-associated virus serotype 6 (AAV6). A porcine model of HF subsequent to left ventricular (LV) myocardial infarction (MI) was used to study the effects of retrograde injection into the anterior interventricular vein of either AAV6.βARKct or AAV6.luciferase as a control 2 weeks after MI. Echocardiography and LV hemodynamics were performed before and 6 weeks after gene transfer. Robust and long-term βARKct expression was found after AAV6-mediated delivery, leading to significant amelioration of LV haemodynamics and contractile function in HF pigs compared with AAV6.luciferase-treated control animals that showed a continued decline in cardiac function. Interestingly, the neurohormonal axis was virtually normalized in AVV6.βARKct-treated HF animals, represented by reductions in plasma norepinephrine levels, whereas AAV6.luciferase-treated pigs showed further increases in plasma catecholamine levels. As a result, LV remodelling and foetal gene expression was reversed by AVV6.βARKct gene therapy. These data--showing sustained amelioration of cardiac function in a post-MI pig HF model--demonstrate the therapeutic potential of βARKct gene therapy for HF.

Abstract 17362: Regulation of Oxidative Stress and Apoptotic Signaling in Cardiac Myocytes by G Protein-Coupled Receptor Kinase-2: The Role of NADPH Oxidases

Oxidative stress due to generation of reactive oxygen species (ROS) is an important mechanism of cardiac myocyte death following ischemia-reperfusion (I-R) injury and can lead to heart failure. NADPH oxidases (Nox) are major cellular enzymatic sources for superoxide (O 2 ) radical production and Nox2 and Nox4 are the two predominant isoforms expressed in the heart. The mechanisms by which these Nox isoforms are regulated in the heart after various stresses are unclear. G protein-coupled receptor kinase-2 (GRK2) is a critical regulator of β-adrenergic signaling and cardiac function. We hypothesized that GRK2 may play a role in regulating cardiac myocyte oxidative stress and apoptosis. Cardiac myocytes were exposed to 3 hours of hypoxia (2% O 2 , 5% CO 2 , 93% N 2 ) followed by 3 hours of reoxygenation (5% CO 2 , 95% air). Hypoxia-reoxygenation (H-R) led to significant 3.5-fold upregulation of GRK2 activity as well as ROS production (4.7±0.7 vs 1.1±0.4 nmoles/min/mg protein) and apoptosis measured by caspase-3 activity (6.2±1.5 vs 1.8±0.6 fluorimetric units/mg protein) compared to normoxic controls. Interestingly, 4-fold overexpression of GRK2 by adenoviral infection led to an identical 3-fold increase in ROS production under basal conditions (3.5±1.2 vs 1.3±0.5 nmoles/min/mg protein with null vector controls) which was completely inhibited by pre-treatment with 10μM apocyanin, a Nox inhibitor. Inhibition of GRK2 by adenoviral-mediated expression of βARKct led to a significant reduction in ROS production and caspase 3 activity following H-R which was similar to that observed by knocking down Nox4. Immunoprecipitated Nox4 demonstrated a strong total serine phosphorylation under H-R conditions. Finally, confocal imaging of Nox4 and GRK2 in cardiac myocytes exposed to H-R demonstrated a primarily peri-nuclear co-localization that was absent under control normoxic conditions. These data provide evidence that GRK2 may be an important regulator of oxidative stress via NADPH oxidases in cardiac myocytes. Further investigation of this novel interaction between GRK2 and Nox4 will provide additional insight into the potential role of GRK2 as a regulator of oxidative stress and apoptotic signaling in the heart.

A Role for GRK2 in Myocardial Ischemic Injury: Indicators of a Potential Future Therapy and Diagnostic

Morbidity and mortality of myocardial infarction remains significant with resulting left ventricular function presenting as a major determinant of clinical outcome. Protecting the myocardium against ischemia reperfusion injury has become a major therapeutic goal and the identification of key signaling pathways has paved the way for various interventions, but until now with disappointing results. This article describes the recently discovered new role of G-protein-coupled receptor kinase-2 (GRK2), which is known to critically influence the development and progression of heart failure, in acute myocardial injury. This article focuses on potential applications of the GRK2 peptide inhibitor βARKct in ischemic myocardial injury, the use of GRK2 as a biomarker in acute myocardial infarction and discusses the challenges of translating GRK2 inhibition as a cardioprotective strategy to a possible future clinical application.

Dysfunction of endothelium-dependent relaxation to insulin via PKC-mediated GRK2/Akt activation in aortas of ob/ob mice

In diabetic states, hyperinsulinemia may negatively regulate Akt/endothelial nitric oxide synthase (eNOS) activation. Our main aim was to investigate whether and how insulin might negatively regulate Akt/eNOS activities via G protein-coupled receptor kinase 2 (GRK2) in aortas from ob/ob mice. Endothelium-dependent relaxation was measured in aortic rings from ob/ob mice (a type 2 diabetes model). GRK2, β-arrestin2, and Akt/eNOS signaling-pathway protein levels and activities were mainly assayed by Western blotting. Plasma insulin was significantly elevated in ob/ob mice. Insulin-induced relaxation was significantly decreased in the ob/ob aortas [vs. age-matched control (lean) ones]. The response in ob/ob aortas was enhanced by PKC inhibitor or GRK2 inhibitor. Akt (at Thr(308)) phosphorylation and eNOS (at Ser(1177)) phosphorylation, and also the β-arrestin2 protein level, were markedly decreased in the membrane fraction of insulin-stimulated ob/ob aortas (vs. insulin-stimulated lean ones). These membrane-fraction expressions were enhanced by GRK2 inhibitor and by PKC inhibitor in the ob/ob group but not in the lean group. PKC activity was much greater in ob/ob than in lean aortas. GRK2 protein and activity levels were increased in ob/ob and were greatly reduced by GRK2 inhibitor or PKC inhibitor pretreatment. These results suggest that in the aorta in diabetic mice with hyperinsulinemia an upregulation of GRK2 and a decrease in β-arrestin2 inhibit insulin-induced stimulation of the Akt/eNOS pathway and that GRK2 overactivation may result from an increase in PKC activity.

G Protein-coupled Receptor Kinase-2 Is a Novel Regulator of Collagen Synthesis in Adult Human Cardiac Fibroblasts

Cardiac fibroblasts (CF) make up 60-70% of the total cell number in the heart and play a critical role in regulating normal myocardial function and in adverse remodeling following myocardial infarction and the transition to heart failure. Recent studies have shown that increased intracellular cAMP can inhibit CF transformation and collagen synthesis in adult rat CF; however, mechanisms by which cAMP production is regulated in CF have not been elucidated. We investigated the potential role of G protein-coupled receptor kinase-2 (GRK2) in modulating collagen synthesis by adult human CF isolated from normal and failing left ventricles. Baseline collagen synthesis was elevated in failing CF and was not inhibited by β-agonist stimulation in contrast to normal controls. β-adrenergic receptor (β-AR) signaling was markedly uncoupled in the failing CF, and expression and activity of GRK2 were increased 3-fold. Overexpression of GRK2 in normal CF recapitulated a heart failure phenotype with minimal inhibition of collagen synthesis following β-agonist stimulation. In contrast, knockdown of GRK2 expression in normal CF enhanced cAMP production and led to greater β-agonist-mediated inhibition of basal and TGFβ-stimulated collagen synthesis versus control. Inhibition of GRK2 activity in failing CF by expression of the GRK2 inhibitor, GRK2ct, or siRNA-mediated knockdown restored β-agonist-stimulated inhibition of collagen synthesis and decreased collagen synthesis in response to TGFβ stimulation. GRK2 appears to play a significant role in regulating collagen synthesis in adult human CF, and increased activity of this kinase may be an important mechanism of maladaptive ventricular remodeling as mediated by cardiac fibroblasts.

Reduction of Sympathetic Activity via Adrenal-targeted GRK2 Gene Deletion Attenuates Heart Failure Progression and Improves Cardiac Function after Myocardial Infarction

Chronic heart failure (HF) is characterized by sympathetic overactivity and enhanced circulating catecholamines (CAs), which significantly increase HF morbidity and mortality. We recently reported that adrenal G protein-coupled receptor kinase 2 (GRK2) is up-regulated in chronic HF, leading to enhanced CA release via desensitization/down-regulation of the chromaffin cell alpha(2)-adrenergic receptors that normally inhibit CA secretion. We also showed that adrenal GRK2 inhibition decreases circulating CAs and improves cardiac inotropic reserve and function. Herein, we hypothesized that adrenal-targeted GRK2 gene deletion before the onset of HF might be beneficial by reducing sympathetic activation. To specifically delete GRK2 in the chromaffin cells of the adrenal gland, we crossed PNMTCre mice, expressing Cre recombinase under the chromaffin cell-specific phenylethanolamine N-methyltransferase (PNMT) gene promoter, with floxedGRK2 mice. After confirming a significant ( approximately 50%) reduction of adrenal GRK2 mRNA and protein levels, the PNMT-driven GRK2 knock-out (KO) offspring underwent myocardial infarction (MI) to induce HF. At 4 weeks post-MI, plasma levels of both norepinephrine and epinephrine were reduced in PNMT-driven GRK2 KO, compared with control mice, suggesting markedly reduced post-MI sympathetic activation. This translated in PNMT-driven GRK2 KO mice into improved cardiac function and dimensions as well as amelioration of abnormal cardiac beta-adrenergic receptor signaling at 4 weeks post-MI. Thus, adrenal-targeted GRK2 gene KO decreases circulating CAs, leading to improved cardiac function and beta-adrenergic reserve in post-MI HF. GRK2 inhibition in the adrenal gland might represent a novel sympatholytic strategy that can aid in blocking HF progression.

Abstract 339: Long-term AAV6-βARKct Myocardial Gene Therapy Improves Cardiac Function Via Restoration Of β-Adrenergic Receptor Signaling And Neurohormonal Status Of The Failing Heart

BACKGROUND: The up-regulation of G protein-coupled receptor kinase-2 (GRK2) that is present in compromised myocardium contributes to dysfunctional β-adrenergic receptor (βAR) signaling and cardiac function in heart failure (HF). The peptide βARKct, which inhibits the activation of GRK2, has been shown in acute gene transfer experiments to rescue HF. This study was designed to evaluate chronic βARKct expression in post-myocardial infarction (MI) induced HF using stable myocardial gene delivery with adeno-associated virus serotype-6 (AAV6). METHODS AND RESULTS: In 12 week post-MI HF rats, we delivered βARKct or as a control, Green Fluorescent Protein, via direct intramyocardial injection. We also treated groups with concurrent administration of metoprolol. We found robust and long-lasting (up to 12 weeks post-delivery) transgene expression in the left ventricle (LV) and βARKct expression resulted in significantly improved global heart function as LV ejection fraction and ±dP/dt were increased, whereas LV end diastolic diameter and pressure were decreased. At the molecular level, cardiac βAR density and cAMP accumulation significantly improved over control groups. Fibrotic and hypertrophy markers, as well as heart-to-body weight ratio were markedly decreased by βARKct gene therapy indicating active reversal of adverse LV remodeling. For the first time, we found that chronic βARKct expression and normalization of cardiac βAR signaling led to a reduction of circulating levels of cardiotoxic neurohormones (catecholamines and aldosterone) demonstrating a potential additive mechanism of GRK2 inhibition. Concomitant metoprolol administration preserved the gain in inotropy achieved by βARKct, suggesting compatibility of these two therapeutic modalities, however, metoprolol alone only prevented the deterioration of cardiac function in HF. CONCLUSIONS : Chronic cardiac βARKct gene therapy for HF treatment via AAV6-mediated intracardiac gene delivery is feasible and results in improved cardiac function accompanied by restoration of βAR molecular abnormalities and amelioration of neurohormonal status of HF. These findings suggest βARKct gene therapy might be clinically applicable and of significant value for human HF treatment. This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania &amp; West Virginia).

Abstract 1629: Adrenal-Targeted GRK2 Gene Deletion Ameliorates Sympathetic Overstimulation and Improves Function of the Failing Heart

Chronic heart failure (HF) is characterized by sympathetic overactivity reflected by enhanced circulating catecholamines (CAs), which contribute to increased morbidity and mortality. We recently reported that adrenal G-protein coupled receptor kinase-2 (GRK2) is up-regulated in chronic HF, leading to enhanced CA release via desensitization/downregulation of the chromaffin cell a 2 -adrenergic receptors (a 2 ARs) that normally inhibit CA secretion. We also showed that adrenal GRK2 inhibition via adenoviral-mediated in vivo gene therapy acutely decreases circulating CAs and improves cardiac inotropic reserve and function ( Lymperopoulos, A. et al., Nat. Med. 13 : 315 –323, 2007 ). In the present study, we hypothesized that adrenal-targeted GRK2 gene deletion might be beneficial in chronic HF by means of normalizing sympathetic activity. Methods: To specifically delete GRK2 only in the chromaffin cells of the adrenal gland, we utilized the Cre/loxP technology, crossing PNMT-Cre mice, which express Cre recombinase under the gene promoter of phenylethanolamine N-methyl transferase (PNMT), a chromaffin cell-specific enzyme. We crossed these mice with floxGRK2 +/+ mice, which have the GRK2 gene flanked by loxP sites, thus targeted for deletion by Cre. At 2 months of age, offspring underwent surgical myocardial infarction (MI) to induce HF, and 4 weeks later, cardiac function, adrenal physiology and CA levels were assessed. Results: Genetic screening and western blotting in adrenal extracts confirmed a significant (54 ± 10 % vs. control, non-crossed floxGRK2 +/+ ) reduction of adrenal GRK2 protein levels in PNMTCre/floxGRK2 mice. Adrenal weight-to-body weight ratio was also significantly lower at 4 weeks post-MI, indicating prevention of post-MI adrenal hypertrophy. Additionally, plasma circulating CAs were reduced, and, importantly, cardiac ejection fraction of PNMTCre/floxGRK2 mice was markedly higher than that of floxGRK2 +/+ mice at 4-weeks post-MI (39.6 ± 1.0%, n = 9, vs. 30.9 ± 1.6%, n = 11, respectively, p = 0.0005). Moreover, contractile function and βAR signaling significantly improved. Conclusions: Adrenal-targeted GRK2 gene knockout decreases circulating CAs, leading to improved cardiac inotropic/adrenergic reserve in post-MI HF. This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania &amp; West Virginia).

Inhibition of vascular smooth muscle G protein-coupled receptor kinase 2 enhances alpha1D-adrenergic receptor constriction.

G protein-coupled receptor kinase 2 (GRK2) is a serine/theorinine kinase that phosphorylates and desensitizes agonist-bound G protein-coupled receptors. GRK2 is increased in expression and activity in lymphocytes and vascular smooth muscle (VSM) in human hypertension and animal models of the disease. Inhibition of GRK2 using the carboxyl-terminal portion of the protein (GRK2ct) has been an effective tool to restore compromised beta-adrenergic receptor (AR) function in heart failure and improve outcome. A well-characterized dysfunction in hypertension is attenuation of betaAR-mediated vasodilation. Therefore, we tested the role of inhibition of GRK2 using GRK2ct or VSM-selective GRK2 gene ablation in a renal artery stenosis model of elevated blood pressure (BP) [the two-kidney, one-clip (2K1C) model]. Use of the 2K1C model resulted in a 30% increase in conscious BP, a threefold increase in plasma norepinephrine levels, and a 50% increase in VSM GRK2 mRNA levels. BP remained increased despite VSM-specific GRK2 inhibition by either GRK2 knockout (GRK2KO) or peptide inhibition (GRK2ct). Although betaAR-mediated dilation in vivo and in situ was enhanced, alpha(1)AR-mediated vasoconstriction was also increased. Further pharmacological experiments using alpha(1)AR antagonists revealed that GRK2 inhibition of expression (GRK2KO) or activity (GRK2ct) enhanced alpha(1D)AR vasoconstriction. This is the first study to suggest that VSM alpha(1D)ARs are a GRK2 substrate in vivo.