Inhibition Of G Protein-coupled Receptor Kinase 2 Research Articles

Abstract 24047: G Protein-coupled Receptor Kinase 2 Negatively Regulates Fatty Acid Utilization and Mitochondrial Bioenergetics

During cardiac injury or stress, G protein-coupled receptor (GPCR) kinase 2 (GRK2) expression levels and activity are increased, leading to a desensitization of myocardial β-adrenergic receptors (βARs) and contributing to the loss of contractile reserve. Up-regulated GRK2 has been shown to be pathogenic in the post-injured heart and is involved in the promotion of heart failure (HF). There is evidence that GRK2 has other, non-GPCR dependent pathological functions within cardiomyocytes. For example, GRK2 localizes to the mitochondria following oxidative stress, where it acts as a pro-death kinase and decreases fatty acid utilization. As metabolic substrate utilization and bioenergetics are key parameters in the maintenance of cardiomyocyte contractility, our objective is to examine the role of GRK2 on metabolism and bioenergetics in the adult heart. We hypothesize that desensitization of βARs via an increase in GRK2 will result in decreased fatty acid-fueled respiration and will compromise cardiomyocyte function. Conversely, ablation of GRK2 will result in increased respiration and function, under these conditions. Our results show that basal respiration, maximal respiration, and reserve respiratory capacity (RRC) are highest in the presence of palmitate versus glucose (1.6, 3, and 7.2-fold, respectively), accompanied by increased (1.3-fold) ATP levels. Moreover, basal and maximal respiration was decreased (1.6 and 1.2-fold, respectively) cardiomyocytes isolated from cardiac-specific GRK2 transgenic mice. This correlates with a decrease in ATP levels and in vivo cardiac fatty acid uptake (1-fold and 1.6-fold, respectively). Conversely, cardiomyocytes isolated from βARKct, a peptide inhibitor of GRK2, transgenic mice or GRK2 knockout mice, show increased maximal respiration (1.5 and 1.4-fold, respectively) and RRC (2.7 and 2.2-fold, respectively) with fatty acids. This correlates with an increase in ATP levels (1.2-1.9-fold). Thus, we propose that increased GRK2, as seen during heart failure, compromises fatty acid-driven mitochondrial respiration, while GRK2 inhibition under these conditions enhances RRC, which is known to improve cellular survival during stress.

Opsin 3 and 4 mediate light-induced pulmonary vasorelaxation that is potentiated by G protein-coupled receptor kinase 2 inhibition.

We recently demonstrated that blue light induces vasorelaxation in the systemic mouse circulation, a phenomenon mediated by the nonvisual G protein-coupled receptor melanopsin (Opsin 4; Opn4). Here we tested the hypothesis that nonvisual opsins mediate photorelaxation in the pulmonary circulation. We discovered Opsin 3 (Opn3), Opn4, and G protein-coupled receptor kinase 2 (GRK2) in rat pulmonary arteries (PAs) and in pulmonary arterial smooth muscle cells (PASMCs), where the opsins interact directly with GRK2, as demonstrated with a proximity ligation assay. Light elicited an intensity-dependent relaxation of PAs preconstricted with phenylephrine (PE), with a maximum response between 400 and 460 nm (blue light). Wavelength-specific photorelaxation was attenuated in PAs from Opn4-/- mice and further reduced following shRNA-mediated knockdown of Opn3. Inhibition of GRK2 amplified the response and prevented physiological desensitization to repeated light exposure. Blue light also prevented PE-induced constriction in isolated PAs, decreased basal tone, ablated PE-induced single-cell contraction of PASMCs, and reversed PE-induced depolarization in PASMCs when GRK2 was inhibited. The photorelaxation response was modulated by soluble guanylyl cyclase but not by protein kinase G or nitric oxide. Most importantly, blue light induced significant vasorelaxation of PAs from rats with chronic pulmonary hypertension and effectively lowered pulmonary arterial pressure in isolated intact perfused rat lungs subjected to acute hypoxia. These findings show that functional Opn3 and Opn4 in PAs represent an endogenous "optogenetic system" that mediates photorelaxation in the pulmonary vasculature. Phototherapy in conjunction with GRK2 inhibition could therefore provide an alternative treatment strategy for pulmonary vasoconstrictive disorders.

Design, Synthesis, and Evaluation of the Highly Selective and Potent G-Protein-Coupled Receptor Kinase 2 (GRK2) Inhibitor for the Potential Treatment of Heart Failure.

A novel class of therapeutic drug candidates for heart failure, highly potent and selective GRK2 inhibitors, exhibit potentiation of β-adrenergic signaling in vitro studies. Hydrazone derivative 5 and 1,2,4-triazole derivative 24a were identified as hit compounds by HTS. New scaffold generation and SAR studies of all parts resulted in a 4-methyl-1,2,4-triazole derivative with an N-benzylcarboxamide moiety with highly potent activity toward GRK2 and selectivity over other kinases. In terms of subtype selectivity, these compounds showed enough selectivity against GRK1, 5, 6, and 7 with almost equipotent inhibition to GRK3. Our medicinal chemistry efforts led to the discovery of 115h (GRK2 IC50 = 18 nM), which was obtained the cocrystal structure with human GRK2 and an inhibitor of GRK2 that potentiates β-adrenergic receptor (βAR)-mediated cAMP accumulation and prevents internalization of βARs in β2AR-expressing HEK293 cells treated with isoproterenol. Therefore, 115h appears to be a novel class of therapeutic for heart failure treatment.

G protein-coupled receptor kinase 2 promotes cardiac hypertrophy.

The increase in protein activity and upregulation of G-protein coupled receptor kinase 2 (GRK2) is a hallmark of cardiac stress and heart failure. Inhibition of GRK2 improved cardiac function and survival and diminished cardiac remodeling in various animal heart failure models. The aim of the present study was to investigate the effects of GRK2 on cardiac hypertrophy and dissect potential molecular mechanisms. In mice we observed increased GRK2 mRNA and protein levels following transverse aortic constriction (TAC). Conditional GRK2 knockout mice showed attenuated hypertrophic response with preserved ventricular geometry 6 weeks after TAC operation compared to wild-type animals. In isolated neonatal rat ventricular cardiac myocytes stimulation with angiotensin II and phenylephrine enhanced GRK2 expression leading to enhanced signaling via protein kinase B (PKB or Akt), consecutively inhibiting glycogen synthase kinase 3 beta (GSK3β), such promoting nuclear accumulation and activation of nuclear factor of activated T-cells (NFAT). Cardiac myocyte hypertrophy induced by in vitro GRK2 overexpression increased the cytosolic interaction of GRK2 and phosphoinositide 3-kinase γ (PI3Kγ). Moreover, inhibition of PI3Kγ as well as GRK2 knock down prevented Akt activation resulting in halted NFAT activity and reduced cardiac myocyte hypertrophy. Our data show that enhanced GRK2 expression triggers cardiac hypertrophy by GRK2-PI3Kγ mediated Akt phosphorylation and subsequent inactivation of GSK3β, resulting in enhanced NFAT activity.

GRK2/β-arrestin mediates arginine vasopressin-induced cardiac fibroblast proliferation.

Cardiac fibrosis is a pathological feature commonly found in hearts exposed to haemodynamic orneurohormonal stress. Elevated levels of arginine vasopressin (AVP) are closely associated with the progression of heart failure and could be an underlying cause of cardiac fibrosis. The aim of this study is to characterize the effect of AVP on neonatal rat cardiac fibroblasts (NRCFs) and to illustrate its signalling mechanism. The proliferative effect of AVP was assessed by methylthiazolyldiphenyl-tetrazolium assay and 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, and the amounts of cellular signalling proteins α-smooth muscle actin (α-SMA), matrix metalloproteinase (MMP) 2, MMP9, and phosphorylated ERK1/2 were determined by western blotting. AVP, in a time- and concentration-dependent manner, promoted NRCF proliferation and the expression of MMP2 and MMP9. Inhibition of G protein-coupled receptor kinase2 (GRK2) by the inhibitory peptide GRK2-Ct or knock-down of GRK2 suppressed AVP-induced BrdU incorporation and the expression of MMP2 and α-SMA in NRCFs. Moreover, shRNA-mediated silencing of β-arrestin1 or β-arrestin 2 abolished AVP-induced BrdU incorporation and MMP2 expression. AVP-induced NRCF proliferation depended on the phosphorylation of ERK1/2 , and inhibition of GRK2 or silencing of β-arrestins blocked AVP-induced ERK1/2 phosphorylation. The effects of AVP on NRCF proliferation and α-SMA expression were blocked by SR45059, a vasopressin receptor type1A (V1A R) selective antagonist. In conclusion, AVP promotes NRCF proliferation through V1A R-mediated GRK2/β-arrestin/ERK1/2 signalling.

G protein coupled receptor kinase-2 upregulation causes κ-opioid receptor desensitization in diabetic heart

Activation of κ-opioid receptor (KOR) ameliorates myocardial ischemia/reperfusion (I/R) injury; however, cardioprotective effects of KOR stimulation disappear in type 1 diabetic subjects with hyperglycemia. The molecular mechanisms underlying this phenomenon remain unknown. Here we found that KOR expression was obviously downregulated and KOR agonism-induced contractile-regulatory and cardioprotective effects were significantly impaired in hearts isolated from streptozotocin (STZ) injection-induced diabetic mice. These in vivo data identified cardiac KOR desensitization as a novel characteristic of the diabetic heart. In cultured cardiomyocytes, high glucose (HG) caused obvious KOR downregulation, accompanied by an upregulation of G protein coupled receptor kinase-2 (GRK2). We found that HG exposure increased the interaction between GRK2 and KOR. More importantly, HG-induced KOR downregulation was reversed by small interfering RNA (siRNA)-mediated GRK2 inhibition. GRK2 knockdown also restored KOR agonism-mediated protection against simulated I/R injury in cardiomyocytes. These in vitro data revealed an essential role of GRK2 in HG-induced KOR desensitization. Finally, cardiac-specific GRK2 knockdown by intramyocardial siRNA injection blocked KOR downregulation and restored contractile-regulatory and cardioprotective effects of KOR agonism in hearts of diabetic mice. In conclusion, these data for the first time demonstrate that GRK2 upregulation is largely responsible for cardiac KOR desensitization in diabetic individuals, which provides novel insights into the management of myocardial I/R injury in patients with diabetes.

Inhibiting Insulin-Mediated β 2 -Adrenergic Receptor Activation Prevents Diabetes-Associated Cardiac Dysfunction

Type 2 diabetes mellitus (DM) and obesity independently increase the risk of heart failure by incompletely understood mechanisms. We propose that hyperinsulinemia might promote adverse consequences in the hearts of subjects with type-2 DM and obesity. High-fat diet feeding was used to induce obesity and DM in wild-type mice or mice lacking β2-adrenergic receptor (β2AR) or β-arrestin2. Wild-type mice fed with high-fat diet were treated with a β-blocker carvedilol or a GRK2 (G-protein-coupled receptor kinase 2) inhibitor. We examined signaling and cardiac contractile function. High-fat diet feeding selectively increases the expression of phosphodiesterase 4D (PDE4D) in mouse hearts, in concert with reduced protein kinase A phosphorylation of phospholamban, which contributes to systolic and diastolic dysfunction. The expression of PDE4D is also elevated in human hearts with DM. The induction of PDE4D expression is mediated by an insulin receptor, insulin receptor substrate, and GRK2 and β-arrestin2-dependent transactivation of a β2AR-extracellular regulated protein kinase signaling cascade. Thus, pharmacological inhibition of β2AR or GRK2, or genetic deletion of β2AR or β-arrestin2, all significantly attenuate insulin-induced phosphorylation of extracellular regulated protein kinase and PDE4D induction to prevent DM-related contractile dysfunction. These studies elucidate a novel mechanism by which hyperinsulinemia contributes to heart failure by increasing PDE4D expression and identify β2AR or GRK2 as plausible therapeutic targets for preventing or treating heart failure in subjects with type 2 DM.

Abstract 106: G Protein-coupled Receptor Kinase 2 Associates With Mitochondrial Proteins and Negatively Alters Mitochondrial Respiration After Myocardial Infarction

During cardiac injury or stress, G protein-coupled receptor (GPCR) kinase 2 (GRK2) expression levels and activity are increased, leading to a desensitization of myocardial β-adrenergic receptors (βARs) and contributing to the loss of contractile reserve. Up-regulated GRK2 has been shown to be pathogenic in the post-injured heart and is involved in the promotion of heart failure (HF). Consequently, inhibition of GRK2 has been shown to rescue several animal models of HF, as demonstrated by cardiac expression of the βARKct, a peptide inhibitor of GRK2. There is new evidence that GRK2 has other, non-GPCR dependent pathological functions within cardiomyocytes. For example, during conditions that increase oxidative stress, GRK2 has been shown to localize to the mitochondria, where it is a pro-death kinase. βARKct has also been shown to inhibit the mitochondrial translocation of GRK2 due to inhibition of Hsp90 binding, a chaperone found to bind and localize GRK2 to mitochondria. Our objective is to identify the functions and targets of GRK2 in the mitochondria and its role in the development of HF. We hypothesize that GRK2 has mitochondrial targets that impair mitochondrial function in vivo, contributing to the pathogenesis of HF. Our results show that ADP-stimulated and maximal respiration are significantly decreased in mitochondria isolated from mouse hearts 2 weeks post-myocardial infarction (MI). Cardiac-specific expression of the βARKct rescued the reduction in mitochondrial respiration post-MI, in the presence of pyruvate or palmitoylcarnitine. To identify the mitochondrial targets of GRK2, we performed interactome and phospho-interactome analyses on GRK2 immunoprecipitates from neonatal rat cardiomyocytes subjected to oxidative stress. These results revealed that GRK2 interacts with several mitochondrial proteins, including those involved in metabolism, oxidative stress, calcium handling and cell death. Thus we propose that during ischemia, an increase in mitochondrial GRK2 results in its association with proteins that compromise respiration and contribute to myocardial damage and HF. This demonstrates that understanding both the canonical and non-canonical roles of GRK2 in HF is essential for more targeted intervention for therapy.

G-Protein-Coupled Receptor Kinase 2 (GRK2) Inhibitors: Current Trends and Future Perspectives.

G-protein-coupled receptor kinase 2 (GRK2) is a G-protein-coupled receptor kinase that is ubiquitously expressed in many tissues and regulates various intracellular mechanisms. The up- or down-regulation of GRK2 correlates with several pathological disorders. GRK2 plays an important role in the maintenance of heart structure and function; thus, this kinase is involved in many cardiovascular diseases. GRK2 up-regulation can worsen cardiac ischemia; furthermore, increased kinase levels occur during the early stages of heart failure and in hypertensive subjects. GRK2 up-regulation can lead to changes in the insulin signaling cascade, which can translate to insulin resistance. Increased GRK2 levels also correlate with the degree of cognitive impairment that is typically observed in Alzheimer's disease. This article reviews the most potent and selective GRK2 inhibitors that have been developed. We focus on their mechanism of action, inhibition profile, and structure-activity relationships to provide insight into the further development of GRK2 inhibitors as drug candidates.

Inhibition of G-protein-coupled Receptor Kinase 2 Prevents the Dysfunctional Cardiac Substrate Metabolism in Fatty Acid Synthase Transgenic Mice

Impairment of myocardial fatty acid substrate metabolism is characteristic of late-stage heart failure and has limited treatment options. Here, we investigated whether inhibition of G-protein-coupled receptor kinase 2 (GRK2) could counteract the disturbed substrate metabolism of late-stage heart failure. The heart failure-like substrate metabolism was reproduced in a novel transgenic model of myocardium-specific expression of fatty acid synthase (FASN), the major palmitate-synthesizing enzyme. The increased fatty acid utilization of FASN transgenic neonatal cardiomyocytes rapidly switched to a heart failure phenotype in an adult-like lipogenic milieu. Similarly, adult FASN transgenic mice developed signs of heart failure. The development of disturbed substrate utilization of FASN transgenic cardiomyocytes and signs of heart failure were retarded by the transgenic expression of GRKInh, a peptide inhibitor of GRK2. Cardioprotective GRK2 inhibition required an intact ERK axis, which blunted the induction of cardiotoxic transcripts, in part by enhanced serine 273 phosphorylation of Pparg (peroxisome proliferator-activated receptor γ). Conversely, the dual-specific GRK2 and ERK cascade inhibitor, RKIP (Raf kinase inhibitor protein), triggered dysfunctional cardiomyocyte energetics and the expression of heart failure-promoting Pparg-regulated genes. Thus, GRK2 inhibition is a novel approach that targets the dysfunctional substrate metabolism of the failing heart.

β Adrenergic Receptor Kinase C-Terminal Peptide Gene-Therapy Improves β2-Adrenergic Receptor-Dependent Neoangiogenesis after Hindlimb Ischemia.

After hindlimb ischemia (HI), increased catecholamine levels within the ischemic muscle can cause dysregulation of β2-adrenergic receptor (β2AR) signaling, leading to reduced revascularization. Indeed, in vivo β2AR overexpression via gene therapy enhances angiogenesis in a rat model of HI. G protein-coupled receptor kinase 2 (GRK2) is a key regulator of βAR signaling, and β adrenergic receptor kinase C-terminal peptide (βARKct), a peptide inhibitor of GRK2, has been shown to prevent βAR down-regulation and to protect cardiac myocytes and stem cells from ischemic injury through restoration of β2AR protective signaling (i.e., protein kinase B/endothelial nitric oxide synthase). Herein, we tested the potential therapeutic effects of adenoviral-mediated βARKct gene transfer in an experimental model of HI and its effects on βAR signaling and on endothelial cell (EC) function in vitro. Accordingly, in this study, we surgically induced HI in rats by femoral artery resection (FAR). Fifteen days of ischemia resulted in significant βAR down-regulation that was paralleled by an approximately 2-fold increase in GRK2 levels in the ischemic muscle. Importantly, in vivo gene transfer of the βARKct in the hindlimb of rats at the time of FAR resulted in a marked improvement of hindlimb perfusion, with increased capillary and βAR density in the ischemic muscle, compared with control groups. The effect of βARKct expression was also assessed in vitro in cultured ECs. Interestingly, ECs expressing the βARKct fenoterol, a β2AR-agonist, induced enhanced β2AR proangiogenic signaling and increased EC function. Our results suggest that βARKct gene therapy and subsequent GRK2 inhibition promotes angiogenesis in a model of HI by preventing ischemia-induced β2AR down-regulation.

GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.

The Role of GRK2 and Its Potential as a New Therapeutic Target in Diabetic Vascular Complications

A decrease in nitric oxide (NO) production may induce pathological conditions associated with endothelial dysfunction and diabetes. Although a decrease in NO production caused by impaired Akt/endothelial nitric oxide synthesis (eNOS) signaling has been demonstrated at the aorta in the presence of diabetic vascular complications, little is known regarding the details of the mechanism. We identified G-protein-coupled receptor kinase 2 (GRK2) as a critical factor in diabetic endothelial dysfunction. GRK2 plays a role in many physiological functions including regulation of G-protein-coupled receptors (GPCRs). We found that the vasculature affected by type 2 diabetes expresses high levels of GRK2, which may induce endothelial dysfunction caused by impaired Akt/eNOS signaling. GRK2 activation also induces changes in the subcellular localization of GRK2 and β-arrestin 2, a downstream protein, from the cytosol to membrane. In mouse aorta GRK2 may be, on translocation, a key negative regulator and an important regulator of β-arrestin 2/Akt/eNOS signaling, which has been implicated in diabetic endothelial dysfunction. Furthermore, in the aortic membrane of type 2 diabetic model mice under insulin stimulation, the impaired Akt/eNOS signaling was improved by a selective GRK2 inhibitor. These results suggest that in diabetes the GRK2 inhibitor ameliorates vascular endothelial dysfunction via Akt/eNOS signaling by inhibiting GRK2 activity and enhancing β-arrestin 2 translocation to the membrane under GPCR or non-GPCR stimulation, thereby contributing to blood pressure- and blood glucose-lowering effects. We propose that the GRK2 inhibitor may be a promising therapeutic target for cardiovascular complications in type 2 diabetes.

Erythropoietin Reverses Sepsis-Induced Vasoplegia to Norepinephrine Through Preservation of α1D-Adrenoceptor mRNA Expression and Inhibition of GRK2-Mediated Desensitization in Mouse Aorta.

We investigated the effect of erythropoietin (EPO) posttreatment on survival time and vascular functions in a mouse model of sepsis. Sepsis was induced by cecal ligation and puncture. After 20 ± 2 hours of sepsis, thoracic aorta was isolated for assessing its reactivity to norepinephrine (NE) and acetylcholine (ACh). We also measured the tissue nitric oxide (NO) level, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), G protein-coupled receptor kinase 2 (GRK2), and α1D adrenoceptor messenger RNA (mRNA)/protein expression. In septic mice, EPO moderately improved the survival time from 19.68 ± 0.75 to 34.7 ± 3.2 hours. Sepsis significantly decreased the aortic contractile response to NE along with reduced α1D mRNA and protein expression. Erythropoietin significantly preserved the α1D receptor expression and restored NE-induced contractions to control levels in septic mice. Further, it attenuated the aortic α1D receptor desensitization in sepsis which was evident from reduced GRK2 mRNA expression. Accordingly, a selective GRK2 inhibitor markedly restored the contractile responses to NE in sepsis. Erythropoietin treatment attenuated iNOS mRNA expression and iNOS-induced overproduction of NO, but improved endothelium-dependent relaxation to ACh associated with increased eNOS mRNA expression. In conclusion, EPO seems to reverse sepsis-induced vasoplegia to NE through the preservation of α1D adrenoceptor mRNA/protein expression, inhibition of GRK2-mediated desensitization, and attenuation of NO overproduction in the mouse aorta.

LINKING CARDIAC INSULIN RESISTANCE AND HEART FAILURE: GRK2 AS AN INTEGRATIVE NODE

G protein-coupled receptor kinase 2 (GRK2) has arisen as a signaling hub with critical regulatory roles in cardiac function and dysfunction. Cardiac GRK2 levels are increased during hypertension, cardiac ischemia and heart failure both in humans and in experimental models, whereas genetic inhibition of GRK2 is cardioprotective in different animal models of these pathologies. However, the mechanistic basis of these effects is not fully understood. Recent recent research has indicated that GRK2 not only phosphorylates and desensitizes G protein-coupled receptors (GPCR) that are important for heart function such as β-adrenergic or angiotensin type 1, but also modulates other pathways relevant for cardiac physiology such as the insulin signaling cascade. Using GRK2 hemizygous (GRK2+/-) mice, we have unveiled that a lower GRK2 dosage promotes enhanced insulin sensitivity in adult murine hearts what correlates with a cardioprotective gene expression profile. Also, GRK2 levels increase in cardiac tissue in well-established experimental models of systemic insulin resistance, such as high fat diet (HFD) feeding or ob/ob mice what parallels the impaired cardiac insulin sensitivity observed in these conditions. Our findings suggest that pathological inputs of different etiology such as increased catecholamine levels or high dietary fat converge in common important nodes key to the progress of the pathology. One such key connecting hub is GRK2, whose enhanced cardiac expression would promote progression towards maladaptive myocardial remodeling and heart failure.

Abstract 13917: Inhibition of GRK2 Prevents Cardiac Fibroblast-Mediated Maladaptive Ventricular Remodeling

Remote (non-infarct) territory fibrosis is a significant cause of post-myocardial infarction (MI) heart failure (HF). We have previously shown that increased activity of G protein-coupled receptor kinase-2 (GRK2) in adult human cardiac fibroblasts (CF) isolated from failing hearts is an important mechanism of cardiac fibrosis. This study investigates the potential therapeutic role of GRK2 inhibition on CF biology in vivo. Adult male rats underwent LAD ligation to induce post-MI HF. GRK2 was inhibited by intra-coronary adenoviral-mediated delivery of a GRK2 inhibitor (Ad-GRK2ct) immediately following LAD ligation (n=11). Control rats received a null adenovirus (n=10). Animals were studied prior to and up to 12 weeks (wks) post-MI and adenoviral delivery. There was a significant decline in LV function at 2 wks post-MI which was present through 12 wks [Fractional shortening: 0.35±0.01 vs. 0.52±0.01, p<0.01] in Ad-null rats vs. pre-MI. Remote territory (non-infarct area) fibrosis increased by 2 wks post-MI [6±1% vs. 2±1% fibrosis, p<0.01] progressing by 12 wks to 12% fibrosis [p<0.01], consistent with adverse remodeling. Collagen synthesis was upregulated 2.9-fold in CF isolated 12 wks post-MI [p<0.03] and GRK2 activity was increased 1.4-fold [p=0.002]. There was a 42% decrease in intracellular cAMP [p<0.05] and loss of β-agonist-stimulated inhibition of collagen synthesis characteristic of normal CF [3969±1058 vs. 708±95 cmp/mg protein, p<0.01]. Intra-coronary delivery of Ad-GRK2ct following LAD ligation significantly inhibited post-MI LV dysfunction vs. Ad-Null as measured by improved fractional shortening [0.42±0.01 vs. 0.30±0.02, p<0.01] and ejection fraction [72±1% vs. 57±2%, p<0.01]. Ad-GRK2ct also decreased peri-infarct and remote territory fibrosis by 60% [p<0.001]. Consistent with these findings, Ad-GRK2ct resulted in decreased a-SMA, collagen I, and collegen III expression in CF isolated 12 wks post-MI vs. Ad-Null providing evidence of decreased post-MI CF activation and myofibroblast transformation with Ad-GRK2ct. Targeted inhibition of GRK2 and restoration of β-adrenergic signaling/cAMP production in CF may represent a novel therapeutic approach to prevent pathological fibrosis and maladaptive remodeling post-MI.

The adrenergic system in cardiovascular pathophysiology: a translational science point of view.

Heart failure (HF) is one of the leading causes for mortality and morbidity worldwide. Despite advantages in the management and treatment of this syndrome, nowadays, it is estimated that 50% of HF patients die within 5 years from diagnosis (McMurray et al., 2012). Thus, a better understanding of the molecular mechanisms underlying structural, functional, neuro-hormonal, and metabolic alterations of the failing heart is necessary for the identification of new therapeutic targets and strategies. The adrenergic system is crucial for cardiac function, and even more critical in diseased states characterized by elevated sympathetic nervous system (SNS) hyperactivity (Lymperopoulos et al., 2013). SNS hyperactivity is a salient characteristic of chronic HF and causes cardiac up-regulation of G protein-coupled receptor kinase 2 (GRK2), which in turn induces beta-adrenergic receptor dysregulation in the heart (Rengo et al., 2009, 2012). The present Research Topic aims to present some of the more relevant and recent acquisitions on the molecular abnormalities of the adrenergic system occurring in HF. Dr. Lymperopoulos has reported the molecular mechanisms of regulation of SNS in HF patho-physiology, discussing their therapeutic implications for the failing heart (Lymperopoulos, 2013). The importance of SNS hyperactivity as a main therapeutic target in HF, represents the rationale for the use of beta-blockers, as discussed by Drs. Barrese and Taglialatela. These authors also reported the molecular bases explaining the differences in response to beta-blocker therapy among HF patients (Barrese and Taglialatela, 2013). Dr. Ferrara and collaborators showed the molecular similarities between physiological aging and HF. Both these conditions are characterized by SNS hyperactivity and cardiac beta-adrenergic receptor signaling dysfunction, and this may help to explain why HF is more frequent and its manifestation more severe in the elderly patients (Ferrara et al., 2014). The interconnections between adrenergic system and cardiac metabolism, oxidative stress and nitric oxide signaling have also been discussed in this Research Topic. It is known from several years that the adrenergic system has a profound effect on the regulation of cardiac metabolism. In this regard, Ciccarelli et al. reported the most updated discoveries in the molecular mechanisms involved in the interactions between adrenergic system hyperactivity and metabolic abnormalities, such as insulin resistance and altered glucose metabolism (Ciccarelli et al., 2013). The effects of beta-adrenoceptors on Reactive Oxygen Species generation are described by Corbi et al.; these authors reported also a fascinating hypothesis of the involvement of sirtuins on beta-adrenergic receptors signaling with a potential role in HF pathophysiology (Corbi et al., 2013). Dr. Conti and collaborators reported the mechanisms of the crosstalk between nitric oxide and beta-adrenergic receptor system, in particular in the control of endothelial function and vascular tone (Conti et al., 2013). Evidences accumulated over the past 20 years support the pathogenic key role of cardiac GRK2 levels/activity in determining HF-related beta-adrenergic receptor dysfunction and cardiac inotropic reserve reduction. All these data indicate GRK2 inhibition, via gene therapy, as a new HF therapeutic approach that has been shown to be compatible and, in some models, also synergistic to beta-blockers. Cannavo et al. provide a contemporary update of this field by describing the therapeutic potentialities of this approach and its beneficial effects not only on beta-adrenergic receptor signaling, but also on cardiac metabolism, apoptosis, and mitochondrial dysfunction (Cannavo et al., 2013). De Lucia and collaborators extended the therapeutic potentialities of GRK2 inhibition to the adrenal glands and to the control of HF-related SNS outflow (de Lucia et al., 2014). Since its first demonstration by Lymperopoulos et al. (2007), GRK2 appeared to be critical in the regulation of adrenal alfa2 adrenergic receptor function, extending also to other organs the therapeutic benefits of GRK2 inhibition in HF. Finally, Dr. Leosco in his review explained the molecular mechanisms involved in the beneficial effects of exercise training in curbing SNS hyperactivity and beta-adrenergic receptor dysfunction observed in HF. A crucial role seems to be played by the ability of physical activity to reduce GRK2 levels both in the heart and in the adrenal medulla, with relevant effects on cardiovascular function (Leosco et al., 2013). The overall Research Topic indicates that the great advances achieved in the last decades in understanding the molecular alterations involved in the pathophysiology of HF are opening new opportunities for the treatment of this syndrome and, potentially, their future application to the clinical practice might result to further improvements of patient care. Moreover, the interesting new findings, discussed herein, will hopefully stimulate further research on these arguments.

GRK2 in the Heart: A GPCR Kinase and Beyond

Heart failure (HF) is a common end point for many underlying cardiovascular diseases. Down-regulation and desensitization of β-adrenergic receptors (β-AR) caused by G-protein-coupled receptor (GPCR) kinase 2 (GRK2) are prominent features of HF. Recent Advances and Critical Issues: Significant progress has been made to understand the pathological role of GRK2 in the heart both as a GPCR kinase and as a molecule that can exert GPCR-independent effects. Inhibition of cardiac GRK2 has proved to be therapeutic in the failing heart and may offer synergistic and additional benefits to β-blocker therapy. However, the mechanisms of how GRK2 directly contributes to the pathogenesis of HF need further investigation, and additional verification of the mechanistic details are needed before GRK2 inhibition can be used for the treatment of HF. The newly identified characteristics of GRK2, including the S-nitrosylation of GRK2 and the localization of GRK2 on mitochondria, merit further investigation. They may contribute to it being a pro-death kinase and result in HF under stressed conditions through regulation of intracellular signaling, including cardiac reduction-oxidation (redox) balance. A thorough understanding of the functions of GRK2 in the heart is necessary in order to finalize it as a candidate for drug development.

Tonic Inhibition by G Protein–Coupled Receptor Kinase 2 of Akt/Endothelial Nitric-Oxide Synthase Signaling in Human Vascular Endothelial Cells under Conditions of Hyperglycemia with High Insulin Levels

G protein-coupled receptor kinase 2 (GRK2) participates together with β-arrestins in the regulation of G protein-coupled receptor signaling, but emerging evidence suggests that GRK2 can interact with a growing number of proteins involved in signaling mediated by other membrane receptor families under various pathologic conditions. We tested the hypothesis that GRK2 may be an important contributor to vascular endothelial dysfunction in diabetes. Human umbilical venous endothelial cells (HUVECs) were exposed to high glucose and high insulin (HG/HI) to mimic insulin-resistant diabetic conditions. GRK2 expression and membrane translocation were up-regulated under HG/HI conditions. HG/HI did not modify activation of Akt or endothelial nitric-oxide synthase (eNOS), but GRK2 inhibitor or small interfering RNA (siRNA) resulted in an increase in Akt and eNOS activation in HUVECs exposed to HG/HI. Extracellular signal-regulated kinase 1/2 (ERK1/2) activation was increased after exposure to HG/HI, which was prevented by GRK2 inhibitor or siRNA. ERK1/2-mediated GRK2 phosphorylation at Ser-670 confirmed that ERK1/2 participated in a negative feedback regulatory loop. In human embryonic kidney 293T cells that overexpressed GRK2, Akt activity was unchanged, whereas ERK1/2 activity was raised. The effect of GRK inhibitor treatment on Akt/eNOS signaling was associated with membrane translocation of β-arrestin 2. The experiments with β-arrestin 2 siRNA showed that β-arrestin 2 may act as a positive modulator of Akt/eNOS signaling. Our studies reveal that GRK2, which is up-regulated by HG/HI, leads to a tonic inhibition of the insulin Akt/eNOS pathway in endothelial cells. We provide new insights into the pathogenesis of diabetes-associated vascular endothelial dysfunction.

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.