G Protein-coupled Receptor Kinase 2 Protein Levels Research Articles

Cardiac GRK2 Protein Levels Show Sexual Dimorphism during Aging and Are Regulated by Ovarian Hormones.

Cardiovascular disease (CVD) risk shows a clear sexual dimorphism with age, with a lower incidence in young women compared to age-matched men. However, this protection is lost after menopause. We demonstrate that sex-biased sensitivity to the development of CVD with age runs in parallel with changes in G protein-coupled receptor kinase 2 (GRK2) protein levels in the murine heart and that mitochondrial fusion markers, related to mitochondrial functionality and cardiac health, inversely correlate with GRK2. Young female mice display lower amounts of cardiac GRK2 protein compared to age-matched males, whereas GRK2 is upregulated with age specifically in female hearts. Such an increase in GRK2 seems to be specific to the cardiac muscle since a different pattern is found in the skeletal muscles of aging females. Changes in the cardiac GRK2 protein do not seem to rely on transcriptional modulation since adrbk1 mRNA does not change with age and no differences are found between sexes. Global changes in proteasomal or autophagic machinery (known regulators of GRK2 dosage) do not seem to correlate with the observed GRK2 dynamics. Interestingly, cardiac GRK2 upregulation in aging females is recapitulated by ovariectomy and can be partially reversed by estrogen supplementation, while this does not occur in the skeletal muscle. Our data indicate an unforeseen role for ovarian hormones in the regulation of GRK2 protein levels in the cardiac muscle which correlates with the sex-dependent dynamics of CVD risk, and might have interesting therapeutic applications, particularly for post-menopausal women.

Calpains mediate isoproterenol-induced hypertrophy through modulation of GRK2.

Inhibition of the Ca2+-dependent proteases calpains attenuates post-infarction remodeling and heart failure. Recent data suggest that calpain activity is elevated in non-ischemic cardiomyopathies and that upregulation of the key cardiac G-protein-coupled receptor kinase 2 (GRK2) signaling hub promotes cardiac hypertrophy. However, the functional interactions between calpains and GRK2 in this context have not been explored. We hypothesized that calpain modulates GRK2 levels in myocardial hypertrophy of non-ischemic cause, and analyzed the mechanisms involved and the potential therapeutic benefit of inhibiting calpain activity in this situation. The oral calpain inhibitor SNJ-1945 was administered daily to male Sprague-Dawley rats or wild-type and hemizygous GRK2 mice treated with 5mg/Kg/day isoproterenol intraperitoneally for 1week. In isoproterenol-treated animals, calpains 1 and 2 were overexpressed in myocardium and correlated with increased calpain activity and ventricular hypertrophy. Oral co-administration of SNJ-1945 attenuated calpain activation and reduced heart hypertrophy as assessed using morphological and biochemical markers. Calpain activation induced by isoproterenol increased GRK2 protein levels, while genetic downregulation of GRK2 expression prevented isoproterenol-mediated hypertrophy independently of calpain inhibition. GRK2 upregulation was associated to calpain-dependent degradation of the GRK2 ubiquitin ligase MDM2 and to enhanced NF-κB-dependent GRK2 gene expression in correlation with calpain-mediated IĸB proteolysis. These results demonstrate that calpain mediates isoproterenol-induced myocardial hypertrophy by modulating GRK2 protein content through mechanisms involving the control of GRK2 stability and expression. Sustained calpain inhibition attenuates isoproterenol-induced myocardial hypertrophy and could be an effective therapeutic strategy to limit ventricular remodeling of non-ischemic origin.

Involvement of G protein-coupled receptor kinase 2 (GRK2) in the development of non-alcoholic steatosis and steatohepatitis in mice and humans

Insulin resistance (IR) and obesity are important risk factors for non-alcoholic fatty liver disease (NAFLD). G protein-coupled receptor kinase 2 (GRK2) is involved in the development of IR and obesity in vivo. However, its possible contribution to NAFLD and/or non-alcoholic steatohepatitis (NASH) independently of its role on IR or fat mass accretion has not been explored. Here, we used wild-type (WT) or GRK2 hemizygous (GRK2±) mice fed a high-fat diet (HFD) or a methionine and choline-deficient diet (MCD) as a model of NASH independent of adiposity and IR. GRK2± mice were protected from HFD-induced NAFLD. Moreover, MCD feeding caused an increased in triglyceride content and liver-to-body weight ratio in WT mice, features that were attenuated in GRK2± mice. According to their NAFLD activity score, MCD-fed GRK2± mice were diagnosed with simple steatosis and not overt NASH. They also showed reduced expression of lipogenic and lipid-uptake markers and less signs of inflammation in the liver. GRK2± mice preserved hepatic protective mechanisms as enhanced autophagy and mitochondrial fusion and biogenesis, together with reduced endoplasmic reticulum stress. GRK2 protein was increased in MCD-fed WT but not in GRK2± mice, and enhanced GRK2 expression potentiated palmitic acid-triggered lipid accumulation in human hepatocytes directly relating GRK2 levels to steatosis. GRK2 protein and mRNA levels were increased in human liver biopsies from simple steatosis or NASH patients in two different human cohorts. Our results describe a functional relationship between GRK2 levels and hepatic lipid accumulation and implicate GRK2 in the establishment and/or development of NASH.

Abstract 318: MicroRNA Regulation of G Protein-Coupled Receptor Kinase 2 After Cardiac Injury

The increase in myocardial expression and activity of G Protein-Coupled Receptor (GPCR) Kinase 2 (GRK2) during the development and progression of heart failure (HF) has been documented to be pathological. Upregulation of GRK2 that is involved with deactivating agonist-activated β-adrenergic receptors (βARs) results in weakened contractile responsiveness through decreases in βAR density, coupling, and sensitivity. Non-GPCR activity of GRK2 has also been shown to be involved in cardiac dysfunction. Studies with knockout or blockade of GRK2 are able to significantly reduce and/or prevent HF phenotypes. Conversely, models with over-expressed GRK2 produce increased injury and accelerated HF development. Being such an important kinase in cardiac disease, regulation of GRK2 expression and activity is critical. Of interest, there is not much known about how GRK2 is regulated (mRNA or protein) in the heart and mechanisms for its up-regulation after cardiac injury are not well understood. There is evidence of GRK2 being regulated by microRNAs (miRNAs) in endothelial and blood cells. We believe cardiac GRK2 may also be regulated in a similar miRNA-mediated fashion. MiRNA profiles become dysregulated with the onset of stress/injury and contribute to orchestrating many classical HF phenotypes, including βAR aberrations. We hypothesize that the expression of a GRK2-regulating miRNA is altered in HF, which contributes to changes in GRK2 regulation resulting in its pathological increase. MiRNA microarray analysis was done on 2-week post-myocardial infarction (MI) murine hearts to examine miRNA profiles compared to sham-operated. Cardiac miRNA species were examined for their expression levels, seed sequence, and TargetScan prediction and alignment to mouse and human GRK2 3’UTR. Candidate miRNAs underwent 3’UTR analysis against GRK2 3’UTR and transfection into AC16 cardiac cells. Two miRNAs were identified to significantly reduce GRK2 protein and/or RNA levels as well directly bind to the 3’UTR of GRK2. Further studies will be done to elucidate the mechanism these miRNAs are able to regulate GRK2 levels. Results from these studies can be applied towards GRK2 inhibition via miRNA-based therapeutics.

GRK2-S670A Mice reveal cardioprotection post ischemia-reperfusion

Background: β-adrenergic receptors (βARs) are critical regulators of cardiac contractility whose function are drastically impaired during heart failure (HF). Activated βARs are regulated via phosphorylation by G-protein coupled receptor kinase-2 (GRK2) and subsequent interaction with β-arrestin. Animal and human studies have shown that GRK2 is elevated in HF pathogenesis. In addition to GRK2 being involved in pathological βAR our lab has uncovered non-canonical actions of this kinase in HF development. One such novel action of GRK2 is within mitochondria where we have shown increased GRK2 localization after oxidative stress. Our lab has shown that in the heart, phosphorylation of GRK2 via Map kinase at residue Ser670 promotes binding to Hsp90 and further enhances translocation of GRK2 to the mitochondria after ischemia. Methods: To determine the ultimate role of this pathway in the post-ischemic heart we have developed and generated novel GRK2-S670A knock-in mice where all endogenous GRK2 cannot be regulated by phosphorylation at this residue. Baseline characterization of these mice show a minimal phenotype, however we have found significant differences after ischemia-reperfusion (I/R) injury. Results: Compared to control mice (WT) expressing wild-type GRK2, GRK2-S670A mice have significantly less infarction size and improved cardiac function post-24h I/R. GRK2-S670A showed improved ejection fraction post-IR (53.56%+/- 2.08 S670A vs 42.38%+/- 1.6 WT) and smaller infarcted areas (13.67%+/- 1.976 S670A vs 20.78%+/- 3.579 WT) when compared to control mice. Further, we found that mitochondrial GRK2 protein levels were lower in the GRK2-S670A mice at the area at risk post I/R when compared to WT mice (0.8103 +/- 0.1458 S670A vs 1.610 +/- 0.1983 WT). Conclusion: Overall, our data suggest that phosphorylation at Ser670 of GRK2 promotes translocation to the mitochondria leading to detrimental effects including cell death. This mechanism suggests a novel way to develop pharmacological interventions to aid in the treatment of HF.

Suppression of GRK2 expression reduces endothelial dysfunction by restoring glucose homeostasis

Despite the associations between diabetic complications and vascular endothelial dysfunction, a direct therapeutic method targeting endothelial dysfunction remains poorly characterized. We have previously shown that chemical inhibition of G-protein-coupled receptor kinase 2 (GRK2) slightly enhances insulin sensitivity and reduces endothelial dysfunction in type 2 diabetic mice. In this study, we identified GRK2 as a novel therapeutic target of diabetic endothelial dysfunction and investigated the effect on diabetic endothelial dysfunction through the systemic administration of GRK2 siRNA using a hydrodynamic-based procedure, resulting in suppression of increased GRK2 protein levels in the liver. Suppressed GRK2 levels in the liver markedly improved glucose homeostasis, as well as improved the impaired endothelial Akt/eNOS-dependent signal activation (insulin-stimulated phosphorylation of Akt and eNOS) and vascular responses (clonidine-induced and insulin-induced endothelial-dependent relaxation response and phenylephrine-induced contractile response) in type 2 diabetic aortas. Interestingly, insulin-stimulated Akt/eNOS signaling was increased only by normalizing the glucose concentration in human umbilical vein endothelial cells (HUVECs) with GRK2 overexpression, suggesting of an important role of hepatic GRK2. Our results clarified the relationship among hepatic GRK2, glucose homeostasis, and vascular endothelial function. Liver-targeting GRK2 siRNA delivery represents a novel therapeutic tool to restore glucose homeostasis and reduce endothelial dysfunction.

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.

Obesity-induced cardiac lipid accumulation in adult mice is modulated by G protein-coupled receptor kinase 2 levels.

BackgroundThe leading cause of death among the obese population is heart failure and stroke prompted by structural and functional changes in the heart. The molecular mechanisms that underlie obesity-related cardiac remodeling are complex, and include hemodynamic and metabolic alterations that ultimately affect the functionality of the myocardium. G protein-coupled receptor kinase 2 (GRK2) is an ubiquitous kinase able to desensitize the active form of several G protein-coupled receptors (GPCR) and is known to play an important role in cardiac GPCR modulation. GRK2 has also been recently identified as a negative modulator of insulin signaling and systemic insulin resistance.MethodsWe investigated the effects elicited by GRK2 downregulation in obesity-related cardiac remodeling. For this aim, we used 9 month-old wild type (WT) and GRK2+/− mice, which display circa 50% lower levels of this kinase, fed with either a standard or a high fat diet (HFD) for 30 weeks. In these mice we studied different parameters related to cardiac growth and lipid accumulation.ResultsWe find that GRK2+/− mice are protected from obesity-promoted cardiac and cardiomyocyte hypertrophy and fibrosis. Moreover, the marked intracellular lipid accumulation caused by a HFD in the heart is not observed in these mice. Interestingly, HFD significantly increases cardiac GRK2 levels in WT but not in GRK2+/− mice, suggesting that the beneficial phenotype observed in hemizygous animals correlates with the maintenance of GRK2 levels below a pathological threshold. Low GRK2 protein levels are able to keep the PKA/CREB pathway active and to prevent HFD-induced downregulation of key fatty acid metabolism modulators such as Peroxisome proliferator-activated receptor gamma co-activators (PGC1), thus preserving the expression of cardioprotective proteins such as mitochondrial fusion markers mitofusin MFN1 and OPA1.ConclusionsOur data further define the cellular processes and molecular mechanisms by which GRK2 down-regulation is cardioprotective during diet-induced obesity, reinforcing the protective effect of maintaining low levels of GRK2 under nutritional stress, and showing a role for this kinase in obesity-induced cardiac remodeling and steatosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-016-0474-6) contains supplementary material, which is available to authorized users.

Effects of paroxetine-mediated inhibition of GRK2 expression on depression and cardiovascular function in patients with myocardial infarction.

BackgroundParoxetine is a selective serotonin reuptake inhibitor utilized in the treatment of depression and anxiety disorders. Recent studies have identified paroxetine as a G protein-coupled receptor kinase-2 (GRK2) inhibitor capable of reversing cardiac dysfunction and remodeling in experimental models of acute myocardial infarction (AMI). We determine the clinical importance of paroxetine on cardiac functions in patients having AMI with depression (AMID) in comparison with fluoxetine, an unrelated selective serotonin reuptake inhibitor that does not inhibit GRK2.MethodsDiagnosis of depression was based on the 17-item Hamilton Depression Scale and Self-rating Depression Scale in AMI patients after hospital admission. AMID patients were randomly assigned to paroxetine or fluoxetine for treatment of depression. Heart rate variability and cardiac function were evaluated. GRK2 protein levels were measured using peripheral lymphocytes and Western blot.ResultsGRK2 expression in AMID patients was significantly higher than that in AMI patients without depression. In AMID patients, GRK2 levels were positively correlated with the 17-item Hamilton Depression Scale and the Self-rating Depression Scale scores, and negatively correlated with heart rate variability. Treatment of AMID patients with paroxetine significantly reduced the expression of GRK2, normalized the autonomic nervous system function, and improved cardiac performance. In contrast, fluoxetine normalized the autonomic nervous system but did not reduce the expression of GRK2 nor improved cardiac performance.ConclusionThis study suggests that paroxetine is effective for improving cardiac function in patients with AMID and such effect correlates with GRK2 reduction.

Abstract 449: GRK2-S670A Mice Reveal Cardioprotection Post Ischemia-reperfusion

β-adrenergic receptors (βARs) are critical regulators of cardiac contractility whose function are drastically impaired during heart failure (HF). Activated βARs are regulated via phosphorylation by G-protein coupled receptor kinase-2 (GRK2) and subsequent interaction with β-arrestin. Numerous studies have shown that GRK2 is elevated in human HF and animal models demonstrate that it contributes to HF pathogenesis after ischemic injury (IR). Further, our lab has uncovered non-canonical actions of GRK2 that are involved in HF development. One such novel action of GRK2 is within mitochondria where we have shown increased GRK2 localization after oxidative stress. Our lab has shown that in the heart, phosphorylation of GRK2 via Map kinase at residue Ser670 promotes binding to Hsp90 and further enhances translocation of GRK2 to the mitochondria after oxidative stress such as ischemia. To determine the ultimate role of this pathway in the post-ischemic heart we have generated novel GRK2-S670A mice where all endogenous GRK2 cannot be phosphorylated at this residue. Baseline characterization of these mice along with their controls, show a minimal phenotype, however we have found significant differences post-IR. Compared to control mice (NLC), GRK2-S670A have significantly less infarction size 24 hrs after IR and echocardiography and hemodynamics revealed significantly improved cardiac function in GRK2-S670A mice post-IR. GRK2-S670A mice showed improved ejection fraction (52.83%+/- 2.826 S670A vs 42.38%+/- 1.603 NLC) and smaller infarcted areas (9.942%+/- 1.763 S670A vs 20.78%+/- 3.579 NLC) when compared to NLC mice. Further, we found that mitochondrial GRK2 protein levels were lower in the GRK2-S670A mice at the area at risk (0.1428+/-0.02485 S670A vs 0.2327+/- 0.023 NLC). Overall, our data suggest that phosphorylation at Ser670 of GRK2 might act as a “biological switch” in cardiomyocytes, where un-phosphorylated GRK2 is readily available in the cytoplasm to desensitize receptors such as βARs, but upon phosphorylation, GRK2 translocates to the mitochondria leading to detrimental effects including cell death. This mechanism suggests a novel way to develop pharmacological interventions to aid in the treatment of HF.

Prognostic Value of Lymphocyte G Protein-Coupled Receptor Kinase-2 Protein Levels in Patients With Heart Failure.

Sympathetic nervous system hyperactivity is associated with poor prognosis in patients with heart failure (HF), yet routine assessment of sympathetic nervous system activation is not recommended for clinical practice. Myocardial G protein-coupled receptor kinase-2 (GRK2) is upregulated in HF patients, causing dysfunctional β-adrenergic receptor signaling. Importantly, myocardial GRK2 levels correlate with levels found in peripheral lymphocytes of HF patients. The independent prognostic value of blood GRK2 measurements in HF patients has never been investigated; thus, the purpose of this study was to evaluate whether lymphocyte GRK2 levels predict clinical outcome in HF patients. We prospectively studied 257 HF patients with mean left ventricular ejection fraction of 31.4±8.5%. At the time of enrollment, plasma norepinephrine, serum NT-proBNP, and lymphocyte GRK2 levels, as well as clinical and instrumental variables were measured. The prognostic value of GRK2 to predict cardiovascular (CV) death and all-cause mortality was assessed using the Cox proportional hazard model including demographic, clinical, instrumental, and laboratory data. Over a mean follow-up period of 37.5±20.2 months (range, 3-60 months), there were 102 CV deaths. Age, left ventricular ejection fraction, New York Heart Association class, chronic obstructive pulmonary disease, chronic kidney disease, N-terminal-pro brain natriuretic peptide, and lymphocyte GRK2 protein levels were independent predictors of CV mortality in HF patients. GRK2 levels showed an additional prognostic and clinical value over demographic and clinical variables. The independent prognostic value of lymphocyte GRK2 levels was also confirmed for all-cause mortality. Lymphocyte GRK2 protein levels can independently predict prognosis in patients with HF.

Impact of diabetes mellitus on lymphocyte GRK2 protein levels in patients with heart failure.

Diabetes mellitus (DM) is associated with impaired prognosis in patients with heart failure (HF), but pathogenic mechanisms are unclear. In the failing heart, elevated β-adrenergic receptor (β-AR) activation by catecholamines causes G-protein-coupled receptor kinase-2 (GRK2) upregulation which is responsible for β-AR signalling dysfunction. Importantly, GRK2 expression, measured in peripheral lymphocytes of HF patients, correlates with levels of this kinase in the failing myocardium reflecting the loss of hemodynamic function. Moreover, HF-related GRK2 protein overexpression promotes insulin resistance by interfering with insulin signalling. The aim of this study was to assess lymphocyte GRK2 protein levels in HF patients with and without DM. Patients with a diagnosis of HF were enrolled in the study. All subjects underwent a complete clinical examination (including NYHA functional class assessment and echocardiography) and blood draw for serum N-terminal pro-brain natriuretic peptide (NT-proBNP), lymphocyte GRK2 and plasma norepinephrine (NE) levels. Demographic data including age, sex, medications, cardiovascular risk factors and presence of comorbidities were also collected. Two hundred and sixty-eight patients with HF (left ventricular ejection fraction [LVEF] 30.6 ± 7.6%) with and without DM were enrolled. No differences between the two groups were found in terms of demography, HF aetiology, LVEF, NYHA class, NE and NT-proBNP. GRK2 was significantly higher in patients with DM compared to non-DM. At multivariate linear regression analysis, LVEF, NE, NT-proBNP and diabetes came out to be independent predictors of GRK2 levels in the overall study population. In HF patients, DM is associated with significantly more elevated lymphocyte GRK2 protein levels, likely reflecting more compromised cardiac β-AR signalling/function, despite similar hemodynamic status and neuro-hormonal activation compared to patients without DM. These findings contribute to explain the negative prognostic impact of DM in patients with HF.

Abstract 13888: Long-term Intermittent Fasting Treatment Improves Cardiac Function and Inotropic Reserve by Restoration of Cardiac Beta-adrenergic Signaling in an Experimental Model of Chronic Heart Failure

Introduction and Hypothesis: Restricted diets are effective interventions to enhance cardiovascular function and metabolic profile and are known to improve life spam. IF (Intemittent fasting) dietary regimen has a cardioprotective effect in a rat model of myocardial infarction (MI) when diet is started before MI induction. In heart failure (HF), upregulation of G protein-coupled receptor kinase 2 (GRK2) contributes to dysfunctional beta-adrenergic receptor (βAR) signaling and to decrease cardiac inotropic reserve. Moreover, it has been shown that caloric restriction has positive effects on the development of left ventricular hypertrophy and improves ischemic tolerance. Hence, we will test whether a long-term restricted diet, started late after MI, is beneficial in HF and how it could affect cardiac adrenergic signaling. Methods: forty rats were randomly assigned to MI or sham operation. Four weeks later, a time point when post-ischemic HF was established, HF and sham rats were further randomized to a one year IF dietary restriction or ad libitum diet. Thus, our final animal population consisted in 4 groups: Sham normal diet, Sham IF diet, HF normal diet and HF IF diet. Results: One year of IF diet induced a robust decrease in body weight. In HF groups, restricted diet resulted in improved cardiac systolic function and reduced left ventricle end diastolic diameter compared to HF rats fed with normal diet, as measured by echocardiography at the end of the study period. No differences in cardiac function and dimension were observed between sham groups treated with different diets. Consistently, invasive hemodynamic showed that both LV contractility and relaxation in response to βAR stimulation were significantly increased in IF HF rats compared to HF normal diet animals. IF diet resulted in improved cardiac βAR density and adenylyl cyclase activity in HF rats when compared to HF rats treated with standard diet. Restoration of βAR signaling was associated to a dramatic reduction in cardiac GRK2 protein levels. Conclusions: We have demonstrated for the first time that IF, started when HF is already established, ameliorates cardiac function and inotropic reserve in an experimental model of HF. At the molecular level, IF diet significantly improves βAR signaling in HF.

Dexmedetomidine alleviates rat post-ischemia induced allodynia through GRK2 upregulation in superior cervical ganglia.

A transient decrease in G protein-coupled receptor kinase 2 (GRK2) in nociceptors can produce long-lasting neuroplastic changes in nociceptor function, eventually enhancing and prolonging inflammatory hyperalgesia. Here, we investigated the effects of selective α2-adrenoceptor agonist dexmedetomidine (DMED) on GRK2 expression in superior cervical ganglion (SCG) in a rat model of complex regional pain syndrome type I (CRPS-I). The ipsilateral 50% paw withdrawal thresholds (PWTs) to mechanical stimuli decreased significantly starting from 24 h after ischemia-reperfusion (I/R) injury, and lasted for over 3 weeks; the ipsilateral cold allodynia scores, GRK2 protein and mRNA levels in SCGs all increased significantly. No significant differences were found in the contralateral side except GRK2 mRNA reduced significantly after 48 h I/R injury, but still higher than those in the ipsilateral side. Following daily injection of 10 μg/kg of DMED for a maximum of 7 days, the ipsilateral PWTs on days 1, 2, 7, 14, and 21 after DMED administration were significantly higher than those in control group; the GRK2 protein and mRNA expressions in the ipsilateral SCGs were also significantly upregulated; the ipsilateral cold allodynia scores were significantly reduced. No significant differences were found in the contralateral 50%PWTs, cold allodynia scores, and GRK2 protein level except GRK2 mRNA levels increased significantly on days 1 to 7 after DMED administration. Therefore, a transient decrease of GRK2 expression in SCG neurons might be involved in the development and maintenance of allodynia in CRPS-I and DMED might alleviate this allodynia through GRK2 upregulation in SCG neurons.

Abstract 51: Aldosterone Induces Cardiac Deleterious Effects Through A Grk2 And Grk5 Dependent Pathway

High Aldosterone (Aldo) plasma levels are present in heart failure patients and are associated with increased cardiac fibrosis and hypertrophy. These effects have been shown to be, in part, related to the transactivation of the angiotensin II receptor (AT1R), a G-protein coupled receptor (GPCR). In this regard, the GPCR kinase 2 (GRK2) and 5 (GRK5) are both critical regulators of cardiac function through GPCR regulation. Since these GRKs have also been shown to play a role in cardiac hypertrophy and HF development we investigated whether these kinases may play a role in myocardial Aldo signaling. To test this, we first treated neonatal rat ventricular cardiomyocytes (NRVMs) with Aldo (1 μM) and looked for GRK regulation. Following 12 hrs of Aldo-stimulation we observed a significant increase in both GRK2 and GRK5 protein levels. We also found that Aldo induced the localization of GRK2 to mitochondria, which the lab has previously found to occur following ischemic injury leading to increased cell death and mitochondrial dysfuntion. Indeed, high Aldo on NRVMs led to a significant increase in ROS generation and cell death, as observed by Mitosox Red and TUNEL staining, respectively. Notably, all these effects were abolished respectively by Spironolactone (Spiro, an Aldo receptor blocker) or Losartan (Los, an AT1R antagonist) pre-treatment. Next, in nuclear fractions, purified from Aldo-treated NRVMs, we observed a consistent increase in GRK5 nuclear localization that consequently induced a significant activation of hypertrophic genes, which is consistent with previous studies showing GRK5 being a key regulator of maladaptive cardiac hypertrophy after pressure-overload. Importantly, Spiro pre-treatment efficiently abolished these effects of GRK5. Finally, we treated wild-type mice in vivo with Aldo for two and four weeks and not only found that this induced significant cardiac hypertrophy and fibrosis compared to saline-treated mice, we also found significant increases in levels of GRK2 and GRK5. Therefore, our study provide, for the first time, data demonstrating that GRK2 and GRK5 are potential regulators of Aldo-mediated cardiac pathology acting either down-stream of mineralocorticoid receptor or transactivated AT1Rs.

Reduction of lymphocyte G-protein coupled receptor kinase-2 (GRK2) after exercise training predicts survival in patients with heart failure

Background: Increased cardiac G-protein coupled receptor kinase-2 (GRK2) expression has a pivotal role at inducing heart failure (HF)-related β-adrenergic receptor (βAR) dysfunction. Importantly, abnormalities of βAR signaling in the failing heart, including GRK2 protein levels, are mirrored in circulating lymphocytes and are directly correlated with HF severity. Exercise training has been demonstrated to exert several beneficial effects on the failing heart, including normalization of cardiac βAR function and GRK2 protein levels. In the present study we evaluated whether lymphocyte GRK2 and its changes after an exercise training program can predict long-term survival in HF patients. Methods and results: At this aim, we prospectively studied 193 HF patients who underwent a 3-month exercise training program. Lymphocyte GRK2 protein levels, plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) and norepinephrine were measured at baseline and after training along with clinical and functional parameters (left ventricular ejection fraction, NYHA class, and peak-VO2). Cardiac-related mortality was evaluated during a mean follow up period of 37±20 months. Exercise induced a significant reduction of lymphocyte GRK2 protein levels. Importantly, exercise related changes of GRK2 (delta values) robustly predicted survival in our study population. Interestingly, the lack of any significant effect of exercise to reduce lymphocyte GRK2 protein levels identified those HF patients with the poorest outcome. ![Figure][1] Conclusions: Our data offer the first demonstration that changes of lymphocyte GRK2 induced by exercise can strongly predict outcome in patients with advanced HF. [1]: pending:yes

Reduction of lymphocyte G protein-coupled receptor kinase-2 (GRK2) after exercise training predicts survival in patients with heart failure

Increased cardiac G protein-coupled receptor kinase-2 (GRK2) expression has a pivotal role at inducing heart failure (HF)-related β-adrenergic receptor (βAR) dysfunction. Importantly, abnormalities of βAR signalling in the failing heart, including GRK2 overexpression, are mirrored in circulating lymphocytes and correlate with HF severity. Exercise training has been shown to exert several beneficial effects on the failing heart, including normalization of cardiac βAR function and GRK2 protein levels. In the present study, we evaluated whether lymphocyte GRK2 levels and short-term changes of this kinase after an exercise training programme can predict long-term survival in HF patients. For this purpose, we prospectively studied 193 HF patients who underwent a 3-month exercise training programme. Lymphocyte GRK2 protein levels, plasma N-terminal pro-brain natriuretic peptide, and norepinephrine were measured at baseline and after training along with clinical and functional parameters (left ventricular ejection fraction, NYHA class, and peak-VO2). Cardiac-related mortality was evaluated during a mean follow-up period of 37 ± 20 months. Exercise was associated with a significant reduction of lymphocyte GRK2 protein levels (from 1.29 ± 0.52 to 1.16 ± 0.65 densitometric units, p < 0.0001). Importantly, exercise related changes of GRK2 (delta values) robustly predicted survival in our study population. Interestingly, HF patients who did not show reduced lymphocyte GRK2 protein levels after training presented the poorest outcome. Our data offer the first demonstration that changes of lymphocyte GRK2 after exercise training can strongly predict outcome in advanced HF.

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.

Angiotensin II causes endothelial dysfunction via the GRK2/Akt/eNOS pathway in aortas from a murine type 2 diabetic model

Nitric oxide (NO) production and endothelial function are mediated via the Akt/eNOS pathway. We investigated the reductions of these mechanism(s) in type 2 diabetes. Diabetic model (nicotinamide+streptozotocin-induced) mice were fed for 4 weeks on a normal diet either containing or not containing losartan, an AT1 R antagonist. Relaxations and NO productions were measured in isolated aortas. G-protein coupled receptor kinase 2 (GRK2) protein levels and activities in the Akt/eNOS signaling-pathway were mainly assayed by Western blotting. Clonidine- and insulin-induced relaxations and NO productions, all of which were significantly decreased in aortas isolated from the diabetics, were normalized by 4 weeks’ losartan administration. Plasma angiotensin II (Ang II) and GRK2 protein levels were increased in diabetes, and each was normalized by 4 week's losartan administration. Additionally, there was a direct correlation between the plasma Ang II and aortic GRK2 protein levels. In the diabetics, the clonidine-induced responses (but not the insulin-induced ones) were enhanced by GRK2-inhibitor. Akt phosphorylation was markedly below control in the clonidine-stimulated diabetes. The phosphorylation of Akt at Thr308 was significantly normalized and the phosphorylation of eNOS at Ser1177 tended to be increased by GRK2-inhibitor in the clonidine-stimulated diabetics. Our data suggest that (a) the Akt/eNOS pathway is downstream of GRK2, and that GRK2 inhibits Akt/eNOS activities, and (b) this pathway underlies the impaired NO production seen in type 2 diabetes, in which there are defective phosphorylations of Akt and eNOS that may be caused by an upregulation of GRK2 secondary to a high plasma Ang II level. Inhibitors of GRK2 warrant further investigation as potential new therapeutic agents in diabetes.

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.