Cardiac G Protein-coupled Receptor Kinase 2 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.

Aldosterone Jeopardizes Myocardial Insulin and β-Adrenergic Receptor Signaling via G Protein-Coupled Receptor Kinase 2.

Hyperaldosteronism alters cardiac function, inducing adverse left ventricle (LV) remodeling either via increased fibrosis deposition, mitochondrial dysfunction, or both. These harmful effects are due, at least in part, to the activation of the G protein-coupled receptor kinase 2 (GRK2). In this context, we have previously reported that this kinase dysregulates both β-adrenergic receptor (βAR) and insulin (Ins) signaling. Yet, whether aldosterone modulates cardiac Ins sensitivity and βAR function remains untested. Nor is it clear whether GRK2 has a role in this modulation, downstream of aldosterone. Here, we show in vitro, in 3T3 cells, that aldosterone impaired insulin signaling, increasing the negative phosphorylation of insulin receptor substrate 1 (ser307pIRS1) and reducing the activity of Akt. Similarly, aldosterone prevented the activation of extracellular signal-regulated kinase (ERK) and the production of cyclic adenosine 3′,5′-monophosphate (cAMP) in response to the β1/β2AR agonist, isoproterenol. Of note, all of these effects were sizably reduced in the presence of GRK2-inhibitor CMPD101. Next, in wild-type (WT) mice undergoing chronic infusion of aldosterone, we observed a marked GRK2 upregulation that was paralleled by a substantial β1AR downregulation and augmented ser307pIRS1 levels. Importantly, in keeping with the current in vitro data, we found that aldosterone effects were wholly abolished in cardiac-specific GRK2-knockout mice. Finally, in WT mice that underwent 4-week myocardial infarction (MI), we observed a substantial deterioration of cardiac function and increased LV dilation and fibrosis deposition. At the molecular level, these effects were associated with a significant upregulation of cardiac GRK2 protein expression, along with a marked β1AR downregulation and increased ser307pIRS1 levels. Treating MI mice with spironolactone prevented adverse aldosterone effects, blocking GRK2 upregulation, and thus leading to a marked reduction in cardiac ser307pIRS1 levels while rescuing β1AR expression. Our study reveals that GRK2 activity is a critical player downstream of the aldosterone signaling pathway; therefore, inhibiting this kinase is an attractive strategy to prevent the cardiac structural disarray and dysfunction that accompany any clinical condition accompanied by hyperaldosteronism.

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.

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.

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 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.

Downregulation of G protein-coupled receptor kinase 2 levels enhances cardiac insulin sensitivity and switches on cardioprotective gene expression patterns

G protein-coupled receptor kinase 2 (GRK2) has recently emerged as a negative modulator of insulin signaling. GRK2 downregulation improves insulin sensitivity and prevents systemic insulin resistance. Cardiac GRK2 levels are increased in human heart failure, while genetically inhibiting GRK2 leads to cardioprotection in mice. However, the molecular basis underlying the deleterious effects of GRK2 up-regulation and the beneficial effects of its inhibition in the heart are not fully understood. Therefore, we have explored the interconnections among a systemic insulin resistant status, GRK2 dosage and cardiac insulin sensitivity in adult (9month-old) animals. GRK2+/− mice display enhanced cardiac insulin sensitivity and mild heart hypertrophy with preserved systolic function. Cardiac gene expression is reprogrammed in these animals, with increased expression of genes related to physiological hypertrophy, while the expression of genes related to pathological hypertrophy or to diabetes/obesity co-morbidities is repressed. Notably, we find that cardiac GRK2 levels increase in situations where insulin resistance develops, such as in ob/ob mice or after high fat diet feeding. Our data suggest that GRK2 downregulation/inhibition can help maintain cardiac function in the face of co-morbidities such as insulin resistance, diabetes or obesity by sustaining insulin sensitivity and promoting a gene expression reprogramming that confers cardioprotection.

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.

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

Myocardial Ablation of G Protein–Coupled Receptor Kinase 2 (GRK2) Decreases Ischemia/Reperfusion Injury through an Anti-Intrinsic Apoptotic Pathway

Studies from our lab have shown that decreasing myocardial G protein–coupled receptor kinase 2 (GRK2) activity and expression can prevent heart failure progression after myocardial infarction. Since GRK2 appears to also act as a pro-death kinase in myocytes, we investigated the effect of cardiomyocyte-specific GRK2 ablation on the acute response to cardiac ischemia/reperfusion (I/R) injury. To do this we utilized two independent lines of GRK2 knockout (KO) mice where the GRK2 gene was deleted in only cardiomyocytes either constitutively at birth or in an inducible manner that occurred in adult mice prior to I/R. These GRK2 KO mice and appropriate control mice were subjected to a sham procedure or 30 min of myocardial ischemia via coronary artery ligation followed by 24 hrs reperfusion. Echocardiography and hemodynamic measurements showed significantly improved post-I/R cardiac function in both GRK2 KO lines, which correlated with smaller infarct sizes in GRK2 KO mice compared to controls. Moreover, there was significantly less TUNEL positive myocytes, less caspase-3, and -9 but not caspase-8 activities in GRK2 KO mice compared to control mice after I/R injury. Of note, we found that lowering cardiac GRK2 expression was associated with significantly lower cytosolic cytochrome C levels in both lines of GRK2 KO mice after I/R compared to corresponding control animals. Mechanistically, the anti-apoptotic effects of lowering GRK2 expression were accompanied by increased levels of Bcl-2, Bcl-xl, and increased activation of Akt after I/R injury. These findings were reproduced in vitro in cultured cardiomyocytes and GRK2 mRNA silencing. Therefore, lowering GRK2 expression in cardiomyocytes limits I/R-induced injury and improves post-ischemia recovery by decreasing myocyte apoptosis at least partially via Akt/Bcl-2 mediated mitochondrial protection and implicates mitochondrial-dependent actions, solidifying GRK2 as a pro-death kinase in the heart.

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.

Effects of exercise training on cardiovascular adrenergic system

In heart failure (HF), exercise has been shown to modulate cardiac sympathetic hyperactivation which is one of the earliest features of neurohormonal derangement in this syndrome and correlates with adverse outcome. An important molecular alteration related to chronic sympathetic overstimulation in HF is represented by cardiac β-adrenergic receptor (β-AR) dysfunction. It has been demonstrated that exercise reverses β-AR dysfunction by restoring cardiac receptor membrane density and G-protein-dependent adenylyl cyclase activation. In particular, several evidence indicate that exercise reduces levels of cardiac G-protein coupled receptor kinase-2 (GRK2) which is known to be involved in both β1-AR and β2-AR dysregulation in HF. Similar alterations of β-AR system have been described also in the senescent heart. It has also been demonstrated that exercise training restores adrenal GRK2/α-2AR/catecholamine (CA) production axis. At vascular level, exercise shows a therapeutic effect on age-related impairment of vascular reactivity to adrenergic stimulation and restores β-AR-dependent vasodilatation by increasing vascular β-AR responsiveness and reducing endothelial GRK2 activity. Sympathetic nervous system overdrive is thought to account for >50% of all cases of hypertension and a lack of balance between parasympathetic and sympathetic modulation has been observed in hypertensive subjects. Non-pharmacological, lifestyle interventions have been associated with reductions in SNS overactivity and blood pressure in hypertension. Several evidence have highlighted the blood pressure lowering effects of aerobic endurance exercise in patients with hypertension and the significant reduction in sympathetic neural activity has been reported as one of the main mechanisms explaining the favorable effects of exercise on blood pressure control.

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.

Adrenal GRK2 lowering is an underlying mechanism for the beneficial sympathetic effects of exercise training in heart failure

Exercise training has been reported to exert beneficial effects on cardiac function and to reduce morbidity and mortality of chronic heart failure (HF). Augmented sympathetic nervous system (SNS) activity, leading to elevated circulating catecholamine (CA) levels, is a hallmark of chronic HF that significantly aggravates this disease. Exercise training has been shown to also reduce SNS overactivity in HF, but the underlying molecular mechanism(s) remain unidentified. We recently reported that adrenal G protein-coupled receptor kinase-2 (GRK2), an enzyme that regulates the sympathoinhibitory alpha(2)-adrenoceptors (alpha(2)-ARs) present in the CA-producing adrenal medulla, is upregulated in HF, contributing to the chronically elevated CA levels and SNS activity of the disease. In the present study, we tested whether exercise training can affect the adrenal GRK2-alpha(2)-AR-CA production system in the context of HF. For this purpose, a 10-wk-long exercise training regimen of adult male Sprague-Dawley rats starting at 4 wk postmyocardial infarction (post-MI) was employed, and examination at the end of this treatment period revealed significant amelioration of beta-AR-stimulated contractility in response to exercise training, accompanied by cardiac GRK2 reduction and restoration of circulating plasma CA levels. Importantly, adrenal GRK2 expression (72 + or - 5% reduction vs. post-MI untrained) and alpha(2)-AR number were also restored after exercise training in post-MI animals. These results suggest that exercise training restores the adrenal GRK2-alpha(2)-AR-CA production axis, and this might be part of the mechanism whereby this therapeutic modality normalizes sympathetic overdrive and impedes worsening of the failing heart.

Cardiac hypertrophy in neonatal nephrectomized rats: the role of the sympathetic nervous system

Cardiac hypertrophy is frequently encountered in patients with renal failure and represents an independent risk factor for cardiovascular morbidity and mortality. The pathogenesis of cardiac hypertrophy is related to multiple factors, including excess adrenergic activity. This study investigated how renal injury in the early stages of life affects the adrenergic system and thereby potentially influences cardiac growth. Biomarkers of cardiac hypertrophy were used to assess adrenergic function. Newborn male Sprague-Dawley rats were allocated to three groups of five rats each: 5/6 nephrectomy (Nx), pair-fed controls (PF), and sham-operated (SH). Nx animals had significantly higher plasma urea nitrogen, serum creatinine, and mean arterial blood pressure. The heart-weight/body-weight ratio of the Nx cohort was higher than SH and PF (p < 0.001) groups. Plasma norepinephrine (NE) of Nx animals was almost twofold higher than SH and PF (p < 0.01) animals. Compared with SH and PF, Nx animals had higher alpha1A-receptor protein expression, lower cardiac beta1- and beta2-receptor protein expression (p < 0.05), but higher G-protein-coupled receptor kinase-2 (GRK2) expression (p < 0.05). Norepinephrine transporter protein (NET) and renalase protein expression in cardiac tissue from Nx pups were significantly lower than SH and PF. Our data suggest that early age Nx animals have increased circulating catecholamines due to decreased NE metabolism. Enhancement of cardiac GRK2 and NE can contribute to cardiac hypertrophy seen in Nx animals. Furthermore, AKT (activated via alpha1A receptors), as well as increased alpha1A receptors and their agonist NE, might contribute to the observed hypertrophy.

Uncoupling of Myocardial β-Adrenergic Receptor Signaling During Coronary Artery Bypass Grafting: The Role of GRK2

Cardiopulmonary bypass (CPB) and cardioplegic arrest during cardiac surgery leads to desensitization of myocardial beta-adrenergic receptors (beta-ARs). Impaired signaling through this pathway can have a detrimental effect on ventricular function and increased need for inotropic support. The mechanism of myocardial beta-AR desensitization during cardiac surgery has not been defined. This study investigates the role of G protein-coupled receptor kinase-2 (GRK2), a serine-threonine kinase which phosphorylates and desensitizes agonist-occupied beta-ARs, as a primary mechanism of beta-AR uncoupling during coronary artery bypass grafting (CABG) with CPB and cardioplegic arrest. Forty-eight patients undergoing elective CABG were enrolled in this study. Myocardial beta-AR signaling was assessed by measuring total beta-AR density and adenylyl cyclase activity in right atrial biopsies obtained before CPB and just before weaning from CPB. Myocardial GRK2 expression and activity were also measured before CPB and just before weaning from CPB. Myocardial beta-AR signaling was significantly impaired after CPB and cardioplegic arrest during CABG. Cardiac GRK2 expression was not altered; however, there was a twofold increase in GRK2 activity during CABG. There was an even greater elevation in cardiac GRK2 activity in patients with severely depressed ventricular function. Increased myocardial GRK2 activity appears to be the primary mechanism of impaired beta-AR signaling during CABG with CPB and cardioplegic arrest. This may contribute to the greater need for inotropic support in patients with severe ventricular dysfunction. Strategies to inhibit activation of GRK2 during CABG may decrease morbidity in this patient population.