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Background The Persistent Left (PL) Superior Vena Cava (SVC) is a congenital malformation of the thoracic venous return that has an incidence of 0.2-3% in the general population and an incidence of about 0.5% in patients undergoing cardiac implantable electronic device. In this case, the placement of the leads, especially for the Right Ventricle (RV) and Left Ventricle (LV) through one of the branches could be challenging. Methods A 66-year-old man with hypertension, diabetes and dyslipidemia, in optimal pharmacologic therapy for congestive heart failure (NYHA II), hospitalized for recurrent syncopal episodes, with extreme sinus bradycardia, first-degree AV block and Left Bundle Branch Block, The patient had a LV hypertrophy and mild global systolic function (LV ejection fraction of 40%) for these we opted for a pacemaker CRT device implantation. Results Isolation of left cephalic vein was used as venous access for right atrium (RA) and RV leads while Seldinger technique via subclavian vein was used as venous access for LV lead. The guidewire through the subclavian vein pointed out the presence of PLSVC. Left subclavian venogram confirmed PLSVC and revealed a bridging innominate vein. To pass through the innominate vein and reach the SVC, a 0.035-inch hydrophilic guidewire was used. A 6Fr-sheath was advanced through the hydrophilic guidewire, subsequently a second metallic guidewire was advanced through the same introducer by removing the dilator. The RV and RA leads were placed respectively on low right interventricular septum and RA appendage passing through bridging innominate vein. Electrical parameters were normal. For the placement of the LV lead a 9.5Fr sheath introducer was advanced over the guidewire through the PLSVC. A venogram showed a lateral and a postero-lateral branch of the coronary sinus with acute angulation. With a 6Fr quadripolar Josephson catheter the postero-lateral branch was entered, eventually a 9Fr peelable outer guide catheter was advanced through the electrophysiology catheter becoming selective with the interested branch. A quadripolar S shaped lead was used, it was advanced into the targeted vein through a 0,014fr hydrophilic guidewire. Adequate electrical parameters and stability were achieved. Conclusion Lead implantation in patient with PLSVC is challenging. Placing the RA and RV leads through SVC ensure a better stability along time. The cannulation of CS is easily achieved by approach via PLSVC while selective vein cannulation can be difficult due to the modified acute angulation.

Background and aim β 2 -Adrenoceptors (β 2 -ARs) are G protein-coupled receptors (GPCRs) expressed in the major insulin target tissues. The interplay between β 2 -AR and insulin pathways is involved in the maintenance of glucose homeostasis. The aim of this study was to explore the consequences of β 2 -ARs deletion on insulin sensitivity and insulin signaling cascade in metabolically active tissues. Methods and results We evaluated glucose homeostasis in skeletal muscle and liver of β 2 -AR-null mice (β 2 -AR −/− ) by performing in vivo (glucose tolerance test and insulin tolerance test) and ex vivo (glucose uptake and glycogen determination) experiments. β 2 -AR gene deletion is associated with hepatic insulin resistance and preserved skeletal muscle insulin sensitivity. Importantly, we demonstrate that hepatic β 2 -AR regulates insulin-induced AKT activation via Grb2-mediated SRC recruitment through a G i -independent mechanism. Conclusions β-AR stimulation contributes to the development of early stages of insulin resistance progression in the liver. Our findings indicate that the cross-talk between β 2 -AR and insulin signaling represents a fundamental target towards the development of novel therapeutic approaches to treat type 2 diabetes and metabolic syndrome.

Aims The effects of GRK2 inhibition on myocardial metabolism in heart failure (HF) are unchartered. In this work, we evaluated the impact of pharmacological inhibition of GRK2 by a cyclic peptide, C7, on metabolic, biochemical, and functional phenotypes in experimental HF. Methods and results C7 was initially tested on adult mice ventricular myocyte from wild type and GRK2 myocardial deficient mice (GRK2‐cKO), to assess the selectivity on GRK2 inhibition. Then, chronic infusion of 2 mg/kg/day of C7 was performed in HF mice with cryogenic myocardial infarction. Cardiac function in vivo was assessed by echocardiography and cardiac catheterization. Histological, biochemical, and metabolic studies were performed on heart samples at time points. C7 induces a significant increase of contractility in wild type but not in adult ventricle myocytes from GRK2‐cKO mice, thus confirming C7 selectivity for GRK2. In HF mice, 4 weeks of treatment with C7 improved metabolic features, including mitochondrial organization and function, and restored the biochemical and contractile responses. Conclusions GRK2 is a critical molecule in the physiological regulation of cardiac metabolism. Its alterations in the failing heart can be pharmacologically targeted, leading to the correction of metabolic and functional abnormalities observed in HF.

Despite the availability of several therapies for the management of blood glucose in diabetic patients, most of the treatments do not show benefits on diabetic cardiomyopathy, while others even favor the progression of the disease. New pharmacological targets are needed that might help the management of diabetes and its cardiovascular complications at the same time. GRK2 appears a promising target, given its established role in insulin resistance and in systolic heart failure. Using a custom peptide inhibitor of GRK2, we assessed in vitro in L6 myoblasts the effects of GRK2 inhibition on glucose extraction and insulin signaling. Afterwards, we treated diabetic male mice (db/db) for 2 weeks. Glucose tolerance (IGTT) and insulin sensitivity (ITT) were ameliorated, as was skeletal muscle glucose uptake and insulin signaling. In the heart, at the same time, the GRK2 inhibitor ameliorated inflammatory and cytokine responses, reduced oxidative stress, and corrected patterns of fetal gene expression, typical of diabetic cardiomyopathy. GRK2 inhibition represents a promising therapeutic target for diabetes and its cardiovascular complications.

G protein coupled receptor kinase type 2 (GRK2) plays an important role in the development and maintenance of cardiac hypertrophy and heart failure even if its exact role is still unknown. In this study, we assessed the effect of GRK2 on the regulation of cardiac hypertrophy. In H9C2 cells, GRK2 overexpression increased atrial natriuretic factor (ANF) activity and enhanced phenylephrine-induced ANF response, and this is associated with an increase of NFκB transcriptional activity. The kinase dead mutant and a synthetic inhibitor of GRK2 activity exerted the opposite effect, suggesting that GRK2 regulates hypertrophy through upregulation of NFκB activity in a phosphorylation-dependent manner. In two different in vivo models of left ventricle hypertrophy (LVH), the selective inhibition of GRK2 activity prevented hypertrophy and reduced NFκB transcription activity. Our results suggest a previously undisclosed role for GRK2 in the regulation of hypertrophic responses and propose GRK2 as potential therapeutic target for limiting LVH.

Objectives Although blood pressure is considered the major determinant of left ventricular hypertrophy in hypertension, genetic variability is increasingly being considered among the factors influencing this complication. β2-Adrenergic receptors (β2ARs) are up-regulated in hypertension and largely polymorphic within the human population. Recently, we have shown that the Glu27 β2AR variant is strongly associated with cardiac hypertrophy in hypertension. The objective of this study is to verify whether this polymorphism also affects hypertrophy regression in response to antihypertensive therapy. Methods In a prospective follow-up study we screened 970 hypertensive patients of Caucasian descent for the Gly16Arg, Gln27Glu, and Thr164Ile β2AR polymorphisms and left ventricular echocardiographic hypertrophy and assigned selected patients to enalapril or atenolol to assess left ventricular hypertrophy regression after 2-year follow-up. Results were stratified according to treatment and the Glu27Gln polymorphism of the β2AR. In cells with stable overexpression of the Glu27 or Gln27 variant of β2AR, we also explored the implications of this polymorphism on hypertrophy-related intracellular signal transduction. Results Among hypertensive patients, the Gly16 allele was found in 63% of patients and the Glu27 allele was found in 40.6%. Both polymorphisms were in linkage disequilibrium, as expected. Four hundred forty-one hypertrophic hypertensive patients completed the 2-year follow-up. At baseline, patients carrying at least 1 allele of the Glu27 variant presented with a larger cardiac size despite similar blood pressure levels (142.9 ± 22.5 g/m2 in Glu27 carriers versus 138.2 ± 18.4 g/m2 in Gln27 carriers, P < .02). Blood pressure normalization was achieved by both drugs. At follow-up, compared with the Gln27 patients, the Glu27 patients showed a larger reduction in hypertrophy when treated with enalapril (percent change in left ventricular mass, −6.3% ± 7.7% in Glu27 carriers versus −2.18% ± 7.9% in Gln27 carriers; P < .05) but not with atenolol therapy (−2.8% ± 8.9% in Glu27 carriers versus −2.4% ± 8.8% in Gln27 carriers, P = not significant). In in vitro studies the activation of p38 and extracellular signal-regulated kinase (ERK-) 1/2 (data not shown) and the activity of the atrial natriuretic factor (ANF) promoter after isoproterenol (INN, isoprenaline) stimulation were larger in Glu27 β2AR overexpressing cells than in Gln27 β2AR overexpressing cells (fold difference compared with unstimulated cells, 9.7 ± 2.9 for Glu27 β2AR versus 4.2 ± 0.3 for Gln27 β2AR; P < .05). Conclusions The Glu27 variant of β2AR enhances hypertension-induced left ventricular hypertrophy. In these patients angiotensin-converting enzyme inhibitors are more efficient than β-blockers in reducing cardiac size. Clinical Pharmacology & Therapeutics (2006) 80, 633–645; doi: 10.1016/j.clpt.2006.09.006

Background and aims Atherosclerosis is a degenerative process of the arterial wall implicating activation of macrophages and proliferation of vascular smooth muscle cells. Calcium-calmodulin dependent kinase type II (CaMKII) in vascular smooth muscle cells (VSMCs) regulates proliferation, while in macrophages, this kinase governs diapedesis, infiltration and release of extracellular matrix enzymes. We aimed at understanding the possible role of CaMKII in atherosclerosis plaques to regulate plaque evolution towards stability or instability. Methods Clinically defined stable and unstable plaques obtained from patients undergoing carotid end arteriectomy were processed for evaluation of CaMKs protein expression, activity and localization. Results The larger content of CaMKII was found in CD14 + myeloid cells that were more abundant in unstable rather than stable plaques. To test the biological effect of activated CD14 + myeloid cells, VSMCs were exposed to the conditioned medium (CM) of macrophages extracted from carotid plaques. CM induced attenuation of CaMKs expression and activity in VSMCs, leading to the reduction of VSMCs proliferation. This appears to be due to the CaMKII dependent release of cytokines. Conclusions These results indicate a pivotal role of CaMKs in atherosclerosis by regulating activated myeloid cells on VSMCs activity. CaMKII could represent a possible target for therapeutic strategies based on macrophages specific inhibition for the stabilization of arteriosclerotic lesions.

Vitamin D has long been established as an elemental factor of bone physiology. Beyond mineral metabolism, the expression of the vitamin D receptor has been identified throughout the cardiovascular (CV) system. Experimental studies showed beneficial effects of vitamin D on heart and vessels, but vitamin D intoxication in animals also led to hypercalcemia and vascular calcification. Our knowledge has been extended by epidemiological studies that showed that 25-hydroxyvitamin D (25(OH)D) levels are inversely associated with an increased CV risk itself, but also with established CV risk factors, such as arterial hypertension, endothelial dysfunction and atherosclerosis. Conversely, randomized controlled trials could not document significant and consistent effects of vitamin D supplementation on CV risk or events. Potential explanations may lie in differences in reference ranges or the possibility that low vitamin D in CV disease is only an epiphenomenon. In the latter case, the key question is why low 25(OH)D levels are such a strong predictor of health. While we wait for new data, the current conclusion is that vitamin D is a strong risk marker for CV risk factors and for CV diseases itself.

Metabolic stimuli such as insulin and insulin like growth factor cause cellular accumulation of G protein Coupled Receptor Kinase 2 (GRK2), which in turn is able to induce insulin resistance. Here we show that in fibroblasts, GRK2 is able to increase ATP cellular content by enhancing mitochondrial biogenesis; also, it antagonizes ATP loss after hypoxia/reperfusion. Interestingly, GRK2 is able to localize in the mitochondrial outer membrane, possibly through one region within the RGS homology domain and one region within the catalytic domain. In vivo, GRK2 removal from the skeletal muscle results in reduced ATP production and impaired tolerance to ischemia. Our data show a novel sub-cellular localization of GRK2 in the mitochondria and an unexpected role in regulating mitochondrial biogenesis and ATP generation.

G protein receptor kinase 2 (GRK2) has been for years mainly considered the negative regulator of the cardiac β adrenergic signaling. However GRK2 is a ubiquitous molecule and its kinase activity and scaffold properties brought to several investigations which have evidenced its involvement in pathophysiology of extra-cardiac diseases. Later discoveries, moreover, indicated that this molecule is also able to influence other pathways such as insulin signaling by an inhibitory role similar to what described years before on βAR signaling. The importance of this novel function is in particular related to the possibility that this molecule can regulate the cellular metabolism, modifying the ability of cells to utilize different substrates. This hypothesis has been recently investigated in animal model of Heart Failure, evidencing that upregulation of GRK2 leads to alterations of cardiac glucose metabolism in the early stages of the disease. However GRK2 shows increased level also in the early stages of others chronic disease such as Alzheimer's Disease, indicating that these findings could be possibly applied to others cellular system and supporting the emerging idea of GRK2 as master regulator of cellular metabolism. © 2012 Bentham Science Publishers.

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

AIMS: Insulin (Ins) resistance (IRES) associates to increased cardiovascular risk as observed in metabolic syndrome. Chronic stimulation of beta-adrenergic receptors (betaAR) due to exaggerated sympathetic nervous system activity is involved in the pathogenesis of IRES. The cellular levels of G protein coupled receptor kinase 2 (GRK2) increase during chronic betaAR stimulation, leading to betaAR desensitization. We tested the hypothesis that GRK2 plays a role in betaAR-induced IRES. METHODS AND RESULTS: We evaluated Ins-induced glucose uptake and signalling responses in vitro in cell overexpressing the beta(2)AR, the GRK2, or the catalytically dead mutant GRK2-DN. In a model of increased adrenergic activity, IRES and elevated cellular GRK2 levels, the spontaneously hypertensive rats (SHR) we performed the intravenous glucose tolerance test load. To inhibit GRK2, we synthesized a peptide based on the catalytical sequence of GRK2 conjugated with the antennapedia internalization sequence (Ant-124). Ins in human kidney embryonic (HEK-293) cells causes rapid accumulation of GRK2, tyrosine phosphorylation of Ins receptor substrate 1 (IRS1) and induces glucose uptake. In the same cell type, transgenic beta(2)AR overexpression causes GRK2 accumulation associated with significant deficit of IRS1 activation and glucose uptake by Ins. Similarly, transgenic GRK2 overexpression prevents Ins-induced tyrosine phosphorylation of IRS1 and glucose uptake, whereas GRK2-DN ameliorates glucose extraction. By immunoprecipitation, GRK2 binds IRS1 but not the Ins receptor in an Ins-dependent fashion, which is lost in HEK-GRK2 cells. Ant-124 improves Ins-induced glucose uptake in HEK-293 and HEK-GRK2 cells, but does not prevent GRK2/IRS1 interaction. In SHR, Ant-124 infusion for 30 days ameliorates IRES and IRS1 tyrosine phosphorylation. CONCLUSION: Our results suggest that GRK2 mediates adrenergic IRES and that inhibition of GRK2 activity leads to increased Ins sensitivity both in cells and in animal model of IRES.

ObjectivesThis study investigated the relationship between beta2-adrenergic receptor (B2AR) Ile164 polymorphism and coronary artery disease (CAD).BackgroundB2ARs are crucial to the regulation of vascular tone, and neoangiogenesis is impaired in the presence of isoleucine at position 164 (Ile164) B2AR gene polymorphism. No data deal with the role of the variants at position 164 of the B2AR gene in the setting of CAD.MethodsThe study population consisted of 330 patients undergoing percutaneous coronary intervention (PCI).ResultsThe Ile164 polymorphism frequency was higher in CAD (12.1% vs. 3%, p = 0.008) with respect to the control population. We divided our population into 2 groups: group 1 (290 patients, threonine/threonine genotype at position 164 [164Thr/Thr]) and group 2 (40 patients, threonine/isoleucine genotype at position 164 [164Thr/Ile]). Patients of group 2 presented an earlier onset of CAD (56.7 ± 7.8 vs. 59.5 ± 10, p = 0.04) and a higher incidence of multivessel disease (25.4% vs. 41%, p = 0.044). At follow-up, group 2 showed a higher incidence of new acute myocardial infarction (17.5% vs. 4.5%, p = 0.001), new PCI (37.5% vs. 13.1%, p < 0.0001), and cardiac death (10% vs. 3.1%, p = 0.036). Cox regression analysis identified Ile164 as an independent predictor of cardiac death (odds ratio [OR]: 3.731, 95% confidence interval [CI]: 1.004 to 13.867, p = 0.049) and an overall major adverse cardiac event (OR: 4.100, 95% CI: 1.945 to 8.640, p = 0.0001). A replication study was done on a population of 150 patients with peripheral artery disease. The presence of the Ile164 allele was associated with a higher incidence of acute myocardial infarction (54.5% vs. 25.2%, p = 0.035) or combined events (acute myocardial infarction, PCI, or coronary artery bypass graft) (63.6% vs. 30.9%, p = 0.027).ConclusionsOur study suggests that the B2AR Ile164 mutant is associated with a more aggressive CAD and adversely affects prognosis in patients undergoing PCI.

Cardiovascular disease and heart failure (HF) still collect the largest toll of death in western societies and all over the world. A growing number of molecular mechanisms represent possible targets for new therapeutic strategies, which can counteract the metabolic and structural changes observed in the failing heart. G protein-coupled receptor kinase 2 (GRK2) is one of such targets for which experimental and clinical evidence are established. Indeed, several strategies have been carried out in place to interface with the known GRK2 mechanisms of action in the failing heart. This review deals with results from basic and preclinical studies. It shows different strategies to inhibit GRK2 in HF in vivo (βARK-ct gene therapy, treatment with gallein, and treatment with paroxetine) and in vitro (RNA aptamer, RKIP, and peptide-based inhibitors). These strategies are based either on the inhibition of the catalytic activity of the kinase (“Freeze!”) or the prevention of its shuttling within the cell (“Don’t Move!”). Here, we review the peculiarity of each strategy with regard to the ability to interact with the multiple tasks of GRK2 and the perspective development of eventual clinical use.

AIMS: Activation of Ca2+/Calmodulin protein kinase II (CaMKII) is an important step in signaling of cardiac hypertrophy. The molecular mechanisms by which CaMKII integrates with other pathways in the heart are incompletely understood. We hypothesize that CaMKII association with extracellular regulated kinase (ERK), promotes cardiac hypertrophy through ERK nuclear localization. METHODS AND RESULTS: In H9C2 cardiomyoblasts, the selective CaMKII peptide inhibitor AntCaNtide, its penetratin conjugated minimal inhibitory sequence analog tat-CN17β, and the MEK/ERK inhibitor UO126 all reduce phenylephrine (PE)-mediated ERK and CaMKII activation and their interaction. Moreover, AntCaNtide or tat-CN17β pretreatment prevented PE induced CaMKII and ERK nuclear accumulation in H9C2s and reduced the hypertrophy responses. To determine the role of CaMKII in cardiac hypertrophy in vivo, spontaneously hypertensive rats were subjected to intramyocardial injections of AntCaNtide or tat-CN17β. Left ventricular hypertrophy was evaluated weekly for 3 weeks by cardiac ultrasounds. We observed that the treatment with CaMKII inhibitors induced similar but significant reduction of cardiac size, left ventricular mass, and thickness of cardiac wall. The treatment with CaMKII inhibitors caused a significant reduction of CaMKII and ERK phosphorylation levels and their nuclear localization in the heart. CONCLUSION: These results indicate that CaMKII and ERK interact to promote activation in hypertrophy; the inhibition of CaMKII-ERK interaction offers a novel therapeutic approach to limit cardiac hypertrophy.

Aims The G protein-coupled receptor kinase-2 (GRK2 or b-ARK1) regulates b-adrenergic receptors (b-ARs) in the heart, and its cardiac expression is elevated in human heart failure (HF). We sought to determine whether myocardial levels and activity of GRK2 could be monitored using white blood cells, which have been used to study cardiac b-ARs. Moreover, we were interested in determining whether GRK2 levels in myocardium and lymphocytes may be associated with b-AR dysfunction and HF severity. Methods and results In myocardial biopsies from explanted failing human hearts, GRK activity was inversely correlated with b-AR-mediated cAMP production (R 2 ¼ 20.215, P , 0.05, n ¼ 24). Multiple regression analysis confirmed that GRK activity participates with b-AR density to regulate catecholaminesensitive cAMP responses. Importantly, there was a direct correlation between myocardial and lymphocytes GRK2 activity (R 2 ¼ 0.5686, P , 0.05, n ¼ 10). Lymphocyte GRK activity was assessed in HF patients with various ejection fractions (EFs) (n ¼ 33), and kinase activity was significantly higher in patients with lower EFs and was higher with increasing NYHA class (P , 0.001). Conclusion Myocardial GRK2 expression and activity are mirrored by lymphocyte levels of this kinase, and its elevation in HF is associated with the loss of b-AR responsiveness and appears to increase with disease severity. Therefore, lymphocytes may provide a surrogate for monitoring cardiac GRK2 in human HF.

OBJECTIVE: Cardiac and vascular remodeling occur in response to hypertension. Genetic background appears to modify the development of target organ damage (TOD). We evaluated the impact on hypertension-associated TOD of a highly polymorphic gene with elevated significance for the regulation of the cardiovascular system, the beta2AR gene. METHODS: We recruited 775 hypertensives (mean +/- SE: age 53.5 +/- 0.5, from 20 to 84 years; female 32.7\%; systolic (SBP)/diastolic (DBP) blood pressure: 159 +/- 1.2/101 +/- 0.6 mmHg) referred to the departmental outpatient clinic and screened them for the Arg16Gly, Gln27Glu, and Ile164Thr variants of beta2AR gene. We performed association analyses on clinical, anamnesis, anthropometrical and biochemical parameters as well as cardiac and vascular ultrasound. RESULTS: We found that the three polymorphisms did not affect blood pressure levels. Cardiac TOD appeared to be related to the Glu27 variant. In fact, the Glu27 allele associates with a 1.4-fold higher risk of developing cardiac hypertrophy, and directly correlated with larger systolic and diastolic left ventricle internal diameters. Vascular TOD was not affected by the three polymorphisms. Ancillary to our finding we observed that the Glu27 variant is associated with a higher incidence of dyslipidemia. CONCLUSIONS: Our data indicate that beta2AR gene polymorphisms participate in the determination of cardiac TOD associated with hypertension.

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

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

Objective— The role of endothelial G protein–coupled receptor kinase 2 (GRK2) was investigated in mice with selective deletion of the kinase in the endothelium (Tie2-CRE/GRK2 fl/fl ). Approach and Results— Aortas from Tie2-CRE/GRK2 fl/fl presented functional and structural alterations as compared with control GRK2 fl/fl mice. In particular, vasoconstriction was blunted to different agonists, and collagen and elastic rearrangement and macrophage infiltration were observed. In primary cultured endothelial cells deficient for GRK2, mitochondrial reactive oxygen species was increased, leading to expression of cytokines. Chronic treatment with a reactive oxygen species scavenger in mice corrected the vascular phenotype by recovering vasoconstriction, structural abnormalities, and reducing macrophage infiltration. Conclusions— These results demonstrate that GRK2 removal compromises vascular phenotype and integrity by increasing endothelial reactive oxygen species production.

B2 Adrenergic Receptor Polymorphisms and Treatment- Outcomes in Cardiovascular Diseases Cardiovascular diseases (CVD) represent the major health problem in the western world and heavy social and economic burden. Under this name an eterogenous group of multifactorial conditions is included. There is appreciable inter-individual variability in the susceptibility to cardiovascular disease and in the response to the associated pharmacological treatments. Genetic polymorphism may be, at least in part, responsible for both susceptibility to disease and inter-individual variability in response to pharmacological treatments.

Dopamine DRD1 receptor regulates renal function and vascular resistance. It plays a role in the pathogenesis of hypertension in animal models. In humans, the DRD1 gene presents a A-48G polymorphism associated to hypertension in a Japanese population. To explore the role of this polymorphism on blood pressure and renal function in Caucasian hypertensive patients (H), we evaluated the allele frequencies in a populations of 697 H and 100 blood volunteers, and found no difference in the distribution of the alleles between the two groups (AA;AG;GG: 13%;50%;37%; and 12%; 51%;36% respectively). In H, we found a significant difference between AA and GG in serum creatinine (AA: 1.06±.08 mg/dl; GG:0.97±0.02 mg/dl, p

Background The expression of calcium/calmodulin‐dependent kinase IV ( CaMKIV ) was hitherto thought to be confined to the nervous system. However, a recent genome‐wide analysis indicated an association between hypertension and a single‐nucleotide polymorphism ( rs10491334 ) of the human CaMKIV gene ( CaMK4 ) , which suggests a role for this kinase in the regulation of vascular tone. Methods and Results To directly assess the role of CaMKIV in hypertension, we characterized the cardiovascular phenotype of CaMK4 −/− mice. They displayed a typical hypertensive phenotype, including high blood pressure levels, cardiac hypertrophy, vascular and kidney damage, and reduced tolerance to chronic ischemia and myocardial infarction compared with wild‐type littermates. Interestingly, in vitro experiments showed the ability of this kinase to activate endothelial nitric oxide synthase. Eventually, in a population study, we found that the rs10491334 variant associates with a reduction in the expression levels of CaMKIV in lymphocytes from hypertensive patients. Conclusions Taken together, our results provide evidence that CaMKIV plays a pivotal role in blood pressure regulation through the control of endothelial nitric oxide synthase activity.

Vascular smooth muscle cell (VSMC) proliferation contributes to vascular remodeling in atherosclerosis and hypertension. Calcium-dependent signaling through calcium/calmodulin-dependent kinase II (CaMKII) and ERK1/2 activation plays an important role in the regulation of VSMC proliferation by agents such as α-adrenergic receptor agonists. Nevertheless, how the CaMKII and ERK pathways interact in VSMCs has yet to be characterized. The aim of the present study was to clarify this interaction in response to α1-adrenergic receptor-mediated VSMC proliferation. We discovered that phenylephrine stimulation resulted in complex formation between CaMKII and ERK in a manner that facilitated phosphorylation of both protein kinases. To assess the effects of CaMKII/ERK association on VSMC proliferation, we inhibited endogenous CaMKII either pharmacologically or by adenoviral-mediated gene transfer of a kinase-inactive CaMKII mutant. Inhibition of CaMKII activation but not CaMKII autonomous activity significantly decreased formation of the CaMKII/ERK complex. On the contrary, the expression of constitutively active CaMKII enhanced VSMC growth and CaMKII/ERK association. In addressing the mechanism of this effect, we found that CaMKII could not directly phosphorylate ERK but instead enhanced Raf1 activation. By contrast, ERK interaction with CaMKII facilitated CaMKII phosphorylation and promoted its nuclear localization. Our results reveal a critical role for CaMKII in VSMC proliferation and imply that CaMKII facilitates assembly of the Raf/MEK/ERK complex and that ERK enhances CaMKII activation and influences its subcellular localization.

Cellular growth and glucose uptake are regulated by multiple signals generated by the insulin receptor. The mechanisms of individual modulation of these signals remain somewhat elusive. We investigated the role of CaMKII in insulin signalling in a rat skeletal muscle cell line, demonstrating that CaMKII modulates the insulin action on DNA synthesis and the negative feedback that down regulates glucose uptake. Insulin stimulation generated partly independent signals leading to the rapid activation of Akt, Erk-1/2 and CaMKII. Akt activation was followed by Glut-4 translocation to the plasma membrane and increase of glucose uptake. Then, IRS-1 was phosphorylated at S612, the IRS-1/p85PI3K complex was disrupted, Akt was no more phosphorylated and both Glut-4 translocation and glucose uptake were reduced. Inhibition of CaMKII abrogated the insulin-induced Erk-1/2 activation, DNA synthesis and phosphorylation of IRS-1 at S612. Inhibition of CaMKII also abrogated the down-regulation of insulin-stimulated Akt phosphorylation, Glut-4 membrane translocation and glucose uptake. These results demonstrate that: 1 - CaMKII modulates the insulin-induced Erk-1/2 activation and cell proliferation; 2 - after the initial stimulation of the IRS-1/Akt pathway, CaMKII mediates the down-regulation of stimulated glucose uptake. This represents a novel mechanism in the selective control of insulin signals, and a possible site for pharmacological intervention.

Increased levels of G protein coupled receptor kinase GRK2 appear to participate in hypertension presumably through the desensitization of beta adrenergic receptors (betaARs) that mediate vasodilatation. There are contrasting data on the occurrence of betaAR desensitization in the vasculature, we therefore investigated betaAR vasodilatation and desensitization in normotensives and in hypertensive humans. In blood lymphocytes, we assessed betaAR signaling and GRK2 expression and found betaAR signaling alterations and, consistent with desensitization, ncreased GRK2 levels in hypertensives. We studied in vivo vasodilatation to the betaAR agonist isoproterenol (ISO) injected in the brachia artery in control conditions and during the concomitant infusion of heparin, a known in vitro nonspecific GRK inhibitor. ISO induced a dose-dependent vasorelaxation that was attenuated in hypertensives indicating a loss of betaAR signaling. Intra-arterial infusion of heparin nhibited lymphocyte GRK2 activity and prevented desensitization of betaAR vasodilatation in normotensives. In hypertensives, heparin restored vasodilatation to ISO, to levels observed in normotensives. Our results suggest that betaAR desensitization does indeed occur at the vascular levels in vivo, and that heparin by acting as a GRK inhibitor prevents this in normotensives and restores impaired betaAR vasodilation in hypertensives. We conclude that desensitization participates to impaired betaAR vasodilation in hypertension.

We have recently demonstrated that endothelial beta(2) adrenergic receptors beta(2)AR regulate eNOS activity and consequently vascular tone, through means of PKB/AKT. In this work we explored the signal transduction pathway leading to AKT/eNOS activation in endothelial cells (EC). Using pharmacological and molecular inhibitors both in cultured EC cells and in ex vivo rat carotid preparations, we found that G(i) coupling of the beta 2AR is needed for AKT activation and vasorelaxation. Since endothelial activation is sensitive to pertussis toxin but not to G(i beta gamma), inhibition by beta ARKct, we conclude that G(alpha i) mediates beta AR induced AKT activation. Downstream, beta AR signalling requires the soluble tyrosine kinase SRC, as both in cultured EC and rat carotid, the mutant dominant negative of SRC prevent beta(2)AR induced endothelial activation and vasodilation. In EC, G(alpha i) directly interacts with SRC and this interaction leads to SRC activation and phosphorylation in a manner that is regulated by Balk stimulation. We propose a novel signal transduction pathway for beta(2)AR stimulation trough G(alpha i) and SRC, leading to activation of AKT. (c) 2007 Elsevier Inc. All rights reserved.

β 2 -Adrenergic receptors (β 2 ARs) are widely expressed, although their physiological relevance in many tissues is not yet fully understood. In vascular endothelial cells, they regulate NO release and vessel tone. Here we provide novel evidence that β 2 ARs can regulate neoangiogenesis in response to chronic ischemia. We used in vivo adenoviral-mediated gene transfer of the human β 2 AR to the endothelium of the rat femoral artery and increased β 2 AR signaling resulting in ameliorated angiographic blood flow and hindlimb perfusion after chronic ischemia. Histological analysis confirmed that β 2 AR overexpression also produced benefits on capillary density. The same maneuver partially rescued impaired angiogenesis in spontaneously hypertensive rats (SHR), whereas gene delivery of the G-protein–coupling defective mutant Ile164 β 2 AR failed to provide ameliorations. Stimulation of endogenous and overexpressed β 2 AR on endothelial cells in vitro was found to regulate cell number by inducing proliferation and [ 3 H]-thymidine incorporation through means of extracellular receptor-activated kinase and vascular endothelial growth factor. The β 2 AR also has novel effects on endothelial cell number through stimulation of proapoptosis and antiapoptosis pathways involving p38 mitogen-activated protein kinase and PI3-kinase/Akt activation. Therefore, β 2 ARs play a critical role in endothelial cell proliferation and function including revascularization, suggesting a novel and physiologically relevant role in neoangiogenesis in response to ischemia.

Background — Impaired β-adrenergic receptor (AR)–mediated vasorelaxation in hypertension plays a role in increased peripheral vascular resistance and blood pressure. Because the β 2 AR is the most abundant vascular AR subtype, we sought to enhance βAR vasorelaxation by overexpressing β 2 ARs via adenoviral-mediated gene transfer (ADβ 2 AR) to the vascular endothelium of the carotid artery. Methods and Results — In normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats, we exposed the right common carotid artery to ADβ 2 AR in situ for 15 minutes by injection into the lumen while the blood flow was interrupted. Control carotids received an empty vector (ADempty). Three days later, transgene expression and selective endothelial localization were confirmed in infected vessels. Vasoregulation after β 2 AR overexpression (2-fold) was studied in isolated organ baths. ADβ 2 AR carotid responses to α 1 AR and α 2 AR agonists were not affected, whereas responses to epinephrine were altered and βAR-mediated vasorelaxation was enhanced after β 2 AR overexpression. As expected, βAR-mediated vasodilatation in control carotids of SHR rats was significantly less than in similar control WKY carotid arteries. ADβ 2 AR treatment enhanced βAR vasorelaxation in SHR to levels similar to those seen in ADβ 2 AR WKY carotids. Conclusions — Our results demonstrate a critical role for the endothelium in βAR-mediated vasorelaxation and suggest that impaired βAR signaling may account for dysfunctional βAR vasorelaxation in hypertension rather than impaired endothelium-dependent nitric oxide metabolism.

Aging is associated with alterations in β-adrenergic receptor (β-AR) signaling and reduction in cardiovascular responses to β-AR stimulation. Because exercise can attenuate age-related impairment in myocardial β-AR signaling and function, we tested whether training could also exert favorable effects on vascular β-AR responses. We evaluated common carotid artery responsiveness in isolated vessel ring preparations from 8 aged male Wistar-Kyoto (WKY) rats trained for 6 wk in a 5 days/wk swimming protocol, 10 untrained age-matched rats, and 10 young WKY rats. Vessels were preconstricted with phenylephrine (10-6 M), and vasodilation was assessed in response to the β-AR agonist isoproterenol (10-10-3 × 10-8 M), the α2-AR agonist UK-14304 (10-9-10-6 M), the muscarinic receptor agonist ACh (10-9-10-6 M), and nitroprusside (10-8-10-5 M). β-AR density and cytoplasmic β-AR kinase (β-ARK) activity were tested on pooled carotid arteries. β-ARK expression was assessed in two endothelial cell lines from bovine aorta and aorta isolated from a 12-wk WKY rat. β-AR, α2-AR, and muscarinic responses, but not that to nitroprusside, were depressed in untrained aged vs. young animals. Exercise training restored β-AR and muscarinic responses but did not affect vasodilation induced by UK-14304 and nitroprusside. Aged carotid arteries showed reduced β-AR number and increased β-ARK activity. Training counterbalanced these phenomena and restored β-AR density and β-ARK activity to levels observed in young rat carotids. Our data indicate that age impairs β-AR vasorelaxation in rat carotid arteries through β-AR downregulation and desensitization. Exercise restores this response and reverts age-related modification in β-ARs and β-ARK. Our data support an important role for β-ARK in vascular β-AR vasorelaxation.