Pharmacological inhibition of GRK2 improves cardiac metabolism and function in experimental heart failure.

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.

Antidiabetic and Cardioprotective Effects of Pharmacological Inhibition of GRK2 in db/db Mice.

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.

Vitamin-D concentrations, cardiovascular risk and events - a review of epidemiological evidence.

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.

Cellular subtype expression and activation of CaMKII regulate the fate of atherosclerotic plaque.

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.

"Freeze, Don't Move": How to Arrest a Suspect in Heart Failure - A Review on Available GRK2 Inhibitors.

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 (ßARK-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.

Integrating GRK2 and NFkappaB in the Pathophysiology of Cardiac Hypertrophy.

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.

Targeting the CaMKII/ERK Interaction in the Heart Prevents Cardiac Hypertrophy.

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-CN17ß, and the MEK/ERK inhibitor UO126 all reduce phenylephrine (PE)-mediated ERK and CaMKII activation and their interaction. Moreover, AntCaNtide or tat-CN17ß 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-CN17ß. 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.

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

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

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

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

Endothelial G protein-coupled receptor kinase 2 regulates vascular homeostasis through the control of free radical oxygen species.

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.

CaMK4 Gene Deletion Induces Hypertension.

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. (J Am Heart Assoc. 2012;1:e001081 doi: 10.1161/JAHA.112.001081.).

GRK2 at the control shaft of cellular metabolism.

G protein receptor kinase 2 (GRK2) has been for years mainly considered the negative regulator of the cardiac ß 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 ßAR 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.

Mitochondrial localization unveils a novel role for GRK2 in organelle biogenesis.

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.

A Polymorphism within the Promoter of the Dopamine Receptor D1 (DRD1 -48A/G) Associates with Impaired Kidney Function in White Hypertensive Patients.

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<0.03). Treatment restored serum creatinine at levels comparable between genotypes (AA: 0.99±0.03 mg/dl; GG: 0.94±0.02 mg/dl, n.s.). To replicate the finding, in a case control study of 8 AA and 7 GG hypertensive patients matched for age, sex and body mass index, in pharmacological wash out for 30 days, we evaluated serum (Creatinine, Na, Uric Acid, Urea) and urinary (volume/24h, protein/24h, creatinine clearance/24h) biochemistry and renal hemodynamic assessed by ultrasound. Once again, the AA group showed higher serum creatinine, Na, Uric acid and urea, reduced creatinine clearance and a higher level of urinary protein excretion. These changes occurred while no differences were observed in diuresis and renal vascular resistances. In conclusions, the DRD1 A-48G polymorphism identifies a class of H that is prone to hypertension related kidney alterations.

Association study on long-living individuals from Southern Italy identifies rs10491334 in the CAMKIV gene that regulates survival proteins.

Long-living individuals (LLIs) are used to study exceptional longevity. A number of genetic variants have been found associated in LLIs to date, but further identification of variants would improve knowledge on the mechanisms regulating the rate of aging. Therefore, we performed a genome-wide association study on 410 LLIs and 553 young control individuals with a 317K single-nucleotide polymorphism (SNP) chip to identify novel traits associated with aging. Among the top (p < 1 × 10(-4)) SNPs initially identified, we found rs10491334 (CAMKIV) (odds ratio [OR] = 0.55; 95% confidence interval [CI] 0.42-0.73; p = 2.88 × 10(-5)), a variant previously reported associated with diastolic blood pressure, associated also in a replication set of 116 LLIs and 160 controls (OR = 0.54; 95% CI 0.32-0.90; p = 9 × 10(-3)). Furthermore, in vitro analysis established that calcium/calmodulin-dependent protein kinase IV (CAMKIV) activates the survival proteins AKT, SIRT1, and FOXO3A, and we found that homozygous carriers of rs10491334 have a significant reduction in CAMKIV expression. This, together with the observed reduction in minor-allele carriers among centenarians, points to a detrimental role for the SNP. In conclusion, prolongevity genes are activated by CAMKIV, the levels of which are influenced by rs10491334, a SNP associated with human longevity.

Impaired neoangiogenesis in ß2-adrenoceptor gene-deficient mice: restoration by intravascular human ß2-adrenoceptor gene transfer and role of NFκB and CREB transcription factors.

BACKGROUND AND PURPOSE: There is much evidence supporting the role of ß2-adrenoceptors (ß2AR) in angiogenesis but the mechanisms underlying their effects have not been elucidated. Hence, we studied post-ischaemic angiogenesis in the hindlimb (HL) of ß2AR knock-out mice (ß2AR-/-) in vivo and explored possible molecular mechanisms in vitro. EXPERIMENTAL APPROACH: Femoral artery resection (FAR) was performed in wild-type and ß2AR-/- 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 ß2AR (ADß2AR) was used to reinstate ß2ARs in ß2AR-/- 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 ß2AR-/- mice subjected to FAR, but was restored by gene therapy with ADß2AR. The proangiogenic responses to a variety of stimuli were impaired in ß2AR-/- EC in vitro. Moreover, removal of ß2ARs impaired the activation of NFκB, a transcription factor that promotes angiogenesis; neither isoprenaline (stimulates ßARs) nor TNFα induced NFκB activation in ß2AR(-/-) EC. Interestingly, cAMP response element binding protein (CREB), a transcription factor that counter regulates NFκB, was constitutively increased in ß2AR(-/-) ECs. ADß2AR administration restored ß2AR membrane density, reduced CREB activity and reinstated the NFκB response to isoprenaline and TNFα. CONCLUSIONS AND IMPLICATIONS: Our results suggest that ß2ARs control angiogenesis through the tight regulation of nuclear transcriptional activity.

The G protein coupled receptor kinase 2 plays an essential role in beta-adrenergic receptor-induced insulin resistance.

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.

Calcium-calmodulin-dependent kinase II (CaMKII) mediates insulin-stimulated proliferation and glucose uptake.

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.

Effects of Ile164 polymorphism of beta2-adrenergic receptor gene on coronary artery disease.

OBJECTIVES: This study investigated the relationship between beta(2)-adrenergic receptor (B2AR) Ile164 polymorphism and coronary artery disease (CAD). BACKGROUND: B2ARs 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. METHODS: The study population consisted of 330 patients undergoing percutaneous coronary intervention (PCI). RESULTS: The 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). CONCLUSIONS: Our study suggests that the B2AR Ile164 mutant is associated with a more aggressive CAD and adversely affects prognosis in patients undergoing PCI.

Enhanced GRK2 expression and desensitization of betaAR vasodilatation in hypertensive patients.

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.