Fermí Montó

The impact of alpha1-adrenoceptors up-regulation accompanied by the impairment of beta-adrenergic vasodilatation in hypertension.

In human and animal hypertension models, increased activity of G-protein-coupled receptor kinase (GRK) 2 determines a generalized decrease of β-adrenergic vasodilatation. We analyzed the possibility of differential changes in the expression and functionality of α1A, α1B, α1D, β1, β2, and β3-ARs also being involved in the process. We combined the quantification of mRNA levels with immunoblotting and functional studies in aortas of young and adult spontaneously hypertensive rats (SHRs) and their controls (Wistar Kyoto). We found the expression and function of β1-adrenoceptors in young prehypertensive SHRs to be higher, whereas a generalized increase in the expression of the six adrenoceptors and GRK2 was observed in aortas of adult hypertensive SHRs. α1D- and β3-Adrenoceptors, the subtypes that are more resistant to GRK2-mediated internalization and mostly expressed in rat aorta, exhibited an increased functional role in hypertensive animals, showing two hemodynamic consequences: 1) an increased sensitivity to the vasoconstrictor stimulus accompanied by a decreased sensitivity to the vasodilator stimulus (α1D-ARs are the most sensitive to agonists, and β3-ARs are the least sensitive to agonists); and 2) a slower recovery of the basal tone after adrenergic stimulus removal because of the kinetic characteristic of the α1D subtype. These functional changes might be involved in the greater sympathetic vasoconstrictor tone observed in hypertension.

Different expression of adrenoceptors and GRKs in the human myocardium depends on heart failure ethiology and correlates to clinical variables

Downregulation of β(1)- adrenergic receptors (β(1)-ARs) and increased expression/function of G-protein-coupled receptor kinase 2 (GRK2) have been observed in human heart failure, but changes in expression of other ARs and GRKs have not been established. Another unresolved question is the incidence of these compensatory mechanisms depending on heart failure etiology and treatment. To analyze these questions, we quantified the mRNA/protein expressions of six ARs (α(1A), α(1B), α(1D), β(1), β(2), and β(3)) and three GRKs (GRK2, GRK3, and GRK5) in left (LV) and right ventricle (RV) from four donors, 10 patients with ischemic cardiomyopathy (IC), 14 patients with dilated cardiomyopathy (DC), and 10 patients with nonischemic, nondilated cardiopathies (NINDC). We correlated the changes in the expressions of ARs and GRKs with clinical variables such as left ventricular ejection fraction (LVEF) and left ventricular end-systolic and left ventricular end-diastolic diameter (LVESD and LVEDD, respectively). The main findings were 1) the expression of the α(1A)-AR in the LV positively correlates with LVEF; 2) the expression of GRK3 and GRK5 inversely correlates with LVESD and LVEDD, supporting previous observations about a protective role for both kinases in failing hearts; and 3) β(1)-AR expression is downregulated in the LV and RV of IC, in the LV of DC, and in the RV of NINDC. This difference, better than an increased expression of GRK2 (not observed in IC), determines the lower LVEF in IC and DC vs. NINDC.

Myocardial G protein receptor-coupled kinase expression correlates with functional parameters and clinical severity in advanced heart failure.

BACKGROUND In heart failure (HF), sympathetic hyperactivation induces deleterious effects in myocardial β-adrenergic signaling, with receptor down-regulation and desensitization mediated by G protein receptor-coupled kinases (GRKs). We hypothesised that changes in GRK isoforms may be associated with clinical status in advanced HF, using the Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) scale. METHODS We included 31 patients with advanced HF undergoing transplantation. According to INTERMACS profiles, mRNA and protein levels of GRK isoforms in left ventricular (LV) myocardium were analyzed and compared with nonfailing LV samples. RESULTS In failing LV myocardium, GRK2 and GRK5 (but not GRK3) protein was up-regulated compared with control samples. Among HF patients, an increase in GRK2 and GRK5 mRNA and protein abundance was observed in β-agonist-treated patients (vs β-blockers: P < .05) and in higher-risk INTERMACS status (profiles 2 and 3 vs 4 and 5: P < .05). A significant negative correlation of GRK2 expression with LV stroke volume supported these findings. CONCLUSIONS Increased GRK2 correlates with clinical severity using the INTERMACS scale and LV stroke volume, supporting it as a potential target in advanced HF. These changes are paralleled by GRK5 expression in the failing myocardium, suggesting a relevant role in human HF.

The three α1-adrenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation.

We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α1-adrenoceptor (α1-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting β-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α1A- and α1B-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α1A-AR, but not α1B-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by β-arrestin 2 knockdown or concanavalin A treatment. α1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α1D antagonist BMY7378. Following activation by phenylephrine, α1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α1D-AR subtype was not observed via CypHer5 technology. The three α1-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on β-arrestin 2 mediated internalization.

beta-Adrenoceptor and GRK3 expression in human lymphocytes is related to blood pressure and urinary albumin excretion.

Objective The objective of our work was to analyze if changes in the expression of β-adrenoceptors (β-ARs) and G-protein-coupled receptor kinases (GRKs) in human lymphocytes – a practical surrogate for myocardial or vascular cells – are related to the hypertensive state and its clinical consequences. Methods Real-time quantitative RT-PCR was employed to evaluate the expression of the three β-ARs (β1, β2, β3) and three GRKs (GRK2, GRK3, GRK5) in human lymphocytes obtained from both normotensive and hypertensive patients, some of whom had been treated with blockers of the renin–angiotensin system. Office blood pressure, 24-h ambulatory blood pressure, urinary albumin excretion and serum biochemical profile were also recorded. Results and conclusions β1-AR expression levels were higher in circulating lymphocytes from hypertensive patients (2-ΔΔCt = 2.135 ± 0.4252*, vs. control group), but this difference was not observed when these patients were treated with blockers of the renin–angiotensin system. β1-AR levels directly correlated (r2 = 0.5711, P = 0.0185) with urinary albumin excretion in microalbuminuric patients, which relates alterations of this receptor to cardiovascular risk. An inverse correlation was observed between the expression levels of β2-AR and diastolic blood pressure (r2 = 0.2078, P = 0.0031), suggesting that β2-AR levels in lymphocytes mirror their expression in vascular cells, in which β2-AR-mediated relaxation regulates vascular resistance. mRNA levels for GRK3 were inversely correlated with systolic and diastolic blood pressure (day, night and 24 h), which suggests a protective role for GRK3 in the regulation of human blood pressure, as supported by previous findings in transgenic mice.

Myocardial and Peripheral Lymphocytic Transcriptomic Dissociation of β-adrenoceptors and G Protein-coupled Receptor Kinases in Heart Transplantation

BACKGROUND The genetic expression of adrenergic receptors plays an important pathophysiologic role in heart failure. G protein-coupled receptor kinases (GRKs) desensitize the beta-receptor to catecholaminergic stimulation. It has been suggested that their mRNA expression in peripheral lymphocytes could mirror the changes in their myocardial expression in the failing heart, but this relationship between the myocyte and lymphocyte has not been studied in heart transplantation (HT). The objective of this study was to analyze adrenergic receptor and GRK mRNA expression in myocardium and lymphocytes and their correlation. METHODS Twenty-three HT patients without evidence of acute rejection or echocardiographic dysfunction were assessed. Myocardial biopsy samples and peripheral blood lymphocytes were obtained, and alpha(1)- and beta-adrenoceptor subtype and GRK subtype mRNA was analyzed using reverse transcript-polymerase chain reaction (RT-PCR). RESULTS Mean age was 45 +/- 15 years, with a median of time since HT of 205 (351) days. In biopsies, the beta(1)/beta(2)-adrenoceptor ratio was 57%/42%, and GRK5 was the most commonly expressed, followed by GRK2. In lymphocytes, the beta(1)/beta(2) ratio was 3%/96%, whereas GRK2 mRNA expression was greater than that of other subtypes. There was no correlation between myocardial and lymphocyte parameters. There were no correlations with clinical variables, but lymphocyte beta(2) and GRK2 were increased with time since HT. CONCLUSIONS In the transplanted heart, there is no correlation between mRNA expression of adrenoceptors and GRKs in myocardium and peripheral lymphocytes. With time since transplant, mRNA expression of lymphocyte but not myocardial beta(2)-adrenoceptor and GRK2 increases. Therefore, this dissociation between myocardial and lymphocyte mRNA expression limits the potential use of peripheral blood samples for diagnosis of graft dysfunction.

β2- and β1-Adrenoceptor Expression Exhibits a Common Regulatory Pattern With GRK2 and GRK5 in Human and Animal Models of Cardiovascular Diseases.

Abstract: To explore if genic expression of &bgr;1- or &bgr;2-adrenoceptors (ARs) exhibits a common regulatory pattern with G protein–coupled receptor kinase (GRK) 2, GRK3, or GRK5 expression, we determined messenger RNA levels for these genes in different tissues from human and animal models of cardiovascular disease. We measured genic expression by qRT polymerase chain reaction in the left and right ventricles or peripheral blood mononuclear cells from healthy (n = 21), hypertensive (n = 20), heart failure (n = 24), and heart transplanted patients (n = 17) or in left ventricle, peripheral blood mononuclear cells, and kidney from spontaneously hypertensive rats or L-N-methyl-arginine–induced hypertensive rats and their respective controls (n = 4–5). In diseased versus healthy subjects and rats, parallel changes in messenger RNA levels of GRK2 and &bgr;2-AR or GRK5 and &bgr;1-AR were observed in each territory. Therefore, without excluding other regulatory mechanisms, the parallelism observed suggests a common regulatory pattern for the &bgr;1-AR/GRK5 and &bgr;2-AR/GRK2 genes, which is independent of cellular type or pathology. This highlights the need to focus not only on GRKs but also on &bgr;1- or &bgr;2-AR changes to completely understand the involvement of &bgr;-AR/GRK pathways in cardiovascular diseases.

Differences in the Signaling Pathways of α1A- and α1B-Adrenoceptors Are Related to Different Endosomal Targeting

Aims To compare the constitutive and agonist-dependent endosomal trafficking of α1A- and α1B-adrenoceptors (ARs) and to establish if the internalization pattern determines the signaling pathways of each subtype. Methods Using CypHer5 technology and VSV-G epitope tagged α1A- and α1B-ARs stably and transiently expressed in HEK 293 cells, we analyzed by confocal microscopy the constitutive and agonist-induced internalization of each subtype, and the temporal relationship between agonist induced internalization and the increase in intracellular calcium (determined by FLUO-3 flouorescence), or the phosphorylation of ERK1/2 and p38 MAP kinases (determined by Western blot). Results and Conclusions Constitutive as well as agonist-induced trafficking of α1A and α1B ARs maintain two different endosomal pools of receptors: one located close to the plasma membrane and the other deeper into the cytosol. Each subtype exhibited specific characteristics of internalization and distribution between these pools that determines their signaling pathways: α1A-ARs, when located in the plasma membrane, signal through calcium and ERK1/2 pathways but, when translocated to deeper endosomes, through a mechanism sensitive to β-arrestin and concanavalin A, continue signaling through ERK1/2 and also activate the p38 pathway. α1B-ARs signal through calcium and ERK1/2 only when located in the membrane and the signals disappear after endocytosis and by disruption of the membrane lipid rafts by methyl-β-cyclodextrin

Myocardial and lymphocytic expression of eNOS and nNOS before and after heart transplantation: relationship to clinical status.

AIMS The present study investigates the expression and clinical relevance of the constitutive NO synthases in heart and peripheral blood mononuclear cells (PBMCs) obtained from heart failure patients. MAIN METHODS mRNA and protein levels (qRT-PCR and immunoblot) of eNOS and nNOS were determined in: i) Left ventricle (LV, n=4) and PBMCs (n=10) from healthy donors; ii) LV, right ventricle (RV) and PBMCs of heart failure (HF) patients (n=32); and iii) biopsies and PBMCs of the HF patients after cardiac transplant (n=15). KEY FINDINGS Expression of constitutive NOS isoforms in heart exhibits wide variability in HF patients, but this variability was not related to aetiology, disease severity, concomitant pathologies or drug regimes. A significantly increased eNOS expression was found in LV from HF patients without vs. with pulmonary hypertension. Overall, higher eNOS expression in this chamber was associated with lower pulmonary arterial pressure. Furthermore, a higher eNOS expression in HF is associated with smaller LV diameter, whereas, a higher post-transplant eNOS expression is related to greater cardiac distensibility. In the RV, nNOS increased after transplant. The positive correlation found between the nNOS expression in the LV of HF patients and the cardiac index suggests a role for this isoform in facilitating cardiac work. A decreased expression of eNOS was observed in PBMCs from HF patients vs. healthy donors, which recovers after transplant. SIGNIFICANCE A selective up-regulation of the cardiac expression of each NOS isoform in the failing heart, which is not mirrored by PBMCs, is related to an improved health status.

Glucocorticoids as modulators of expression and activity of Antithrombin (At): Potential clinical relevance

INTRODUCTION An inverse relationship has been reported between decreased postoperative Antithrombin (AT) plasmatic levels and the incidence of complications. We hypothesized that Nuclear Hormone Receptors could modulate the expression of SERPINC1, encoding AT, through a Hormone Regulatory Element present in its promoter, and thus hormone analogs could be a pharmacological complement in surgical procedures to activate endogenous AT synthesis. MATERIALS AND METHODS The expression of SERPINC1 was analyzed in HepG2 cells by quantitative RT-PCR and Western Blot. Two studies were conducted with (a) patients submitted to cardiac surgery with cardiopulmonary bypass receiving (n =17) or not (n=321) glucocorticoids (GCs) as part of their pharmacological treatment, and (b) patients who received (n =20) or not (n=16) GCs as part of their surgery (exodontia or knee arthroscopic, respectively). AT activity in plasma was determined by Innovance Antithrombin Test on a BCS XP System hemostasis analyzer. RESULTS 13 nuclear hormone receptor ligands were assayed, being GW4064 (FXR ligand) the most potent activator. Retinoids, activating RXR, and GCs (Dexamethasone, cortisone and methylprednisolone) also resulted in increased AT expression. Chronic GC treatment mitigates the decreased AT activity observed after cardiac surgery. In patients who received two acute GC doses, pre-operative and post-operative AT activity was similar, whereas a significant decrease was observed after surgery in untreated patients. CONCLUSIONS Whereas retinoids and FXR ligands are investigational compounds, regulation of AT by GCS could have a higher potential for translation to clinical practice, pre-conditioning the patient against complications related to reduced AT levels. Larger prospective studies are needed to define the exact role of GCs and their potential clinical utility in cardiac surgery.