Genetic interactions in the ß-adrenoceptor/G-protein signal transduction pathway and survival after coronary artery bypass grafting: a pilot study.

BACKGROUND: In heart failure, ß-adrenergic receptor (ßAR) stimulation desensitizes the receptor, uncouples the downstream Gαs protein, and diminishes signal transduction. We tested the hypotheses that haplotype-tagging single-nucleotide polymorphisms (htSNPs) within the Gαs gene (GNAS) (i) are functionally active and alter Gαs expression, (ii) influence survival after coronary artery bypass grafting (CABG), and (iii) interact with ßAR SNPs. METHODS: Amplification of GNAS intron 1 was followed by cloning, reporter assays, electrophoretic mobility shift assays, and western blots. In a pilot study, 185 patients on ßAR blockade undergoing CABG were studied prospectively. The primary endpoint was cardiac-related mortality at 1 yr. RESULTS: Two htSNPs defined three common haplotypes with altered reporter activity, allele-specific transcription factor binding, and Gαs protein expression (highest in *3 carriers followed by *2 and *1 haplotypes, P=0.013). After CABG, mortality was GNAS diplotype-dependent: *3/*3: 0%; *3/*2: 2.4%; *3/*1: 2.9%; *2/*2: 4.5%; *2/*1: 9.1%; and *1/*1: 20.0% (P=0.004). While ß(1)AR SNPs were not associated with mortality, ß(2)AR Arg16 allele carriers were at higher risk than Gly16 allele carriers (P=0.008). Gene-gene interaction using gene-related risk alleles demonstrated the number of risk alleles to be independently associated with death (hazard ratio 2.3; 95% confidence interval: 1.5-3.5; P=0.0003). Carriers of the no-risk allele had higher maximum isoproterenol-stimulated adenylyl cyclase activities than risk allele carriers (P=0.003). CONCLUSIONS: Interactions in the ßAR/Gαs pathway may be associated with altered mortality after CABG. This could reconcile previously inconclusive data regarding the effects of ßAR SNPs on cardiovascular prognosis.

A prospective cohort study to identify and evaluate endotypes of venous thromboembolism: Rationale and design of the Genotyping and Molecular Phenotyping in Venous ThromboEmbolism project (GMP-VTE).

Several clinical, genetic and acquired risk factors for venous thromboembolism (VTE) have been identified. However, the molecular pathophysiology and mechanisms of disease progression remain poorly understood. This is reflected by uncertainties regarding the primary and secondary prevention of VTE and the optimal duration of antithrombotic therapy. A growing body of literature points to clinically relevant differences between VTE phenotypes (e.g. deep vein thrombosis (DVT) versus pulmonary embolism (PE), unprovoked versus provoked VTE). Extensive links to cardiovascular, inflammatory and immune-related morbidities are testament to the complexity of the disease. The GMP-VTE project is a prospective, multi-center cohort study on individuals with objectively confirmed VTE. Sequential data sampling was performed at the time of the acute event and during serial follow-up investigations. Various data levels (e.g. clinical, genetic, proteomic and platelet data) are available for multi-dimensional data analyses by means of advanced statistical, bioinformatic and machine learning methods. The GMP-VTE project comprises n = 663 individuals with acute VTE (mean age: 60.3 ±â€¯15.9 years; female sex: 42.8%). In detail, 28.4% individuals (n = 188) had acute isolated DVT, whereas 71.6% subjects (n = 475) had PE with or without concomitant DVT. In the study sample, 28.9% (n = 129) of individuals with PE and 30.1% (n = 55) of individuals with isolated DVT had a recurrent VTE event at the time of study enrolment. The systems-oriented approach for the comprehensive dataset of the GMP-VTE project may generate new biological insights into the pathophysiology of VTE and refine our current understanding and management of VTE.

[Methods to evaluate aspirin and clopidogrel resistance]. / Methoden zur Messung der Azetylsalizylsäure- bzw. Clopidogrelresistenz.

Based on the concept that the so-called resistance to anti-platelet drugs is meant to describe a phenomenon where the drug does not hit its direct pharmacodynamic target, assays, used to evaluated the effects of anti-platelet drugs, should as closely as possible measure the direct pharmacodynamic effect of a particular drug. Thus, for the detection of aspirin effects, thromboxane concentrations or arachidonic acid-induced responses (light aggregometry, whole-blood aggregometry) should be measured. For the detection of clopidogrel actions, VASP phosphorylation (flow cytometry) or ADP-induced responses (light aggregometry, whole blood aggregometry) should be analysed.

Is cyclic AMP formation desensitized in patients with end-stage renal failure?

1 Cyclic AMP formation has consistently been reported to be desensitized in various tissues including heart of animal models of end-stage renal failure (ESRF). In contrast, reports on desensitization of cAMP formation in ESRF patients remain contradictory. Whether this discrepancy results from a difference between human ESRF and its animal models or from the use of circulating blood cells in the human and various solid tissues in the animal studies, remains unclear. Therefore, we performed three studies with heart and platelets of ESRF patients undergoing haemodialysis or continuous ambulatory peritoneal dialysis and age- and gender-matched controls with normal renal function (n = 11-13 each). 2 In platelets from haemodialysis patients adenylyl cyclase activity in response to receptor-dependent and -independent agonists was reduced by approximately 30%, and this could be explained by an alteration at the level of adenylyl cyclase itself. However, no such desensitization was seen in platelets from peritoneal dialysis patients. 3 In hearts from ESRF patients undergoing haemodialysis, beta-adrenoceptor density and subtype distribution, cAMP formation in response to the beta-adrenoceptor agonist isoprenaline or various receptor-independent stimuli, were very similar to those in control patients but activity of G-protein-coupled receptor kinase was increased by approximately 20%. 4 We conclude that conflicting reports on the desensitization of cAMP formation between ESRF patients and ESRF animal models are not explained by the use of solid tissues in animal studies vs. circulating blood cells in patient studies. Rather desensitization of cAMP formation seems to be a less consistent feature of human ESRF than of its animal models.

Chamber-specific alterations of noradrenaline uptake (uptake(1)) in right ventricles of monocrotaline-treated rats.

1. In rats a single injection of the alkaloid monocrotaline (60 mg MCT kg(-1) body weight, i.p.) caused right ventricular hypertrophy and heart failure. The aim of this study was to find out whether, in these MCT-treated rats, the cardiac neuronal noradrenaline uptake (uptake(1)) might undergo chamber-specific alterations. 2. For this purpose we assessed in right and left ventricular slices, uptake(1) activity (by [(3)H]-noradrenaline accumulation), and in right and left ventricular membranes, uptake(1) carrier protein density (by [(3)H]-nisoxetine binding). 3. Uptake(1)-inhibitors blocked [(3)H]-noradrenaline accumulation in ventricular slices and [(3)H]-nisoxetine binding in ventricular membranes with the order of potency: desipramine > nisoxetine >> cocaine > or = GBR 12909, indicating that with both approaches noradrenaline uptake(1) was determined. 4. In right ventricular slices of MCT-treated rats uptake(1) activity was significantly lower than in control rats (84.7+/-8.2 vs 145.1+/-6.2 pmol noradrenaline mg(-1) tissue 15 min(-1); P<0.05). This was accompanied by a significant decrease in the density of [(3)H]-nisoxetine binding sites (73.7+/-14.4 vs 125.9+/-9.1 fmol mg(-1) protein; P:<0.05). 5. In left ventricular slices of MCT-treated rats uptake(1) activity was not significantly altered (131.2+/-10.5 vs 116.1+/-5.2 pmol noradrenaline mg(-1) tissue 15 min(-1)); similarly, also the density of [(3)H]-nisoxetine binding sites was unchanged (108+/-9.7 vs 123+/-7.7 fmol mg(-1) protein). 6. We conclude that in MCT-treated rats with right ventricular hypertrophy and heart failure uptake(1) activity is chamber-specifically reduced possibly due to a decrease in carrier protein density.

Beta-adrenoceptor polymorphisms.

There can be no doubt that beta(1)-, beta(2)- and beta(3)-adrenoceptor genes have genetic polymorphisms. Two single nucleotide polymorphisms have been described for the beta(1)- (Ser49Gly; Gly389Arg), three for the beta(2)- (Arg16Gly; Gln27Glu; Thr164Ile) and one for the beta(3)-adrenoceptor subtype (Trp64Arg) that might be of functional importance. The possibility that changes in expression or properties of the beta-adrenoceptors due to single nucleotide polymorphisms might have phenotypic consequences influencing their cardiovascular or metabolic function or may contribute to the pathophysiology of several disorders like hypertension, congestive heart failure, asthma or obesity is an idea that has attracted much interest during the last 10 years. At present, it appears that these beta-adrenoceptor polymorphisms are very likely not disease-causing genes, but might be risk factors, might modify disease and/or might influence progression of disease. The aim of this review is to provide an overview of the functional consequences of such beta-adrenoceptor polymorphisms in vitro, ex vivo and in vivo.

Presynaptic alpha-2C adrenoceptor-mediated control of noradrenaline release in humans: genotype- or age-dependent?

In vitro alpha-2CDel322-325 adrenoceptor (AR) polymorphism exhibits reduced functional responsiveness. We studied whether this is true also in vivo in humans. We assessed in nine young wild-type (WT) alpha-2C AR subjects (aged 23 years), 10 elder WT alpha-2C AR subjects (aged 63 years), and nine alpha-2CDel AR subjects (aged 28 years) clonidine (1 microg/kg intravenous (i.v.) bolus)-evoked plasma noradrenaline (pNA), heart rate (HR), and blood pressure (BP) changes. Clonidine-evoked pNA decreases were comparable in young WT alpha-2C and in alpha-2CDel AR subjects, but significantly lower (P=0.033) in elder subjects. Similarly, clonidine-evoked HR decreases were significantly larger in young WT alpha-2C and in alpha-2CDel AR subjects than in elder subjects, whereas clonidine-evoked BP decreases were larger in elder subjects. In conclusion, alpha-2CDel AR appears to play only a minor role in presynaptic regulation of NA release and/or to be not hypofunctional in vivo in humans, but functional responsiveness of presynaptic alpha-2 AR declines with ageing.

Presence, distribution and physiological function of adrenergic and muscarinic receptor subtypes in the human heart.

The sympathetic and parasympathetic nervous system play a powerful role in controlling cardiac function by activating adrenergic and muscarinic receptors. In the human heart there exist alpha1-, beta1- and beta2-adrenoceptors and M2-muscarinic receptors and possibly also (prejunctional) alpha2-adrenoceptors. Beta1- and beta2-adrenoceptors are quite evenly distributed in the human heart while M2-receptors are heterogeneously distributed (more receptors in atria than in ventricles). Stimulation of beta1- and beta2-adrenoceptors causes increases in heart rate and force of contraction while stimulation of M2-receptors decreases heart rate (directly in atria) and force of contraction (indirectly in ventricles). Pathological situations (such as heart failure) or pharmacological interventions (for example, beta-blocker treatment) can alter the distribution of beta1- and beta2-adrenoceptors in the human heart, while M2-receptors are only marginally affected. On the other hand, relatively little is known on distribution and functional role of alpha1- and alpha2-adrenoceptor subtypes in the human heart.

The cardiac beta-adrenoceptor-G-protein(s)-adenylyl cyclase system in monocrotaline-treated rats.

In rats, injection of the alkaloid monocrotaline (MCT) causes right ventricular hypertrophy and cardiac failure. In order to study whether, in MCT-treated rats, changes in the cardiac beta -adrenoceptor-G-protein(s)-adenylyl cyclase system might be comparable to those found in human primary pulmonary hypertension, we assessed in right and left ventricles from MCT-treated rats the components of the beta -adrenoceptor system: the receptor number and subtype distribution (by (-)-[(125)I]iodocyanopindolol binding), the G-proteins (by quantitative Western blotting), and the activity of adenylyl cyclase. A single injection of 60 mg/kg i.p. MCT caused in rats right ventricular hypertrophy (RVH); part of the rats developed cardiac failure (RVF). In these rats the cardiac beta -adrenoceptor-G-protein(s)-adenylyl cyclase system was markedly changed beta -adrenoceptors were desensitized due to a decrease in receptor number, an uncoupling of the receptor from the G(s)-adenylyl cyclase system, a decrease in G(s)and a decrease in the activity of the catalytic unit of adenylyl cyclase. In general, these changes were more pronounced in right ventricles v left ventricles, and in rats with RVF v rats with RVH. On the other hand, cardiac muscarinic receptors and G(i)appeared not to be altered. We conclude that in MCT-treated rats changes in the cardiac beta -adrenoceptor-G-protein(s)-adenylyl cyclase system occur that resemble those observed in human primary pulmonary hypertension. Thus, MCT-treated rat appears to be a suitable animal model to study in more detail the pathophysiology of the development of right heart failure, and to identify new therapeutic possibilities.