Petra Schnabel

Expression of Endothelin-1, Endothelin-Converting Enzyme, and Endothelin Receptors in Chronic Heart Failure

BACKGROUND Elevated plasma levels of endothelin (ET)-1 have been reported in association with heart diseases, including heart failure. Furthermore, it has been suggested that ET-1 acts as a local autocrine/paracrine factor with biological activities such as vasoconstriction, mitogenesis, and inotropic effects on the heart. This study investigated alterations of ET-1, ET receptor, and endothelin-converting enzyme (ECE) expression in left ventricular myocardium from patients with end-stage heart failure. METHODS AND RESULTS mRNA concentrations of ETA and ETB receptors, prepro-ET-1 (ppET-1), and ECE in left ventricles from nonfailing donors hearts (NF) and from patients with end-stage chronic heart failure (NYHA functional class IV) due to dilated cardiomyopathy (DCM) were compared by use of a competitive reverse transcription-polymerase chain reaction technique. There was no significant difference in mRNA expression for ppET-1, ECE-1, and ETA receptors, whereas a significant reduction of ETB-receptor mRNA was observed in DCM hearts. 125I-labeled ET-1 radioligand binding studies demonstrated a significant downregulation of ETB receptors, whereas ETA-receptor density was increased in membranes from DCM hearts. Phosphoramidon-sensitive ECE activity and immunodetectable amounts of ECE protein in left ventricular membrane preparations did not differ between NF and DCM hearts. Finally, immunoreactive ET-1 concentrations were increased in DCM hearts. CONCLUSIONS The present study demonstrates changes in the ET-receptor expression pattern in favor of the ETA receptor in human end-stage heart failure. Furthermore, activation of the cardiac ET system with increased tissue ET-1 concentrations in the failing myocardium is observed. This is more likely due to decreased clearance than to increased synthesis, because ppET-1 gene expression and ECE activity are unchanged.

Phosphatidylinositol 3-Kinase-dependent Membrane Recruitment of Rac-1 and p47phox Is Critical for α-Platelet-derived Growth Factor Receptor-induced Production of Reactive Oxygen Species

Platelet-derived growth factor (PDGF) plays a critical role in the pathogenesis of proliferative diseases. NAD(P)H oxidase (Nox)-derived reactive oxygen species (ROS) are essential for signal transduction by growth factor receptors. Here we investigated the dependence of PDGF-AA-induced ROS production on the cytosolic Nox subunits Rac-1 and p47phox, and we systematically evaluated the signal relay mechanisms by which the αPDGF receptor (αPDGFR) induces ROS liberation. Stimulation of the αPDGFR led to a time-dependent increase of intracellular ROS levels in fibroblasts. Pharmacological inhibitor experiments and enzyme activity assays disclosed Nox as the source of ROS. αPDGFR activation is rapidly followed by the translocation of p47phox and Rac-1 from the cytosol to the cell membrane. Experiments performed in p47phox(-/-) cells and inhibition of Rac-1 or overexpression of dominant-negative Rac revealed that these Nox subunits are required for PDGF-dependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant αPDGFRs that lack specific binding sites for αPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase Cγ, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase Cγ, or SHP-2) completely abolished PDGF-dependent p47phox and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant αPDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110α (but not p110β) was identified as the crucial isoform that elicits αPDGFR-mediated production of ROS. Finally, bromodeoxyuridine incorporation and chemotaxis assays revealed that the lack of ROS liberation blunted PDGF-AA-dependent chemotaxis but not cell cycle progression. We conclude that PI3K/p110α mediates growth factor-dependent ROS production by recruiting p47phox and Rac-1 to the cell membrane, thereby assembling the active Nox complex. ROS are required for PDGF-AA-dependent chemotaxis but not proliferation.

β-Adrenergic signal transduction in the failing and hypertrophied myocardium

Abstract A strong sympathetic activation has been observed in heart failure and is the cause of β-adrenergic desensitization in this condition. On the receptor level there is downregulation of β1-adrenergic receptors and uncoupling of β2-adrenoceptors. The latter mechanism has been related to an increased activity and gene expression of β-adrenoceptor kinase in failing myocardium, leading to phosphorylation and uncoupling of receptors. β3-Adrenoceptors mediate negative inotropic effects, but alterations in these receptors are not known. In addition, an increase in inhibitory G protein α subunits (Giα) has been suggested to be causally linked to adenylyl cyclase desensitization in heart failure. In contrast, the catalytic subunit of adenylyl cyclase, stimulatory G protein α and βγ subunits, have been observed to be unchanged. Recent evidence shows that increases in Giα also depress adenylyl cyclase in compensated cardiac hypertrophy both in monogenic and polygenic and in secondary hypertension. These increases of Giα can suppress adenylyl cyclase in the absence of β-adrenergic receptor downregulation. Since cardiac hypertrophy in pressure overload is a strong predictor of cardiac failure, these observations indicate that adenylyl cyclase desensitization by Giα may be a pathophysiologically relevant mechanism contributing to the progression from compensated cardiac hypertrophy to heart failure.

The Arg389Gly β1-adrenoceptor gene polymorphism determines contractile response to catecholamines

OBJECTIVES Recently, the Arg389Gly beta1-adrenoceptor (beta1AR) gene polymorphism has been detected. The Arg variant exhibited increased responsiveness to agonist-induced stimulation in vitro. Functional studies in isolated human atrial muscle strips and in-vivo studies revealed contradictory results regarding the functional relevance of this polymorphism. We sought to characterize the functional consequences of the Arg389Gly beta1-AR polymorphism in 30 consecutive healthy male volunteers in vivo. METHODS beta1-AR genotype was determined by PCR and restriction analysis, which was confirmed by DNA sequencing. We compared heart rate, blood pressure, and contractile response of the various genotype carriers with a modified dobutamine stress echocardiography protocol. RESULTS Subjects homozygous for the Arg389 beta1AR showed a significantly higher increase in fractional shortening upon cumulative doses of dobutamine as compared to subjects carrying one or two copies of the Gly389 allele. A statistically significant difference was observed at a dobutamine dose of 10 microg/kg/min (46.5 +/- 1.3 vs. 41.8 +/- 1.0 %; P = 0.023) and was maximal at 40 microg/kg/min (61.9 +/- 1.4 vs. 52.8 +/- 1.6; P = 0.001). As a result, the systolic blood pressure response to dobutamine was significantly enhanced in individuals homozygous for the Arg389 allele, whereas the effect on heart rate did not differ between the two groups. Normalization for changing afterload conditions by calculating the pressure-dimension ratio revealed similar effects, indicating that the beta1AR-mediated effects are mainly a result of increased myocardial inotropy. CONCLUSION These data indicate that the Arg389Gly beta1AR polymorphism is functionally relevant in vivo and determines contractile responsiveness to catecholamines in humans.

Molecular aspects of adrenergic signal transduction in cardiac failure

Abstract Abnormal β-adrenergic signal transduction and intracellular Ca2+ handling appear to be a major cause of systolic and diastolic dysfunction in humans with heart failure. The precise mechanisms which cause an alteration in Ca2+ handling have been a subject of investigation in recent years. Several lines of evidence suggest that activation of neurohormonal systems plays a central role. Altered Ca2+-handling (increased diastolic concentrations, reduced systolic Ca2+ release) have a strong impact on diastolic and systolic performance of failing hearts. Sarcoplasmic reticulum Ca2+ ATPase is reduced in activity and in steady-state mRNA concentration. The Na+-Ca2+ exchanger is upregulated at the mRNA and protein levels. Phospholamban depends strongly on cAMP-dependent phosphorylation. A strong sympathetic activation has been shown to desensitize the cAMP system. At the receptor level, there is downregulation of β1-adrenergic receptors. An uncoupling of β2-adrenoceptors has been attributed to an increased activity and gene expression of β-adrenergic receptor kinase in failing myocardium, leading to phosphorylation and uncoupling of receptors. Finally, recent evidence suggests that cAMP-dependent transcription mechanisms may play a role during β-adrenergic stimulation and cardiomyopathy with heart failure – by means of altered actions of cAMP response element binding protein, the cAMP response element modulator, or the activating transcription factor 1. The exact characterization of signal transduction defects could offer novel approaches to the pharmacological treatment of heart failure.

Characterization of β1-selectivity, adrenoceptor-Gs-protein interaction and inverse agonism of nebivolol in human myocardium

Intrinsic activity and β1‐selectivity are important features of β‐blockers in the treatment of patients with coronary syndromes and heart failure. In human myocardium, intrinsic activity and β1‐selectivity of the novel β‐adrenoceptor antagonist nebivolol have not yet been determined. The study examines intrinsic activity, β‐adrenoceptor‐G‐protein coupling and β1‐selectivity of nebivolol and bisoprolol in human ventricular myocardium. Furthermore, intrinsic activity of both compounds is compared to the one of bucindolol, carvedilol and metoprolol in human atrial myocardium. Radioligand binding studies ([125I]‐lodocyanopindolol) were performed on membrane preparations of human failing and nonfailing myocardium and on COS‐7 cells transfected with human β1‐ and β2‐adrenoceptors, respectively. Functional experiments were carried out on isolated muscle preparations of human left ventricular and right atrial myocardium from failing and nonfailing hearts. Radioligand binding studies reveal 3 – 4 fold β1‐selectivity for nebivolol and 16 – 20 fold β1‐selectivity for bisoprolol in human myocardium. In COS‐7‐cells, β1‐selectivity is 3 fold for nebivolol and 15 fold for bisoprolol. Neither the binding of nebivolol nor of bisoprolol is affected by the presence of guanylylimidodiphosphate (Gpp(NH)p). Nebivolol and bisoprolol exert similar inverse agonist activity in human ventricular as well as atrial myocardium. In atrial myocardium, inverse agonism of both compounds is higher compared to bucindolol, equal to carvedilol and lower compared to metoprolol. Favourable haemodynamic effects of nebivolol in humans are not due to β1‐selectivity or partial agonist activity of this agent. Other mechanisms, i.e. the production of nitric oxide, may thus be responsible for its unique haemodynamic profile.

Down‐regulation of aortic and cardiac AT1 receptor gene expression in transgenic (mRen‐2) 27 rats

Transgenic(TG) (mRen‐2) rats overexpressing the mouse renin gene develop fulminant hypertension and cardiac hypertrophy. Since the activation of AT1 receptor by angiotensin II is involved in blood pressure regulation, cardiac performance and myocardial growth, we investigated the biological effects of angiotensin II and the regulation of the AT1 receptor in the heart and aorta of TGR (mRen‐2)27 rats in comparison to control animals. Contraction studies on isolated cardiac muscle strips reveal that angiotensin II exerts no positive inotropic effect on the left ventricular myocardium of both, transgenic and control rats. In contrast, angiotensin II leads via AT1 receptor activation in the left atrium of control rats to a significant contraction (130±5% of basal contraction) which is not detectable in left atrium preparations of the transgenic animals. Furthermore, AT1 receptor activation causes a profound contraction of aortic rings isolated from control rats amounting to 1.39±0.2 mN mg−1 wet weight, whereas aortic rings from TGR (mRen‐2)27 rats contract only minimally upon angiotensin II stimulation (0.2±0.02 mN mg−1 wet weight). These altered physiological responses of angiotensin II in the transgenic rats are in part due to a marked down‐regulation of the AT1 receptor in atrial, ventricular and aortic tissue of these transgenic animals in comparison to control Sprague‐Dawley rats, as shown by radioligand binding assays and quantitative polymerase chain reaction (PCR) experiments. The AT1 receptor density Bmax in the left atrium was 1.3±0.08 fmol mg−1 protein in control rats (KD 1.1±0.18 nmol l−1) and 0.94±0.15 fmol mg−1 protein (KD 2.1±0.3 nmol l−1. In the aorta Bmax values were 15.1±0.5 fmol mg−1 protein (KD 1.9±0.27 nmol l−1) for control rats and 11.3±0.76 fmol mg−1 protein (KD 1.9±0.27 nmol l−1) for the TGR(mRen‐2)27 rats AT1 receptor mRNA was reduced in the transgenic animals to 46±3% in the left atrium, 50±11% in the left ventricle and 40±3% in the aorta, respectively. Together, the AT1 receptor is down‐regulated in TGR (mRen‐2)27 rats in comparison to wildtype Sprague Dawley rats leading to a profoundly decreased response of cardiac and aortic tissue upon stimulation with angiotensin II.

Binding properties of beta-blockers at recombinant beta1-, beta2-, and beta3-adrenoceptors.

The human heart contains at least four distinct beta-adrenoceptor subtypes, three of which have been cloned. However, the binding properties of beta-blockers to the different beta-adrenoceptor subpopulations are not yet thoroughly characterized. Human beta1-, beta2- and beta3-adrenoceptors were expressed in COS-7 cells and [125I]iodocyanopindolol saturation binding, and competition experiments with commonly used beta-blockers were performed in the respective membrane preparations. Atenolol and metoprolol were about fivefold selective for beta1- versus beta2- and beta3-adrenoceptors. Bisoprolol was approximately 15-fold selective for beta1- versus beta2- and approximately 31-fold selective for beta1- versus beta3-adrenoceptors. Carvedilol was nonselective for any beta-adrenoceptor subtype. We conclude that the beta1-selectivities of atenolol, metoprolol, and bisoprolol are lower in COS cell membranes compared with previous investigations performed in native membranes. All beta-blockers investigated bind to beta3-adrenoceptors. Differential binding properties to beta3-adrenoceptors might imply different responses as to body weight, cardiac contractility, heart rate, and growth regulation. This might imply differential indications for the drugs investigated.

Reduced effects of dopexamine on force of contraction in the failing human heart despite preserved β2-adrenoceptor subpopulation

The results of the present study show that the reduction of the total number of β adrenoceptors affected the β1-adrenoceptor subpopulation, whereas the β2 adrenoceptors were not detectably altered in the failing heart. Dopexamine had a 9.8-fold greater affinity to β2 adrenoceptors than to β1adrenoceptors. In nonfailing myocardium, dopexamine increased force of contraction concentration-dependently. However, dopexamine alone had no effect in papillary muscle strips from moderately (NYHA II-III) and severely (NYHA IV) failing myocardium. However, in the presence of milrinone, it concentration-dependently increased force of contraction. Under this condition, the effectiveness was slightly less pronounced in NYHA IV than in NYHA II-III. Dopexamine concentration-dependently stimulated adenylate cyclase activity. Experiments with the β1-selective antagonist CGP 207.12 A and the β2-selective antagonist ICI 118.551 showed that both stimulation of adenylate cyclase and the increase of force of contraction are mediated by β2 adrenoceptors. It is concluded that although the number of β2 adrenoceptors is preserved in the failing myocardium, dopexamine alone does not increase force of contraction. However, the positive inotropic effect of dopexamine, which is observed in the presence of milrinone and the stimulation of adenylate cyclase activity by dopexamine are mediated by R2 adrenoceptors. Therefore, β2 adrenoceptors exist in the human myocardium, are coupled to adenylate cyclase, and are capable of increasing force of contraction. These results provide evidence for an impaired coupling of β2 adrenoceptors to mechanisms beyond receptor occupation in terminal heart failure.

Effects of Angiotensin II Type 1 Receptor Blockade and Angiotensin-Converting Enzyme Inhibition on Cardiac β-Adrenergic Signal Transduction

Inhibition of the renin-angiotensin system has been shown to improve symptoms and prognosis in heart failure. We compared the effects of inhibition of angiotensin-converting enzyme or blockade of angiotensin II type 1 (AT1) receptors in a model with renin-induced hypertension that is known to exhibit similar changes in sympathetic activation and beta-adrenergic desensitization, as observed in heart failure. Treatment with captopril (100 mg/kg of feed) or the AT1-antagonist Bay 10-6734 (100 mg/kg of feed) was performed in transgenic rats harboring the mouse renin 2d gene [TG(mREN2)27]. Neuropeptide Y and angiotensin II levels, adenylyl cyclase activity, beta-adrenergic receptors, G(salpha), and G(ialpha) were investigated. TG(mREN2)27 showed a depletion of myocardial neuropeptide Y stores and an increase in myocardial angiotensin II concentrations. Isoprenaline- and guanylylimidodiphosphate-stimulated adenylyl cyclase activities and beta-adrenergic receptor density were reduced, whereas the catalyst and G(salpha)-function were unchanged. G(ialpha) protein and mRNA concentrations were increased. All alterations were normalized by both treatments. Systolic left ventricular pressures, plasma atrial natriuretic peptide, and myocardial steady state atrial natriuretic peptide mRNA concentrations and heart weights were similarly reduced by both treatments. Sympathetic neuroeffector defects are similarly reversed by angiotensin-converting enzyme inhibition or AT1 antagonism. The data support the concept that pharmacological interventions in the myocardial renin-angiotensin system significantly reverse local sympathetic neuroeffector defects. This could be important for the beneficial effects of these agents.