Markus Flesch

Unchanged Protein Levels of SERCA II and Phospholamban but Reduced Ca2+ Uptake and Ca2+-ATPase Activity of Cardiac Sarcoplasmic Reticulum From Dilated Cardiomyopathy Patients Compared With Patients With Nonfailing Hearts

BACKGROUND The aim of the present study was to investigate whether Ca2+ uptake into the sarcoplasmic reticulum (SR) is altered in failing human myocardium resulting from dilated cardiomyopathy. METHODS AND RESULTS Ca(2+)-ATPase (SERCA II) activity and Ca(2+)-dependent 45Ca2+ uptake (oxalate supported, steady state) in isolated vesicles from the SR (VSR) and in crude membrane preparations (CSR) (free Ca2+, 0.01 to 100 mumol/L) from nonfailing (donor hearts, n = 13) and terminally failing (heart transplants, dilated cardiomyopathy, n = 17) human myocardium were studied. In the same hearts, protein levels (Western blot analysis) and mRNA levels (Northern blot analysis) of SERCA II and phospholamban were measured. Increasing concentrations of Ca2+ were followed by an increased Ca(2+)-ATPase activity and Ca2+ uptake. Ca2+ uptake activity and Ca(2+)-ATPase activity in CSR preparations from failing myocardium were significantly reduced compared with nonfailing hearts (Ca(2+)-ATPase, 163 +/- 8 and 125 +/- 7 nmol ATP/mg protein per minute for nonfailing tissue and failing tissue in New York Heart Association [NYHA] class IV, respectively; Ca2+ uptake, 7.1 +/- 0.8 and 3.5 +/- 0.3 nmol/mg protein per minute in CSR from nonfailing and NYHA class IV hearts, respectively P < .05). In contrast, no significant difference was measured in VSR. In the same preparations (CSR and VSR), both SERCA II and phospholamban levels (Western blot technique with monoclonal antibodies) were unchanged in failing compared with nonfailing tissue. mRNA expression relative to GAPDH mRNA for SERCA IIa and for phospholamban was significantly reduced in failing human myocardium (P < .05). CONCLUSIONS These findings provide evidence that in failing human myocardium caused by dilated cardiomyopathy, protein levels of SERCA II and phospholamban are unchanged even though mRNA levels for SERCA II and phospholamban and the SERCA II function are reduced compared with nonfailing myocardium.

Effect of β-Blockers on Free Radical–Induced Cardiac Contractile Dysfunction

Background—We examined the effects of hydroxyl radicals (OH·) on human myocardial contractility and on sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity and the effects of the β-receptor antagonists metoprolol, carvedilol, and its metabolite BM-910228. Methods and Results—Isometric force of contraction was determined in isolated human myocardium. H2O2 1 mmol/L and Fe3+-nitrilotriacetic acid (Fe3+-NTA) 0.1 mmol/L used for generation of OH· induced a decrease in basal force of contraction and an increase in diastolic tension in atrial and left ventricular myocardial preparations. After challenge with OH·, the maximum positive inotropic response to Ca2+ 1.8 to 15 mmol/L was decreased by 60% and by 39%, respectively. The effects of OH· could be blocked by catalase. Carvedilol and its metabolite BM-910228 attenuated the OH·-induced impairment of the inotropic response to Ca2+ in atrial myocardial preparations. Metoprolol had no significant effect. The stimulation frequency (0.5 to 3.0 Hz)–dependent increase ...

Decreased number of circulating progenitor cells in obesity: beneficial effects of weight reduction

AIMS Cardiovascular risk factors are associated with decreased levels of circulating progenitor cells (CPC). The aim of this study was to determine whether the number of CPC is an independent correlate of body mass index (BMI) and whether weight loss leads to an increase in CPC. METHODS AND RESULTS CD34 positive and KDR/CD34, CD133/CD34, and CD117/CD34 double positive cells were measured by fluorescence activated cell sorting (FACS) analysis in peripheral blood of 149 volunteers (52.5 +/- 12.0 years, BMI 21.5-52.7 kg/m(2), mean 31.6 +/- 5.1 kg/m(2)) participating in a weight reduction program offered by German pharmacies. In addition, carotid intima media thickness (IMT) and brachial artery flow-mediated dilatation were determined. After a diet and sports program for 6 months, 86 representing subjects were re-evaluated (mean weight loss 5.8 +/- 5.2 kg). There was an inverse correlation between BMI as well as waist circumference and CPC, especially CD34 positive, KDR/CD34 positive, CD133/CD34 positive, and CD117/CD34 positive cells. This decrease in CPC in obesity held true not only for the absolute cell numbers, but also for the relative fractions of KDR, CD133, and CD117 positive cells within the CD34 positive cells, indicating a specific down regulation of these progenitor cell types. Multiple regression analysis revealed that BMI was a more prominent predictor of CPC regulation than blood pressure, LDL cholesterol, triglycerides, fasting glucose, and smoking. IMT increased in dependence on BMI (P < 0.001) and was inversely correlated with the number of CD34 positive cell (P < 0.05). After diet, there was a significant increase of CD34 and CD117/CD34 positive cells, which correlated with the decrease in BMI. Also, weight loss was accompanied by a decrease in IMT (P = 0.015), which also correlated with the increase in CPC (P < 0.001). The increase in the number of CPC was independent from whether weight loss was achieved by increased physical exercise or by reduced calorie intake only. CONCLUSION Obesity is associated with decreased numbers of CPC and increased IMT. Diet and weight loss lead to an increase in CPC count, which might contribute to regression of IMT.

Different intrinsic activities of bucindolol, carvedilol and metoprolol in human failing myocardium

Clinical studies have shown different effects of β‐blockers on the β‐adrenergic system, tolerability and outcome in patients with heart failure. The study examines β‐adrenoceptor‐G‐protein coupling and intrinsic activity of bucindolol, carvedilol and metoprolol in human ventricular myocardium. Radioligand binding studies ([125I]‐Iodocyanopindolol) were performed in membrane preparations of human failing and nonfailing myocardium. Functional experiments were carried out in isolated muscle preparations of human left ventricular myocardium from failing hearts. Bucindolol and carvedilol bound non‐selectively to β1‐ and β2‐adrenoceptors and exerted guanine nucleotide modulatable binding. Metoprolol was 35‐fold β1‐selective and lacked guanine nucleotide modulatable binding. All β‐blockers antagonized isoprenaline‐induced enhancement of contractility. In preparations in which the coupling of the stimulatory G‐protein to adenylate cyclase was facilitated by forskolin, bucindolol increased force of contraction in three and decreased it in five experiments. Carvedilol increased force in one and decreased it in six experiments. Metoprolol decreased force in all experiments by 89.4±2.2% (P<0.01 metoprolol vs carvedilol and bucindolol). The negative inotropic effect of metoprolol was antagonized by bucindolol. It is concluded that differences in intrinsic activity can be detected in human myocardium and have an impact on cardiac contractility. In human ventricular myocardium, bucindolol displays substantially higher intrinsic activity than metoprolol and carvedilol. Bucindolol can behave as partial agonist or partial inverse agonist depending on the examined tissue. Differences in intrinsic activity may contribute to differences in β‐adrenoceptor regulation and possibly to differences in tolerability and outcomes of patients with heart failure.

Alcohol and the risk of myocardial infarction

Abstract Epidemiological studies have repeatedly demonstrated a beneficial effect of moderate alcohol consumption on the incidence of coronary heart disease, myocardial infarction and overall mortality. The latter increases with excessive alcohol consumption. Although most epidemiological studies demonstrate a beneficial effect of alcohol consumption independent from the specific kind of alcoholic beverage, there is increasing evidence that wine and in particular red wine might contain pharmacological substances, which prevent atherosclerosis and myocardial infarction independent from the wine ethanol. Pathophysiological mechanisms mediating these beneficial effects include effects of wine phenols and tannins on LDL-cholesterol oxidation status, thrombocyte aggregation, endothelial function and smooth muscle cell proliferation. Identification and characterization of the pharmacologically active substances might provide the stage for the development of new substances to be used in the prevention of coronary artery disease and myocardial infarction.

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.

Central role for ornithine decarboxylase in β-adrenoceptor mediated hypertrophy

Objective: TGF-β stimulation of cardiac myocytes induces a hypertrophic responsiveness to β-adrenoceptor stimulation. This study investigates whether this β-adrenoceptor mediated effect depends on induction of ornithine decarboxylase (ODC). Methods: Isolated adult ventricular cardiomyocytes from rats were used as an experimental model. Cells were either cultured in 20% (v/v) FCS to activate autocrine released TGF-β or used without pre-treatment. The hypertrophic response was characterized by an increased 14C-phenylalanine incorporation, RNA and protein mass or by an increased expression of atrionatriurectic factor and ODC. The results on cell cultures were compared to those achieved by isoprenaline perfused mice hearts from transgenic mice overexpressing TGF-β1. Results: ODC activity and expression increased within 2 h in TGF-β1 pre-treated cells under isoprenaline. In the presence of ODC inhibitors (α-methylornithine or difluoromethylornithine) this increase remained absent and the increases in 14C-phenylalanine incorporation, protein and RNA mass under isoprenaline were abolished. In cells not exposed to TGF-β no induction of ODC was observed. Isoprenaline also induced ODC in isolated perfused ventricles from transgenic mice overexpressing TGF-β1, but not in ventricles from their nontransgenic counterparts. Conclusions: This study shows first, a pivotal role for ODC induction in the hypertrophic response of cardiomyocytes to β-adrenoceptor stimulation and second, that ODC induction in vivo and in vitro requires pre-treatment of cardiomyocytes with TGF-β. It is concluded that TGF-β induces a hypertrophic responsiveness to β-adrenoceptor stimulation that is characterized by ODC induction.

Dissociation of blood pressure reduction from end-organ damage in TGR(mREN2)27 transgenic hypertensive rats

Objective Since the biochemical disturbance underlying hypertension may be an important determinant of patient outcome, we compared the effects of early treatment with different antihypertensive drugs on end-organ damage in the TGR(mREN2)27 transgenic rat (REN-2). In these REN-2 rats, hypertension is primarily caused by increased activity of the tissue renin–angiotensin system. Design and methods Seven-week-old REN-2 rats were either untreated or treated orally with an optimal daily dose of carvedilol (30 mg/kg), hydralazine (30 mg/kg), losartan (10 mg/kg) or quinapril (15 mg/kg). Nontransgenic littermates served as normotensive controls. After 11 weeks of treatment, we determined plasma norepinephrine concentrations, left ventricular atrial natriuretic factor messenger RNA and cardiac and vascular function and hypertrophy. Results Chronic treatment with carvedilol and hydralazine significantly decreased blood pressure to a similar level but failed to normalize it, whereas both losartan and quinapril completely normalized blood pressure. Despite a blood pressure reduction in all treatment groups, only losartan, quinapril and hydralazine preserved endothelial function, while carvedilol did not. Furthermore, losartan and quinapril prevented cardiac and medial hypertrophy. The expression of atrial natriuretic factor messenger RNA paralleled the hemodynamic changes. Plasma norepinephrine levels were normalized by losartan or quinapril but remained increased after carvedilol and hydralazine treatment. Conclusions In REN-2 hypertensive rats, end-organ damage can be prevented by both inhibition of the angiotensin converting enzyme and blockade of the angiotensin II type 1 receptor, but not by merely lowering blood pressure. When blood pressure is not fully normalized, the effects on end-organs are clearly dissociated from the antihypertensive effects.

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.

Differential effects of carvedilol and metoprolol on isoprenaline-induced changes in β-adrenoceptor density and systolic function in rat cardiac myocytes

OBJECTIVE beta-Blockers improve cardiac function and survival in heart failure patients. The underlying mechanisms are not completely elucidated. Differences between agents might be important for the development of more specific therapeutical approaches. This study investigated whether metoprolol or carvedilol alter beta-adrenergic signaling differently. METHODS beta-Adrenoceptor density and systolic function were determined in rat adult ventricular cardiac myocytes. RESULTS 12 h isoprenaline-treatment (Iso, 1 micromol/l) reduced beta-adrenoceptor density by 33% (P<0.01). The effect was abolished by incubation with isoprenaline plus metoprolol (3 micromol/l), but was more pronounced after coincubation with carvedilol (0.003 micromol/l, P<0.05 Carv vs. Iso). Metoprolol alone had no effect on beta-adrenoceptor density, but carvedilol induced a decrease in receptor density even in absence of isoprenaline (P<0.05 Carv vs. ctr.). The isoprenaline (0.0003-10 micromol/l) induced concentration-dependent increase in myocyte shortening was blunted after 12 h preincubation with Iso (1 micromol/l, P<0.001). This reduction was abolished or partly prevented by coincubation with metoprolol or carvedilol, respectively. Carvedilol decreased the number of receptors which had to be occupied by isoprenaline in order to obtain 50% and 90% increase in myocyte cell shortening. Comparison of guanine nucleotide-dependent binding characteristics of isoprenaline, carvedilol and metoprolol revealed beta-receptor agonist like binding characteristics for carvedilol, but antagonist like binding characteristics for metoprolol. CONCLUSION Metoprolol but not carvedilol prevents isoprenaline-induced downregulation of myocyte beta-adrenoceptors. The difference might be due to specific binding properties of the beta-blockers. Restoration of isoprenaline responsiveness by carvedilol might be due to improved coupling of beta-receptors to postreceptor effects.