István Szokodi

Apelin, the Novel Endogenous Ligand of the Orphan Receptor APJ, Regulates Cardiac Contractility

Abstract— The orphan receptor APJ and its recently identified endogenous ligand, apelin, exhibit high levels of mRNA expression in the heart. However, the functional importance of apelin in the cardiovascular system is not known. In isolated perfused rat hearts, infusion of apelin (0.01 to 10 nmol/L) induced a dose-dependent positive inotropic effect (EC50: 33.1±1.5 pmol/L). Moreover, preload-induced increase in dP/dtmax was significantly augmented (P <0.05) in the presence of apelin. Inhibition of phospholipase C (PLC) with U-73122 and suppression of protein kinase C (PKC) with staurosporine and GF-109203X markedly attenuated the apelin-induced inotropic effect (P <0.001). In addition, zoniporide, a selective inhibitor of Na+-H+ exchange (NHE) isoform-1, and KB-R7943, a potent inhibitor of the reverse mode Na+-Ca2+ exchange (NCX), significantly suppressed the response to apelin (P <0.001). Perforated patch-clamp recordings showed that apelin did not modulate L-type Ca2+ current or voltage-activated K+ currents in isolated adult rat ventricular myocytes. Apelin mRNA was markedly downregulated in cultured neonatal rat ventricular myocytes subjected to mechanical stretch and in vivo in two models of chronic ventricular pressure overload. The present study provides the first evidence for the physiological significance of apelin in the heart. Our results show that apelin is one of the most potent endogenous positive inotropic substances yet identified and that the inotropic response to apelin may involve activation of PLC, PKC, and sarcolemmal NHE and NCX.

Circulating and cardiac levels of apelin, the novel ligand of the orphan receptor APJ, in patients with heart failure.

The orphan receptor APJ and its recently identified endogenous ligand, apelin, are expressed in the heart. However, their importance in the human cardiovascular system is not known. This study shows that apelin-like immunoreactivity is abundantly present in healthy human heart and plasma. Gel filtration HPLC analysis revealed that atrial and plasma levels of high molecular weight apelin, possibly proapelin, were markedly higher than those of mature apelin-36 itself. As assessed by quantitative RT-PCR analysis, left ventricular apelin mRNA levels were increased 4.7-fold in chronic heart failure (CHF) due to coronary heart disease (p<0.01) and 3.3-fold due to idiopathic dilated cardiomyopathy (p<0.05), whereas atrial apelin mRNA levels were unchanged. Atrial and plasma apelin-like immunoreactivity as well as atrial and ventricular APJ receptor mRNA levels were significantly decreased in CHF. Our results suggest that a new cardiac regulatory peptide, apelin, and APJ receptor may contribute to the pathophysiology of human CHF.

Evidence for cAMP-Independent Mechanisms Mediating the Effects of Adrenomedullin, a New Inotropic Peptide

BACKGROUND Adrenomedullin (ADM), a new vasorelaxing and natriuretic peptide, may function as an endogenous regulator of cardiac function, because ADM and its binding sites have been found in the heart. We characterize herein the cardiac effects of ADM as well as the underlying signaling pathways in vitro. METHODS AND RESULTS In isolated perfused, paced rat heart preparation, infusion of ADM at concentrations of 0.1 to 1 nmol/L for 30 minutes induced a dose-dependent, gradual increase in developed tension, whereas proadrenomedullin N-20 (PAMP; 10 to 100 nmol/L), a peptide derived from the same gene as ADM, had no effect. The ADM-induced positive inotropic effect was not altered by a calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP8-37, or H-89, a cAMP-dependent protein kinase inhibitor. ADM also failed to stimulate ventricular cAMP content of the perfused hearts. Ryanodine (3 nmol/L), a sarcoplasmic reticulum Ca2+ release channel opener, suppressed the overall ADM-induced positive inotropic effect. Pretreatment with thapsigargin (30 nmol/L), which inhibits sarcoplasmic reticulum Ca2+ ATPase and depletes intracellular Ca2+ stores, attenuated the early increase in developed tension produced by ADM. In addition, inhibition of protein kinase C by staurosporine (10 nmol/L) and blockade of L-type Ca2+ channels by diltiazem (1 micromol/L) significantly decreased the sustained phase of ADM-induced increase in developed tension. Superfusion of atrial myocytes with ADM (1 nmol/L) in isolated left atrial preparations resulted in a marked prolongation of action potential duration between 10 and -50 mV transmembrane voltage, consistent with an increase in L-type Ca2+ channel current during the plateau. CONCLUSIONS Our results show that ADM enhances cardiac contractility via cAMP-independent mechanisms including Ca2+ release from intracellular ryanodine- and thapsigargin-sensitive Ca2+ stores, activation of protein kinase C, and Ca2+ influx through L-type Ca2+ channels.

Adrenomedullin gene expression in the rat heart is stimulated by acute pressure overload: blunted effect in experimental hypertension.

The levels of adrenomedullin (ADM), a newly discovered vasodilating and natriuretic peptide, are elevated in plasma and ventricular myocardium in human congestive heart failure suggesting that cardiac synthesis may contribute to the plasma concentrations of ADM. To examine the time course of induction and mechanisms regulating cardiac ADM gene expression, we determined the effect of acute and short-term cardiac overload on ventricular ADM mRNA and immunoreactive ADM (ir-ADM) levels in conscious rats. Acute pressure overload was produced by infusion of arginine8-vasopressin (AVP, 0.05μ g/kg/min,iv) for 2 h into 12-week-old hypertensive TGR(mREN-2)27 rats and normotensive Sprague-Dawley (SD) rats. Hypertension and marked left ventricular hypertrophy were associated with 2.2-times higher ir-ADM levels in the left ventricular epicardial layer (178 ± 36 vs. 81 ± 23 fmol/g, P < 0.05) and 2.6-times higher ir-ADM levels in the left ventricular endocardial layer (213 ± 23 vs. 83 ± 22 fmol/g, P < 0.01). The infusio...Somatostatin (SRIF) acts on specific membrane receptors to inhibit exocrine and endocrine pancreatic functions. Five SRIF receptor genes have been cloned, producing six receptor proteins (sst-s). We used a recently developed antibody to localize the sst2A splice variant in the rat pancreas. Western blots identified the sst2A receptor as an 90 kDa glycosylated protein in pancreatic tissue. In tyramide-amplified immunostainings all acinar cells, and the glucagon and pancreatic polypeptide immunoreactive cells (A and PP, respectively) were intensely labeled for sst2A, while no signal was detected in SRIF producing (D) cells. A very few insulin immunoreactive (B) cells were also labeled for sst2A, but the signal in these cells was lower than in exocrine, A or PP cells. Absorption of the sst2A antibody with the receptor peptide abolished specific staining in both immunoblots and tissue sections (negative control). These studies are the first to localize any SRIF receptor subtype in the rat pancreas. The specific localization of sst2A receptor in acinar, A and PP cells if confirmed in humans, would suggest that subtype specific analogs will be useful for the therapeutic regulation of exocrine and/or endocrine pancreatic secretion.

Evidence for a Functional Role of Angiotensin II Type 2 Receptor in the Cardiac Hypertrophic Process In Vivo in the Rat Heart

Background—The precise function of angiotensin II type 2 receptor (AT2-R) in the mammalian heart in vivo is unknown. Here, we investigated the role of AT2-R in cardiac pressure overload. Methods and Results—Rats were infused with vehicle, angiotensin II (Ang II), PD123319 (an AT2-R antagonist), or the combination of Ang II and PD123319 via subcutaneously implanted osmotic minipumps for 12 or 72 hours. Ang II–induced increases in mean arterial pressure, left ventricular weight/body weight ratio, and elevation of skeletal &agr;-actin and &bgr;-myosin heavy chain mRNA levels were not altered by PD123319. In contrast, AT2-R blockade resulted in a marked increase in the gene expression of c-fos, endothelin-1, and insulin-like growth factor-1 in Ang II–induced hypertension. In parallel, Ang II–stimulated mRNA and protein expression of atrial natriuretic peptide were significantly augmented by AT2-R blockade. Moreover, PD123319 markedly increased the synthesis of B-type natriuretic peptide. Furthermore, the expression of vascular endothelial growth factor and fibroblast growth factor-1 was downregulated by Ang II only in the presence of AT2-R blockade. Conclusions—Our results provide evidence that AT2-R plays a functional role in the cardiac hypertrophic process in vivo by selectively regulating the expression of growth-promoting and growth-inhibiting factors.

Apelin Increases Cardiac Contractility via Protein Kinase Cε- and Extracellular Signal-Regulated Kinase-Dependent Mechanisms

Background Apelin, the endogenous ligand for the G protein-coupled apelin receptor, is an important regulator of the cardiovascular homoeostasis. We previously demonstrated that apelin is one of the most potent endogenous stimulators of cardiac contractility; however, its underlying signaling mechanisms remain largely elusive. In this study we characterized the contribution of protein kinase C (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2) and myosin light chain kinase (MLCK) to the positive inotropic effect of apelin. Methods and Results In isolated perfused rat hearts, apelin increased contractility in association with activation of prosurvival kinases PKC and ERK1/2. Apelin induced a transient increase in the translocation of PKCε, but not PKCα, from the cytosol to the particulate fraction, and a sustained increase in the phosphorylation of ERK1/2 in the left ventricle. Suppression of ERK1/2 activation diminished the apelin-induced increase in contractility. Although pharmacological inhibition of PKC attenuated the inotropic response to apelin, it had no effect on ERK1/2 phosphorylation. Moreover, the apelin-induced positive inotropic effect was significantly decreased by inhibition of MLCK, a kinase that increases myofilament Ca2+ sensitivity. Conclusions Apelin increases cardiac contractility through parallel and independent activation of PKCε and ERK1/2 signaling in the adult rat heart. Additionally MLCK activation represents a downstream mechanism in apelin signaling. Our data suggest that, in addition to their role in cytoprotection, modest activation of PKCε and ERK1/2 signaling improve contractile function, therefore these pathways represent attractive possible targets in the treatment of heart failure.

Endothelin-1 Contributes to the Frank-Starling Response in Hypertrophic Rat Hearts

Abstract—Endothelin-1 is involved in mechanical load–induced cardiac growth processes; it also has effects on contractility. The interaction of endothelin-1 and the Frank-Starling response is unknown. The present study aimed to characterize the role of endothelin-1 in the regulation of the Frank-Starling response, one of the major mechanisms regulating cardiac contractile force, in both normal and hypertrophied hearts. Nontransgenic rat hearts and hypertrophic hearts of hypertensive double transgenic rats harboring human angiotensinogen and renin genes were studied in a Langendorff isolated heart setup with a liquid-filled balloon inside the left ventricle used to measure contractile parameters. The rats were studied at compensated phase, before showing any signs of heart failure. Compensated hypertrophy in double transgenic rat hearts resulted in improved contractility at a given level of preload when compared with nontransgenic rat hearts. Hearts of both rat lines showed preserved Frank-Starling responses, that is, increased contractile function in response to increased end-diastolic pressure. The mixed endothelin A/B receptor antagonist bosentan attenuated the Frank-Starling response by 53% (P <0.01) in the double transgenic hearts but not in nontransgenic hearts. The diastolic parameters remained unaffected. The left ventricles of the double transgenic rat hearts showed an 82% higher level of endothelin type A receptor mRNA and a 25% higher level of immunoreactive endothelin-1 compared with nontransgenic rat hearts. The type 1 angiotensin II receptor antagonist CV-11974 had no significant effect on contractile function in response to load in either strain. These results show that endogenous endothelin-1 contributes to the Frank-Starling response in hypertrophied rat hearts by affecting systolic performance.

Functionally Opposing Roles of Extracellular Signal-Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase in the Regulation of Cardiac Contractility

Background— Extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38-MAPK) have been shown to regulate various cellular processes, including cell growth, proliferation, and apoptosis in the heart. However, the function of these signaling pathways in the control of cardiac contractility is unclear. Here, we characterized the contribution of ERK1/2 and p38-MAPK to the inotropic effect of endothelin-1 (ET-1). Methods and Results— In isolated perfused rat hearts, infusion of ET-1 (1 nmol/L) for 10 minutes increased contractility and phosphorylation of ERK1/2 and their downstream target p90 ribosomal S6 kinase (p90RSK). Suppression of ERK1/2 activation prevented p90RSK phosphorylation and attenuated the inotropic effect of ET-1. Pharmacological inhibition of epidermal growth factor receptor kinase activity abolished ET-1–induced epidermal growth factor receptor transactivation and ERK1/2 and p90RSK phosphorylation and reduced ET-1–mediated inotropic response. Moreover, inhibition of the p90RSK target Na+-H+ exchanger 1 attenuated the inotropic effect of ET-1. In contrast to ERK1/2 signaling, suppression of p38-MAPK activity further augmented ET-1–enhanced contractility, which was accompanied by increased phosphorylation of phospholamban at Ser-16. Conclusions— MAPKs play opposing roles in the regulation of cardiac contractility in that the ERK1/2-mediated positive inotropic response to ET-1 is counterbalanced by simultaneous activation of p38-MAPK. Hence, selective activation of ERK1/2 signaling and inhibition of p38-MAPK signaling may represent novel means to support cardiac function in disease.

Intrapericardial infusion of endothelin-1 induces ventricular arrhythmias in dogs.

OBJECTIVES Recently, extremely high levels of endothelin-1 (ET-1) were detected in the pericardial fluid of patients with heart disease; however, the pathophysiological importance of this finding is not known. The present study was designed to characterize ET-1 levels in canine pericardial fluid and to investigate the effects of local high concentrations of exogenous ET-1 in vivo. METHODS In anesthetized, open-chest dogs ET-1 (Groups 1 and 2: 11 and 33 pmol.kg-1.min-1; n = 6 and 6, respectively) or physiological saline (Group 3, n = 5) were infused into the closed pericardial sac for 40 min. In serial pericardial fluid and aortic blood plasma samples, ET-1 levels were measured by radioimmunoassay, and analysed by high-performance liquid chromatography (HPLC). Systemic arterial blood pressure, heart rate, cardiac output (CO), standard ECG and right ventricular endocardial monophasic action potentials (MAPs) were recorded. RESULTS Basal pericardial fluid ET-1 levels were significantly higher than respective plasma levels (342 +/- 210 vs. 8.0 +/- 5.2 pmol.l-1, n = 14, P < 0.001. In HPLC analysis pericardial fluid ET-1 was indistinguishable from ET-1(1-21). Infusion of exogenous ET-1 into the pericardial space induced ventricular arrhythmias in all instances, which were associated with 9.7-fold increase in pericardial fluid ET-1 levels. Ventricular tachycardias developed in 9 of 12 animals. The arrhythmogenic effect of ET-1 was more apparent in dogs with the larger dose. Before the onset of arrhythmias, intrapericardial infusion of ET-1 increased QT time (Group 1: 207 +/- 18 to 230 +/- 23 ms, P < 0.01; Group 2: 220 +/- 12 to 277 +/- 17 ms, P < 0.01) and MAP duration at 90% repolarization (at 300 ms cycle length) (Group 1: 192 +/- 9 to 216 +/- 9 ms, P < 0.01; Group 2: 205 +/- 9 to 255 +/- 9 ms, P < 0.001). Hemodynamic variables did not change significantly prior to the onset of ventricular tachyarrhythmias. In Group 3, arrhythmias were not observed and all electrophysiological and hemodynamic parameters remained unchanged. CONCLUSIONS Administration of exogenous ET-1 into the pericardial space induces ventricular arrhythmias associated with prolongation of QT time and MAP duration. Whether pericardial fluid ET-1 under pathophysiological conditions can ever reach sufficiently high levels to induce ventricular arrhythmias remains to be elucidated.

Role of reactive oxygen species in the regulation of cardiac contractility

Increased production of reactive oxygen species (ROS) has been linked to the pathogenesis of contractile dysfunction in heart failure. However, it is unclear whether ROS can regulate physiological cellular processes in the myocardium. Here, we characterized the role of endogenous ROS production in the acute regulation of cardiac contractility in the intact rat heart. In isolated perfused rat hearts, endothelin-1 (ET-1, 1nmol/L) stimulated ROS formation in the left ventricle, which was prevented by the antioxidant N-acetylcysteine and the NAD(P)H oxidase inhibitor apocynin. N-acetylcysteine, the superoxide dismutase mimetic MnTMPyP, and apocynin significantly attenuated ET-1-mediated inotropic effect, which was accompanied by inhibition of extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. Moreover, the mitochondrial K(ATP) channel blocker 5-HD, and the mitochondrial large conductance calcium activated potassium channel blocker paxilline, but not the sarcolemmal K(ATP) channel blocker HMR 1098 attenuated the inotropic response to ET-1. However, ET-1-induced ROS generation was not abolished by inhibiting mitochondrial K(ATP) channel opening. In contrast to ET-1 stimulation, the positive inotropic effect of β(1)-adrenergic receptor agonist dobutamine (250nmol/L) was significantly augmented by N-acetylcysteine and apocynin. Moreover, dobutamine-induced phospholamban phosphorylation was markedly enhanced by apocynin. In conclusion, NAD(P)H oxidase-derived ROS play a physiological role in the acute regulation of cardiac contractility in the intact rat heart. Our results reveal that ET-1-induced increase in cardiac contractility is partially dependent on enhanced ROS generation, which in turn, activates the ERK1/2 pathway. On the other hand, β-adrenergic receptor-induced positive inotropic effect and phospholamban phosphorylation is enhanced by NAD(P)H oxidase inhibition.