Heikki Ruskoaho

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.

Endothelial Dysfunction and Salt-Sensitive Hypertension in Spontaneously Diabetic Goto-Kakizaki Rats

Endothelial dysfunction is associated with hypertension, hypercholesterolemia, and heart failure. We tested the hypothesis that spontaneously diabetic Goto-Kakizaki (GK) rats, a model for type 2 diabetes, exhibit endothelial dysfunction. Rats also received a high-sodium diet (6% NaCl [wt/wt]) and chronic angiotensin type 1 (AT1) receptor blockade (10 mg/kg PO valsartan for 8 weeks). Compared with age-matched nondiabetic Wistar control rats, GK rats had higher blood glucose levels (9.3±0.5 versus 6.9±0.2 mmol/L for control rats), 2.7-fold higher serum insulin levels, and impaired glucose tolerance (all P <0.05). Telemetry-measured mean blood pressure was 15 mm Hg higher in GK rats (P <0.01) compared with control rats, whereas heart rates were not different. Heart weight– and kidney weight–to–body weight ratios were higher in GK rats (P <0.05), and 24-hour albuminuria was increased 50%. Endothelium-mediated relaxation of noradrenaline-precontracted mesenteric arterial rings by acetylcholine was impaired compared with the control condition (P <0.05), whereas the sodium nitroprusside–induced relaxation was similar. Preincubation of the arterial rings with the NO synthase inhibitor NG-nitro-l-arginine methyl ester and the cyclooxygenase inhibitor diclofenac inhibited relaxations to acetylcholine almost completely in GK rats but not in Wistar rats, suggesting that endothelial dysfunction can be in part attributed to reduced relaxation via arterial K+ channels. Perivascular monocyte/macrophage infiltration and intercellular adhesion molecule-1 overexpression were observed in GK rat kidneys. A high-sodium diet increased blood pressure by 24 mm Hg and 24-hour albuminuria by 350%, induced cardiac hypertrophy, impaired endothelium-dependent relaxation further, and aggravated inflammation (all P <0.05). The serum level of 8-isoprostaglandin F2&agr;, a vasoconstrictor and antinatriuretic arachidonic acid metabolite produced by oxidative stress, was increased 400% in GK rats on a high-sodium diet. Valsartan decreased blood pressure in rats fed a low-sodium diet and prevented the inflammatory response. In rats fed a high-sodium diet, valsartan did not decrease blood pressure or improve endothelial dysfunction but protected against albuminuria, inflammation, and oxidative stress. As measured by quantitative autoradiography, AT1 receptor expression in the medulla was decreased in GK compared with Wistar rats, whereas cortical AT1 receptor expression, medullary and cortical angiotensin type 2 (AT2) receptor expressions, and adrenal ACE and neutral endopeptidase expressions were unchanged. A high-sodium diet did not influence renal AT1, AT2, ACE, or neutral endopeptidase expressions. In valsartan-treated GK rats, the cortical and medullary AT1 receptor expressions were decreased in the presence and absence of a high-sodium diet. A high-sodium diet increased plasma brain natriuretic peptide concentrations in presence and absence of valsartan treatment. We conclude that hypertension in GK rats is salt sensitive and associated with endothelial dysfunction and perivascular inflammation. AT1 receptor blockade ameliorates inflammation during a low-sodium diet and partially protects against salt-induced vascular damage by blood pressure–independent mechanisms.

Distinct Upregulation of Extracellular Matrix Genes in Transition From Hypertrophy to Hypertensive Heart Failure

Cardiac hypertrophy in response to pressure overload is initially beneficial but eventually leads to heart failure, a major cause of morbidity and mortality in the Western countries. Although abnormalities in left ventricular (LV) diastolic filling are early features associated with pressure overload-induced LV hypertrophy, the molecular mechanisms regulating transition to diastolic heart failure are poorly understood. We analyzed global changes in gene expression in 12-, 16-, and 20-month-old spontaneously hypertensive rats (SHR) and their age-matched controls, Wistar Kyoto rats, using DNA microarrays. In SHR, a progressive LV hypertrophy was associated with increased expression of hypertrophy-associated genes including contractile protein and natriuretic peptide genes. Echocardiography indicated that 16-month-old SHR had features of diastolic dysfunction leading to diastolic failure at age 20 months without significant changes in LV systolic function. Comparison analysis revealed that the extracellular matrix genes strikingly dominated the list of altered genes after transition to the heart failure, whereas there was no major shift in gene expression patterns involved in calcium homeostasis and neurohumoral activation, as well as myofilament contractile and cytoskeletal proteins. The microarray analysis also revealed differential gene expression of several novel factors, such as thrombospondin-4 and matrix Gla protein, as well as unknown expressed sequence tags. Our data show that transition from LV hypertrophy to diastolic hypertensive heart failure is almost exclusively associated with progressive remodeling of the extracellular matrix and provide new insights into the pathogenesis of hypertrophy by suggesting existence of novel regulators of LV remodeling.

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.

Gene Structure of a New Cardiac Peptide Hormone: A Model for Heart-Specific Gene Expression1

Volume excess and mechanical load lead to the induction of the endocrine activity of the heart. The increased production and secretion of A- and B-type natriuretic peptides (ANP and BNP), in turn, unload the heart due to their physiological effects. To find out the mechanisms of cardiac-specific expression and sensitivity to mechanical stimuli of the natriuretic peptide genes, we have used salmon (Salmo salar) as our model organism, because osmoregulating fish have a particularly well developed defense mechanism against volume excess. We have previously cloned a complementary DNA from salmon heart encoding a novel vasorelaxant cardiac hormone, salmon cardiac peptide (sCP). Its production is restricted to the heart, and its release is very sensitive to mechanical load. We have now cloned the gene encoding sCP. The structure of the gene suggests that sCP may represent an ancestral form of the mammalian natriuretic peptides. Remarkably, despite the large phylogenetic distance, the sCP promoter is as effective as mammalian ANP promoters in cultured neonatal rat atrial cardiomyocytes. Therefore, structural and functional comparisons of the promoters of sCP and ANP provide an excellent means of identifying the elements and transcription factors required for atrial-specific gene expression and the regulation of the endocrine function of the heart. Isolation of the protein product of sCP gene from salmon atrium demonstrated that the storage form of sCP is the prohormone of 126 amino acids. The final processing of the prohormone appears to take place during exocytosis of the secretory granules, as the released and circulating form is the biologically active 29-amino acid sCP.

Endothelin-1 Is Involved in Stretch-Induced Early Activation of B-Type Natriuretic Peptide Gene Expression in Atrial but Not in Ventricular Myocytes Acute Effects of Mixed ETA/ETB and AT1 Receptor Antagonists In Vivo and In Vitro

BACKGROUND The precise role of paracrine and autocrine factors in mechanical load-induced activation of cardiac gene expression is unknown. Here we report the effects of endothelin-1 (ET-1) and angiotensin II (Ang II) receptor antagonism on acute pressure overload-induced activation of cardiac B-type natriuretic peptide (BNP) gene expression in spontaneously hypertensive rats (SHRs) in vivo and on mechanical stretch-induced increase in atrial BNP gene expression in vitro. METHODS AND RESULTS Acute pressure overload produced in conscious SHRs by infusion of arginine8-vasopressin (0.05 microg x kg(-1) x min(-1)) for 2 hours resulted in an increase in BNP mRNA levels in the left ventricle as well as in the atrium. Bolus injections of bosentan (mixed ET(A)/ET(B) receptor antagonist, 10 mg/kg I.V.) but not losartan (AT1 receptor antagonist, 10 mg/kg I.V.) blocked the increase of the BNP mRNA levels produced by pressure overload in the left atria, whereas the elevation of BNP mRNA levels was similar (a 1.9-fold increase) in the left ventricles of vehicle-, losartan-, and bosentan-infused SHRs. In an isolated perfused rat heart preparation, infusion of bosentan (1 micromol/L) for 2 hours inhibited the mechanical stretch-induced increase in BNP mRNA levels in the right atria, whereas an AT1 receptor antagonist, CV-11974 (10 nmol/L), had no effect. CONCLUSIONS The findings of the present study demonstrate that Ang II and ET-1 are not obligatorily required for stretch to trigger the increased BNP gene expression in ventricular myocytes in vivo. In contrast, mechanical load on the atrial myocytes did initiate an ET-1-dependent expression of BNP gene showing that endogenous ET-1 production differentially regulates BNP gene expression in atrial and ventricular myocytes.

Involvement of Transcriptional and Posttranscriptional Mechanisms in Cardiac Overload–Induced Increase of B-Type Natriuretic Peptide Gene Expression

The induction of atrial and ventricular B-type natriuretic peptide (BNP) gene expression is one of the earliest events occurring during hemodynamic overload. To examine the molecular mechanisms for increased BNP gene expression during cardiac overload, we studied the induction of the BNP gene expression compared with that of atrial natriuretic peptide (ANP) in a modified perfused rat heart preparation. An increase in right atrial pressure of 5 mm Hg resulted in a 1.4-fold (P < .05) and 2.2-fold (P < .01) increase in BNP mRNA levels after 1 and 2 hours, respectively, whereas ANP mRNA levels remained unchanged. Stretching for up to 2 hours also significantly increased right atrial immunoreactive BNP (ir-BNP) levels (from 15.8 +/- 2.2 to 20.1 +/- 1.2 ng/mg, P < .05). Actinomycin D (10 micrograms/mL), a transcriptional inhibitor, completely inhibited the stretch-induced increase in atrial BNP mRNA levels at 1 hour (P < .05) and 2 hours (P < .001), whereas a protein synthesis inhibitor, cycloheximide (90 micrograms/mL), had no effect on basal or direct mechanical stretch-induced increase in right atrial BNP mRNA levels. Furthermore, we examined the role of tyrosine kinase and protein kinase C activities in acute mechanical stretch-induced increase in BNP synthesis. Tyrosine kinase inhibitors lavendustin A (1 mumol/L) and tyrphostin A25 (3 mumol/L) and protein kinase C inhibitors staurosporine (30 nmol/L) and chelerythrine (1 mumol/L) prevented the stretch-induced increase in right atrial ir-BNP concentrations at 2 hours. In addition, chelerythrine inhibited the increase of right atrial BNP mRNA levels stimulated by cardiac overload. These resuls demonstrate that the early increase of BNP mRNA levels by mechanical stretch results from increased transcriptional activation and is independent of protein synthesis. Our results also suggest that protein kinase C and tyrosine kinases activities may be involved in coupling cardiac overload to alterations in atrial BNP synthesis.

Pressure Overload Increases GATA4 Binding Activity via Endothelin-1

Background —The signaling cascades responsible for the activation of transcription factors in the hypertrophic growth of cardiac myocytes during hemodynamic overload are largely unknown. Several of the genes upregulated in the hypertrophied heart, including B-type natriuretic peptide (BNP) gene, are controlled by the cardiac-restricted zinc finger transcription factor GATA4. Methods and Results —An in vivo model of intravenous administration of arginine8-vasopressin (AVP) for up to 4 hours in conscious normotensive rats was used to study the signaling mechanisms for GATA activation in response to pressure overload. Gel mobility shift assays were used to analyze the trans-acting factors that interact with the GATA motifs of the BNP promoter. AVP-induced increase in mean arterial pressure was followed by a significant increase in the BNP and c-fos mRNA levels in both the endocardial and epicardial layers of the left ventricle, whereas GATA4 and GATA6 mRNA levels remained unchanged. Pressure overload within 15 to 60 minutes produced an increase in left ventricular BNP GATA4 but not GATA5 and GATA6 binding activity, and at 30 minutes a 2.2-fold increase (P <0.001) in GATA4 binding was noted. The mixed endothelin-1 ETA/ETB receptor antagonist bosentan but not the angiotensin II type 1 receptor antagonist losartan completely inhibited the pressure overload–induced increase in left ventricular BNP GATA4 binding activity. Bosentan alone had no statistically significant effect on GATA4 binding activity of the left ventricle in conscious animals. Conclusions —ET-1 is a signaling molecule that rapidly upregulates GATA4 DNA binding activity in response to pressure overload in vivo.