Atrial Natriuretic Peptide mRNA Research Articles

Heart and lung VEGF mRNA expression in rats with monocrotaline- or hypoxia-induced pulmonary hypertension.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that is upregulated during exposure to hypoxia. In this study, we analyzed heart and lung VEGF mRNA expression and examined pulmonary vascular remodeling as well as myocardial capillary density in two rat models of pulmonary hypertension involving exposure to chronic hypoxia (CH) and treatment with monocrotaline (MCT), respectively. The rats were studied after 0.5, 1, 3, 15, and 30 days of exposure to 10% O2 or 1, 6, and 30 days after a subcutaneous MCT injection (60 mg/kg). Both CH and MCT induced pulmonary hypertension and hypertrophy of the right ventricle (RV) with increased RV weight and atrial natriuretic peptide mRNA expression. VEGF mRNA expression as assessed by Northern blot analysis was potently induced after 12 h of hypoxia in both the right and left ventricles. After prolonged exposure to hypoxia, VEGF mRNA returned to baseline in the left ventricle (LV) but remained increased in the RV, where it peaked after 30 days. In MCT rats, VEGF mRNA was unchanged in the LV but decreased by 50% in the RV and by 90% in the lungs after 30 days. VEGF mRNA remained unchanged in the lungs from CH rats. Pulmonary vascular remodeling was more pronounced in MCT than in CH rats. The number of capillaries per RV myocyte was increased in rats exposed to 30 days of hypoxia, whereas it remained unchanged in MCT rats despite a similar degree of RV hypertrophy. Our results suggest that the sustained increase in VEGF expression in the hypertrophied RV during CH may account for the increased number of capillaries per myocyte. In contrast, reduced VEGF expression in the lungs and RV of MCT rats may aggravate pulmonary vascular remodeling and compromise RV myocardial perfusion.

Increased renal ANP synthesis, but decreased or unchanged cardiac ANP synthesis in water-deprived and salt-restricted rats

Experiments were performed to examine the effect of water deprivation and salt restriction on ANP synthesis in the kidneys and hearts of normal rats. A 4-day water deprivation (WD) and 7-day salt restriction (SR; 0.01% NaCl) were performed in 12 and 14 rats, respectively. Atrial natriuretic peptide (ANP) mRNA expression in the kidney was assessed with reverse transcription-polymerase chain reaction coupled with Southern blot hybridization, while the ANP mRNA in the hearts was measured by Northern blot hybridization. ANP and angiotensin II concentrations in the extracted plasma were measured by radioimmunoassay. The molecular form of renal ANP-like protein was characterized by reverse phase-high-performance liquid chromatography (RP-HPLC). Renal outer and inner medullary ANP mRNA showed a respective 11-fold and ninefold increase in WD rats, and an eightfold and fivefold increase in SR rats as compared to corresponding control groups. Inversely, cardiac atrial ANP mRNA and plasma ANP were decreased in WD rats, whereas they did not change in the SR group. Plasma angiotensin II concentration increased in conjunction with the decrease of urine sodium excretion in both groups. RP-HPLC analysis revealed a 45% extraction of ANP in the WD rat kidneys, whereas only 3% ANP in the control kidneys migrated in a molecular form similar to cardiac atrial proANP. Our results demonstrate that water deprivation and salt restriction markedly enhance renal ANP mRNA, whereas water deprivation suppresses cardiac atrial ANP mRNA and plasma ANP concentrations. The current study indicates that renal ANP and cardiac atrial ANP appear to be two distinct systems regulated by different mechanisms and possibly exhibiting different intra-renal paracrine and systemic endocrine functions.

Changing Patterns of Gene Expression in the Pulmonary Trunk-banded Rat Heart

A pressure-overload model in the rat by banding the pulmonary trunk (PT) was developed to investigate alterations in gene expression in left- and right-ventricular compartments during the transition from compensated right-ventricular (RV) hypertrophy to right heart failure. Right heart failure in rat is characterized by liver cirrhosis, hydrothorax and ascites. The diameter of constriction was found to determine the time course of heart failure development. Only the RV free wall and the right atrium increased in weight, without a difference between compensated and failing RV. An increase in circulating ANP revealed a hypertrophic response of the myocardium, while increased circulating ammonia levels discriminated between compensated hypertrophy and failure. As parameters for stress, fibrosis and Ca2+-handling, changes in the pattern and level of the mRNAs encoding atrial natriuretic peptide (ANP), collagenIIIα1, and sarcoplasmic endoplasmic reticular calcium ATPase 2 (SERCA2), phospholamban (PLB) and calsequestrin (CSQ) were studied by Northern blot andin situhybridization analyses. Pulmonary trunk banding resulted in an induction of ANP mRNA, a moderate increase in collagenIIIα1mRNA and a decrease in SERCA2 and PLB mRNA levels in both the left and right ventricles, but changes were most pronounced in the myocardium surrounding the RV cavity. Increased ammonia blood levels are a promising prognostic marker to detect the development of right heart failure.

Effect of chronic GH overproduction on cardiac ANP expression and circulating ANP levels

Sodium and water retention are common in acromegaly and upon GH administration. The underlying mechanisms, however, have not been clearly characterized as yet. Therefore, the aim of this study was to examine possible alterations of atrial natriuretic peptide (ANP), an endogenous regulator of volume homeostasis, in response to chronic elevated GH. We used GH-transgenic mice (GH-TM) as a model for chronic hypersomatotropinemia and moreover investigated 7 and 27 week old animals, respectively, in order to discriminate between short and long term effects of GH overexpression. Hematocrit values were reduced in GH-TM compared to control animals and it is known that plasma volume is increased in these animals. Structural lesions of the kidney were found in the GH-TM, however, in the animals studied there were no signs of renal insufficiency as evidenced by serum creatinine and urea levels. The serum concentration of immunoreactive ANP (IR-ANP) determined by RIA was significantly ( P<0.005) elevated in the young GH-TM as compared to control littermates (81.7±13.3 vs. 50.9±10.8 fmol/ml). The increase in serum IR-ANP of 27 week old GH-TM, however did not reach the level of significance (57.13±16.3 vs. 50.25±16.4 fmol/ml). Serum samples of control mice as well as of the 7 week old animals mainly contained ANP 99-126, known to be the circulating form of ANP. In contrast, serum of 27 week old GH-TM predominantly showed the cardiac storage form of ANP, ANP 1–126. Cardiac expression of ANP was quantified by Northern blot analysis. mRNA coding for ANP was found 1.2- and 2-fold increased in the atria of 7 and 27 week old GH-TM, respectively. In parallel, a 2.2-fold (7 week) and 2-fold (27 week) increase of IR-ANP was observed in transgenic atria compared to tissue of control animals. In contrast, no significant difference of ANP mRNA expression or of content of IR-ANP was observed in the ventricles of both groups of animals. In conclusion, GH-TM show various alterations in their ANP status suggesting an influence of the peptide on the effect of GH in fluid retention.

Left-Ventricular Structural and Functional Remodeling in the Mouse after Myocardial Infarction: Assessment with the Isovolumetrically-contracting Langendorff Heart

The goal of this study was to determine whether the isovolumically-contracting Langendorff heart could be used to assess changes in left-ventricular volume and contractile reserve in the mouse heart after myocardial infarction. Myocardial infarction (40±3% of the left ventricle by weight) was induced in CD-1 mice by ligation of the left-anterior descending coronary artery. Two weeks after infarction there was compensatory hypertrophy of the non-infarcted ventricle as indicated by increases in heart-to-body weight ratio (5.5±0.2v4.9±0.2 mg/g;P<0.05;n=12) and the expression of atrial natriuretic peptide mRNA (4.4±1.4-fold;P<0.001;n=4). Left-ventricular pressure–volume relationships were assessedin vitroin isovolumically-contracting hearts perfused with red cell-supplemented buffer (hematrocrit=40%). Myocardial infarction caused left-ventricular dilation with a rightward-shift of the diastolic pressure–volume relationship. This was associated with reduced left-ventricular contractile function, as evidenced by a decrease in developed pressure over a range of left-ventricular volumes. Thus, it is feasible to use the isovolumically-contracting Langendorff preparation to assess the structural and functional consequences of left-ventricular remodeling in the mouse after a myocardial infarction.

β-Adrenoceptor antagonist propranolol potentiates hypotensive action of natriuretic peptides

β-Adrenoceptor antagonists are known to increase plasma atrial natriuretic peptide (ANP) levels despite their hypotensive action. The aim of the present study was to examine the role of the ANP system in the antihypertensive effects of a β-adrenoceptor antagonist. We investigated the effects of propranolol (75 mg kg −1 day −1, p.o., 4 weeks) on the ANP system in stroke-prone spontaneously hypertensive rats. Plasma ANP levels were significantly higher in the propranolol group than in the control group. Both receptor densities and mRNA levels of ANP C receptor were significantly decreased in the lung as the major site of ANP clearance from the circulation. In contrast, both central venous pressure and ANP mRNA levels in the heart were not significantly different between the two groups. Under both basal and ANP-stimulated conditions, the cGMP content in the aorta was significantly greater in the propranolol group than in the control group, whereas the basal and stimulated cGMP content of the kidney was similar in the two groups. Inhibition of endogenous ANP action by a specific ANP receptor antagonist, HS-142-1, produced a greater increase of blood pressure in the propranolol group than in the control group. These results suggest potentiation of natriuretic peptide activity as a new antihypertensive mechanism of the β-adrenoceptor antagonist propranolol.

Differential Regulation of Ventricular Adrenomedullin and Atrial Natriuretic Peptide Gene Expression in Pressure and Volume Overload in the Rat

1. Adrenomedullin is a recently discovered vasodilating and natriuretic peptide whose physiological and pathophysiological roles remain to be established. Like atrial natiuretic peptide adrenomedullin is expressed in the left ventricle. Ventricular expression of atrial natriuretic peptide is known to be markedly increased by volume or pressure overload. In this study we investigated whether ventricular expression of adrenomedullin is similarly stimulated under such conditions. 2. Ventricular adrenomedullin and atrial natriuretic peptide mRNA levels as well as those of a loading control mRNA (glyceraldehyde-3-phosphate dehydrogenase) were quantified by Northern blot analysis in (a) rats with severe post-infarction heart failure induced by left coronary ligation at 30 days post-surgery and (b) in rats with pressure-related cardiac hypertrophy induced by aortic banding at several time points (0.5, 1 and 4 h, and 1, 4, 7 and 28 days) after surgery. Levels were compared with those in matched sham-operated controls. 3. The mRNA level of atrial natriuretic peptide was markedly increased (8-10-fold) in the left ventricle of animals with post-infarction heart failure. In contrast, there was only a modest (40%) increase in the level of adrenomedullin mRNA. In rats with pressure-induced cardiac hypertrophy the ventricular level of atrial natriuretic peptide mRNA was again markedly increased (maximum 10-fold). The increase was first noticeable at 24 h post-banding and persisted until 28 days. In contrast, there was no change in adrenomedullin mRNA level compared with sham-operated rats at any time point. 4. Despite having similar systemic effects, the expression of adrenomedullin and atrial natriuretic peptide in the left ventricle is differently regulated. The findings imply distinct roles for the two peptides. The results do not support an important role for ventricular adrenomedullin expression in the remodelling process that occurs during the development of cardiac hypertrophy but suggest that ventricular adrenomedullin participates in the local and/or systemic response to heart failure.

Expression of Atrial and Brain Natriuretic Peptides and Their Genes in Hearts of Patients With Cardiac Amyloidosis

Objectives. We investigated the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) and their genes in the hearts of patients with cardiac amyloidosis and those with isolated atrial amyloidosis.Background. The expression of ANP and BNP is augmented in the ventricles of failing or hypertrophied hearts, or both. The expression of ANP and BNP in the ventricles of hearts with cardiac amyloidosis, which is hemodynamically similar to restrictive cardiomyopathy, is not yet known. ANP is the precursor protein of isolated atrial amyloid.Methods. We analyzed the immunohistocytochemical localizations of ANP and BNP as well as the expression of their mRNAs by in situ hybridization in the myocardium and measured the plasma levels of ANP and BNP in patients with cardiac amyloidosis.Results. Four of the five right and all six left ventricular endomyocardial biopsy specimens obtained from six patients with cardiac amyloidosis were immunohistochemically positive for both ANP and BNP; none of the biopsy specimens from eight normal subjects were positive for ANP or BNP. All four of the right atria obtained at operation showed positive immunoreactions for both peptides. Electron microscopy identified specific secretory granules in ventricular myoctyes of the patients with cardiac amyloidosis, but not in ventricular myocytes from the normal control subjects. Double immunocytochemical analysis revealed the co-localization of ANP and BNP in the same granules and that isolated atrial amyloid fibrils were immunoreactive for ANP and BNP, whereas ventricular amyloid fibrils were negative for both peptides. Both ANP mRNA and BNP mRNA were expressed in the ventricles of the patients with cardiac amyloidosis but not in the normal ventricles. The autopsy study of four patients with cardiac amyloidosis revealed an almost transmural distribution of ANP and BNP, with predominance in the endocardial side. Plasma BNP levels in the patients were markedly elevated ([mean ± SD] 1,165.1 ± 561.2 pg/ml) compared with those in the control subjects (8.9 ± 6.0 pg/ml, p < 0.05).Conclusions. Expression of ANP and BNP and their genes was augmented in the ventricular myocytes of the patients with cardiac amyloidosis. Both regional mechanical stress by amyloid deposits and hemodynamic stress by diastolic dysfunction may be responsible for the expression of the peptides in patients with cardiac amyloidosis.

RT-PCR analysis of mRNA expression of natriuretic peptide family and their receptors in rat inner ear

To assess the possible physiological role of the atrial natriuretic peptide (ANP) family, we investigated the expression of mRNA of ANP, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and their receptors in rat inner ear using the reverse transcription-polymerase chain reaction method. ANP and CNP message bands were detected in the inner ear, but the BNP message band was not. Amplification products of the expected sizes of ANP-A, ANP-B, and ANP-C receptors were detected in the inner ear. These results suggest that natriuretic peptide family may influence the function of the inner ear through the ANP-A, ANP-B, and ANP-C receptors.

Effects of angiotensin II type 1 receptor blockade and angiotensin-converting enzyme inhibition on cardiac beta-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.

Preserved Frank-Starling mechanism in human end stage heart failure.

The goal of the present study was to examine the ability of failing myocardium to respond to enhanced preload with an increase in force development. The effect of various preload conditions (2.5-15 mN) on force development was studied in right ventricular trabeculae carneae from explanted human failing hearts with ischemic cardiomyopathy (ICM, n = 5, 42 preparations) or idiopathic dilated cardiomyopathy (DCM, n = 9, 77 preparations). To determine the severity of cardiac impairment we measured the positive inotropic effect of beta-adrenoceptor stimulation and calcium (ISO/Ca2+ ratio) and the expression of atrial natriuretic peptide (ANP) mRNA in all hearts. (1) Force of contraction increased with stepwise augmentation of preload (length at 2.5 mN preload to length of maximal force development) from 3.7 +/- 0.5 (ICM) and 2.7 +/- 0.4 (DCM) to 8.3 +/- 0.9 and 6.5 +/- 0.8 mN/mm2, respectively (p < 0.05). (2) The ISO/Ca2+ ratio was 0.40 +/- 0.04 (ICM) and 0.35 +/- 0.03 (DCM), respectively. (3) ANP mRNA was expressed in all preparations, albeit at greatly varying levels (ICM 22.5 +/- 6.1 and DCM 18.7 +/- 4.7 normalized arbitrary units). (4) Contraction experiments performed in left ventricular tissue (n = 3, 32 preparations) essentially confirmed the results. The Frank-Starling mechanism is preserved in terminally failing human hearts irrespective of the underlying etiology. We found no relation between the severity of cardiac impairment as assessed by either ANP expression or the ISO/Ca2+ ratio and the ability of failing human myocardium to respond to enhanced preload with an increase in force development.

Analysis of gene expression patterns in small amounts of human ventricular myocardium by a multiplex RNase protection assay.

End-stage human heart failure is associated with changes in expression of steady-state messenger RNA (mRNA) levels. These changes correspond to alterations in protein levels and myocardial function and may have clinical implications regarding etiology, clinical state, or prognosis. However, analysis of mRNA levels in endomyocardial biopsies can be accomplished only by the quantitative polymerase chain reaction, which is difficult to standardize. The aim of the study was to evaluate whether the RNase protection assay is applicable to measure mRNAs of multiple genes simultaneously in small amounts of ventricular myocardium comparable to myocardial biopsies. Total RNA was prepared from left ventricular myocardium from terminally failing hearts with idiopathic (n=9) or ischemic cardiomyopathy (n=7) and from nonfailing control hearts (n=10). mRNA was measured by an optimized RNase protection assay for the beta1-adrenoceptor, the stimulatory G protein alpha-subunit (Gsalpha), phospholamban, the calcium ATPase of the sarcoplasmic reticulum (SERCA), beta-myosin heavy chain (beta-MHC), and the atrial natriuretic peptide (ANP). We extracted 10.7+/-2.1 microg total RNA from three myocardial biopsies taken in vitro. All of the six genes were measurable in duplicate in a total of 7 microg RNA. mRNAs of beta1-adrenoceptor, phospholamban, and SERCA were lower in failing than in nonfailing myocardium by 50%, 33%, and 42% respectively, whereas beta-MHC and Gsalpha mRNAs were unchanged. mRNA of ANP was expressed at high levels only in the failing myocardium, providing a highly specific and sensitive marker for discriminating nonfailing and failing hearts. A direct comparison with ANP and Gsalpha levels obtained by Northern blot analysis with 7.5 microg total RNA showed a good correlation between the two methods. The RNase protection assay is thus a suitable method for simultaneous measurements of multiple mRNA levels in human myocardial biopsies. Changes in mRNA levels closely reflected those identified by other methods using larger amounts of RNA. Increased myocardial ANP mRNA levels determined by the RNase protection assay may serve as a molecular marker of heart failure.

Effect of manidipine hydrochloride, a calcium antagonist, on isoproterenol-induced left ventricular hypertrophy.

We examined the effect of a calcium antagonist, manidipine hydrochloride, on cardiac hypertrophy and the expression of the atrial natriuretic peptide (ANP), transforming growth factor beta 1 (TGF-beta 1), and extracellular matrix protein genes in rats with isoproterenol-induced cardiac hypertrophy. Rats were continuously infused with saline or isoproterenol (0.5 mg/kg per day) for 7 days using an osmotic minipump. Treatment with manidipine hydrochloride (once a day at 3 mg/kg) began 1 day before minipump implantation and continued until the end of the experiments (each group; n = 6). After treatment, left ventricular weight was measured and mRNA was extracted and analyzed by Northern blot hybridization. Isoproterenol increased left ventricular weight (2.40 +/- 0.04 g/kg; p < 0.01) without increasing blood pressure. ANP, collagen type I and type III, and fibronectin mRNAs were increased 1.5-(p < 0.01), 1.9- (p < 0.01), 2.7- (p < 0.01), and 3.2-fold (p < 0.01), respectively, by isoproterenol infusion. However, TGF-beta 1, collagen type IV, and laminin B1 and B2 mRNA levels were unchanged by isoproterenol. Manidipine hydrochloride prevented isoproterenol-induced left ventricular hypertrophy (2.26 +/- 0.02 g/kg; p < 0.01) and expression of mRNA of ANP (0.9-fold of the control value; p < 0.01), collagen types I (1.1-fold; p < 0.01) and type III (1.6-fold; p < 0.01), and fibronectin (1.1-fold; p < 0.01). Thus, manidipine hydrochloride prevented cardiac hypertrophy and changes in the expression of genes for ANP and interstitial components of extracellular matrix induced by isoproterenol.

Phenylephrine, endothelin, prostaglandin F2alpha' and leukemia inhibitory factor induce different cardiac hypertrophy phenotypes in vitro.

In these studies, we show that endothelin (ET), leukemia inhibitory factor (LIF), phenylephrine (PE), and prostaglandin F2alpha (PGF2alpha), which are all hypertrophic for neonatal rat cardiac myocytes in culture, induce distinct morphological, physiological, and genetic changes after a 48-h treatment. Transmission electron microscopy revealed differences in myofibril organization, with ET-treated cells containing the most mature-looking myofibrils and PGF2alpha- and LIF-treated cells the least. ET- and PE-treated cultures contained the same number of beating cells as control, but LIF and PGF2alpha treatment increased the number of beating cells 180%. Treatment with LIF, PE, and PGF2alpha increased the beat rate to 3.3 times that of control. After exposure to the beta-adrenergic agonist isoproterenol, the beat rate increased 50% for PGF2alpha' 54% for PE, 84% for LIF, and 125% for control. ET treatment did not increase the beat rate, nor did these cells respond to isoproterenol. ET, LIF, and PE increased the production of atrial natriuretic peptide (ANP) by three-fold and PGF2alpha by 18-fold over nontreated cells. Brain natriuretic peptide (BNP) was increased fourfold by ET and PE, 16-fold by LIF, and 29-fold by PGF2alpha. Interestingly, on a pmol/L basis, only LIF induced more BNP than ANP. Treatment with all agents led to a similar pattern of gene induction: increased expression of the embryonic genes for ANP and skeletal alpha-actin, and less than a twofold change in the constitutively expressed gene myosin light chain-2, with the exception that LIF did not induce skeletal alpha-actin. Each agent, however, induced ANP mRNA with a different time-course. We conclude that at least four distinct cardiac myocyte hypertrophy response programs can be induced in vitro. Further studies are necessary to determine whether these correlate to the different types of cardiac hypertrophy seen in vivo.

Regulation of the rat atrial natriuretic peptide gene after acute imposition of left ventricular pressure overload.

The upregulation of left ventricular (LV) atrial natriuretic peptide (ANP) mRNA is a highly conserved marker of cardiac hypertrophy. The aim of this study was to further examine the pathway leading to ANP induction during pressure overload of the heart. Systolic wall stress was imposed acutely on isovolumetrically beating rat hearts in a Langendorff apparatus (sigma-=300 x 10[3] dyne/cm2). Northern and Western blots revealed that elevated wall stress induced LV c-fos and c-jun mRNAs (3.5- and 3-fold, P<.05 after 60 minutes), c-Fos and c-Jun proteins (3.9- and 4.3-fold, P<.05 after 120 minutes), as well as ANP mRNA (2.2-fold, P<.05 after 120 minutes). ANP upregulation was prevented by inhibition of protein synthesis (cycloheximide). Electrophoresis mobility shift assays were performed to link c-Fos and c-Jun (ie, components of the heterodimeric transcription factor AP-1) and ANP induction. A putative AP-1 binding site within the rat ANP promoter (nucleotides -512 to -473) bound specifically to nuclear proteins of wall stress-stimulated hearts. Antibodies directed against c-Fos protein resulted in a shift of this DNA/protein complex, suggesting physical interaction between AP-1 and the ANP promoter. Myocardial transfection of promoter constructs revealed that after acute imposition of wall stress, this AP-1 site enhanced a reporter gene (8- to 10-fold compared with a minimal promoter, P<.05). Interestingly, nuclear extracts of stimulated hearts as well as pure AP-1 protein bound to a putative CRE site (nucleotides -613 to -584) as well. Like the AP-1 site, this cAMP-responsible element (CRE) site was found to enhance the transfected ANP promoter/reporter gene significantly (17.5-fold, P<.05). Mutation of either AP-1 or CRE sites did not decrease reporter gene activity, whereas mutation of both resulted in loss of inducibility. These experiments suggest that LV ANP regulation after acute wall stress includes the activation of AP-1 and/or CRE cis acting elements. However, the transient nature of c-fos and c-jun upregulation also suggests that AP-1 is not the only mediator of ANP induction in LV hypertrophy.

Cloning, sequence analysis, tissue-specific expression, and prohormone isolation of Eel atrial natriuretic peptide.

A complementary DNA (cDNA) encoding eel atrial natriuretic peptide (ANP) precursor was specifically amplified from eel atrial mRNAs by rapid-amplification polymerase chain reaction. The sequence analysis of the cDNA using multiple clones revealed that the preproANP consists of 140 amino acid residues carrying a signal sequence at its N-terminus and a mature ANP at its C-terminus. An additional glycine residue was attached to the C-terminus of previously isolated eel ANP. The glycine residue may be used for amidation of the C-terminus or removed after processing. The cleavage site of a signal peptide with 22 amino acid residues was confirmed by isolation of proANP protein from eel atria. The proANP sequence deduced from the cDNA was also confirmed for 71% of the isolated protein. Sequence comparison with other natriuretic peptides revealed that eel ANP is more similar to mammalian ANP than to B-type natriuretic peptide (BNP) at both amino acid and nucleotide sequence levels. The eel ANP gene was a single copy gene as shown by Southern blot analysis. Northern blot analysis showed that eel ANP mRNA is approximately 0.8 kb in size and exclusively detected in the atrium. Thus, eel ANP is a true atrial hormone judging from both the sequence and the site of production. However, reverse transcription-polymerase chain reaction detected ANP message in the brain, gill, cardiac ventricle, red body of swim bladder (rete mirabilis), intestine, head kidney (including interrenal and chromaffin tissues) and kidney. Most of these tissues are involved in ion and/or gas exchange in fishes.

Myocardial osteopontin expression is associated with left ventricular hypertrophy.

Osteopontin (OP) has been identified in cultured rat cardiac fibroblasts, where it contributes to angiotensin II (AII)-induced remodeling processes; in cultured cardiomyocytes; and in macrophages in cardiac tissues with inflammation. However, the presence of OP has not been reported in histological sections of myocardial tissue. In the present study, we investigated (1) the regulation of OP mRNA expression in cultured rat cardiomyocytes; (2) the localization of OP mRNA in neonatal and adult normal and hypertrophied rat hearts; and (3) the histology of OP expression in myocardial specimens from humans either with myocyte hypertrophy or with no pathological changes. Cultured neonatal cardiomyocytes expressed OP mRNA and were immunoreactive for OP. Endothelin-1 (ET-1) and norepinephrine (NE) increased both OP and atrial natriuretic peptide (ANP) mRNA levels twofold to threefold (P<.01). OP mRNA was prominent in ventricular tissue from neonatal and adult rats with renovascular hypertension and aortic banding, whereas barely detectable levels were observed in normal adult cardiac tissue. ANP and OP mRNA levels in normal and hypertrophied ventricles correlated (r2=.87, P<.001). OP immunoreactivity and mRNA transcripts were predominantly found in cardiomyocytes not associated with inflammatory cells in sections from neonatal and adult hypertrophied hearts. No staining was detectable in normal adult hearts. Human myocardium with extensive fibrosis and cardiomyocyte hypertrophy obtained from explanted hearts with either idiopathic (n=5) or ischemic cardiomyopathy (n=7) demonstrated substantial myocyte immunoreactivity for both OP and ANP in right and left ventricles that was not associated with leukocyte infiltration. In situ hybridization identified cardiomyocytes as the major source of OP mRNA transcripts in these hearts. In contrast, OP immunoreactivity was not detectable in four of five endomyocardial biopsies with normal histology. The present study provides the first evidence that cardiomyocytes are a prominent source of OP in vivo and suggests that induction of OP expression is strongly associated with ventricular hypertrophy.

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.

Changes in gene expression in the intact human heart. Downregulation of alpha-myosin heavy chain in hypertrophied, failing ventricular myocardium.

Using quantitative RT-PCR in RNA from right ventricular (RV) endomyocardial biopsies from intact nonfailing hearts, and subjects with moderate RV failure from primary pulmonary hypertension (PPH) or idiopathic dilated cardiomyopathy (IDC), we measured expression of genes involved in regulation of contractility or hypertrophy. Gene expression was also assessed in LV (left ventricular) and RV free wall and RV endomyocardium of hearts from end-stage IDC subjects undergoing heart transplantation or from nonfailing donors. In intact failing hearts, downregulation of beta1-receptor mRNA and protein, upregulation of atrial natriuretic peptide mRNA expression, and increased myocyte diameter indicated similar degrees of failure and hypertrophy in the IDC and PPH phenotypes. The only molecular phenotypic difference between PPH and IDC RVs was upregulation of beta2-receptor gene expression in PPH but not IDC. The major new findings were that (a) both nonfailing intact and explanted human ventricular myocardium expressed substantial amounts of alpha-myosin heavy chain mRNA (alpha-MHC, 23-34% of total), and (b) in heart failure alpha-MHC was downregulated (by 67-84%) and beta-MHC gene expression was upregulated. We conclude that at the mRNA level nonfailing human heart expresses substantial alpha-MHC. In myocardial failure this alteration in gene expression of MHC isoforms, if translated into protein expression, would decrease myosin ATPase enzyme velocity and slow speed of contraction.

Dexamethasone-induced Up-regulation of Adrenomedullin and Atrial Natriuretic Peptide Genes in Cultured Rat Ventricular Myocytes

To determine whether adrenomedullin (AM), a novel 51-residue vasodilator peptide originally isolated from human pheochromocytoma, is expressed by the heart, and whether the expression of cardiac AM gene is regulated by glucocorticoids, the effect of dexamethasone (DEX) on the expression of protein and mRNA of AM and atrial natriuretic peptide (ANP) was tested in cultured ventricular myocytes prepared from the neonatal rats. Northern blot analysis of rat ventricular myocytes with cDNAs for rat AM and ANP as probes revealed distinct bands corresponding to the sizes of rat AM mRNA (1.6 kb) and rat ANP mRNA (1 kb), respectively. Dexamethasone increased steady-state levels of both AM and ANP mRNA as well as secretion of both AM and ANP immunoreactivity in a time- and dose-dependent manner. The stimulatory effects of dexamethasone were completely abolished by a glucocorticoid antagonist (RU38486) and a RNA synthesis inhibitor (actinomycin D). The approximate half-lives of basal and dexamethasone-induced AM mRNA were about 4 h, suggesting that dexamethasone-induced up-regulation of AM mRNA was unlikely due to its decreased degradation. These data suggest that glucocorticoids directly stimulate gene expression of AM as well as ANP in rat ventricular myocytes.