| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1Max-Delbrueck-Center for Molecular Medicine, Berlin 13092; and 2Franz-Volhard-Klinik, Charité Campus Berlin-Buch, Humboldt-University, Berlin 13125, Germany
Submitted 6 October 2003 ; accepted in final form 12 May 2004
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
gene expression; atrial natiuretic factor
The plasma concentrations as well as cardiac tissue concentrations and ANP mRNA expression are increased in experimental heart failure induced by volume overload in the rat (1, 3, 16). However, the ANP response to acute volume expansion is blunted in cardiac hypertrophy as well as heart failure (18, 23, 25). It is unclear whether the impaired ANP release in heart failure is a result of neurohormonal suppression or due to an inhibition of the release mechanism itself.
The critical step during the ANP release is the conversion of pro-ANP to ANP. Recently, corin, a membrane-bound type II serin protease, also known as LRP4, has been identified as a pro-ANP-converting enzyme (21, 27). Corin converts pro-ANP(1–126) into ANP(99–126) by cleaving pro-ANP specifically behind arginine at position 98 in vitro (28) and in a murine cardiac myocytic cell line, HL-5 (26). Corin was shown to be expressed in the heart, testis, and kidney of mice, but cardiac expression was predominant (21, 27). Corin is also expressed in the human heart (11). Corin and NPs are coexpressed during development suggesting that corin might be important for conversion of pro-ANP to ANP at this stage as well (27).
However, no information is available on corin in the rat, neither on the sequence nor on the tissue distribution of corin mRNA expression. It is further unknown as to whether corin mRNA expression is regulated and related to a blunted ANP release in heart failure. Therefore, this study was aimed to clone and sequence rat corin cDNA, determine the expression pattern in several organs, and correlate the corin mRNA level at the site of highest synthesis, the atria, to ANP release upon stretch in experimental heart failure due to chronic volume overload in the rat.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Experimental heart failure model in the rat. The infrarenal aortocaval shunt in male Wistar rats was used as a model of volume overload-induced heart failure as previously described (8). Sham-operated rats used as controls were treated identically except that no puncture of the vessels was performed. Four weeks after surgery, two separate sets of sham- and shunt-operated rats were used for either in vivo hemodynamic measurements and molecular biology studies or working heart studies. The study was carried out in accordance with the local authorities and conformed with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996).
Hemodynamic measurements. Animals were anesthetized with chloralhydrate, and a polyethylene-50 tubing catheter was inserted via the right carotid artery into the left ventricle for measurement of mean arterial and end-diastolic pressures. Central venous pressure was measured by cannulating the left jugular vein. Left ventricular contractility (dP/dtmax) was obtained from the ventricular pressure curves, which were converted with a Gould differentiator (G4615). All pressures were registered with a Statham P23 XL transducer and a Gould AMP 4600 amplifier. Heart rate was derived from the arterial blood pressure signal.
Molecular Biology Studies
RNA preparation. After the rats were euthanized, the hearts were rapidly dissected, washed in ice-cold diethylpyrocarbonate-treated water, and weighed, and the four heart chambers were immediately separated and frozen in liquid nitrogen. The organs were kept at –80°C until RNA extraction. Total RNA was extracted from several organs of control animals as well as from the atria of control and shunt animals with TRIzol reagent (Invitrogen, Life Technologies) according to the manufacturer's protocol.
Molecular cloning of rat corin. First-strand cDNA was generated from 5 µg atrial rat RNA by reverse transcription (RT-PCR Kit, Stratagene) using an oligo-dT primer. Corin was amplified by PCR from 5 µl first-strand cDNA using specific primers (forward primer 5'-CTTGGAGTCGGAGGCTCGGAC-3' and reverse primer 5'-TTACCATAGAGCTCCAGATGTCTC-3', BioTez) and a mixture of polymerases with proofreading activity (AccuTaq, Sigma). The obtained full-length corin cDNA was purified by gel electrophoresis, elongated by incubation with Taq polymerase for 30 min at 72°C, and cloned into the pGEM-T vector (Promega). Corin was sequenced twice and stepwise starting with standard SP6 and T7 primers (Invitek). The obtained corin cDNA sequence was compared with the available Genbank sequence of the rat genome, and three additional corin fragments were amplified by RT-PCR from atrial RNA and sequenced to rule out mistakes in sequencing or PCR amplication (forward primer 1 5'-TTCCTCCTGCTTGTGCTCATC-3' and reverse primer 1 5'-CTTGTCCACACAGTCATGGTC-3'; forward primer 2 5'-GTGGAATGCAGAAGTGGACAG-3' and reverse primer 2 5'-GTGCTCCCATTGAGATTCTC-3'; and forward primer 3 5'-TACCAAGCAAGACTGTGGTC-3' and reverse primer 3 5'-GACACATTGCTGTACACTCC-3', BioTez).
Analysis of corin mRNA expression. Corin (317 nt) and GAPDH (187 nt) were amplified by RT-PCR using the primers corin5 5'-CAAATTCTGCCCTACCACAGCAC-3', corin3 5'-GGCGTCGCTGTTATTCTCAGTG-3', GAPDH5 5'-CCATGGAGAAGGCTGGGG-3', and GAPDH3 5'-CAAAGTTGTCATGGATGACC-3' and cloned into the pGEM-T vector (Promega). The plasmids were digested with PvuII, and antisense probes were [32P]UTP labeled by in vitro transcription (Promega). Corin mRNA expression was measured by a RNase protection assay according to the manufacturer's protocol (RPA III kit, Ambion) using 20 µg total RNA and 35,000 counts/min antisense probe each. Protected fragments were separated on a polyacrylamide gel and detected by a Fuji phosphoimager (Fujix BAS 2000, Fuji). For semiquantitative determination of corin mRNA, the signals were normalized to GAPDH mRNA expression.
Measurement of ANP Release in a Working Heart Preparation
The thorax was opened under chloralhydrate anesthesia, and the heart was rapidly removed. The aorta and left atrium (LA) were cannulated, and the heart was perfused with a modified Krebs-Henseleit solution (2.1 mM MgSO4, 118 mM NaCl, 4.7 mM KCl, 60 µM Na-EDTA, 24.7 mM NaHCO3, 1.5 mM CaCl2, and 11.1 mM glucose, Merck) at 37°C using an isolated heart apparatus (Type 830, Hugo Sachs; March-Hugstetten, Germany). The solution was saturated with 95% O2-5% CO2 (pH 7.4). For equilibration and the baseline period, hearts were perfused retrograde via the aorta at a constant perfusion pressure (65 mmHg). The perfusion pressure was kept constant throughout the experiment. Baseline values for ANP secretion were obtained after 30-min equilibration while the LA pressure (LAP) was 0 mmHg. ANP release was stimulated by an elevation of the LAP to 10 mmHg. Perfusates were collected on ice, immediately frozen, and stored at –80°C. ANP concentration was measured by radioimmunoassay as described (16) and normalized to heart weight. The antibody used in this study was raised agaist the COOH-terminal rat ANP, thus detecting pro-ANP as well as COOH-terminal ANP.
Statistical Analysis
The differences between the two groups were evaluated with the paired Student's t-test using StatView software. The significance level was set at P < 0.05. All data are expressed as means ± SE.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Rat corin cDNA was cloned and sequenced (Genbank accession number AY251285), and the encoded protein was found to be 93% homologous to mouse and 85% homologous to human corin (Fig. 1). Functional important domains such as the putative transmembrane domain and ligand-binding motifs were found to be highly conserved.
|
Tissue Distribution of Corin mRNA
Tissue distribution of corin mRNA was analyzed by Northern blot analysis and RT-PCR. In Northern blot analysis, corin mRNA was exclusively detectable in the heart, and there with highest concentrations in the right atrium (RA; Fig. 2A). RT-PCR confirmed the predominant localization to the heart but also identified additional expression in the kidney, aorta, brain, and testis (Fig. 2B). Expression in the aorta and brain has not previously been demonstrated in mice and humans.
|
Four weeks after shunt induction, heart weight was significantly increased compared with controls (598 ± 18 vs. 310 ± 5 mg/100 g body wt, P < 0.01; Fig. 3A). All four heart chambers contributed to the elevated heart weight, demonstrating biventricular hypertrophy due to chronic volume overload. In parallel, dP/dtmax was impaired (3,650 ± 254 vs. 4,911 ± 242 mmHg/s, P < 0.05; Fig. 3B), whereas left ventricular end-diastolic pressure (LVEDP) as well as central venous pressure (CVP) were elevated, in rats with aortocaval shunt compared with controls (LVEDP: 15.2 ± 2.5 vs. 5.4 ± 1.3 mmHg, P < 0.01; Fig. 3C; CVP: 9.5 ± 0.9 vs. 1.6 ± 0.5 mmHg, P < 0.01; Fig. 3D).
|
To test the hypothesis that ANP release from isolated hearts is blunted in heart failure, LA stretch was applied by elevating the left intra-atrial pressure to 10 mmHg (Fig. 4A). ANP release during baseline was not different between shunt-operated and control rats (control: 359 ± 52 fmol·min–1·g–1, shunt: 355 ± 32 fmol·min–1·g–1; Fig. 4B). LA stretch significantly increased ANP secretion from normal hearts, whereas ANP release was blunted compared with baseline values in hearts from rats with aortocaval shunt (control 1,195 ± 197 fmol·min–1·g–1, shunt 639 ± 99 fmol·min–1·g–1, P < 0.05; Fig. 4B).
|
Regulation of LA and RA corin mRNA levels were studied in experimental heart failure by ribonuclease protection assay. Both LA and RA corin mRNA concentrations were found to be significantly decreased in rats with an aortocaval shunt compared with controls (LA: 0.464 ± 0.009 vs. 0.564 ± 0.009 AU, P < 0.001; Fig. 5; RA: 0.566 ± 0.014 vs. 0.638 ± 0.004 AU, P < 0.001; Fig. 6).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
The major stimulus for ANP release is acute volume expansion (15), leading to atrial stretch. After acute volume load, a rapid increase of circulating ANP plasma levels occurs within a few minutes (12). The coexpression of ANP and corin in both atria points toward a possible function of corin for the cleavage of pro-ANP upon atrial stretch in the rat heart in vivo.
In several models of experimental heart failure as well as in human heart failure, ANP release after either acute volume load or exercise is blunted (7, 13, 17, 18, 24, 25). Willenbrock et al. (25) suggested that the ANP system is impaired in rats with an acute aortocaval shunt and that the activation of the angiotensin system contributes to the impairment of the ANP system. Inadequate ANP secretion upon stimulation may thereby contribute to fluid retention during the progression of heart failure.
However, it is unclear whether the impaired ANP release in heart failure is a result of neurohormonal suppression or due to changes in atrial cardiomyocytes themselves. We investigated the stretch-induced ANP release in isolated hearts from normal rats and rats with chronic volume overload due to aortocaval shunt. These animals were characterized by significant cardiac hypertrophy in parallel with impaired ventricular function. Whereas baseline secretion of ANP was not different between both groups, ANP release after LA stretch was significantly reduced in animals with shunt. Because the pressure was subphysiological at baseline compared with intact animals and thereby the stimulus for ANP release was minimal under these conditions, we assumed that the pro-ANP-converting ability of corin was not used to full capacity. Only after application of atrial stretch we found a significantly blunted ANP release from hearts of heat failure rats, suggesting that reduced corin expression might be the rate-limiting factor for ANP secretion after atrial stretch. The atrial ANP mRNA expression level as well as ANP concentrations have been measured in rats with an aortocaval shunt. Langenickel et al. (16) showed that ANP mRNA was upregulated in the LA of shunted rats, whereas the RA ANP mRNA expression remained unchanged. Willenbrock et al. (25) found unchanged ANP mRNA as well as ANP concentration in both the LA and RA in this heart failure model. These data suggest that the blunted ANP release we observed in shunt animals was neither due to impaired synthesis nor depleted storage vesicles of ANP.
In experimental heart failure, the atrial corin mRNA level was decreased in parallel to a blunted ANP release upon stretch. These data indicate for the first time, that corin, which has been shown to cleave pro-ANP in vitro, might also be a rate-limiting mechanism for ANP release in vivo. It has been shown that pro-ANP is activated during secretion, probably by corin located on the cell surface (9, 11, 19). Thus downregulated corin expression might be responsible for the blunted release of active ANP in heart failure. In fact, increased circulating amounts of unprocessed ANP precursor are found in heart failure patients and animal models (2, 20, 22). It has been shown that ventricles induce ANP expression in heart failure and release mostly the precursor pro-ANP into the circulation (20). The low amount of corin expression (Fig. 2A) may cause this incomplete processing in ventricles. Thus our data indicate that the observed high levels of circulating ANP precursor in heart failure are derived from insufficient availability of corin in atria and ventricles. To date, no mechanisms regulating the gene expression and/or activity of corin have been identified. Thus the relation between congestive heart failure and the downregulation of corin needs to be further investigated.
|
GRANTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
Present address of R. Willenbrock: St. Elisabeth Hospital, Mauerstrasse 5, 06110 Halle (Saale), Germany.
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  D. L. Dries Relevance of Molecular Forms of Brain Natriuretic Peptide for Natriuretic Peptide Research Hypertension, May 1, 2007; 49(5): 971 - 973. [Full Text] [PDF]
|
|
|
|
|
|
D. M. Heublein, B. K. Huntley, G. Boerrigter, A. Cataliotti, S. M. Sandberg, M. M. Redfield, and J. C. Burnett Jr Immunoreactivity and Guanosine 3',5'-Cyclic Monophosphate Activating Actions of Various Molecular Forms of Human B-Type Natriuretic Peptide Hypertension, May 1, 2007; 49(5): 1114 - 1119. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Burnett Jr and T. M. Olson Natriuretic Peptides and Myocardial Structure: Insights From Population Genetics Hypertension, April 1, 2007; 49(4): 765 - 766. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
