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Am J Physiol Heart Circ Physiol 287: H1516 –H1521, 2004. First published May 20, 2004; 10.1152/ajpheart.00947.2003. Rat corin gene: molecular cloning and reduced expression in experimental heart failure Thomas H. Langenickel,1 Ines Pagel,2 Jens Buttgereit,1 Katja Tenner,1 Maren Lindner,1 Rainer Dietz,2 Roland Willenbrock,2 and Michael Bader1 1 Max-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 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 proANP(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. gene expression; atrial natiuretic factor METHODS Animal Studies (NPs) in mammals consist of the three structurally homologous peptide hormones atrial NP (ANP), brain NP (BNP), and C-type NP (CNP). ANP as the first discovered and most important NP is predominantly synthesized in atrial cardiomyocytes (6, 10). It is stored as proANP(1–126) and released as biologically active ANP(99 –126) into the circulation (5, 14). The major stimulus for ANP release is atrial stretch (15). Regarding their actions, NPs are involved in the neurohumoral regulation in heart failure counteracting the angiotensin-aldosterone system and balancing water and electrolyte homeostasis. 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 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). Shamoperated 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 con- Address for reprint requests and present address of T. Langenickel: National Heart, Lung, and Blood Institute, 50 Center Dr., Bldg. 50/4529, Bethesda, MD 20892 (E-mail: [email protected]). 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. THE NATRIURETIC PEPTIDES H1516 0363-6135/04 $5.00 Copyright © 2004 the American Physiological Society http://www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on August 9, 2017 Langenickel, Thomas H., Ines Pagel, Jens Buttgereit, Katja Tenner, Maren Lindner, Rainer Dietz, Roland Willenbrock, and Michael Bader. Rat corin gene: molecular cloning and reduced expression in experimental heart failure. Am J Physiol Heart Circ Physiol 287: H1516 –H1521, 2004. First published May 20, 2004; 10.1152/ajpheart.00947.2003.—Stored cardiac pro-atrial natriuretic peptide (pro-ANP) is converted to ANP and released upon stretch from the atria into the circulation. Corin is a serin protease with pro-ANP-converting properties and may be the rate-limiting enzyme in ANP release. This study was aimed to clone and sequence corin in the rat and to analyze corin mRNA expression in heart failure when ANP release upon stretch is blunted. Full-length cDNA of rat corin was obtained from atrial RNA by RT-PCR and sequenced. Tissue distribution as well as regulation of corin mRNA expression in the atria were determined by RT-PCR and RNase protection assay. Heart failure was induced by an infrarenal aortocaval shunt. Stretch was applied to the left atrium in a working heart modus, and ANP was measured in the perfusates. The sequence of rat corin cDNA was found to be 93.6% homologous to mouse corin cDNA. Corin mRNA was expressed almost exclusively in the heart with highest concentrations in both atria. The aortocaval shunt led to cardiac hypertrophy and heart failure. Stretch-induced ANP release was blunted in shunt animals (control 1,195 ⫾ 197 fmol 䡠 min⫺1 䡠 g⫺1; shunt: 639 ⫾ 99 fmol 䡠 min⫺1 䡠 g⫺1, P ⬍ 0.05). Corin mRNA expression was decreased in both atria in shunt animals [right atrium: control 0.638 ⫾ 0.004 arbitrary units (AU), shunt 0.566 ⫾ 0.014 AU, P ⬍ 0.001; left atrium: control 0.564 ⫾ 0.009 AU, shunt 0.464 ⫾ 0.009 AU, P ⬍ 0.001]. Downregulation of atrial corin mRNA expression may be a novel mechanism for the blunted ANP release in heart failure. RAT CORIN IN EXPERIMENTAL HEART FAILURE H1517 tractility (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 g atrial rat RNA by reverse transcription (RT-PCR Kit, Stratagene) using an oligo-dT primer. Corin was amplified by PCR from 5 l 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 AJP-Heart Circ Physiol • VOL 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 g 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 KrebsHenseleit solution (2.1 mM MgSO4, 118 mM NaCl, 4.7 mM KCl, 60 287 • OCTOBER 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on August 9, 2017 Fig. 1. Amino acid sequences of rat, mouse, and human corin. Consensus shows amino acids identical in at least two corins. The putative transmembrane domain and the ligand binding motifs (SDE and similar) are underlined. H1518 RAT CORIN IN EXPERIMENTAL HEART FAILURE M 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 COOHterminal ANP. 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. 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. Heart Weight and Hemodynamics 4 Wk After Shunt Induction RESULTS Molecular Cloning of Rat Corin 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 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. Fig. 2. Tissue distribution of corin mRNA. A: Northern blot analysis demonstrated corin mRNA expression in the heart, and there with highest concentration in both atria. B: RT-PCR analysis confirmed major expression in the heart but also detected expression in the aorta, kidney, brain, and testis. AJP-Heart Circ Physiol • VOL 287 • OCTOBER 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on August 9, 2017 Statistical Analysis the putative transmembrane domain and ligand-binding motifs were found to be highly conserved. We compared the cDNA with rat genome sequence in GenBank and deduced the structure of the rat corin gene. Despite some uncertainties due to incompleteness of the rat genomic sequence, the gene structure seems to be similar to the human and mouse structure with 22 exons covering about 240 kb on rat chromosome 14. RAT CORIN IN EXPERIMENTAL HEART FAILURE H1519 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). ANP Release From Isolated Heart After LA Stretch Fig. 3. Hemodynamic evaluation of heart failure induced by aortocaval shunt. A: relative heart weight (HW) is increased in animals with an aortocaval shunt. bw, Body weight. **P ⬍ 0.01. B: left ventricular contractility (dP/dtmax) is reduced in rats with an aortocval shunt. *P ⬍ 0.05. C: left ventricular end-diastolic pressure (LVEDP) is increased in animals with an aortocaval shunt. **P ⬍ 0.01. D: central venous pressure (CVP) is increased in animals with an aortocaval shunt. **P ⬍ 0.01. AJP-Heart Circ Physiol • VOL Fig. 4. Atrial natriuretic peptide (ANP) release after stretch. A: after a 30-min baseline period, left atrial pressure (LAP) was increased from 0 to 10 mmHg in a Langendorff isolated heart perfusion, and basal ANP release was measured during the baseline period. B: ANP release due to an increase in LAP is significantly lower in animals with an aortocaval shunt compared with controls. *P ⬍ 0.05. Baseline ANP release was not different in both groups. 287 • OCTOBER 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on August 9, 2017 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䡠min⫺1 䡠g⫺1, shunt: 355 ⫾ 32 fmol䡠min⫺1 䡠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䡠min⫺1 䡠g⫺1, shunt 639 ⫾ 99 fmol䡠min⫺1 䡠g⫺1, P ⬍ 0.05; Fig. 4B). H1520 RAT CORIN IN EXPERIMENTAL HEART FAILURE Atrial Corin mRNA Level in Experimental Heart Failure 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 Fig. 5. Left atrial corin mRNA expression detected by RNase protection assay (bottom) is decreased in animals with aortocaval shunt. AU, arbitrary units. ***P ⬍ 0.001. AJP-Heart Circ Physiol • VOL Fig. 6. Right atrial corin mRNA expression detected by RNase protection assay (bottom) is decreased in animals with aortocaval shunt. ***P ⬍ 0.001. 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. 287 • OCTOBER 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on August 9, 2017 In the present study, the molecular cloning and characterization of rat corin is described. Rat corin is highly homologous to mouse and human corin and shows a similar pattern of expression being detectable mainly in both atria but also in the kidney and testis. In contrast to the mouse and human, this study was able to demonstrate corin mRNA expression also in the aorta and brain. 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. RAT CORIN IN EXPERIMENTAL HEART FAILURE ACKNOWLEDGMENTS Present address of I. Pagel: National Heart, Lung, and Blood Institute, Cardiovascular Branch, Bldg. 10/7B02, 9000 Rockville Pike, Bethesda, MD 20892. Present address of R. Willenbrock: St. Elisabeth Hospital, Mauerstrasse 5, 06110 Halle (Saale), Germany. GRANTS This study was supported by research fellowships from the Max-DelbrueckCenter for Molecular Medicine (to T. H. Langenickel and I. Pagel) and by a grant from the “Verbund Klinische Pharmakologie Berlin-Brandenburg” (to T. H. Langenickel). 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