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Department of Pathology (Y.K., Y.M., C.K., A.D.D., T.S., H.S.), Department of Molecular Biology (K.Ta.), Second Department of Internal Medicine (K.To.), Tohoku University School of Medicine; and Department of Respiratory Oncology/Molecular Medicine (M.E., T.N.), Tohoku University Institute of Development, Aging and Cancer, Sendai, Japan 980-8575
Address all correspondence and requests for reprints to: Yuichiro Kimura, M.D., Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. E-mail: kimuyu{at}patholo2.med.tohoku.ac.jp
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mRNAs were detected in all four chambers. CRF-R1 mRNA was weakly present in some left atria, left ventricles, and in one right ventricle. CRF-R2ß mRNA was detected predominantly in the left atrium. CRF mRNA was not detected in any of the four chambers. Immunostaining for both Ucn and CRF receptors was detected in cardiac myocytes in all four chambers. Ucn-like immunoreactivity was detected in all four chambers by RIA, with the highest concentrations in the left ventricle (1.90 ± 0.5 pmol/g wet weight, mean ± SEM; n = 4). On the other hand, CRF-like immunoreactivity was very low or undetectable in the human heart. Sephadex G-50 column chromatography demonstrated that most of the Ucn-like immunoreactivity in the human heart was eluting earlier than the standard Ucn, with one minor peak in the position for Ucn. Ucn immunoreactivity was not detected in skeletal muscle by immunohistochemistry or RIA. These results suggest that Ucn is produced in the human heart and stored there mainly in the larger molecular weight forms. Endogenously produced Ucn may therefore exert its effects mostly through CRF-R2 in an autocrine and/or paracrine manner in the human heart. |
Introduction |
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CRF receptors are classified into CRF receptor type 1 (CRF-R1) (16) and CRF receptor type 2 (CRF-R2) ( 17). Ucn binds with high affinity to both CRF-R1 and CRF-R2, but this ligand has a much higher affinity for CRF-R2 than CRF (1, 2). The distribution of Ucn nerve fibers in the brain correlates well with that of CRF-R2 but not with that of CRF-R1 (1). CRF-R2 deficient mice have a dramatically attenuated cardiovascular response to Ucn injection (18). CRF-R2 is composed of two different isoforms, CRF-R2 and -R2ß. In the rat, CRF-R2 is expressed predominantly in the brain, whereas CRF-R2ß is expressed in both the brain and periphery, especially in heart and skeletal muscle tissues (19). CRF-R2ß mRNA expression has been shown to be decreased by lipopolysaccharide, corticosterone, and Ucn in the rat heart and in the aorta-derived A7R5 cell line (20). In contrast to rodents, CRF-R2 appears to be the major CRF-R2 isoform in the brain, heart, and skeletal muscle tissues, whereas CRF-R2ß is considered to be the minor isoform in humans (21, 22).
Both Ucn mRNA and peptide are expressed in the rat heart (23, 24). Ucn has also been shown to be expressed in rat cardiomyocytes, its expression levels increased by heat shock and ischemia (12, 13). Nishikimi et al. (25) have recently reported that the human myocardium was immunohistochemically positive for Ucn, the staining of which was more intense in the failing heart. However, endogenous ligand(s) for CRF receptors expressed in the human heart have yet to be elucidated. In addition, Ucn-like immunoreactivity (Ucn-LI) in the heart has not been characterized by chromatography. In this study, we therefore examined the expression of Ucn, CRF, and CRF receptors in the human heart obtained at autopsy by RT-PCR, immunohistochemistry, and RIA.
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Materials and Methods |
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Human hearts were obtained from four patients during an autopsy performed at Tohoku University Hospital in Sendai, Japan. The hearts were obtained from macroscopically normal areas and were immediately separated into their four constituent chambers at the time of autopsy. In all cases, autopsy was performed within 2 h of death. Heart specimens were obtained from a 71-yr-old male who died of hepatic cirrhosis (patient 1), a 21-yr-old male who died of mesothelioma (patient 2), a 75-yr-old male who died of gastric cancer (patient 3), and a 63-year-old female who died of chronic renal failure (patient 4). No significant histopathological changes were detected in these specimens. Thorough review of the patients’ charts revealed that these patients did not have any history of cardiovascular disease. Human skeletal muscle (psoas muscle) tissues were obtained from six patients via the same methodology. These patients also had no history of cardiovascular disease. The families of all patients gave written informed consent. All tissues were flash frozen in liquid nitrogen and stored at -80 C.
RT-PCR
Total RNA was isolated by the guanidine thiocyanate-cesium chloride method. Total RNA was denatured at 65 C for 5 min and then reverse transcribed at 37 C for 60 min in a total volume of 20 µl reaction buffer (Life Technologies, Inc., Grand Island, NY) containing 0.5 µg oligo-deoxythymidine (Amersham Pharmacia Biotech, Uppsala, Sweden), 10 nmol deoxy-NTPs, and 400 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Grand Island, NY). The reaction mixture was then heated to 95 C for 5 min and immediately chilled on ice. Subsequently, the synthesized complementary DNA (cDNA) products (4 µl) were subjected to PCR in a reaction mixture (20 µl) containing 10 mmol/liter Tris HCl pH 8.3, 50 mmol/liter KCl, 2 mmol/liter MgCl2, 0.001% gelatin, 4 nmol deoxy-NTPs, 5 pmol of each primer, respectively, and 0.5 U Taq DNA polymerase (Amersham Biosciences, Inc., Piscataway, NJ). The sequences of the primers employed in this study are summarized in Table 1
. Both Ucn and CRF primers were designed according to the published nucleotide sequences for Ucn (26 ; GenBank accession no. AF038633) and CRF genes (27 ; GenBank accession no. V00571). CRF receptor primers were designed according to the published cDNA nucleotide sequences for CRF-R1 (16 ; GenBank accession no. L23332), -R2 (22 ; GenBank accession no. U34587), and -R2ß (21), respectively. These primers were designed so as to insert the intron between the sense and antisense primers, thereby eliminating the possibility of amplifying any genomic DNA (16, 21, 22, 26, 27). The sense primer for CRF-R2ß was designed to include the site of divergence between CRF-R2 and -R2ß (21). Human placenta was used as a positive control for the expression of both Ucn and CRF mRNA. Human pituitary gland, hypothalamus, and left cardiac atrium were used as positive controls for the expression of CRF-R1, -R2, and -R2ß mRNAs, respectively. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was examined as an internal control. After preheating at 96 C for 2 min, denaturation, annealing, and elongation were carried out at 96 C for 15 sec, at 64 C (CRF-R1, -R2, -R2ß) or 60 C (GAPDH) for 30 sec, and at 72 C for 1 min, respectively. PCR was carried out for 35 cycles (CRF-R1, -R2, -R2ß), or 22 cycles (GAPDH), respectively.
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Immunohistochemistry
Immunostaining for Ucn, CRF, and CRF receptors was performed on serial mirror tissue sections cut at 3 µm from paraffin-embedded specimens. To preserve the antigenicity, the sections were pretreated by hydrated autoclaving in 10 mM sodium citrate buffer (pH 6.0) at 120 C for 5 min, and then allowed to cool for approximately 1 h at room temperature. The slides were then placed in 100% methanol with 0.3% (vol/vol) hydrogen peroxidase activity and then treated with 1% (vol/vol) normal goat serum (Ucn and CRF) or normal rabbit serum (CRF receptors) for 30 min at room temperature in a moisture chamber. Primary antibodies (optimal dilution; 1:2500 for Ucn, 1:2000 for CRF and 1:40 for CRF receptors) were applied onto the tissue sections for 18 h at 4 C. The polyclonal antibodies against Ucn and CRF were raised in rabbits, as previously reported (5, 28). The polyclonal antibody against CRF receptors raised in goat was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). This antibody is reactive with both CRF-R1 and CRF-R2. After washing in 0.01 M PBS, the reacted sections (Ucn and CRF) were incubated with biotinylated antirabbit immunoglobulin for 30 min at room temperature and subsequently incubated with peroxidase-conjugated streptavidin for 20 min, using a Histofine immunostaining kit (Nichirei Co. Ltd, Tokyo, Japan). The reacted section for CRF receptors was incubated with horseradish peroxidase-conjugated antigoat Ig for 60 min at room temperature. These sections were again washed with 0.01 M PBS, and the antigen-antibody complexes were visualized by immersion in 3.3'-diaminobenzidine solution (0.01 M 3.3'-diaminobenzidine in 0.05 M Tris-HCl buffer, pH 7.6; and 0.006% hydrogen peroxide).
An absorption test for Ucn immunoreactivity, which used an antibody-antigen mixture containing an equal volume of the optimally diluted antiserum and Ucn peptide solution (20 µmol/liter, final concentration), was conducted by incubating specimens for 18 h at 4 C. After centrifugation, the resultant supernatants were used as a preabsorbed antibody. A negative control absorption test, in which Ucn peptide was replaced by human CRF solution (20 µmol/liter, final concentration), was performed in parallel.
Peptide extraction and RIA
Tissues were extracted as reported previously (29). Briefly, the tissue (approximately 1 g) was boiled in 2 ml of 1 mol/liter acetic acid for 10 min. Eight milliliters of 50% methanol in 0.5 mol/liter acetic acid were added to each sample, and the tissue was then homogenized. The homogenate was subsequently centrifuged at 15,000 x g for 30 min. The supernatant was separated, dried by air, reconstituted in assay buffer [0.1 mol/liter phosphate buffer, pH 7.5 containing 0.1% (wt/vol) BSA, 0.2% (vol/vol) Triton X-100, and 0.1% (wt/vol) sodium azide] and assayed.
Urocortin-like immunoreactivity (Ucn-LI) and CRF-LI in the tissue extracts were measured by their respective specific RIAs, as previously reported (5, 28). RIA for Ucn has previously been shown to have no significant cross reaction with CRF, ANP, BNP, urotensin I, NPY, or other peptides (5). RIA for CRF has previously been shown to have no significant cross-reaction with Ucn, ANP, BNP, urotensin I, NPY, or other peptides (28).
Chromatography
Chromatographic characterization of the tissue extracts was performed by Sephadex G-50 (superfine) column chromatography and reverse phase HPLC using a µBondapak C18 column (3.9 mm x 300 mm, Waters Corp., Milford, MA). Cardiac tissue extracts obtained from four subjects were pooled and reextracted by a Sep-Pak C18 cartridge (Waters Corp.). The extracts were reconstituted in 1 mol/liter acetic acid containing 0.5% (wt/vol) BSA and loaded onto the Sephadex G-50 column (10 x 400 mm). Peptides on the column were eluted with 1 mol/liter acetic acid containing 0.5% (wt/vol) BSA at a flow rate of 10 ml/hour. Fractions (0.65 ml/fraction) were collected, dried by air, reconstituted in assay buffer and assayed. The elution position of CNP-53 was determined for the molecular weight marker by RIA (30).
For HPLC analysis, the pooled tissue extracts were reextracted with a Sep-Pak C18 cartridge (Waters Corp.) reconstituted in 0.1% (vol/vol) trifluoroacetic acid and loaded onto the column. The HPLC analysis was performed with a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid from 10% to 60% at a flow rate of 1 ml/min per fraction over 50 min. Each fraction (1 ml) was collected, dried by air, reconstituted with assay buffer and assayed.
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Results |
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). However, CRF mRNA was not detected in any of these samples. CRF-R2 mRNA was shown to be expressed in the right atrium, right ventricle, left atrium, and left ventricle in all four cases examined. A weak band for CRF-R1 mRNA was detected in the left atria of patients 2 and 3, in the left ventricles of patients 2 and 4, and in the right ventricle of patient 4. Bands seen in the right atrium of patient 3 and left ventricle of patient 1 had different lengths from that of the specific band for CRF-R1, and may therefore be interpreted as nonspecific. CRF-R2ß mRNA expression was observed in the left atrium in all four cases, and in the right atrium of only one out of four cases studied (patient 2). In addition, the relative level of CRF-R2ß expression in this specimen was very weak. We found that the major CRF receptor subtype expressed in all four chambers of the human heart was CRF-R2. PCR products were further analyzed by direct sequencing and confirmed to be identical to the registered cDNA sequences for Ucn, CRF-R1, -R2, and -R2ß, respectively (data not shown). The mRNAs for Ucn and CRF, CRF-R1, -R2, and -R2ß were detected in all positive controls, human placenta, pituitary gland, hypothalamus, and left atrium (Fig. 1). GAPDH mRNA was similarly expressed in all samples. The negative control showed no band (data not shown).
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). Immunoreactivity for Ucn was abolished by the preabsorption of antiserum with synthetic human Ucn but remained unchanged by the antiserum preabsorbed with CRF (data not shown). No specific immunoreactivity was detected in the slides treated with normal rabbit IgG (Fig. 2, C and F) or normal goat IgG (data not shown) instead of the antiserum against Ucn or CRF receptors. CRF immunoreactivity was not detected in any of the samples of human hearts examined. Ucn, CRF, or CRF receptor immunoreactivity was not detected in human skeletal muscles by immunohistochemistry (data not shown).
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). There was no significant difference in the Ucn-LI levels among these four regions of the heart (ANOVA, P > 0.2). Very low levels of CRF-LI were detectable only in some heart extracts (one left ventricle, two right ventricles, two left atria and one right atrium, 0.032–0.052 pmol/g wet weight), but not detectable in the other samples. Ucn-LI or CRF-LI was not detected in skeletal muscle tissues by RIA (n = 6; Ucn, <0.3 pmol/g wet weight; CRF, <0.03 pmol/g wet weight).
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). There were two minor peaks and one major peak eluting earlier than the synthetic human Ucn. This major peak was observed between the elution positions for synthetic CNP-53 (the 36th fraction) and Ucn (the 50th fraction), and may represent a partially processed form of the Ucn precursor. Reverse phase HPLC of the tissues also showed that most of the Ucn-LI in the heart was eluted earlier than synthetic human Ucn, and only a small amount of the Ucn-LI was eluted in the same position as human Ucn (Fig. 5).
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Discussion |
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The highest concentrations of Ucn-LI were detected in the left ventricle, followed by the right ventricle, the left atrium and the right atrium, respectively. The levels of Ucn were comparable with those of the human brain (5). Chromatographic studies demonstrated that the main form of Ucn-LI in the human heart was the larger molecular weight peptide. Human Ucn is generated from a 124-amino acid Ucn precursor through posttranslational enzymatic processing of Arg-Arg at position 81–82 (2). It is interesting that the 22-kDa Ucn precursor protein was detected in rat cardiomyocytes by Western blot analysis (14). On the other hand, the main peak was detected between the elution positions for CNP-53 (a 53-amino acid peptide) and Ucn (a 40-amino acid peptide) in the Sephadex G50 column chromatograph of the present study. The material eluting in this peak may be a peptide consisting of about 45–50 amino acids and may therefore represent a partially processed form of the Ucn precursor rather than the 22-kDa Ucn precursor. There is another possible proteolytic processing site Arg-Arg at position 64–65 in the human Ucn precursor (2), and the cleavage of this site may generate a 57-amino acid peptide containing the 40-amino acid Ucn sequence. Further studies are required to clarify whether such an incompletely processed Ucn is present in the human heart as well as the mature type of Ucn consisting of 40 amino acids. In addition, two minor peaks were also detected in the larger molecular weight regions. A previous study has reported that intracellular accumulation of the precursor forms for ANP increased in severe chronic human congestive heart failure (31).
Previous studies have demonstrated that CRF and Ucn directly regulate cardiovascular functions in animals. Ucn has also been shown to potently increase cAMP levels in both rat cardiac myocytes and nonmyocytes to a greater degree than did CRF (25). Although the iv injection of CRF produced little change in any of the hemodynamic parameters in conscious sheep, Ucn stimulated cardiac contractility significantly and remained effective over a long period of time (11). In addition, Ucn has been suggested to have protective action against ischemic and reperfusion injury in rats (12, 13, 14). However, physiological ligand(s) to CRF receptors expressed in the heart have, to date, not been elucidated (32). In the present study, CRF mRNA was not detected in the human heart, in contrast to the report by Muglia et al. (33), in which the expression of CRF mRNA was demonstrated in the mouse heart by RT-PCR. Baigent and Lowry reported that CRF expression in the rat heart was only evident after 60 PCR cycles (34), suggesting that the CRF expression in the heart was very low. Although it is known that CRF levels in human plasma are very low (35), the presence of Ucn in plasma has not been clarified. Our results suggest that Ucn or Ucn-like material is produced and secreted by the myocardium, and is the physiological ligand of CRF receptors in the human heart. We, however, cannot completely rule out the possibilities that sympathetic nerves supplying the heart express CRF mRNA and that our samples of human heart tissue are likely to possess some CRF containing nerve fibers. The very low levels of CRF-LI detected in some human heart samples by RIA may reflect the expression of CRF in these nerve fibers. Further investigations are required for clarification.
We were also able to demonstrate a relatively wide distribution of CRF receptors in the human heart using immunohistochemistry, suggestive of the autocrine or paracrine roles of Ucn. The antibody against CRF receptors used in this study recognizes both CRF-R1 and CRF-R2. RT-PCR analysis has demonstrated the differential localization of CRF receptor mRNAs: CRF-R2 was a major isoform expressed in all four chambers of the human heart, which is consistent with a previous report (22). In our study, CRF-R2ß was expressed only in some left atria and one right atrium. Moreover, the pharmacological profiles of the two different isoforms of CRF-R2 splice variants have been reported to be very similar (36). Further studies are therefore required to determine whether CRF-R2 and -R2ß in human heart have different actions based on their differential and unique pattern of expression.
The present study has demonstrated the expression of Ucn and CRF receptor subtypes in the human heart. These findings suggest the presence of a novel humoral regulatory system in human cardiac function.
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Acknowledgments |
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Footnotes |
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Abbreviations: ANP, Atrial natriuretic peptide; BNP, brain natriuretic peptide; CRF, corticotropin-releasing factor; CRF-R1 or -R2, CRF receptor type 1 or type 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ucn, urocortin; UCN-LI, Ucn-like immunoreactivity.
Received January 8, 2001.
Accepted September 5, 2001.
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and CRF2ß receptor mRNAs are differentially distributed between the rat central nervous system and peripheral tissues. Endocrinology 136:4139–4142[Abstract]
and ß splice variants: pharmacological characterization using radioligand binding and a luciferase gene expression assay. Neuropharmacology 38:441–448[CrossRef][Medline]This article has been cited by other articles:
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