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(Circulation. 1995;92:2041-2043.)
© 1995 American Heart Association, Inc.

Articles

cAMP Response Element Binding Protein Is Expressed and Phosphorylated in the Human Heart

Frank Ulrich Müller, MD; Peter Bokník, PhD; Andreas Horst; Jörg Knapp, MD; Bettina Linck, MD; Wilhelm Schmitz, MD; Ute Vahlensieck, MD; Michael Böhm, MD; Mario C. Deng, MD; Hans H. Scheld, MD

From the Institut für Pharmakologie und Toxikologie (F.U.M., P.B., A.H., J.K., B.L., W.S., U.V.) and the Klinik und Poliklinik für Thorax-, Herz-, und Gefäßchirurgie (M.C.D., H.H.S.), Universität Münster (Germany) and the Medizinische Klinik III, Universität Köln (Germany).

Correspondence to Dr Frank Ulrich Müller, Institut für Pharmakologie und Toxikologie, Universität Münster, Domagkstraße 12, D-48129 Münster, Germany. E-mail mullerf@uni-muenster.de.

	Abstract

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Background In end-stage failing human hearts and in rat hearts after prolonged in vivo ß-adrenergic treatment, several proteins involved in the cAMP-dependent signal transduction are altered on the protein, mRNA, or transcriptional level, eg, ß-adrenoceptors, G-proteins, or proteins of Ca²⁺ homeostasis. In many tissues, cAMP-dependent transcriptional regulation occurs through the cAMP response element binding protein (CREB) and related transcription factors binding as dimers to cAMP response elements (CREs) in the promoter regions of regulated genes.

Methods and Results To investigate a possible role of CREB in the human heart, nuclear protein of explanted failing and nonfailing human hearts was used to test for CRE specific binding properties in gel mobility shift assays. CRE specific binding was found in competition studies, and CREB and its phosphorylated form were immunologically identified in supershift experiments. The alternatively spliced CREB isoforms CREB327 and CREB341 were found to be expressed on the mRNA level by the reverse transcriptase–polymerase chain reaction.

Conclusions We conclude that in the failing and nonfailing human heart, CREB is expressed on the protein and mRNA levels and that CREB is phosphorylated and able to bind to CREs, indicating a functional role of CREB in the human heart.

Key Words: cardiomyopathy • molecular biology • signal transduction

	Introduction

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In end-stage human heart failure, ß₁-adrenoceptors, G-proteins, and other proteins involved in the cAMP-dependent signal transduction are altered on the protein or mRNA level.^{1 2 3} Because plasma noradrenaline levels are elevated in patients with end-stage heart failure,⁴ prolonged ß-adrenergic stimulation was hypothesized to play a role in these alterations. Similar findings obtained in a model of rats treated with isoproterenol for 4 days supported this concept.^{5 6} In many cell types, cAMP-dependent transcriptional regulation is mediated by the cAMP response element binding protein (CREB) and related transcription factors. CREB binds to distinct consensus sequences in the promoter regions of regulated genes, activating their transcription after phosphorylation of CREB by the cAMP-dependent protein kinase A.⁷ CREB recently was shown to be expressed and phosphorylated in chick neonatal cardiomyocytes.⁸ To elucidate a possible role of CREB in the human heart, the DNA binding activity of human ventricular nuclear protein to CRE-containing DNA-oligonucleotides was investigated in gel shift assays. For the first time, we demonstrate cAMP response element (CRE) specific DNA binding activity in the failing and nonfailing human heart that was immunologically identified as CREB and its phosphorylated form (pCREB). Moreover, we found two isoforms, CREB327 and CREB341, to be expressed on the mRNA level.

	Methods

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Left ventricular tissue from six patients with dilated cardiomyopathy and from two nonfailing transplant donors not transplanted because of technical reasons was frozen in liquid N₂ directly after explantation and stored at -80°C. The study is in accordance with guidelines from the local ethics committee, and patients gave written informed consent. Unless otherwise noted, all further steps were performed at 4°C to inhibit protease or ribonuclease activities. Ventricular nuclei were prepared as described previously,⁶ and nuclear proteins were extracted according to a method described previously⁹ with modifications. Briefly, isolated nuclei from 12 g tissue were extracted in 600 µL extraction buffer containing (in mmol/L) HEPES 30 (pH 8.5), NaCl 450, MgCl₂ 12, EDTA 0.3, PMSF 1, and DTT 6; 25% glycerol; and 1 µg/µL each leupeptin and aprotinin for 45 minutes. After centrifugation at 14 000g, the supernatant was stored at -80°C. Double-stranded DNA oligonucleotides containing the following transcription factor consensus sequences (in italics) were used in gel shift assays: RSS-CRE, rat somatostatin gene promoter: 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3'; HG-CRE, human chorionic gonadotropin gene promoter: 5'-ATGGTAAAAATTGACGTCATGGTAATTACA-3'; MUTHG-CRE, sequence identical to HG-CRE except four bases are mutated in the CRE: 5'-ATGGTAAAAATTTAAACCATGGTAATTACA-3'; Hß₂AR-CRE, human ß₂-adrenoceptor gene promoter: 5'-CGAAAGTTCCCGTACGTCACGGCGAGGGCA-3'; AP-1, collagenase gene promoter: 5'-CGCTTGATGAGTCAGCCGGAA-3'; and OCT-1, immunoglobulin light-chain enhancer: 5'-TTCTAGTGATTTGCATTCGACA-3'. The oligonucleotides were labeled by T₄-polynucleotide kinase (Promega,) and [-³²P]-ATP (NEN Du Pont). Binding reactions were performed for 10 minutes with 10 µg nuclear protein in 19 µL solution containing (in mmol/L) HEPES 20 (pH 7.9), MgCl₂ 5, EDTA 1, KCl 70, and DTT 5; 10% glycerol; and 1 µg/µL poly[dIdC]poly[dIdC] and nonlabeled competitor DNA as indicated (150-fold excess). After addition of 1 µL labeled DNA (25 000 disintegrations per minute), reactions were incubated for 15 minutes and electrophoresed on native 5% polyacrylamide gels (20:1) in 0.5x TBE containing (in mmol/L) Tris-HCl 44.5 (pH 8.0), boric acid 44.5, and EDTA 1. For supershifts, anti-CREB antiserum or anti-pCREB IgG (UBI)¹⁰ was added after the labeled DNA with a 1-hour incubation before electrophoresis. Anti-jun antibody (Oncogene Science) was used as control. Gels were dried and exposed to Phosphor-Imager (Molecular Dynamics).

Total RNA was isolated as described,¹¹ and 400 ng was reverse-transcribed with Tth-DNA polymerase (Boehringer Mannheim) and 750 nmol/L CREB reverse primer at 70°C according to the manufacturer's specifications. Immediately afterward, the cDNA was amplified in 100 µL with 1.5 mmol/L MgCl₂ by 35 rounds of temperature cycling (denaturation at 95°C, annealing at 60°C, and synthesis at 72°C) with CREB specific primers¹² : forward, 5'-CAGCCAC GATTGCCACATTAGCC-3', starting at base 213; reverse, 5'-GGGAATCAGTTACACTATCC-3', ending at base 447; expected length of amplificates, 235 and 277 bp. Southern-blotted polymerase chain reaction (PCR) products were identified by high-stringent hybridization with a CREB327 cDNA, which was a kind gift from Dr T.E. Meyer.¹²

	Results

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Gel mobility shifts were found with nuclear extract of human failing ventricle and the CRE-containing ³²P-labeled oligonucleotide HG-CRE (Fig 1A). These were suppressed by different nonlabeled CRE-containing oligonucleotides but not by the non-CRE oligonucleotide OCT-1 or MUTHG-CRE (identical to HG-CRE but with a mutated CRE). An incomplete inhibition was found by an AP-1 competitor DNA. One shift was supershifted by anti-CREB and anti-pCREB but not by anti-jun antibodies (Fig 1B) with nuclear protein from failing or nonfailing hearts. Identical results were found in six other competition assays with labeled RSS-CRE and ß₂AR-CRE (data not shown) and in eight other supershift experiments with nuclear proteins of three additional hearts (two failing, one nonfailing).

View larger version (56K): [in this window] [in a new window]   Figure 1. Blots showing gel shift assays with human ventricular nuclear protein from failing (dilated cardiomyopathy [DCM]) and nonfailing (NF) hearts and labeled cAMP response elements (CREs) containing DNA fragment HG-CRE (derived from the human chorionic gonadotropin gene promoter). A, The CRE-containing competitor oligonucleotides RSS-CRE (from the rat somatostatin gene promoter), HG-CRE, and Hß2AR-CRE (human ß2-adrenoceptor gene promoter) inhibited the shifts, but they were not or were incompletely affected by noncompetitors (with AP-1 or OCT-1 element) or MUTHG-CRE (identical to HG-CRE but with a mutated CRE). There were additional nonspecific bands. B, Supershifts (arrow) were formed by anti-CREB and anti-pCREB but not anti-jun antibodies through the use of nuclear proteins from failing and nonfailing hearts.

Two fragments of the expected length (235 and 277 bp) were amplified from total RNA of failing and nonfailing left ventricles with CREB-specific primers. Amplification was found to be linear between 200 and 600 ng RNA and exponential between 25 and 31 cycles through the use of total RNA of six different hearts (failing). Both PCR products hybridized with CREB327 cDNA under high-stringent conditions after Southern blotting (Fig 2).

View larger version (43K): [in this window] [in a new window]   Figure 2. Autoradiography of a Southern blot of cAMP response element binding protein (CREB) specific polymerase chain reaction products amplified from failing (DCM) and nonfailing (NF) ventricular total RNA hybridized with labeled CREB327 cDNA.

	Discussion

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Human ventricular nuclear protein showed a CRE specific DNA binding activity. Some inhibition found with an AP-1 oligonucleotide can be explained by the similarity of the AP-1 element with the CRE. CREB can bind to AP-1 sites but with lower affinity than to CREs.¹³ Shifts were detected by anti-CREB and anti-pCREB antibodies, demonstrating the expression and phosphorylation of CREB protein in failing and nonfailing hearts. The PCR products amplified with CREB primers from human ventricular total RNA were identified as CREB341 and CREB327 by length and hybridization with CREB327 cDNA. Amplification of nonprocessed transcripts can be excluded by the location of the primers in different exons. CREB341 and CREB327 are the most abundant isoforms of CREB arising from alternative splicing of a 42-bp exon coding for a part of the transactivational domain present in CREB341 but not in CREB327.¹² The function of both isoforms is a controversial subject. Yamamoto et al¹⁴ reported CREB327 to be only one-tenth as active as CREB341 in transfection assays, whereas other groups found a similar activity of CREB327 and CREB341.^{15 16} Although we did not precisely quantify both isoforms, CREB327 mRNA appeared to be predominant in nonfailing and failing hearts. This is in accord with data from other cell types.^{14 15 16} Further isoforms of CREB were found to be expressed exclusively in testicular tissue.^{16 17} Accordingly, we found no additional isoforms. We have found CREB mRNA in isolated neonatal rat cardiomyocytes (unpublished observation), giving evidence that CREB is expressed in myocytes. It was shown previously that nuclei isolated by the method used here are primarily from myocytes.⁶ We conclude from our data that CREB might play a role in the transcriptional regulation in the human heart, which might be of considerable clinical interest. Thus, it was suggested recently that an unnatural growth response mediated by CREB and related transcription factors could contribute to the poor prognosis of patients with heart failure and that beneficial effects of ß-blockers in these patients could be explained by blunting this component of unnatural growth response.¹⁸ Although our experiments were not designed to investigate differences between failing and nonfailing hearts and accurate quantification with gel shift experiments is a general unresolved problem, no major differences between failing and nonfailing hearts are apparent. The transplant donors whose heart tissues were used for the gel shifts received ß-adrenergic agents (dopamine or dobutamine), whereas the patients with failing hearts did not. Thus, major effects of the pathological state of the heart or of catecholamines on CREB expression are not likely. However, the present data do not allow conclusions about differences in the expression of CREB in nonfailing versus failing hearts. Small differences between groups or counteracting ß-adrenergic effects in nonfailing hearts cannot be excluded. Even small changes in CREB expression could lead to altered transactivational patterns by affecting the balance of heterodimerization of related transcription factors. Therefore, further studies for precise quantification of CREB are necessary to characterize the role of CREB in the pathophysiology of heart failure.

	Acknowledgments

 
We thank Andrea Walter for her excellent technical assistance.

Received April 18, 1995; revision received July 31, 1995; accepted August 18, 1995.

	References

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