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Published as doi: 10.1096/fj.06-7915com.
 (The FASEB Journal. 2007;21:1884-1892.)
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Cardiomyocyte-specific inactivation of transcription factor CREB in mice

Marek Matus*, Geertje Lewin*, Frank Stümpel*, Igor B. Buchwalow{dagger}, Michael D. Schneider{ddagger}, Günther Schütz§, Wilhelm Schmitz* and Frank U. Müller*,1

* Institute of Pharmacology and Toxicology and

{dagger} Institute of Pathology, University of Münster, Münster, Germany;

{ddagger} Department of Medicine, Center for Cardiovascular Development, Baylor College of Medicine, Houston, Texas, USA;

§ Division of Molecular Biology of the Cell I, German Cancer Research Center, Heidelberg, Germany

1Correspondence: Institute of Pharmacology and Toxicology, University of Münster, Domagkstrasse 12, 48129 Münster, Germany. E-mail: mullerf{at}uni-muenster.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The transcription factor cAMP response element (CRE)-binding protein (CREB, Creb1) plays a critical role in regulating gene expression in response to activation of the cAMP-dependent signaling pathway, which is implicated in the pathophysiology of heart failure. Using the Cre-loxP system, we generated mice with a cardiomyocyte-specific inactivation of CREB and studied in this model whether CREB is critical for cardiac function. CREB-deficient mice were viable and displayed neither changes in cardiac morphology nor alterations of basal or isoproterenol-stimulated left ventricular function in vivo or of important cardiac regulatory proteins. Since CREB was proposed as a negative regulator of cardiomyocyte apoptosis by enhancing the expression of the antiapoptotic protein Bcl-2, we analyzed the fragmentation of DNA, the activity of caspases 3/7 and the expression of Bcl-2 and did not observe any differences between CREB-deficient and CREB-normal hearts. Our results suggest that the presence of CREB is not critical for normal cardiac function in mice.—Matus M., Lewin G., Stümpel F., Buchwalow I. B., Schneider M. D., Schütz G., Schmitz W., and Müller F. U. Cardiomyocyte-specific inactivation of transcription factor CREB in mice.


Key Words: Cre-loxP system • knockout mice • cAMP responsive element binding protein • heart


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE CAMP-RESPONSE ELEMENT BINDING PROTEIN (CREB) is a 43 kDa transcription factor that belongs to the CREB-family of basic leucine zipper (bZip) transcription factors, together with the related proteins CRE modulator (CREM) and the activating transcription factor 1 (ATF-1) (1 2 3 4 5) . CREB binds to the CRE (CRE; TGACGTCA and variants thereof) as a homodimer or as a heterodimer in association with other CREM or ATF-1 proteins (1 , 3) . CREB is phosphorylated at a critical serine residue (Ser-133) by various protein kinases, including protein kinase A in response to elevations in intracellular cAMP (2 , 6) or calmodulin (CaM) kinases in response to elevations in intracellular calcium (7) . This phosphorylation facilitates CREB’s interaction with p300, a histone acetyltransferase (HAT), and its closely related coactivator, the 265 kDa CREB-binding protein (CBP) (8 9 10) . The HAT activity of p300 then modifies relaxing of the chromatin structure, thereby promoting gene activation (11) . In its unphosphorylated state, CREB can bind to DNA but is not able to activate transcription (1 , 2) . A mutant CREB protein, CREBA133, contains a Ser-133 to Ala substitution and acts as a nonphosphorylatable dominant-negative repressor of CREB-dependent transcriptional activity both in vitro and in vivo (12 13 14) . This supported the idea that phosphorylation at Ser-133 is required for CREB’s function as a transcriptional activator.

Human heart failure is the common end-stage of various cardiac diseases, including dilated or ischemic cardiomyopathy, and is among the most frequent causes of mortality and morbidity in Western countries (15) . Heart failure is defined as the heart’s inability to adequately cover the body’s blood demand, and the failing heart displays characteristic morphological and functional features including hypertrophy, dilatation, impaired contraction and relaxation, and a diminished response to ß-adrenoceptor agonists (16 , 17) . It is well documented by clinical and experimental studies that chronic stimulation of the ß1-adrenoceptor (ß1AR) by elevated plasma catecholamines plays a crucial role in both initiation and progression of human heart failure (18 , 19) ; accordingly, ß1AR blockade was identified as a therapeutic option to reduce the mortality of heart failure (19 20 21) . Up to now, little is known about mechanisms underlying expressional changes and successive cardiac dysfunction in response to chronic ß1AR stimulation and subsequent activation of the cAMP-dependent signaling pathway in the failing heart. Different mouse models with gain or loss of CREM function implied CREM as an important regulator of cardiac myocyte function (22 23 24) , whereas the cardiac role of CREB is less clear. Transgenic mice with cardiomyocyte-directed expression of CREBA133 developed a phenotype of dilated cardiomyopathy (25) , including progressive dilatation of cardiac chambers and left ventricular dysfunction. Results from this model led to the hypothesis that CREB represents a central regulator of cardiac function. However, overexpression of the dominant-negative CREBA133 mutant is a rather non-specific approach to inhibit the function of CREB, and it cannot be excluded that the suppression of non-CREB CRE-binding transcription factors contributes to the phenotype of CREBA133-transgenic mice. Therefore, we generated transgenic mice with a cardiomyocyte-directed inactivation of CREB, in order to study the cardiac role of CREB in a more specific genetic mouse model.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental animals
Cardiomyocyte-specific CREB-mutant mice (Creb1{alpha}MHCCre) were obtained by cross-breeding Creb1loxP mice (26) that contain loxP sites around Creb1 exon 10 with mice carrying a transgene for the Cre-recombinase under the control of the cardiomyocyte-specific {alpha}-myosin heavy chain promoter ({alpha}MHCCre+) (27) . Cre-mediated recombination of the Creb1loxP allele leads to a Creb1 null allele encoding a truncated CREB protein. This protein lacks the DNA-binding and dimerization domains, which are encoded in exon 10 of the Creb1 gene. Since this truncated protein is unstable the successful recombination results in a loss of CREB (26) . Mice were bred in a genetic background comprising a mix of 129Sv, C57/BL6, and FVB/N and were genotyped by polymerase chain reaction (PCR) on tail DNA samples using primers specific for the Creb1loxP allele (forward, 5'-TATGTAAAGCAAGGGAAGATAATG-3'; reverse, 5'-TAGACATACTTGACCCATAGCATT-3') and for the {alpha}MHCCre transgene (forward, 5'-GCTGCCACGACCAAGTGACAGCAATG-3'; reverse, 5'-GTAGTTATTCGGATCATCAGCTACAC-3') as described (27 ,28) . The analysis of the Creb1 wild-type, loxP and null alleles in DNA samples from different tissues was performed by PCR using a different reverse primer annealing down-stream of the second loxP site located down-stream of exon 10 (5'-GCCCAAGGTTGTGATTCCAGCACT-3'). Experiments were performed with littermates of Creb1{alpha}MHCCre ({alpha}MHCCre+:Creb1loxP/loxP) mice and control mice (Ctr; {alpha}MHCCre–::Creb1loxP/loxP, lacking the {alpha}MHCCre transgene) at an age of 16–24 wk, in accordance with local animal welfare authorities.

Histological analysis
Tissue samples were fixed in buffered 4% formaldehyde and routinely embedded in paraffin. Paraffin sections (4 µm) of ventricular tissue were dewaxed in xylene, rehydrated in graded alcohols and routinely stained with hematoxylin-eosin. Diameters of at least 100 transversely cut cells were determined per individual heart and mean values of the diameters from 3–4 animals per group were compared.

Immunohistochemistry
Paraffin sections (4 µm) of the blocks were dewaxed in xylene, rehydrated in graded alcohols, and transferred into PBS, which was used for all washings and dilutions. Antigen retrieval (Reveal, Biocarta, Hamburg, Germany) for 5 min in a domestic pressure cooker and blocking of non-specific binding sites with BSA-c basic blocking solution (1:10 in PBS, Aurion, Wageningen, The Netherlands) were performed as described (29) . After immunoreacting with a rabbit polyclonal CREB-specific primary antibody (26) (1:1000) overnight at 4°C and subsequent washing in PBS, the sections were treated for 10 min with methanol containing 0.6% H2O2 to quench endogenous peroxidase. For bright-field microscopy, bound primary antibodies were detected using DAKO EnVision-HRP and 3,3'-diaminobenzidine (DAB) substrate kit (Vector Laboratories, Burlingame, CA, USA), counterstained with Ehrlich hematoxylin for 30 s, and mounted with an aqueous mounting medium GelTol (Immunotech, Marseille, France). For fluorescent visualization of bound primary antibodies, sections were immunoreacted for 1 h at room temperature with a goat-anti-rabbit biotinylated antibody (1:300, Vector Laboratories). The biotin label was visualized with Streptavidin-Alexa-488 (1:200, Molecular Probes, Leiden, The Netherlands) and the sections were mounted with Vectashield (Vector Laboratories). The exclusion of either the primary or the secondary antibody from the immunohistochemical reaction or substitution of primary antibodies with the corresponding IgG at the same final concentration resulted in lack of immunostaining. Immunostained sections were examined on a Zeiss Axiophot2 microscope equipped with appropriate filters. Bright-field microscopy images were captured using an AxioCam 12-bit camera and AxioVision single-channel image processing (Carl Zeiss Vision GmbH, Germany). For fluorescent visualization separate images for fluorophore immunolabeling and for autofluorescence of cardiomyocytes and erythrocytes were captured digitally into color-separated components using the AxioCam digital microscope camera and AxioVision multichannel image processing. Separate color images (red for autofluorescence and green for alexa-488) were merged and imported into PhotoImpact 3.0 (Ulead Systems, Inc. Torrance, CA, USA) for further analysis. Images shown are representative of at least 5 independent experiments which gave similar results.

SDS-PAGE and quantitative immunoblotting
Preparation of protein homogenates from mouse cardiac ventricles, electrophoresis on polyacrylamide gels, transfer onto nitrocellulose membranes, as well as the immunological detection using the ECL detection system (Amersham Biosciences, GE Healthcare, Piscataway, NJ, USA) followed published protocols (22 , 24) . We thank Dr. L.R. Jones, Indianapolis, for providing calsequestrin, SERCA2a and junctin antibodies.

Left ventricular catheterization
Mice were anesthetized and left ventricular (LV) function was assessed as described previously (22 , 24) . The nonselective ß-adrenoceptor agonist isoproterenol was administered via the cannulated left jugular vein.

DNA fragmentation
Genomic DNA was purified from freshly isolated ventricles, precipitated overnight in 70% ethanol at –20°C and small DNA fragments were recovered by centrifugation at 20,000 g for 15 min following standard protocols. To remove traces of RNA the sample was treated with DNase-free RNase for 30 min at 37°C and subsequently fractionated by electrophoresis on ethidium bromide-stained 1.5% agarose gels as described (30) . As a positive control, we used the DNA isolated from a Langendorff-perfused mouse heart which was subjected to 60 min no-flow-ischemia followed by 240 min reperfusion.

Luminometric assay of caspase activity
The proteolytic activity of caspases 3 and 7 was determined using a DEVD-sequence containing substrate (Caspase GloTM 3/7 assay; Promega, Madison, USA) following the manufacturer’s specifications as described (30) . The obtained values were indexed to protein concentration which was determined according to the method of Bradford.

Statistics
Data are presented as mean ± SEM or as mean with 95% confidence interval (survival times). Statistically significant differences were determined using the unpaired Student’s t-test or the log rank test (survival) with a P < 0.05 considered as significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of Creb1{alpha}MHCCre mice
The obtained Creb1{alpha}MHCCre ({alpha}MHCCre+:Creb1loxP/loxP) mice were viable and fertile. The genotypic analysis of offsprings was performed at an age of four weeks and revealed the expected Mendelian ratio of genotypes. Thus, no significant differences were found in the viabilities between young Creb1{alpha}MHCCre and control mice. PCR amplification of Creb1 exon 10 was conducted to verify the presence or absence of the Creb1 wild-type allele, the loxP-flanked Creb1loxP allele and the Creb1 null allele, the latter as a result of a Cre-mediated excision of the loxP-flanked region. The loxP allele was amplified from tail-, heart-, and liver-derived genomic DNA of {alpha}MHCCre-negative Creb1loxP/+ or Creb1loxP/loxP mice, regardless of the presence or absence of the {alpha}MHCCre transgene. The null-allele was only detected in hearts from Creb1loxP/+ or Creb1loxP/loxP mice carrying the {alpha}MHCCre-transgene (Fig. 1 ), in line with a cardiomyocyte-specific Cre-recombinase-driven deletion of Creb1 exon 10. To verify the cardiomyocyte-specific inactivation of CREB on the protein level, immunohistological analyses were performed in cardiac ventricular sections from Creb1{alpha}MHCCre and control mice using a CREB-specific 3',3'-diaminobenzidine-horseradish peroxidase (DAB-HRP) immunostaining (Fig. 2 ). In control hearts, CREB was localized in the nuclei of myocytes and nonmyocytes (brown staining) as obvious from counterstaining with hematoxylin (blue). Here, more than 90% of nuclei were CREB-positive. In contrast, more than 90% of myocyte nuclei in Creb1{alpha}MHCCre hearts were CREB-negative while CREB was detectable in most nuclei from nonmyocytes which were located between the myocytes. Immunofluorescent detection of CREB in histological sections under higher magnification confirmed specific deletion of this protein in Creb1{alpha}MHCCre myocytes (Fig. 3 ). Cardiomyocyte nuclei of Creb1{alpha}MHCCre hearts were CREB negative (lack of green fluorescence), whereas fibroblasts and smooth muscle cells of capillaries showed CREB positive nuclei. The combination of CREB-negative myocytes and CREB-positive nonmyocytes in Creb1{alpha}MHCCre hearts demonstrated the successful cardiomyocyte-specific inactivation of CREB.


Figure 1
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  		Figure 1. Cardiomyocyte-specific deletion of Creb1 exon 10. The sequence around Creb1 exon 10 was amplified by PCR from genomic DNA of different tissues of {alpha}MHCCre-negative or -positive Creb1loxP/+ or Creb1loxP/loxP mice. The 1200 bp wild-type allele was only detected in Creb1loxP/+ animals, together with the 1260 bp loxP-flanked allele. Homozygous Creb1loxP/loxP mice demonstrated only the loxP-flanked allele. The deletion of the loxP-flanked sequence, as shown by the presence of the 700 bp null allele, was dependent on the cardiomyocyte-specific expression of Cre recombinase in ventricles (V), while exon 10 was preserved in tail (T) or liver (L) DNA of {alpha}MHCCre+:Creb1loxP/+ and {alpha}MHCCre+:Creb1loxP/loxP mice. On the right side, a 100bp DNA size marker was loaded (M).


Figure 2
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  		Figure 2. DAB-horseradish peroxidase (DAB-HRP) immunostaining of CREB. The majority of cardiomyocyte nuclei in control hearts was CREB-positive (A, brown staining) while the majority of cardiomyocyte nuclei was CREB-negative in Creb1{alpha}MHCCre hearts as visible by the blue counterstaining with hematoxylin (B). Most noncardiomyocytes displayed CREB-positive nuclei in both groups.


Figure 3
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  		Figure 3. Immunofluorescent detection of CREB. A) Cardiomyocyte nuclei of Ctr animals displayed a CREB-specific staining (green fluorescence). B) In sections from Creb1{alpha}MHCCre hearts, the majority of cardiomyocyte-derived nuclei was CREB-negative, whereas most nuclei in noncardiomyocytes were CREB-positive. Yellow and red-brown staining accounted for autofluorescence of erythrocytes and cardiomyocytes, respectively. Nuclei are marked with arrows.

Morphology
There were no striking alterations in cardiac and noncardiac morphology between both groups. Moreover, no differences were found in mean body wt (data not shown) or in heart wt indexed to body wt between groups (in mg per g body wt. Ctr, 5.1±0.1; Creb1{alpha}MHCCre, 5.2±0.1; n=14–16). Microscopic histopathological analysis of hematoxilin/eosin-stained sections did not reveal myocyte hypertrophy or any changes in cardiac morphology in Creb1{alpha}MHCCre hearts (data not shown).

Survival
We studied the survival starting with 93 Creb1{alpha}MHCCre mice and 95 control mice over a period of up to 1 yr (Fig. 4 ). The mean survival times of Creb1{alpha}MHCCre and Ctr mice were 373 d (95% confidence interval 322–423 d) and 398 d (95% confidence interval 345–452 d), respectively, indicating that the mortality was not affected by cardiomyocyte-specific CREB inactivation.


Figure 4
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  		Figure 4. Kaplan-Meier survival analysis. Kaplan-Meier estimates of survivor functions are shown for Ctr (full line) in comparison with Creb1{alpha}MHCCre mice (dashed line). Groups started with 95 (Ctr) and 93 (Creb1{alpha}MHCCre) mice, of which 5 (Ctr) and 6 (Creb1{alpha}MHCCre) animals died during 1 yr. Survival rates between Ctr and Creb1{alpha}MHCCre lines were not significantly changed.

Invasive hemodynamic assessment of cardiac left ventricular function
Assessment of cardiac function was performed in vivo both under basal conditions and after stimulation with increasing doses of the ß-adrenoceptor agonist isoproterenol. No differences were found in hemodynamic parameters of basal heart function between experimental groups (Table 1 ). Detailed analysis of cardiac function after stimulation with isoproterenol did not show any differences between groups; both the efficacy and potency of isoproterenol in respect to its chronotropic, inotropic, and lusitropic effects were unchanged (Fig. 5 ).


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  		Table 1. In vivo assessment of basal hemodynamic parameters


Figure 5
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  		Figure 5. In vivo assessment of cardiac function. Effects of isoproterenol infusion on hemodynamic parameters. Systolic left ventricular pressure (A, LVP), heart rate (B, HR), maximal rate of contraction (C, dP/dtmax) and maximal rate of relaxation (D, dP/dtmin) were obtained from continuous recordings of left ventricular pressure at basal state (B) and during infusion of increasing doses of isoproterenol. The positive chronotropic, inotropic and lusitropic effects of isoproterenol were not different between Ctr ({circ}, n=5) and Creb1{alpha}MHCCre (•, n=5) mice.

Cardiac ventricular gene expression
Since CREB has been suggested as an important regulator of cardiac gene expression, we tested whether its cardiomyocyte-specific inactivation results in expressional alterations of important cardiac regulatory proteins. The protein levels of the protein kinase A (PKA), the Ca2+/CaM-dependent kinase II (CAMK II), the catalytic subunits of protein phosphatase 1 (PP1{alpha}) and 2A (PP2A) and calcineurin (PP2B) did not differ in both groups (Fig. 6 ). Furthermore, the protein levels of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) and its regulator protein phospholamban (PLB) as well as of the SR proteins calsequestrin (CSQ), junctin (JCN) and triadin (TRD) were not changed in Creb1{alpha}MHCCre hearts as compared to controls.


Figure 6
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  		Figure 6. Expression of cardiac regulatory proteins in ventricular homogenates. The expression of protein kinase A (PKA), the Ca2+/CaM-dependent kinase II (CAMK II), the catalytic subunits of protein phosphatase 1 (PP1{alpha}) and 2A (PP2A) and of calcineurin (PP2B), as well as the expression of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) and its regulator protein phospholamban (PLB), the SR proteins calsequestrin (CSQ), triadin (TRD), and junctin (JCN) as well as of the antiapoptotic protein Bcl-2 did not differ between Ctr ({square}, n=7) and Creb1{alpha}MHCCre ({blacksquare}, n=7) mice.

Apoptosis
Since CREB has been shown to prevent apoptosis by increasing the expression of the antiapoptotic protein Bcl-2 (31 32 33) , we tested the hypothesis that CREB inactivation evokes increased cardiac apoptosis. Creb1{alpha}MHCCre hearts did neither show enhanced fragmentation of genomic DNA nor increased activity of caspases 3/7 (Figs. 7 , 8 ) and expression of Bcl-2 was not different between groups (Fig. 6) . This suggests that CREB inactivation does not lead to increased apoptosis under basal conditions in mouse heart.


Figure 7
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  		Figure 7. DNA fragmentation assay. DNA extracted from control and Creb1{alpha}MHCCre mice was fractionated on 1.5% agarose gels and stained by ethidium bromide. As a positive control, a mouse heart was subjected to 60 min of ischemia followed by 240 min of reperfusion (I/R). Note the lack of low MW DNA laddering in both experimental groups compared to the heart subjected to ischemia and reperfusion. On the left side, a 100bp DNA size marker was loaded for comparison (M).


Figure 8
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  		Figure 8. Activity of caspases 3 and 7. Note the comparable caspase activities in ventricular homogenates from control (Ctr) and Creb1{alpha}MHCCre mice (n=5 per group).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Creb1{alpha}MHCCre mice represent a novel genetic mouse model to study the specific role of CREB in cardiac myocytes. The successful Cre-mediated cardiomyocyte-specific inactivation of CREB was confirmed by the exclusive detection of the null allele in the myocardium of {alpha}MHCCre-transgenic Creb1-floxed mice and by the combination of CREB-negative myocytes and CREB-positive nonmyocytes in Creb1{alpha}MHCCre hearts. In Creb1{alpha}MHCCre hearts, more than 90% of cardiomyocyte nuclei were CREB-negative, in line with similar results from the same {alpha}MHCCre transgenic mouse line in another knockout mouse model (27) . Moreover, this result confirms previous data that the truncated CREB protein—devoid of the domains encoded in exon 10—is unstable and prone to accelerated degradation (26) . Immunohistochemistry in hearts from control mice clearly indicates that CREB is expressed in adult mouse cardiac myocytes using a CREB-specific antibody, which was validated previously (26) and whose specificity was further confirmed by the lack of staining in cardiomyocytes from Creb1{alpha}MHCCre mice. Recently, Husse and Isenberg reported that the expression of CREB is restricted to fibroblasts whereas CREM is predominantly expressed in cardiomyocytes in adult rat hearts (34) . The reason for the discrepancy to our results is not clear, however, it may be due to the different methods of CREB detection (immunohistochemistry on cardiac tissue sections vs. Western blot analysis on primarily isolated cardiomyocytes and fibroblasts), due to the different antibodies used, or due to species differences.

The lacking cardiac phenotype of Creb1{alpha}MHCCre mice indicates that surprisingly the presence of CREB is not required to maintain cardiac function in mice. This stands in sharp contrast to results from transgenic mice with heart-directed expression of the dominant-negative CREB-mutant CREBA133 (25) . CREBA133-transgenic mice developed signs of heart failure, including depressed LV function, hypertrophy of cardiomyocytes, dilatation of cardiac chambers, edema, and premature death, and these data led to the hypothesis that CREB is critical for cardiomyocyte function. The discrepancy to our findings in Creb1{alpha}MHCCre mice can be explained by the different approaches to inhibit CREB function in mouse cardiomyocytes. The CREBA133-mutant binds to the CRE but cannot confer transcriptional activation on phosphorylation of Ser-133 as a consequence of the Ser-133 to Ala substitution (12 13 14) . Hence, CREBA133 competes with CREB as well as with other members of the CREB/CREM/ATF-1 family for available CRE binding sites. In consequence, an excess of CREBA133 results in a non-specific, global suppression of CRE-mediated transcriptional activation rather than in a selective inhibition of CREB which was achieved in Creb1{alpha}MHCCre mice. Therefore, in the light of the results presented here, a suppression of non-CREB transcription factors by CREBA133 likely contributes to the phenotype of CREBA133-transgenic mice, albeit this phenotype underscores the relevance of the CRE-mediated transcriptional activation in the heart. Results from other mouse models support this idea and suggest that—in contrast to other tissues—CREM is more important than CREB in cardiomyocytes. The inactivation of CREM led to left ventricular cardiac dysfunction in two independent mouse lines (22 , 23) indicating that, different to CREB, the presence of CREM is a prerequisite for a regular cardiac function. A down-regulation of SERCA2a possibly contributes to the reduced left ventricular performance in CREM-deficient mice; however, this observation was only made in one of the two mouse models (22) . Previously, we have identified a CREM mRNA variant, CREM-Ib{Delta}C-X, in the human heart as well as small CREM repressor proteins translated from this isoform (35) . These repressor proteins have functional properties akin to CREBA133, in particular they bind to the CRE as homodimers or as heterodimers with CREB, displace CREB homodimers from the CRE and inhibit the CRE-mediated transcriptional activation (35) . Transgenic mice with cardiomyocyte-directed expression of CREM-Ib{Delta}C-X developed a complex cardiac phenotype of increased left ventricular function associated with a selective up-regulation of SERCA2a, hypertrophy of cardiomyocytes, atrial dilatation and fibrillation, and premature death (24) . Hence, it is well conceivable that an inhibition of CREM transcription factors contributes to the cardiac phenotype of CREBA133-transgenic mice and therefore to the discrepancy to the findings in Creb1{alpha}MHCCre mice.

The lacking cardiac phenotype of Creb1{alpha}MHCCre mice either indicates that the function of CREB is compensated within the CREB/CREM/ATF-1 family or that CREB is not active or not relevant under physiological conditions in mouse cardiomyocytes. A functional redundancy within CREB/CREM transcription factors was reported in CREB mutant (CREB{alpha}{Delta}) mice with a deletion of Creb1 exon 2 (36) . CREB{alpha}{Delta} mice exhibited an apparently normal phenotype except abnormalities in learning and memory (37) . While the expression of ATF-1 was not changed, an up-regulation of CREM including the activator CREM-{tau} (36) and of CREBß, an alternatively spliced CREB isoform, was associated with the mild phenotype of CREB{alpha}{Delta} mice (38) . In contrast to CREM, the up-regulation of CREBß was critical for a functional compensation in CREB{alpha}{Delta} mice since CREB null mice with a deletion of Creb1 exon 10—also inactivating CREBß—died perinatally, although CREM was up-regulated in CREB null mice, too (39) . In consequence, a compensation by CREBß can be excluded in Creb1{alpha}MHCCre mice having a cardiomyocyte-specific deletion of Creb1 exon 10, in analogy to CREB null mice. A compensation of CREB by CREM—at least regarding cardiac hypertrophy—can also be excluded since heart-to-body wt ratios did not differ between Crem-normal Creb1{alpha}MHCCre mice and Creb1{alpha}MHCCre mice on a Crem–/– background (22) (Crem–/–:Creb1{alpha}MHCCre; n=3–4; data not shown). The possibility that CREB is not phosphorylated at Ser-133 and therefore not active under physiological conditions is also not a likely explanation for the missing phenotype of Creb1{alpha}MHCCre mice, since the phosphorylated form of CREB was immunologically identified in nuclear extracts from human heart (40) as well as in cardiac homogenates from adult wild-type mice (24) or in neonatal chick cardiomyocytes (41 , 42) . One possible explanation for the missing phenotype of Creb1{alpha}MHCCre mice may be that the transcriptional activation via CREB is tightly controlled, e.g., by CREM repressors, under physiological conditions, and that the stoichiometry of activator and repressor isoforms of CRE-binding factors is critical for a regular function of the cardiomyocyte. Then, the inactivation of CREB in Creb1{alpha}MHCCre mice would be without effect since the function of CREB is predominantly inhibited by CREM repressors, and the inactivation of CREM in CREM-deficient mice would release the negative control of CREB and other activators at the CRE finally resulting in decreased cardiac performance. The phosphorylation of CREB finally leads to a transcriptional activation through an interaction with the transcriptional integrator proteins p300 and CBP, which mediate transcriptional activation via their HAT activity (8 9 10 11) . A cardiac role of p300 was highlighted by results from mice with a global inactivation of p300 (43) . These mice die between days 9 and 11.5 of gestation and exhibit defects in heart development. Most reports on CBP-knockout mouse models did not reveal a function of CBP in cardiac development (43 44 45) with one exception (46) . More important in the context of this study, the overexpression of p300 or CBP can induce hypertrophy and overexpression of dominant-negative mutants of both can inhibit phenylephrine-induced hypertrophy, in isolated cardiomyocytes (47) . Furthermore, transgenic mice with heart-directed expression of p300 develop hypertrophy and cardiac dysfunction (48) . However, paradoxically, the application of inhibitors of histone deacetylases (HDACs; counteracting the function of HATs) was associated with a blockade to hypertrophy and fetal gene activation in cardiomyocytes (49) . Therefore, results from genetic mouse models with gain or loss of p300 or CBP implicate both as important regulators of cardiac growth, which also underscores a functional relevance of a CRE-mediated transcriptional regulation in the heart. Further genetic mouse models, e.g., mice with increased function of CREB in the heart, and the combination thereof are required to evaluate the complex role of CREB and CREM and their interaction with p300 and CBP in the pathophysiology of heart failure.


   ACKNOWLEDGMENTS
 
This work was supported by the DFG Mu1376/10–1 and Mu1376/10–2 and DLR/DMBF/IZKF (Interdisziplinäres Zentrum für Klinische Forschung/Interdisciplinary Centre for Clinical Research) Mu01/021/2004. We thank Andrea Walter for excellent technical assistance.

Received for publication December 18, 2006. Accepted for publication January 18, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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G. S. Huggins, J. J. Lepore, S. Greytak, R. Patten, R. McNamee, M. Aronovitz, P. J. Wang, and G. L. Reed
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