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Cardiovascular Research 2003 59(3):563-572; doi:10.1016/S0008-6363(03)00476-0
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Copyright © 2003, European Society of Cardiology

β-Adrenergic stimulation induces cardiac ankyrin repeat protein expression: involvement of protein kinase A and calmodulin-dependent kinase

Oliver Zolka,*,1, Michael Marxa,1, Elmar Jäckelb, Ali El-Armouchec and Thomas Eschenhagenc

aInstitut für Experimentelle und Klinische Pharmakologie und Toxikologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Fahrstr. 17, 91054 Erlangen, Germany
bDana-Farber Cancer Institute, Boston, MA, USA
cInstitut für Experimentelle und Klinische Pharmakologie, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany

zolk{at}pharmakologie.uni-erlangen.de

* Corresponding author. Tel.: +49-9131-852-2783; fax: +49-9131-852-2773.

Received 4 February 2003; revised 4 June 2003; accepted 4 June 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The cardiac ankyrin repeat protein (CARP), a nuclear transcription co-factor that negatively regulates cardiac gene expression, is increased in human heart failure and in animal models of cardiac hypertrophy. The mechanism by which CARP expression is regulated and the consequences of CARP overexpression on cardiac contractility are unknown. Methods and results: Compared to vehicle treated controls, 4-day treatment of male Wistar rats with the β-adrenoceptor agonist isoprenaline (2.4 mg/kg per day) induced hypertrophy and significantly increased CARP mRNA and CARP protein levels in left ventricles. The signalling pathways were investigated in more detail in isolated neonatal rat cardiomyocytes. Treatment of cells with isoprenaline (1 µmol/l) caused a significant increase in CARP mRNA and protein by ~50%. Combined β1- and β2-adrenoceptor blockade, inhibition of protein kinase A (PKA; Rp-cAMPS, 100 µmol/l), and inhibition of calmodulin-dependent protein kinases (CaMK; KN-62, 10 µmol/l) completely reversed the effects of isoprenaline. To examine the consequences of CARP overexpression on contractile function, an adenovirus encoding human CARP as well as a control virus were constructed. Although the basal force of contraction was not different, contractile response to Ca2+ and isoprenaline was significantly diminished in engineered heart tissue infected with the recombinant adenovirus that carries the CARP gene (Ad.CARP). Conclusions: Our study provides the first evidence that overexpression of CARP, which is thought to act as a transcriptional co-repressor, may deteriorate contractile function of the heart tissue. Furthermore, β-adrenoceptor stimulation and activation of PKA and CaMK have been identified as mechanisms that induce expression of CARP in cardiomyocytes.

KEYWORDS Contractile function; Gene expression; Hypertrophy; Signal transduction

This article is referred to in the Editorial by S. Baudet (pages 529–531) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The cardiac ankyrin repeat protein (CARP) is an early marker of cardiac myogenic differentiation, expressed at high levels in the heart during embryonic and fetal development and at progressively lower levels in the neonate and the adult [1,2]. CARP contains four ankyrin repeats thought to play a role in protein/protein interaction [3]. CARP was isolated from the heart as a co-factor of YB-1, a transcription factor that is required for the activation of the myosin light chain 2v gene during cardiac myogenesis [1]. Although it is not clear how CARP regulates YB-1 function during cardiac fetal development, CARP overexpression in postnatal cardiac myocytes generally inhibits the expression of cardiac genes and is thought to act as a cardiac co-repressor [1,2].

By differential gene expression analysis we have identified CARP as a gene that is upregulated in ventricular myocardium in a canine heart failure model [4]. Subsequent human studies confirmed that CARP is markedly increased in failing left ventricles from patients with end-stage heart failure due to dilated or ischemic cardiomyopathy [4]. Activation of the sympathetic nervous system is decisively involved in the pathophysiology of human heart failure. Increased cardiac norepinephrine is known to induce diverse abnormalities in cardiac gene expression and function. In this regard, it is possible that chronic β-adrenoceptor stimulation in heart failure provides a mechanism leading to increased CARP expression. Since CARP has been reported to act as a transcriptional co-repressor, its activation during heart failure might contribute to deregulation of gene expression and progressive functional deterioration.

In the present study we tested the hypothesis that chronic activation of the β-adrenergic system augments CARP expression in the heart. Therefore, an in vivo model of chronic β-adrenoceptor stimulation was investigated. Because CARP expression might be altered by direct or indirect endocrine or paracrine mechanisms, we addressed the question whether β-adrenoceptor stimulation affects CARP expression directly in isolated cardiac myocytes. Moreover, intracellular signaling pathways involved in CARP regulation were studied. Although it is well established that CARP alters expression of genes which are decisively involved in maintenance of contractile function [1,2], it has not been demonstrated whether CARP overexpression in fact changes cardiac contractility. To address this question, the consequences of adenoviral overexpression of CARP on isometric force development were studied in engineered rat heart tissue (EHT).


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Materials
Isoprenaline, ICI 118,551, and CGP 207 12A were purchased from Sigma-Aldrich. Rp-cyclic-3',5'-adenosine-monophosphothioate (Rp-cAMPS) and KN-62 were purchased from Calbiochem-Novabiochem (Schwalbach, Germany).

2.2 Animals
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Male Wistar rats (220–260 g) were treated by 4-day subcutaneous infusions with osmotic minipumps (Alzet ML2) as described previously [5]. Mean rate of infusion was 5 µl/h (varying between 4.4 and 5.6 µl/h). Two groups of animals were treated with NaCl 0.9% (n=10) or (±)-isoprenaline–HCl (dissolved in 0.002 N HCl, 2.4 mg/kg per day; Boehringer Ingelheim, n=6). The dose of isoprenaline was taken from our previous studies [6,7]. The animals had free access to food and tap water. Heart rate was measured daily by recording surface ECG in conscious rats 3 days before and during treatment. Body weight was measured daily. The rats were killed by a blow to the neck and bleeding from the carotid arteries. Hearts were rapidly removed and exsanguinated in ice-cold 0.9% NaCl. Atria and ventricles were dissected and frozen immediately in liquid nitrogen for further analysis.

2.3 Cell culture, inhibitor studies
Primary ventricular myocytes were prepared from 1–4-day-old neonatal Wistar rats as described previously [8]. After enzymatic dissociation of the ventricular tissue, the cells were plated onto uncoated plastic dishes in DMEM/10% FCS for 2 h during which time most of the fibroblasts adhered to the dish. The recovered cells were then plated on fibronectin-coated plastic dishes (60-mm diameter, 2x106 cells) and maintained for ~48 h in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 µmol/l 5-bromo-2'-deoxyuridine. We obtained myocyte cultures in which more than 90% were myocytes, as assessed by immunofluorescence staining with an antibody against sarcomeric {alpha}-actinin (Sigma). The cells were incubated for 24 h in serum-free medium and then exposed to isoprenaline in serum-free medium. For inhibitor studies, the cells were preincubated with the inhibitors of β1- and β2-adrenoceptors, protein kinase A (PKA), calmodulin-dependent protein kinases (CaMK), or with vehicle for 20 min before treatment with isoprenaline. The selective β1-adrenoceptor blocker CGP 207 12A at 300 nmol/l was chosen since the expected KD for this antagonist was 0.3–1 nmol/l at the β1-adrenoceptor but 10,000 times higher at the β2-adrenoceptor [9]. The concentration used would be predicted to shift the response through the β1-adrenoceptor by >3 log units. The selective β2-adrenoceptor antagonist ICI 118,551 (β2/β1-selectivity ratio>100 [10]) was used at 100 nmol/l. Care was taken to exclude light, as the ICI 118,551 compound is light sensitive. Protein kinase A and the calmodulin-dependent protein kinases were selectively blocked by Rp-cAMPS and KN-62, respectively. KN-62 is reported to inhibit CaMKII with an IC50 of 500 nmol/l by interacting with the calmodulin binding site of the enzyme [11]. KN-62 inhibits glycogen synthase kinase 3 and p38 activated/regulated kinase 10-fold less potently and MAPK-activated protein kinase 2 ~30-fold less strongly than CaMKII, and other major protein kinases were inhibited minimally or not at all [11]. Rp-cyclic-3',5'-adenosine-monophosphothioate (Rp-cAMPS) has been used to specifically inhibit PKA. This compound acts through competitive binding to the cAMP-binding sites on the regulatory subunits of the kinase (Ki=11 µmol/l [12]). Rp-cAMPS is cell permeable and resistant to hydrolysis by phosphodiesterases as well as to metabolic degradation by other nucleotide-degrading enzymes which are present in intact cells [13].

2.4 Northern blot analysis
Total cellular RNA was prepared from rat left ventricular myocardium or isolated neonatal cardiac myocytes and separated as described previously [14]. [32P]dCTP random prime-labeled 608-bp fragment of human GAPDH cDNA and the full-length human CARP cDNA, respectively, were used as a specific probe. Where the number of RNA samples to be analysed exceeded the capacity of a single gel, an additional standard RNA preparation was run on each blot that allowed normalisation and pooling of data from different blots.

2.5 Western blot analysis
Proteins from isolated rat cardiac myocytes and from rat myocardium were extracted in SDS homogenization buffer (25 mmol/l Tris–HCl, pH 7.5, 250 mmol/l sucrose, 75 mmol/l urea, 1 mmol/l DTT). Protein concentrations in the supernatants were determined according to the method of Lowry. First 10% SDS–PAGE was carried out and proteins were transferred to a nitrocellulose membrane. Membranes were blocked with 5% (w/v) dried milk in 100 mmol/l Tris, pH 7.5, containing 0.1% (v/v) Tween 20 and 150 mmol/l NaCl (TBST) for 1 h at room temperature prior to incubation with polyclonal antibodies against rat CARP (1:5000), human CARP (1:5000) or calsequestrin (CSQ; Dianova, Germany, 1:2500) overnight at 4°C. The rabbit antibody against the synthetic N-terminal peptide VLRVEELVTGKKC of rat CARP was produced by coupling the peptide to keyhole limpet hemocyanin via a carboxy-terminal Cys [3]. For detection of adenoviral overexpressed human CARP, a polyclonal antiserum was used that did not cross-react with rat CARP. Immunoblots were washed with TBST and incubated with anti-rabbit IgG coupled to horseradish peroxidase in TBST for 1 h at room temperature. Immunoreactive bands were visualised using the ECL detection system (Amersham, Braunschweig, Germany). Calsequestrin was used as a standard protein to normalise for equal loading.

2.6 Recombinant adenoviruses
Recombinant adenovirus that carries the CARP gene (Ad.CARP) was generated by cloning of the full-length human cDNA of CARP into the shuttle vector pAdTrack-CMV and subsequent cotransformation of this plasmid with pAdEasy-1 into Escherichia coli as described by He et al. [15]. Expression of CARP is driven by the constitutive active CMV promoter. The virus also encodes for the wild-type green fluorescent protein (GFP) as a reporter gene expressed under the control of a separate CMV promoter. A GFP-only virus (Ad.GFP) was used as the appropriate control adenovirus. For titration of infectious units cardiomyocytes were infected with different Ad.CARP or Ad.GFP dilutions. Then 48 h later the number of GFP-stained, successfully infected cardiomyocytes was determined by fluorescence microscopy and the ratio of infected to total cells (multiplicity of infection, MOI) was calculated. For localisation studies, human CARP was tagged with GFP at the N-terminus by PCR-cloning of the GFP and the full-length CARP cDNA into the shuttle vector pAdShuttle-CMV before recombinant adenovirus was generated as described.

2.7 Force measurement
Circular engineered heart tissue (EHT) was prepared as described in detail previously [16]. Freshly isolated cardiac cells from neonatal rats were mixed with collagen type I, matrigel (Becton Dickinson) and DMEM culture medium containing horse serum and chick embryo extract. The reconstitution mix was pipetted into circular casting moulds and culture was performed as described earlier [16]. After 12 days in culture, EHTs were treated with Ad.CARP or Ad.GFP control virus (MOI 25) and isometric force of contraction was measured 48 h later in organ baths containing gassed Tyrode’s solution. Electrically stimulated EHTs were stretched to the length at which force of contraction was maximal and inotropic responses to cumulative concentrations of calcium (0.2–2.8 mmol/l) and isoprenaline (0.1–1000 nmol/l) were recorded.

2.8 Statistical analysis
Data are presented as mean±S.E.M. Statistical analysis was performed using Students t-test to compare two groups and one-way ANOVA followed by the Bonferroni procedure for multiple-group comparisons. Two-way ANOVA was used for statistical analysis of time–response and concentration–response curves. A value of P<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Chronic isoprenaline infusion increases CARP expression in rats
CARP protein and mRNA levels were studied in rats treated with the β-adrenoceptor agonist isoprenaline for 4 days and compared with sham treated controls. Consistent with previous studies [5,17], isoprenaline induced cardiac hypertrophy with an 1.4-fold increase in heart weight/body weight ratio (Fig. 1B). As expected, heart rates were significantly increased with a maximum at day 2 of treatment (Fig. 1A). As shown in Fig. 1C and D, CARP mRNA and protein levels were significantly elevated in the hypertrophied myocardium from isoprenaline-treated rats compared to saline-treated controls (CARP mRNA/GAPDH mRNA 3.1±1.3, n=6 vs. 1.2±0.1, n=10; CARP/calsequestrin 2.4±0.3, n=6 vs. 1.6±0.1, n=10).


Figure 1
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  		Fig. 1 CARP expression in rats infused with isoprenaline. (A) Effect of isoprenaline infusion on heart rate (HR). NaCl, saline-treated control rats (n=10); ISO, isoprenaline-treated rats (n=6); P<0.001 (two-way ANOVA). (B) Heart weight to body weight ratio is increased in isoprenaline-treated rats (ISO, n=6) compared to saline-treated control rats (n=10). *P<0.05. (C) Western blot analysis of CARP in left ventricular myocardium. Lysates from neonatal cardiomyocytes which overexpressed human CARP by adenoviral gene transfer (Ad.CARP) served as a positive control. The internal standard protein calsequestrin (CSQ) was used to normalise for equal protein loading. (D) Total RNA (10 µg) from left ventricles was subjected to blot hybridization analysis. Blots were hybridised sequentially with radiolabelled CARP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. ISO, isoprenaline treated rats (n=6); NaCl, vehicle treated controls (n=10). *P<0.01 versus NaCl.

 
3.2 Adenoviral gene transfer: CARP is predominantly localised in the nucleus
A bicistronic adenovirus vector was constructed, which allowed the concomitant expression of human CARP and GFP. The efficiency of gene transfer was evaluated by confocal fluorescence microscopy of GFP expression in cardiac myocytes 48 h after infection. In monolayer cultures, the number of cells infected increased with increasing virus titers (Fig. 2A). In parallel, average CARP protein increased as assessed by Western blot analysis. In an attempt to identify the subcellular localisation of CARP in rat cardiac myocytes, we generated virus encoding for GFP-tagged full-length CARP. We found that CARP–GFP fusion protein was localised at high levels in the nucleus and at much lower levels in the cytosol (Fig. 2B).


Figure 2
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  		Fig. 2 Effect of Ad.CARP on gene expression in rat cardiac myocytes. (A) Isolated rat cardiac myocytes were infected with Ad.CARP at different multiplicity of infection (MOI). Co-expression of the green fluorescent protein (GFP) tracer indicates that the CARP gene is expressed in all myocytes at MOI>1. Right: Western immunoblot analysis shows increasing CARP protein levels in Ad.CARP infected cardiac myocytes with increasing virus concentrations. Protein levels of the internal standard protein calsequestrin (CSQ) are unchanged. Note that the antibody against CARP used in this experiment only recognises the human protein, not the endogenous rat homologue. (B) Localisation of CARP in primary rat cardiomyocytes. Myocytes were infected with Ad.CARP-GFP and overexpressed CARP-GFP fusion protein was detected by confocal microscopy. Right: co-staining of sarcomeric {alpha}-actinin.

 
3.3 CARP depresses contractile function in neonatal rat cardiomyocytes
To estimate the implications of CARP overexpression on myocardial contractile function, experiments in rat engineered heart tissue (EHT) were performed. Unlike papillary muscle, EHTs are easily infected by adenovirus resulting in a high transduction efficacy [18]. Nevertheless, compared to cardiac myocytes grown in a monolayer ~25-fold higher virus titers were necessary to infect all cells in EHTs as verified by GFP staining. Basal force of contraction was not significantly different between EHTs infected with Ad.CARP and Ad.GFP (0.12±0.03 mN, n=10 vs. 0.16±0.03 mN, n=10). Addition of cumulative concentrations of calcium to the organ bath enhanced twitch tension to 0.69±0.09 mN in Ad.CARP versus 1.06±0.10 mN in Ad.GFP control-infected EHTs (P<0.05; Fig. 3). In parallel, a depressed contractile response to cumulative concentrations of the β-adrenoceptor agonist isoprenaline was observed in Ad.CARP treated EHTs compared to Ad.GFP controls (isoprenaline 1 µmol/l: 0.45±0.05 mN, n=10 vs. 0.69±0.08 mN, n=10, P<0.05). The EC50 values for the effect of isoprenaline on force of contraction were unchanged (log EC50: Ad.CARP –9.09±0.08, n=10, Ad.GFP –9.09±0.08, n=10).


Figure 3
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  		Fig. 3 Effects of adenoviral CARP overexpression on contractile function in EHTs. (A) Isometric force of contraction in response to calcium and isoprenaline (ISO) is depressed in EHT infected with Ad.CARP (n=10) compared to EHTs treated with the Ad.GFP control virus (n=10). *P<0.001 (two-way ANOVA). (B) Representative twitch recordings (basal and after stimulation with 1 µmol/l isoprenaline from EHTs infected with Ad.CARP or Ad.GFP control virus.

 
3.4 Increased CARP expression in response to β-adrenergic agonists: involvement of PKA and CaMK
To gain further insights into the mechanisms by which CARP expression is regulated, the effects of isoprenaline treatment were studied in isolated cardiac myocytes. CARP protein levels were significantly increased after 24-h treatment with isoprenaline (CARP/calsequestrin 161±10% of control, n=4, P<0.05; Fig. 4A). For CARP mRNA expression, a time–response curve for isoprenaline was performed which demonstrated that the plateau was reached after an incubation period of 6 h. Expression of CARP mRNA remained enhanced for at least 24 h. For all subsequent experiments, an incubation time of 6 h was chosen. CARP mRNA levels were significantly increased by isoprenaline (CARP mRNA/GAPDH mRNA 148±10% of control, n=9, P<0.05; Fig. 4B). Selective blockade of either the β1- or the β2-adrenoceptor by CGP 207 12A (300 nmol/l) or ICI 118,551 (100 nmol/l), respectively, reversed in part ISO-induced increase in CARP mRNA (Fig. 4C). Combined administration of ICI and CGP, as well as inhibition of protein kinase A (PKA) by the selective inhibitor Rp-cAMPS (100 µmol/l), and inhibition of calmodulin-dependent kinases (CaMK) by KN-62 (10 µmol/l) completely reversed the effects of ISO (Fig. 5). Rp-cAMPS and KN-62 themselves had no significant effect on basal CARP gene expression.


Figure 4
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  		Fig. 4 Effect of β-adrenoceptor stimulation on CARP protein and CARP mRNA levels in neonatal rat ventricular myocytes. Cells were treated with isoprenaline (ISO, 1 µmol/l) or vehicle (Ctr). (A) Cellular protein was collected and subjected to Western blot analysis. (B) Cells were treated for varying periods of time and 10 µg of total RNA was subjected to blot hybridization analysis. Blots were hybridised sequentially with radiolabelled CARP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. *P<0.01 (two-way ANOVA). (C) Cells were treated for 6 h with isoprenaline (1 µmol/l) or vehicle. In addition, cells were pretreated for 20 min with the selective β1-adrenoceptor blocker CGP 207 12A (CGP, 300 nmol/l) and the selective β2-adrenoceptor blocker ICI 118,551 (ICI, 100 nmol/l) before addition of isoprenaline. Pooled data from 7–12 independent experiments. *P<0.05 versus Ctr; #P<0.05 versus ISO.

 

Figure 5
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  		Fig. 5 Effect of PKA and CaMK inhibitors on isoprenaline-induced CARP gene expression in neonatal rat ventricular myocytes. Cells were treated with isoprenaline (ISO, 1 µmol/l) in the presence or absence of specific inhibitors of protein kinase A (Rp-cAMPS, 100 µmol/l) or calmodulin kinase (KN-62, 10 µmol/l), and 10 µg of total RNA was subjected to Northern blot hybridization analysis for CARP mRNA and GAPDH mRNA. Ctr, vehicle treated cells. *P<0.05 versus Ctr; #P<0.05 versus ISO.

 
To determine whether isoprenaline increases the steady-state levels of CARP mRNA by increasing rate of degradation, we measured CARP mRNA levels in the presence of actinomycin D (5 µg/ml) in cardiac myocytes, as shown in Fig. 6. The CARP half-life was 8 h in the absence of isoprenaline. These values were significantly affected in the presence of isoprenaline; the CARP half-life was 12 h. Thus, the adrenoceptor agonist-mediated increase in the levels of CARP mRNA in cardiac myocytes was due, at least in part, to an increase in the stability of the mRNA. As expected, the half-life of GAPDH mRNA levels was comparable between adrenoceptor agonist-treated and untreated cells (not shown).


Figure 6
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  		Fig. 6 Post-transcriptional effects of the β-adrenoceptor agonist isoprenaline on CARP gene expression in neonatal rat ventricular myocytes. (A) Cells were pretreated with actinomycin D (Act D; 10 µmol/l) for 1 h to arrest transcriptional activity and then subjected to isoprenaline (ISO) for varying periods of time. Controls (Ctr) were cultured for 12 h in the absence of actinomycin D. A 10-µg amount of total RNA was subjected to blot hybridisation analysis. Shown is a representative autoradiograph depicting the time course of CARP mRNA decay in the presence or absence of the adrenoceptor agonists. (B) The decay rates were plotted as a percentage of the 0-h value against time. CARP mRNA levels are expressed as the ratio of CARP to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal. The data presented represent means±S.E.M. from four separate experiments; P<0.05 versus control (two-way ANOVA).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Progression of heart failure is related to local and systemic neuroendocrine activation. Especially, activation of the sympathetic nervous system seems to play an important role. In the context of chronic heart failure, the initially appropriate compensatory adrenergic response to diminished myocardial performance ultimately results in an inappropriate or maladaptive response [19]. Several mechanisms by which these maladaptive responses may occur have been discussed. In addition to desensitisation of adenylyl cyclase, promotion of apoptosis, and production of cytotoxicity, β-adrenergic stimulation is a major stimulus to altered gene expression and pathologic hypertrophy [19]. The present study identifies the cardiac ankyrin repeat protein (CARP) as a novel target gene of chronic β-adrenoceptor stimulation in the heart. The primary new finding was that long-term β-adrenergic stimulation in rats by infusion of isoprenaline leads to a marked increase in cardiac CARP mRNA and protein concentrations.

In the rat isoprenaline infusion model, levels of growth factors and mediators that are thought to induce cardiac hypertrophy become elevated a few days after starting treatment [20,21]. Therefore, it might be speculated that regulation of CARP expression results from one of these activated mediators rather than from direct action of isoprenaline. To address this question, we performed in vitro studies in isolated rat cardiac myocytes. The finding that isoprenaline treatment caused a rapid and sustained increase in CARP gene expression suggests that direct activation of β-adrenoceptors is involved. Experiments with selective inhibitors of the β1- and the β2-adrenoceptor reveal that both β-adrenoceptor subtypes equally contribute to increased CARP mRNA content. We also addressed the question which intracellular signalling mechanisms may contribute to augmented CARP expression. Using an in vitro mRNA decay assay we showed that the isoprenaline-induced CARP expression is regulated, at least in part, through increased stability of the CARP mRNA. Moreover, among distinct signalling pathways known to be activated by β-adrenoceptor agonists, PKA and CaMK seem to be involved.

Involvement of PKA is not surprising since it has been known for many years that β-adrenergic receptor stimulation activates the classic cAMP/PKA pathway to regulate intracellular Ca2+ handling and control muscle contraction, but also to regulate gene expression. More recently, CaMKII, a member of a family of Ca2+/calmodulin regulated enzymes that is activated by β-adrenoceptor stimulation [22,23], has attracted much attention. Zhu et al. demonstrated that CaMKII constitutes a novel linkage of β1-adrenoceptor stimulation to cardiomyocyte apoptosis independent of PKA signalling [22]. Transgenic expression of CaMKII induces cardiac hypertrophy and results in dilated cardiomyopathy and heart failure [24,25]. In vitro studies have implicated CaMKII in the regulation of hypertrophy-associated genes, such as ANP, beside its regulatory function on cellular Ca2+ [26,27]. The present study extends these findings supporting a role for CaMK in the regulation of CARP expression.

Previous studies demonstrated that stimuli such as interleukin-1, TNF-{alpha}, or LPS failed to regulate endogenous CARP in primary rat cardiomyocytes [2] whereas other stimuli augment CARP expression. For example, CARP is increased by {alpha}1-adrenergic signalling in neonatal cardiac myocytes [28]. Activation of CARP expression by {alpha}-adrenergic agonists occurs largely at the level of transcription and involves the transcription factor GATA4 [28]. TGF-β also induced the expression of CARP mRNA in C2/2 cells derived from rabbit aortic smooth muscle cells [29]. The latter study demonstrated that changes in CARP transcript stability provide an additional mechanism by which CARP mRNA expression might be regulated. The CARP half-life was 11.3 and 8.5 h in the presence and absence of TGF-β, respectively [29]. Similarly to the effects of TGF-β in C2/2 cells, the present study demonstrates that isoprenaline increased CARP mRNA stability in neonatal rat cardiomyocytes. Thus, agonist-induced regulation of CARP expression may occur at the level of transcription as well as at the level of transcript turnover or degradation.

Increased CARP expression has been noted before in other animal models of cardiac hypertrophy and heart failure. CARP expression was markedly increased in three distinct models of cardiac hypertrophy due to acute or chronic pressure overload, namely the aortic constriction model, the spontaneously hypertensive rats, and the Dahl salt sensitive rats [30]. Most recently, by representational difference analysis of cDNA, we identified CARP as a gene which is up-regulated in a canine model of pacing induced heart failure. Similarly, in human heart failure CARP was increased ~2-fold in left ventricles [4]. Together, these studies demonstrate that CARP is increased in cardiac hypertrophy or heart failure from different aetiology [30]. From initial observations it has been hypothesised that CARP may act as a factor that modulates gene expression [1], and subsequent studies have addressed this question. Now it is well established that CARP negatively regulates many of the cardiac genes such as β-myosin heavy chain, myosin light chain, and cardiac troponin C [1]. These findings suggest that augmented expression of CARP may change expression of sarcomeric proteins and thereby may change contractility. Indeed, the present study demonstrates that CARP overexpression induces contractile dysfunction in engineered heart tissue.

In summary, we have demonstrated that β-adrenoceptor stimulation increases CARP mRNA and protein expression in vivo and in vitro. Given that the sympathetic nervous system becomes activated in patients with congestive heart failure, our previous observation demonstrating augmented CARP expression in failing human hearts [4] closely corresponds to the present experimental findings. In engineered heart tissue, CARP overexpression induces contractile dysfunction. From this observation it could be speculated that increased CARP may also promote contractile dysfunction in human heart failure and thus accelerate disease progression. However, model-specific limitations, such as species differences and different expression levels of CARP in engineered heart tissue compared to human failing myocardium, have to be considered. Future transgenic studies will have to substantiate the hypothesis that CARP is a factor significantly involved in the pathogenesis of heart failure.

Time for primary review 30 days.


   Acknowledgements
 
We thank Pico Caroni, Friedrich Miescher Institute, Basel, Switzerland and Franz-Werner Kluxen, Merck Kg, Darmstadt, Germany for the kind gift of CARP antibodies.


   Notes
 
1 Oliver Zolk and Michael Marx contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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M. Torrado, E. Lopez, A. Centeno, A. Castro-Beiras, and A. T. Mikhailov
Left-right asymmetric ventricular expression of CARP in the piglet heart: regional response to experimental heart failure
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