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European Journal of Heart Failure 2008 10(4):334-342; doi:10.1016/j.ejheart.2008.02.013
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© 2008 European Society of Cardiology

Celecoxib modulates hypertrophic signalling and prevents load-induced cardiac dysfunction

Claudius Jacobshagena,*, Meike Grübera, Nils Teucherb, Albrecht G. Schmidta, Bernhard W. Unsölda, Karl Toischera, Phuc Nguyen Vana, Lars S. Maiera, Harald Köglera and Gerd Hasenfussa

a Department of Cardiology, University of Göttingen Germany
b Department of Cardiothoracic Surgery, University of Göttingen Germany

* Corresponding author. Department of Cardiology Georg-August-University Robert-Koch-Strasse 40 D-37075 Göttingen, Germany. Tel.: +49 551 39 6380; fax: +49 551 39 2953. E-mial address: jacobshagen{at}med.uni-goettingen.de (C. Jacobshagen).


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
In human hearts, the transition from cardiac hypertrophy to advanced heart failure (HF) is accompanied by a tremendous increase in Akt phosphorylation. In non-myocardial tissue, the cyclooxygenase (COX)-2 inhibitor celecoxib has been shown to COX-independently inhibit Akt signalling.

We studied the effects of celecoxib on Akt signalling and hypertrophic response in myocardium. In rabbit isolated cardiac myocytes celecoxib concentration-dependently (10–100 µmol/L) inhibited the insulin-induced increase in phosphorylation of Akt and its downstream targets, GSK-3β and p70 S6 kinase, by reducing the phosphorylation level of the upstream regulator PTEN. Inhibition of Akt signalling was accompanied by a significant suppression of characteristic features of cardiac hypertrophy: Celecoxib concentration-dependently suppressed the agonist-induced enhancement of total protein synthesis and BNP mRNA expression.

In mice (C57BL/6NCrl) subjected to left ventricular (LV) pressure overload by aortic banding, celecoxib treatment (50 mg·kg–1·d–1) significantly attenuated LV dilation and contractile dysfunction compared with placebo-treated mice. Moreover, celecoxib significantly reduced mortality 8 weeks after banding.

Thus, celecoxib can be used to titrate Akt signalling and hypertrophic response in myocardium. It reduces load-induced LV dilation, contractile dysfunction and mortality in vivo. This may have clinical implications for the prevention and treatment of maladaptive hypertrophy and its progression to HF in humans.

Key Words: Hypertrophy • Heart failure • Celecoxib • Akt signalling • Aortic banding

Received July 17, 2007; Revised November 30, 2007; Accepted February 19, 2008


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Myocardial hypertrophy is defined as an enlargement of the cardiac muscle, characterized by an increase in the size of individual cardiac myocytes. It has traditionally been considered an adaptive response to compensate for an increased workload, such as in valvular heart disease, arterial hypertension or myocardial infarction [1-4]. However, sustained hypertrophy caused by these stimuli is associated with a significantly increased risk of progression to heart failure and malignant arrhythmia, leading to increased cardiovascular mortality [5-7]. Therefore, this type of hypertrophy can be classified as maladaptive or pathological.

However, not all forms of cardiac hypertrophy are detrimental. Cardiac growth induced by extensive exercise training or pregnancy is associated with preserved cardiac function in the long term [2,3,8,9]. Therefore, it can be classified as adaptive or physiological hypertrophy. The molecular mechanisms determining the difference between physiological and pathological hypertrophy are still poorly understood. Whereas some authors hypothesize distinct signalling systems for the different types of hypertrophy [1,10-12], there is now growing evidence that identical pathways can induce both, adaptive and maladaptive hypertrophy, depending on the intensity and duration of their activation [13,13-15].

In a conditional transgenic mouse model it has been shown that short term overexpression of Akt, a proximal regulator of a crucial hypertrophic signalling pathway, led to a physiological form of cardiac hypertrophy [14]. However, with longer periods of high level overexpression, Akt causes maladaptive hypertrophy with contractile dysfunction and left ventricular (LV) dilation. In contrast, prolonged but lower levels of Akt overexpression resulted in a moderate hypertrophy with preserved cardiac function [14]. Thus, the intensity and duration of Akt activation appear to determine the character of hypertrophy. Consequently, a pharmacologically controlled limitation of an overwhelming Akt activity could be an attractive approach for the prevention and treatment of maladaptive hypertrophy and its progression to heart failure.

In vascular and tumour tissue the cyclooxygenase (COX)-2 inhibitor celecoxib has been shown to COX-independently inhibit Akt signalling, thereby mediating antiproliferative and antitumour effects [16-18].

In the present study, we tested the hypothesis that celecoxib would modify hypertrophic signalling in cardiac myocytes. Furthermore, we hypothesized that moderate inhibition of Akt activity would preserve ventricular function in a model of pressure overload-induced hypertrophy and heart failure in mice.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Primary culture of rabbit ventricular myocytes and pharmacological interventions
This study was designed and carried out in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).

Female chinchilla bastard rabbits (1.5-2 kg, 3-4 months old, Charles River Laboratories, Kisslegg, Germany) were heparinized and anaesthetized with thiopental sodium (50 mg.kg–1 i.v.). Ventricular myocytes were isolated through enzymatic digestion as described previously [19]. Myocytes were counted and plated on laminin-coated 100-mm dishes (Sarstedt, Newton, USA) at a density of 5x105 rod-shaped cells in 5 mL/dish. The basic culture medium consisted of M199 (Sigma, Steinheim, Germany) supplemented with 5 mmol/L D,L-carnitine, 5 mmol/L taurine, 5 mmol/L creatine, 2 mmol/L glutamine, 100 IU/mL penicillin and 0.1 mg/mL streptomycin. 3 h after plating, unattached cells were removed by a single washing step. Since insulin [20] and phenylephrine [21] are well known to stimulate Akt signalling, we used these two agonists to activate this pathway. Stimulation was started with new supplemented medium (10 mL/dish) containing either 10 µmol/L human insulin (Aventis, Frankfurt, Germany) or 10 µmol/L phenylephrine (Sigma, Taufkirchen, Germany), in the presence of indicated celecoxib (Pfizer, Karlsruhe, Germany) and aspirin (Bayer, Leverkusen, Germany) concentrations. Celecoxib stock solution was prepared at 100 mmol/L in dimethyl sulfoxide (DMSO, Sigma, Taufkirchen, Germany). Control cells were treated with DMSO vehicle (highest final concentration 1 mL/L).

2.2. Quantitative mRNA measurement
DNA-free total RNA was extracted from rabbit ventricular myocytes by a standard protocol using SV Total RNA isolation kit (Omega, Madison, USA). First strand cDNA synthesis was carried out with superScript III reverse transcriptase and random primers (Invitrogen, Karlsruhe, Germany) according to manufacturer's instructions. Realtime PCR reactions were performed on a LightCycler (Roche, Mannheim, Germany) in a final volume of 20 µL in glass capillaries. The reaction mixture consisted of 2 µL cDNA, 0.5 µmol/L of each primer, 1 U of Taq DNA polymerase (Invitrogen), 2 µL of 10xPCR buffer (Invitrogen), 0.2 mmol/L of each dNTP (BioLine, Luckenwalde, Germany), 2.5 mM MgCl2, 500 mg/L BSA (New England BioLabs, Frankfurt, Germany), 50 mL/L DMSO (Sigma), and 1 µL of 1/1000 SYBR Green stock (Roche). After an initial denaturation step of 30 s at 95 °C, the cycling program consisted of 45 cycles of 94 °C for 0 s, 60 °C for 5 s, and 72 °C for 10 s. The emission at 530 nm was measured every cycle at 82 °C for brain natriuretic peptide (BNP) and at 85 °C for glycerol aldehyde phosphate dehydrogenase (GAPDH). The following primer pairs were used: BNP sense TGC TCT TCT TGC ACC TGT, antisense GCA GCT GCT GTA TCT CAG AAA; GAPDH sense TGC CGA GTA CGT GGT GGA AT, antisense ATG GCG TGC ACC GTG GTC AT. cDNAs with known concentrations were used to generate calibration standard curves.

2.3. Western immunoblot analysis
Cardiac myocytes were homogenized mechanically in 200 µL ice cold lysis buffer (containing 1% nonidet P 40, 10% glycerol, 137 mmol/L NaCl, 20 mmol/L Tris, pH 7.4, 20 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L Na4P2O7, 50 mmol/L β-glycerophosphate, 10 mmol/L EDTA, pH 8.0, 1 mmol/L EGTA, pH 7.0, 1 mmol/L phenylmethyl-sulfonyl-fluoride (PMSF), 4 µg/mL aprotinin, 4 µg/mL leupeptin, 4 µg/mL pepstatin). After elimination of the cell debris by centrifugation, protein concentrations in the supernatant were determined by the bicinchoninic acid (BCA) method (Pierce, Bonn, Germany). Samples of 30 µg protein were denatured at 95 °C and subjected to SDS-PAGE. Proteins were electroblotted to nitrocellulose membranes and membranes were blocked over night at 4 °C in 5% (w/v) non-fat dry milk in TRIS-buffered saline. Blots were probed with antibodies against phospho-Akt (Ser473, polyclonal, 1:1000, Cell Signaling, Beverly, USA), Akt (polyclonal, 1:1000, Cell Signaling), phospho-GSK-3β (Ser9, polyclonal, 1:5000, Cell Signaling), GSK-3β (monoclonal, 1:2500 BD Biosciences, Franklin Lakes, USA), phospho-p70 S6 kinase (Thr389, polyclonal, 1:1000, Cell Signaling), p70 S6 kinase (polyclonal, 1:1000, Cell Signaling), phospho-PTEN (Ser380, polyclonal, 1:1000, Cell Signaling) and GAPDH (monoclonal, 1:50000, Biotrend Chemikalien, Cologne, Germany). The antibodies for Akt and phospho-Akt were not isoform-specific. They detected total Akt and total phospho-Akt. Immunoreactive bands were visualized using enhanced chemiluminescence (Pierce) and quantified by two-dimensional scans using a CCD camera system (Multiimager, AlphaInnotech Inc., San Leandro, USA). Total Akt, GSK-3β, p70 S6 kinase and GAPDH protein levels were used as standards.

2.4. [3H]-Leucine-incorporation
Enhanced protein synthesis is one of the defining features of cardiac myocyte hypertrophy [3]. We examined the protein synthesis rate by measurement of [3H]-Leucine-incorporation: L-[3,4,5-3H(N)]-leucine (173,0 Ci/mmol, PerkinElmer, Boston, USA) was added to each culture dish at 5 µCi/mL for the last 4 h of the 24-h-stimulation time. Thereafter, the myocytes were washed twice with ice cold phosphate-buffered saline (PBS) lysed in a lysis buffer (composition: 150 mmol/L NaCl, 50 mmol/L Tris, pH 7.4, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet P40; pH 8.0) and incubated with ice cold 20% trichloroacetic acid for 15 min. The precipitate was centrifugated and resuspended in 0.2 N NaOH. 1.5 mL scintillation cocktail (Rotiszint eco plus, Carl Roth, Karlsruhe, Germany) was applied to the solution and the radioactivity was counted in a liquid scintillation counter (Wallac 1409).

2.5. Generation of mice with transversal aortic constriction (TAC)
For aortic-banding experiments, aortic constriction was performed using a minimally invasive approach as described previously [22]. Briefly, C57BL/6NCrl mice (female, 22-25 g, 3-4 months old, Charles River Laboratories) were anesthetized using intraperitoneal injections of ketamine and xylazine (100 mg.kg–1 and 5 mg.kg–1, respectively). A horizontal incision at the jugulum was used to display the transversal aorta. A 27G needle was tied against the aorta using a 5-0 non-absorbable suture. After removal of the 27G needle, skin was closed and the mice were kept on a heating plate until recovered from anaesthesia. Some mice were subjected to a sham operation in which the transversal aorta was visualized but not banded.

The time frame for the development of cardiac hypertrophy and transition to heart failure after aortic constriction depends on many factors such as body weight, mouse background, gender, needle size and location of constriction (ascending, transversal or descending aorta). We intentionally chose a setting with relative rapid development of LV dilation and impaired contractility to investigate specifically the effects of celecoxib on maladaptive hypertrophy and the transition to heart failure.

2.6. Echocardiography
2D guided M-mode echoes (30 MHz) were obtained from short- and long-axis views at the level of the largest left ventricular (LV)-diameter using a VS-VEVO 660/230 High Resolution Imaging System (Visualsonics, Toronto, Canada). Mice were lightly anesthetized with 2.5% tribromoethanol (0.01 ml.g–1 intraperitoneally) and were allowed to breathe spontaneously. The chest was shaved, acoustic coupling gel was applied, and a warming pad was used to maintain normothermia. Mice were imaged in a shallow left lateral decubitus position. LV end-diastolic (LVEDD) and end-systolic (LVESD) dimensions were measured from original tracings by using the leading edge convention of the American Society of Echocardiography. LV percent fractional shortening (FS), LV mass (LVM), and end-diastolic wall thickness/cavity ratio were calculated as previously described [23]. All echocardiographic measurements were performed in a blinded setting.

2.7. Administration of celecoxib and study protocol
All mice underwent echocardiography analysis to assess baseline dimensions and contractility parameters before starting any intervention or treatment. 2 days before banding mice were randomised to receive either celecoxib (50 mg.kg–1.d–1) or vehicle mixed in solid food. 2 weeks after banding LV diameters and fractional shortening (FS) were reanalyzed by echocardiography. At this time point the number of animals in each group was: n=10 for sham and vehicle; n=15 for sham and celecoxib; n=5 for TAC and vehicle; n=10 for TAC and Celecoxib. TAC surgery and echocardiography were performed in a blinded setting.

2.8. Statistical analysis
Data are presented as mean±SEM. Differences between experimental groups were evaluated for statistical significance by use of paired or unpaired Student t tests where appropriate. For multiple comparisons, the significance level was adjusted according to Bonferroni's method to P<0.05/number of tests. Survival curves where calculated using the Kaplan-Meier method with Logrank-test for statistical comparison. One mouse was excluded from the statistics after having excessive blood loss during the surgery caused by an accidental vascular injury. This mouse died within 24 h after surgery. All other mice were statistically included. A value of P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Effect of celecoxib on phosphorylation of Akt, GSK-3β and p70 S6 kinase in isolated cardiac myocytes
After 1 h of incubation with insulin (10 µmol/L) the phospho-Akt/Akt ratio in rabbit cardiac myocytes significantly increased from 0.60±0.15 to 2.08±0.28 (n=8, P<0.0002; Fig. 1A-B). This was accompanied by a significant effect on the two major downstream branches of Akt, GSK-3β and the mTOR-regulated p70 S6 kinase (S6K). The phospho-GSK-3β/GSK-3β and phospho-S6K/S6K ratio increased from 0.56±0.10 to 1.51±0.18 (n=8, P<0.0002; Fig. 1C-D) and from 0.84±0.06 to 1.27±0.07 (n=5, P<0.02; Fig. 1E-F), respectively. In the presence of celecoxib (25-100 µmol/L), the increase in phosphorylation of Akt, GSK-3β and S6K was suppressed in a concentration-dependent manner. Celecoxib (50 µmol/L) significantly attenuated the insulin-dependent gain of phospho-Akt/Akt, phospho-GSK-3β/GSK-3β and phospho-S6K/S6K ratio from 2.08±0.28 to 1.14±0.24 (n=8, P<0.002 vs. insulin; Fig. 1A-B), from 1.51±0.18 to 0.83±0.10 (n=8, P<0.005 vs. insulin; Fig. 1C-D) and from 1.27±0.07 to 0.88±0.10 (n=5, P<0.02 vs. insulin; Fig. 1E-F), respectively. Higher celecoxib concentrations (100 µmol/L) reduced the degree of phosphorylation of Akt and GSK-3β even below the baseline level of the control group (phospho-Akt/Akt: 0.36±0.06, n=8, P<0.001 vs. insulin; phospho-GSK-3β/GSK-3β: 0.52±0.08, n=8, P<0.002 vs. insulin, Fig. 1A-D).


Figure 01
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  		Fig. 1 Concentration-dependent effect of celecoxib on insulin-induced phosphorylation of Akt, GSK-3β, S6K and PTEN. Isolated cardiac myocytes were incubated with (+) and without (–) insulin (10 µmol/L) in the presence of increasing celecoxib concentrations (0-100 µmol/L) as indicated. Treatment time: 1 h. Myocyte protein extracts were analyzed by Western blotting with phosphospecific antibodies against phospho-Akt, phospho-GSK-3β, phospho-S6K and phospho-PTEN. Total Akt, GSK-3β, S6K and GAPDH protein levels were used as standards. Optical density (OD) of each band was measured. (A) Inhibition of Akt phosphorylation by celecoxib. Representative Western blot. (B) Corresponding summary of n=8 experiments. #P<0.0002 vs. control; *P<0.002 vs. insulin. (C) Inhibition of GSK-3β phosphorylation by celecoxib. Representative Western blot. (D) Corresponding summary of n=8 experiments. #P<0.0002 vs. control; *P<0.005 vs. insulin. (E) Inhibition of S6K phosphorylation by celecoxib. Representative Western blot. (F) Corresponding summary of n=5 experiments. #P<0.02 vs. control; *P<0.02 vs insulin. (G) Reduced level of phosphorylated PTEN by celecoxib. Representative Western blot. (H) Corresponding summary of n=5 experiments. *P<0.005 vs. insulin.

 
With longer incubation periods, the insulin-induced increase in Akt phosphorylation could even be suppressed by lower celecoxib concentrations: After 24 h, 10 µmol/L celecoxib significantly reduced the insulin-dependently increased phospho-Akt/Akt ratio from 1.99±0.14 to 0.95±0.14 (n=4, P<0.01 vs. insulin, online supplementary Fig. 5A-B).

In isolated cardiac myocytes from mice (n=2) we observed a comparable attenuation of Akt phosphorylation by celecoxib as seen in rabbit myocytes.

3.2. Potential mechanisms of celecoxib-induced Akt inhibition
3.2.1. Effect of COX inhibition on Akt phosphorylation
To assess whether the Akt-inhibiting effect of celecoxib is caused by the COX-inhibiting properties of the substance, we treated insulin-stimulated cells with aspirin, a well-known COX inhibitor.

At concentrations known to reduce COX activity in vitro (50-100 µmol/L) aspirin did not inhibit Akt phosphorylation (n=4) (online supplementary Fig. 6A-B). Therefore, we conclude that the celecoxib-mediated effect on Akt is COX-independent.

3.2.2. Effect of celecoxib on PTEN activity
Phosphorylation of phosphatidylinositol-4,5-biphosphate (PIP2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3) mediated by the phosphatidylinositol-3-kinase (PI3K) is a crucial upstream step for the activation of Akt [24]. The phosphatase and tensin homolog on chromosome ten (PTEN) reverses this step by converting PIP3 back to PIP2. Phosphorylation of PTEN inhibits its phosphatase activity [24]. Previous studies in tumour cells have suggested that celecoxib may inhibit the phosphorylation of PTEN [17]. Therefore we assessed the influence of celecoxib on the phosphorylation status of PTEN. We found that celecoxib (50 µmol/L) significantly decreased the phospho-PTEN/GAPDH ratio from 1.15±0.09 to 0.86±0.11 (n=5, P<0.005, Fig. 1G-H). These findings indicate that celecoxib suppresses Akt activity through mechanisms involving inhibition of PTEN phosphorylation, thereby increasing its phosphatase activity.

3.3. Effect of celecoxib on BNP expression
Since activation of the Akt pathway is known to induce cardiac hypertrophy [25], we investigated whether inhibition of this pathway by celecoxib could prevent hypertrophic responses.

At the cellular level, cardiac hypertrophy is characterized by a reinduction of fetal genes, such as BNP [26]. We therefore analyzed the effect of celecoxib on the BNP mRNA expression level by realtime PCR. Stimulation of cardiac myocytes with insulin (10 µmol/L) for 24 h significantly increased the BNP/GAPDH mRNA ratio from 0.039±0.002 to 0.051±0.003 (n=6, P<0.01, Fig. 2A). In the presence of celecoxib, this insulin-induced upregulation of BNP was abolished. 25 µmol/L celecoxib significantly reduced the BNP/GAPDH mRNA ratio to 0.022±0.002 (n=6, P<0.0005 vs. insulin, P<0.01 vs. control, Fig. 2A).


Figure 02
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  		Fig. 2 (A) Effect of celecoxib on BNP mRNA expression level. Isolated cardiac myocytes were incubated with (+) and without (–) insulin (10 µmol/L) in the absence and presence of celecoxib (25 µmol/L). After 24 h of treatment, BNP mRNA expression levels were quantitatively determined by real time PCR and normalized to GAPDH mRNA expression levels. n=6. #P<0.006 vs. control; *P<0.0002 vs. insulin; {dagger}P<0.007 vs. control. (B) Effect of celecoxib on protein synthesis measured by [3H]-leucin-incorporation. Isolated cardiac myocytes were incubated with (+) and without (–) insulin (10 µmol/L) in the presence of increasing celecoxib concentrations (10-25 µmol/L) for 24 h. n=as indicated. #P<0.0004 vs. control; {dagger}P<0.02 vs. control; *P<0.01 vs. insulin.

 
3.4. Impact of celecoxib on protein synthesis
An additional characteristic feature of cardiac hypertrophy is the increased rate of protein synthesis. Incubation with insulin (10 µmol/L) for 24 h significantly increased cardiac myocyte [3H]-leucin-incorporation by 36.9±7.3% (n=11, P<0.0005 vs. control, Fig. 2B). Phenylephrine (PE, 10 µmol/L), which is also known to induce cardiac hypertrophy via activation of the Akt pathway, provoked a comparable augmentation in protein synthesis by 26.2±6.9% (n=12, P<0.001 vs. control, online supplementary Fig. 7). Simultaneous incubation with celecoxib (10 µmol/L) completely abolished the increase in protein synthesis induced by both agonists (n=9, P<0.0005 vs. insulin, Fig. 2B; n=10, P<0.0001 vs. PE, online supplementary Fig. 7). With higher celecoxib concentrations (25 µmol/L) the protein synthesis was reduced even below the baseline level of the control group. Thus, celecoxib is able to inhibit the enhancement of protein synthesis induced by 2 different agonists known to stimulate the Akt pathway.

3.5. Effect of celecoxib on pressure overload-induced alterations in LV dimensions and contractile function
To investigate the effect of celecoxib on the development of cardiac hypertrophy in vivo, we treated mice with celecoxib or vehicle and subjected them to LV pressure overload by aortic banding or to a sham operation. Echocardiography analysis demonstrated that in vehicle-treated mice, exposure to pressure overload for 2 weeks induced significant cardiac hypertrophy. The calculated LV mass/body weight (LVM/BW) ratio significantly increased from 4.36±0.12 (n=10) to 6.78±0.27 (n=5) (P<0.001; online supplementary Fig. 8B) and the thickness of the left ventricular (LV) septal wall significantly increased from 0.74±0.01 mm (n=10) to 0.92±0.03 mm (n=5) (P<0.005; online supplementary Fig. 8C). Furthermore, after 2 weeks of pressure overload the mice already exhibited signs of maladaption: The LV end-systolic and end-diastolic diameters (LVESD and LVEDD) were significantly enlarged (LVESD: From 2.05±0.04 mm [n=10] to 3.66±0.08 mm [n=5], P<0.0001; LVEDD: From 3.65±0.03 mm [n=10] to 4.50±0.09 mm [n=5], P<0.0005; Fig. 3B-C), whereas fractional shortening (FS) had significantly decreased (from 43.66±0.99% [n=10] to 18.82±0.47% [n=5], P<0.00001; Fig. 3A), indicating LV dilation and impaired contractility.


Figure 03
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  		Fig. 3 Effect of celecoxib on pressure overload-induced alterations in LV dimensions and contractile function. Mice subjected to LV pressure overload by transversal aortic constriction (TAC) were treated with celecoxib (50 mg.kg–1.d–1) or placebo. Echocardiography was performed 14 days after banding. (A) Fractional shortening (FS), #P<0.00001; *P<0.01. (B) LV end-systolic diameter (LVESD), #P<0.0001; *P<0.005. (C) LV end-diastolic diameter (LVEDD), #P<0.0005; *P<0.01.

 
Treatment with celecoxib significantly suppressed pressure overload-induced end-systolic and end-diastolic LV dilation compared with vehicle-treated mice (LVESD 3.06±0.13 mm [n=10] vs. 3.67±0.08 mm [n=5], P<0.005; LVEDD 4.09±0.09 mm [n=10] vs. 4.50±0.09 mm [n=5], P<0.005; Fig. 3B-C). Moreover, FS was significantly improved in celecoxib-treated mice compared with vehicle-treated mice (25.41±1.80% [n=10] vs. 18.82±0.47% [n=5], P<0.01; Fig. 3A). Thus, celecoxib significantly attenuated characteristic features of maladaptive hypertrophy and the transition to heart failure.

The calculated LVM/BW ratio was slightly but not significantly reduced in celecoxib-treated banding mice compared with vehicle-treated banding mice (6.03±0.28 [n=10] vs. 6.78±0.27 [n=5], P=0.1; online supplementary Fig. 8B).

In sham-operated mice, celecoxib had no impact on LV dimensions and contractility (Fig. 3A-C). Since some COX inhibitors are known to increase sodium and water retention, it is noteworthy that celecoxib did not cause any alterations in body weight (online supplementary Fig. 8D).

3.6. Effect of celecoxib on pressure overload-induced mortality
Since celecoxib suppressed LV dilation and contractile dysfunction after pressure overload, we assessed its effect on survival rates with a Kaplan-Meier analysis. We found that celecoxib significantly decreased mortality at 8 weeks of pressure overload (placebo-group: n=13, celecoxib-group: n=14, P<0.05, Fig. 4).


Figure 04
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  		Fig. 4 Effect of celecoxib on survival after aortic banding in mice treated with celecoxib or placebo. Kaplan-Meier analysis. P<0.05.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
In the present study, we demonstrate for the first time that in cardiac myocytes celecoxib concentration- and time-dependently inhibits phosphorylation of Akt and both of its downstream branches, GSK-3β and mTOR/S6K, by reducing the phosphorylation level of PTEN. This effect is independent of the COX-inhibiting properties of the substance. At the cellular level, suppression of Akt signalling by celecoxib was paralleled by a significant inhibition of characteristic features of cardiac hypertrophy. Under in vivo-conditions celecoxib did not significantly inhibit load-induced increase in heart weight and LV wall thickness, but it significantly attenuated characteristic signs of maladaptive hypertrophy and heart failure, i.e. LV dilation, contractile dysfunction, and reduced mortality.

Several studies have demonstrated deleterious effects of chronically upregulated Akt signalling in the heart [13,14,27,28]. Accordingly, it appears consistent that a pharmacologically controlled inhibition of Akt activity by celecoxib has cardioprotective effects. However, there are also numerous studies documenting beneficial effects of Akt activation in the heart [12,29-31]. Moreover, physiological hypertrophy in response to exercise training is accompanied by higher levels of activated Akt [12]. Transgenic overexpression of PI3K [30], IGF-1 [31], or the IGF-1 receptor [29], resulting in higher Akt activity, are accompanied by preserved cardiac function. Furthermore, activation of Akt has a pivotal regulatory role for normal postnatal growth of the heart [11].

How is it possible that several studies demonstrate beneficial impact of Akt activation, whereas others show deleterious effects? Analyzing the degree of Akt activation may help to resolve this potential paradox. Whereas in the studies demonstrating protective effects the level of Akt phosphorylation was rather moderate (1.5- to 6-fold) [12,29-31], the level of Akt activity in studies with maladaptive effects was much higher (15- to 80-fold) [14,27,28]. Therefore, it appears that the character of hypertrophy is determined by the degree of Akt activity. Thus, the pharmacological intervention on the Akt pathway may provide a possibility to quantitatively control the Akt activity in vivo, thereby inhibiting maladaptive effects of excessive Akt activation, but maintaining a baseline level of phosphorylated Akt to avoid disruption of cardiac homeostatic mechanisms. Our in vitro data clearly demonstrate that celecoxib is an ideal tool to titrate Akt signaling in isolated cardiac myocytes. However, as a limitation of the study we did not identify the exact degree of celecoxib-induced Akt inhibition in the in vivo setting. Furthermore, we cannot exclude that the COX-inhibiting properties of the substance play a role for the morphological and functional effects of celecoxib in vivo.

Although myocardial hypertrophy has long been considered to be an adaptive response compensating states of increased workload [3], prolonged hypertrophy is associated with an increased risk for heart failure and cardiac mortality [5-7]. Moreover, recent studies have suggested that hypertrophic growth may not be necessary to maintain cardiac output in conditions of increased wall stress [32-35]. In genetically engineered mice with selective inhibition of defined signalling pathways, hypertrophy could be prevented without provoking haemodynamic compromise [32-35]. In particular, inhibited phosphorylation of each of the downstream branches of Akt signalling (GSK-3β and mTOR) has been shown to blunt cardiac hypertrophy in response to pressure overload without disturbing cardiac function. Transgenic mice expressing a phosphorylation-resistant mutant of GSK-3β exhibit a diminished hypertrophic response to pressure overload [36,37]. Despite increased wall stress the ventricular performance of these mice was preserved and the animals were clinically healthy with normal longevity [36,37]. Likewise, inhibition of mTOR by rapamycin attenuated load-induced cardiac hypertrophy without deteriorating cardiac function [38]. Moreover, rapamycin was able to regress already established cardiac hypertrophy and reversed characteristic features of maladaption, i.e. LV dilation and contractile dysfunction [39]. In this regard it is remarkable that in the current study we demonstrate that in cardiac myocytes celecoxib inhibits the phosphorylation of GSK-3β and mTOR-regulated S6K. Even though we did not assess the degree of inhibited phosphorylation in vivo, it remains noteworthy that celecoxib reduces LV dilation, contracile dysfunction and mortality.

In human hearts it has been shown that the transition from cardiac hypertrophy to advanced heart failure is accompanied by a tremendous increase in Akt and GSK-3β phosphorylation suggesting that this pathway may have adverse effects over time [40,41]. Since we have now demonstrated that celecoxib reduces the phosphorylation level of Akt and GSK-3β in vitro, it might be attractive to speculate that a pharmacologically titrated inhibition of that pathway could be also a therapeutic approach to prevent maladaptive hypertrophy and its progression to heart failure in humans.

However, the clinical use of selective COX-2 inhibitors in patients with cardiovascular diseases has been questioned because of their thrombogenic potential [42-45]. Several studies report an increased incidence of serious cardiovascular events in patients receiving COX-2 inhibitors [42-48]. On the other hand, there are several clinical studies confirming the adverse effects of rofecoxib, but not of celecoxib [49,50]. In these studies celecoxib was not associated with an elevated risk of vascular occlusion or other cardiovascular events [50,51]. Whether celecoxib increases the risk for thrombembolic events remains controversial at this point. However, regarding heart failure, users of rofecoxib and traditional NSAIDs (diclofenac, naproxen, ibuprofen), but not of celecoxib exhibited a higher risk of hospital admission for congestive heart failure relative to controls [52]. Therefore, it might be speculated that the Akt-inhibiting effect of celecoxib or other components which may even lack COX 2-affecting properties may play a role in preventing heart failure.

In summary, this is the first study to demonstrate that in myocardium celecoxib concentration-dependently inhibits phosphorylation of Akt and both of its downstream targets, GSK-3β and mTOR/S6K, by lowering the phosphorylation level of PTEN. In mice celecoxib attenuates characteristic features of load-induced maladaptive hypertrophy and heart failure and improves survival. Pharmacologically titrated inhibition of excessive Akt activity might therefore be of therapeutic value to prevent maladaptive hypertrophy and its progression to heart failure in humans. Further studies are warranted to answer this question.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
We are grateful to Michael Kothe, Gudrun Müller, Sandra Ott-Gebauer, Jessica Spitalieri and Susanne Burkhardt for excellent technical assistance. Dr. Maier was funded by the Deutsche Forschungsgemeinschaft (DFG) through an Emmy-Noether-grant (MA 1982/1-5) and is currently funded by a DFG Heisenberg fellowship (MA 1982/3-1). Drs. Maier, Kögler and Hasenfuss are funded by DFG Klinische Forschergruppe grants (MA 1982/2-1 & KO 1873/2-1).


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

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