Skip Navigation


Cardiovascular Research Advance Access first published online on January 10, 2008
This version [Corrected Proof] published online on February 12, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn001
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
78/1/79    most recent
cvn001v2
cvn001v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Smeets, P. J.H.
Right arrow Articles by van Bilsen, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smeets, P. J.H.
Right arrow Articles by van Bilsen, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Cardiac hypertrophy is enhanced in PPAR{alpha}–/– mice in response to chronic pressure overload

Pascal J.H. Smeets1, Birgit E.J. Teunissen1, Peter H.M. Willemsen1, Frans A. van Nieuwenhoven1, Agnieszka E. Brouns2, Ben J.A. Janssen2, Jack P.M. Cleutjens3, Bart Staels4,5,6, Ger J. van der Vusse1 and Marc van Bilsen1,*

1 Department of Physiology, Cardiovascular Research Institute Maastricht, Maastricht University, PO Box 616, 6200 MD Maastricht, the Netherlands
2 Department of Pharmacology and Toxicology, Cardiovascular Research Institute Maastricht, Maastricht University, PO Box 616, 6200 MD Maastricht, the Netherlands
3 Department of Pathology, Cardiovascular Research Institute Maastricht, Maastricht University, PO Box 616, 6200 MD Maastricht, the Netherlands
4 Unité 545, INSERM, Lille F-59019, France
5 Department of Atherosclerose, Institute Pasteur de Lille, Lille F-59019, France
6 Faculté de Pharmacie, Université de Lille II, Lille F-59019, France

* Corresponding author. Tel: +31 433881204; fax: +31 433884166. E-mail address: marc.vanbilsen{at}fys.unimaas.nl

Received 31 August 2007; revised 20 December 2007; accepted 28 December 2007

Time for primary review: 37 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Funding
 References
 
Aims: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a nuclear receptor regulating cardiac metabolism that also has anti-inflammatory properties. Since the activation of inflammatory signalling pathways is considered to be important in cardiac hypertrophy and fibrosis, it is anticipated that PPAR{alpha} modulates cardiac remodelling. Accordingly, in this study the hypothesis was tested that the absence of PPAR{alpha} aggravates the cardiac hypertrophic response to pressure overload.

Methods and results: Male PPAR{alpha}–/– and wild-type mice were subjected to transverse aortic constriction (TAC) for 28 days. TAC resulted in a more pronounced increase in ventricular weight and left ventricular (LV) wall thickness in PPAR{alpha}–/– than in wild-type mice. Compared with sham-operated mice, TAC did not affect cardiac function in wild-type mice, but significantly depressed LV ejection fraction and LV contractility in PPAR{alpha}–/– mice. Moreover, after TAC mRNA levels of hypertrophic (atrial natriuretic factor, {alpha}-skeletal actin), fibrotic (collagen 1, matrix metalloproteinase-2), and inflammatory (interleukin-6, tumour necrosis factor-{alpha}, cyclo-oxygenase-2) marker genes were higher in PPAR{alpha}–/– than in wild-type mice. The mRNA levels of genes involved in fatty acid metabolism (long-chain acyl-CoA synthetase, hydroxyacyl-CoA dehydrogenase) were decreased in PPAR{alpha}–/– mice, but were not further compromised by TAC.

Conclusion: The present findings show that the absence of PPAR{alpha} results in a more pronounced hypertrophic growth response and cardiac dysfunction that are associated with an enhanced expression of markers of inflammation and extracellular matrix remodelling. These findings indicate that PPAR{alpha} exerts salutary effects during cardiac hypertrophy.

KEYWORDS PPAR{alpha}; Pressure-overload; Hypertrophy; Metabolism; Fibrosis; Inflammation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Funding
 References
 
Cardiac hypertrophy is considered to be an adaptive mechanism compensating for a chronic increase in workload.1 In the long term, however, it can become maladaptive, fibrosis develops, and compensated hypertrophy evolves into cardiac failure, which is characterized by an inadequate cardiac pumping activity.1,2 Cardiac hypertrophic growth is associated with neurohormonal as well as metabolic changes. In addition, activation of inflammatory signalling pathways has been considered to promote both cardiac hypertrophy and fibrosis.3–6 Conversely, inhibition of inflammation can attenuate the development of cardiac hypertrophy.7,8

Several ligand-activated nuclear hormone receptors, including peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), have been reported to attenuate the activation of inflammatory signalling pathways9,10 and as such may inhibit cardiac hypertrophy and fibrosis. Consistent with this notion, the specific PPAR{alpha} ligand fenofibrate was able to reduce cardiac hypertrophy both in vitro and in vivo.11 Given the diverse effects of PPAR{alpha}, the exact mechanism for this anti-hypertrophic effect remains largely elusive, and may involve both stimulatory effects on cardiac fatty acid metabolism12 as well as anti-inflammatory effects.13–15

Accordingly, the aims of this study were to assess if deletion of the PPAR{alpha} gene affects pressure overload-induced hypertrophy of the heart and to explore which processes may underlie possible PPAR{alpha}-related differences in the hypertrophic growth response. Hereto, male PPAR{alpha}–/– and wild-type mice were subjected to transverse aortic constriction (TAC) and cardiac growth and function were evaluated non-invasively (echocardiography) and invasively [left ventricular (LV) pressure monitoring during a dobutamine stress test] after 4 weeks. To assess whether PPAR{alpha} deletion affected cardiac hypertrophy, inflammation and fibrosis, gene expression was assessed by quantitative PCR (qPCR). Here we report that (i) absence of PPAR{alpha} results in a more pronounced hypertrophic growth response, suggesting that PPAR{alpha} attenuates cardiac remodelling following pressure overload; (ii) mechanical function is depressed in the hypertrophied heart of mice lacking PPAR{alpha} (iii) deficiency of PPAR{alpha} is associated with enhanced inflammatory and fibrotic activity in the hypertrophying heart.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusion
 Funding
 References
 
2.1 Experimental animals
Male PPAR{alpha}–/– mice,16 backcrossed for more than 10 generations into a pure C57BL6/J background, and age-matched wild-type C57BL6/J mice were included in this study. The genotype was confirmed by qPCR (data not shown). Mice were kept on a 12 h/12 h light/dark cycle in temperature-controlled rooms and had ad libitum access to water and standard lab-chow (Ssniff R/M-H, Ssniff, Soest, Germany). The investigation conforms to 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).

2.2 Transverse aortic constriction
The surgical procedure of TAC has been previously described.17 Briefly, 12–14 weeks old mice were anesthetized with xylazine (5 mg/kg s.c.) and ketamine (100 mg/kg i.m.). Anaesthesia was maintained by isoflurane inhalation (1.5–2.5%). After opening the chest, the transverse aorta of PPAR{alpha}–/– (n = 23) and wild-type mice (n = 13) was ligated between the truncus brachiocephalicus and the left common carotid artery by tying a 6–0 silk suture against a 25-gauge needle. Sham-operated PPAR{alpha}–/– (n = 11) and wild-type mice (n = 9) underwent the same procedure without ligation of the aorta. Animals recovered at 30°C for 24 h and were injected with the analgesic buprenorphine (Temgesic; 0.1 mg/kg s.c.). Mice were sacrificed 28 days after TAC or sham operation.

2.3 Echocardiographic and haemodynamic measurements
Transthoracic echocardiography was performed 4 weeks after TAC or sham operation using a Hewlett-Packard 15 MHz linear array transducer interfaced with a Sonos 5500 ultrasound machine (Philips, Eindhoven, the Netherlands) as previously described in De Celle et al.18 Mice were anaesthetized with isoflurane and body temperature was maintained by placing the mice on a heating pad (37°C). LV end-diastolic and end-systolic internal diameter (LVIDd and LVIDs), anterior and posterior wall thickness end-diastolic (AWTd and PWTd) and end-systolic (AWTs and PWTs), fractional shortening (FS), ejection fraction (EF), and end-diastolic and end-systolic volume (EDV and ESV) of the LV were measured using data from short-axis (AWT, PWT, LVID, and FS) and parasternal long-axis (EF, EDV, and ESV) views.

Heart rate (HR), LV systolic pressure, and LV contractility (+dP/dtmax) were also measured 4 weeks after TAC or sham operation under baseline conditions and during a dobutamine stress test as previously described.18 Briefly, mice were anaesthetized with urethane (2.5 mg/kg i.p.) and a Millar catheter (Mikro-tip 1.4F; SPR-671, Millar Instruments, Houston, TX, USA) was inserted in the right carotid artery. In addition, a fluid-filled catheter was inserted in the femoral artery to determine the pressure-gradient across the aortic constriction. Next, the Millar catheter was advanced into the LV to measure LV pressure and its first derivative +dP/dt (mmHg/s) as a measure of LV contractility. Dobutamine (Sigma, St Louis MO, USA) was infused via a catheter in the vena jugularis with increasing doses up to 0.1 mg/kg/min.

2.4 Excision of the heart and cardiac tissue preparation
After completion of the haemodynamic measurements, the heart was excised, the atria were removed, and total ventricular tissue was weighed. Thereafter, ventricles were separated in LV, right ventricle (RV), and septum and snap-frozen for RNA analysis. In a subset of animals, the ventricles were dissected longitudinally. One-half was fixed for 24 h in 4% paraformaldehyde dissolved in phosphate-buffered saline and subsequently embedded in paraffin for further histological analysis. The other half was divided in LV, RV, and septum and snap-frozen in liquid nitrogen. Snap-frozen LV tissue samples were used for RNA analysis.

2.5 Histological analysis
Paraffin sections were deparaffinized and subsequently stained with haematoxylin and eosin (H&E) to determine cardiomyocyte short-axis cross-sectional area (CSA) in the LV.19 Interstitial fibrosis was measured using Sirius red staining.20 Stained H&E and Sirius red sections were quantified using Quantimet 570 software (Leica, the Netherlands).

2.6 Gene expression
LV tissue was homogenized using an UltraTurrax (Janke&Kunkel, Staufen, Germany) and total RNA was isolated with TRI reagent (Sigma) according to the manufacturer’s protocol and complemented with an additional wash step of 70% ethanol to increase the purity of the RNA. Integrity of the RNA was checked by means of the 260 : 280 ratio. cDNA synthesis and qPCR assays were performed as previously described.21 Primers used for analysis are given in Table 1. Results were normalized to the geometric mean of three reference genes, i.e. cyclophilin A (CycloA), acidic ribosomal phosphoprotein P0 (ARBP) and hypoxanthine guanine phosphoribosyl transferase (HPRT) according to Vandesompele et al.22 using qBase software.23 The expression of these reference genes was not affected by genotype and intervention.


View this table:
[in this window]
[in a new window]

  		Table 1 Primers used for quantitative polymerase chain reaction

 
2.7 Statistics
Results are presented as mean ± SEM. Data were analysed by one-way ANOVA and contrast analysis for multiple comparisons using SPSS 12 software (SPSS Inc.). The dose-dependent curves for dobutamine were compared using two-way ANOVA and a post hoc Bonferonni test. A P-value of less than 0.05 was considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusion
 Funding
 References
 
3.1 Transverse aortic constriction-induced hypertrophy
None of the PPAR{alpha}–/– and wild-type sham-operated animals and of the wild-type animals subjected to TAC died during the course of the study. However, 11 out of 23 (48%) of the PPAR{alpha}–/– mice died within 1 h after TAC surgery, indicating that the TAC surgical procedure was poorly tolerated by PPAR{alpha}–/– mice. In sham-operated control animals, total ventricular weight (VW) was slightly higher in PPAR{alpha}–/– mice than in wild-type mice (Table 2). After 28 days of TAC, VW was significantly increased in both wild-type and PPAR{alpha}–/– mice. The increase in VW was more pronounced in PPAR{alpha}–/– TAC than in wild-type TAC, both when expressed as absolute weight, or when normalized to tibia length or body weight (VW/body weight +37% vs. +24%, respectively). In wild-type mice, TAC resulted in an increase in the right carotid artery pressure and a pressure gradient of ~40 mmHg across the constriction as measured after 28 days. In PPAR{alpha}–/– mice, the pressure-gradient was comparable, but the pressures in both the right carotid artery and femoral artery were lower than in wild-type mice.


View this table:
[in this window]
[in a new window]

  		Table 2 Characteristics of wild-type and PPAR{alpha}–/– mice 28 days after transverse aortic constriction (TAC) or sham operation

 
Wet lung weight was approximately 10% higher in PPAR{alpha}–/– than in wild-type mice. However, when normalized to body weight no significant differences were apparent. Moreover, the ratio between wet and dry lung weight was similar for all groups, indicating that the differences in wet lung weight were not related to pulmonary oedema.

3.2 Cardiac dimensions and haemodynamic function
Diastolic and systolic anterior and posterior wall thickness (AWT and PWT) were similar in sham-operated wild-type and PPAR{alpha}–/– mice as determined echocardiographically (Table 3). TAC significantly increased AWT and PWT in both wild-type and PPAR{alpha}–/– mice during diastole. The increase in diastolic AWT and PWT was more pronounced in PPAR{alpha} –/– TAC animals. Histological examination of H&E-stained LV tissue sections revealed that the cardiomyocyte CSA tended to be larger in sham-operated PPAR{alpha}–/– mice when compared with wild-type mice (P = 0.078) (Figure 1). CSA significantly increased in both PPAR{alpha}–/– mice and in wild-type mice after TAC. The increase in CSA after TAC tended to be higher in PPAR{alpha}–/– mice when compared with wild-type (P = 0.081).


Figure 1
View larger version (67K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1 Cardiomyocyte cross-sectional area (CSA) of sham-operated and transverse aortic constriction (TAC)-subjected wild-type and PPAR{alpha}–/– mice. (A) Representative examples of haematoxylin and eosin-stained left ventricular (LV) tissue from sham- or TAC-operated wild-type and PPAR{alpha}–/– mice. (B) Cardiomyocyte CSA in LV tissue sections was quantified using Quantimet 570 software. Results are expressed as mean ±SEM. Asterisk (*) indicates statistically significant difference when compared with corresponding sham-operated animals (P < 0.05).

 


View this table:
[in this window]
[in a new window]

  		Table 3 Left ventricular dimensions of wild-type and PPAR{alpha}–/– mice 28 days after transverse aortic constriction (TAC) or sham operation

 
In sham-operated animals, the diastolic and systolic LV internal diameter (LVIDd and LVIDs) of PPAR{alpha}–/– mice were slightly, but significantly, larger when compared with wild-type mice. LVIDd and LVIDs did not increase in wild-type animals after TAC. However, LVIDs of PPAR{alpha}–/– TAC mice was 9% larger than that of PPAR{alpha}–/– sham mice. In wild-type mice, EF and FS were not affected by TAC (Figure 2A and B). In PPAR{alpha}–/– mice, however, TAC resulted in a significant decline of both EF and FS.


Figure 2
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2 Parameters of cardiac haemodynamic function in wild-type and PPAR{alpha}–/– mice after transverse aortic constriction (TAC). (A) Ejection fraction; (B) fractional shortening; (C) left ventricular systolic pressure; and (D) +dP/dtmax were measured by echocardiographic (A, B) and haemodynamic measurements (C, D) 28 days after TAC. Results are expressed as mean ± SEM of n = 8–11 mice per experimental group. Asterisk (*) symbol indicates statistically significant difference when compared with corresponding sham-operated animals (P < 0.05). Hash (#) symbol indicates statistically significant difference when compared with corresponding wild-type animals (P < 0.05).

 
Invasive haemodynamic analysis showed that LV systolic pressure and +dP/dtmax did not differ between sham-operated wild-type and PPAR{alpha}–/– mice (Figure 2C and D). TAC was associated with a significantly higher LV systolic pressure in wild-type mice, but not in PPAR{alpha}–/– mice, suggesting that the hearts of PPAR{alpha}–/– mice were unable to cope with the increase in afterload to which they were exposed for 28 days. The +dP/dtmax also was lower in PPAR{alpha}–/– mice when compared with wild-type mice after TAC. When hearts were stressed with dobutamine, the increase in +dP/dtmax was less in PPAR{alpha}–/– sham mice than in wild-type sham mice (Figure 3A). In wild-type mice TAC did not influence the dobutamine dose-dependent increase in +dP/dtmax. In contrast, in PPAR{alpha}–/– mice subjected to TAC +dP/dtmax was further compromised. At the highest dobutamine dose tested, +dP/dtmax was almost 20% lower in PPAR{alpha}–/– TAC than in wild-type TAC mice. Heart rate (HR) was similar for all groups under basal conditions as wells as after maximal stimulation with dobutamine (Figure 3B).


Figure 3
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3 Left ventricular (LV) contractility and heart rate in wild-type and PPAR{alpha}–/– mice after transverse aortic constriction (TAC). (A) LV contractility and (B) heart rate were measured during increasing doses of dobutamine infusion in PPAR{alpha}–/– and wild-type mice 28 days after TAC. Results are expressed as mean ± SEM of n = 8–11 mice per experimental group. Asterisk (*) indicates that curve is statistical significant different when compared with corresponding sham-operated animals (P < 0.05). Hash (#) indicates that curve is statistical significant different when compared with wild-type TAC animals (P < 0.05).

 
3.3 Gene expression
The mRNA level of hypertrophic marker genes ANF and {alpha}-skeletal actin ({alpha}-SKA) was similar in both sham-operated groups (Figure 4A and B). TAC increased ANF and {alpha}-SKA mRNA after 28 days both in wild-type and PPAR{alpha}–/– mice. The increase in ANF and {alpha}-SKA mRNA was more pronounced in PPAR{alpha}–/– mice than in wild-type mice, although in the case of {alpha}-SKA the difference did not reach the level of statistical significance.


Figure 4
Figure 4
View larger version (56K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4 Distinct gene expression in hearts of sham-operated and transverse aortic constriction (TAC)-subjected wild-type and PPAR{alpha}–/– mice. mRNA levels were assessed in left ventricular (LV) tissue 28 days after TAC or sham operation. Quantitative polymerase chain reaction assays were performed to determine the expression level of hypertrophic (A, ANF; B, {alpha}-skeletal actin), inflammatory (C, IL-6; D, TNF-{alpha}; E, COX-2; F, MCP-1), fibrotic (G, Col1; H, MMP-2), and metabolic (I, Acsl1; J, Hadha) marker genes. Results are expressed as mean ± SEM of n = 6 per experimental group. Asterisk (*) indicates statistically significant difference when compared with corresponding sham-operated animals (P < 0.05). Hash (#) indicates statistically significant difference when compared with corresponding wild-type animals (P < 0.05).

 
To investigate whether TAC is accompanied by increased cardiac expression of inflammatory marker genes, mRNA levels of the pro-inflammatory cytokines interleukin-6 (IL-6), tumour necrosis factor-{alpha} (TNF-{alpha}), cyclo-oxygenase-2 (COX-2), and monocyte chemo-attractant protein-1 (MCP-1) were determined (Figure 4C–F). Inflammatory gene expression was comparable in sham-operated PPAR{alpha}–/– and wild-type mice. After TAC, IL-6 and COX-2 mRNA were unaltered in wild-type mice, but were markedly increased in PPAR{alpha}–/– animals. TNF-{alpha} mRNA levels also tended to increase in PPAR{alpha}–/– TAC animals (P = 0.093). The mRNA level of MCP-1 was affected neither in wild-type mice nor in PPAR{alpha}–/– mice subjected to TAC.

As markers for fibrosis, we evaluated the expression of collagen 1 (Col1) and matrix metalloproteinase 2 (MMP-2). The expression of these fibrotic markers was similar in the two sham groups. TAC led to a substantial increase of Col1 and MMP-2 expression in PPAR{alpha}–/– mice, but not in wild-type mice (Figure 4G and H). Histological staining for fibrosis, however, did not reveal significant differences in fibrotic area (averaging 2% of total tissue area) related to genotype or in response to TAC (data not shown).

As anticipated, the mRNA level of PPAR-responsive genes involved in fatty acid metabolism, e.g. Acyl-CoA synthetase long-chain family member 1 (Acsl1) and the β-oxidation enzyme hydroxyacyl-Coenzyme A dehydrogenase (Hadha) was decreased in PPAR{alpha}–/– mice. The expression of both genes was not affected by TAC (Figure 4I and J).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusion
 Funding
 References
 
In this study, we demonstrate that the absence of PPAR{alpha} is associated with a more pronounced hypertrophic response of the murine heart following pressure overload. The increased hypertrophy is accompanied by decreased haemodynamic cardiac function as deduced from echocardiographic analysis and dobutamine stress tests. Moreover, gene expression analysis reveals that fibrotic and inflammatory responses are enhanced in the hearts of PPAR{alpha}–/– mice subjected to TAC. The collective findings indicate that loss of PPAR{alpha} activity is detrimental for the heart when subjected to a chronic increase in workload and suggest that the enhanced cardiac inflammatory response contributes to this negative effect.

4.1 Functional characterization of PPAR{alpha}–/– mice
The PPAR{alpha}–/– mouse is a well-established murine model to study the role of PPAR{alpha} in lipid metabolism and the relation between changes in lipid handling and disease progression.24–26 At first sight, these mice develop normally and do not show phenotypical abnormalities. However, after imposition of stress, e.g. in response to a metabolic challenge or exercise, or during ageing, phenotypical differences become readily apparent particularly in tissues with high oxidative capacity such as the heart.25,27,28

In the present study no differences were observed in baseline cardiac performance of sham-operated wild-type and PPAR{alpha}–/– mice. A number of studies reported that cardiac function is already compromised under basal, unstressed conditions as reflected by a reduced fractional shortening,29,30 or a decline in developed pressure of ex vivo perfused hearts.29 Other studies reported no change in baseline haemodynamic performance of ex vivo perfused PPAR{alpha}–/– hearts.31,32 However, when the workload was increased acutely, the myocardial ATP content dropped and cardiac function rapidly deteriorated.31 Consistent with this, we show that the imposition of an acute stress in situ via dobutamine infusion, unveils differences in cardiac function between sham-operated PPAR{alpha}–/– and wild-type mice.

The oxidation of fatty acids (FA) forms the principal source of ATP for the healthy, beating heart.33 Confirming the importance of PPAR{alpha} in the transcriptional control of cardiac FA oxidation capacity, the expression of rate-determining enzymes involved in FA activation (Acsl1) and mitochondrial β-oxidation (Hadha) is reduced in the hearts of sham-operated PPAR{alpha}–/– mice (Figure 4). Accordingly, it is quite conceivable that loss of PPAR{alpha} compromises the energy-generating capacity of the cardiac muscle and may explain some of the dobutamine stress-induced functional abnormalities observed in the sham-operated PPAR{alpha}–/– mice.

The compromised cardiac function in PPAR{alpha}–/– mice has not only been attributed to disturbances in energy production,31 but also to diminished β-adrenergic responsiveness.29 The blunted dobutamine response as observed in the present study would fit with the latter notion. Finally, an increased vulnerability to oxidative stress has been invoked as a cause of functional abnormalities.30 It remains to be established, however, to what extent each of these mechanisms contributes to the impairment of cardiac function under basal condition or after imposition of an acute or chronic stress.

4.2 Cardiac hypertrophy in PPAR{alpha}–/– mice
In the present study, young male PPAR{alpha}–/– and wild-type mice were subjected to a relatively mild chronic increase in afterload as reflected by the modest pressure-gradient created by the TAC (35–40 mm Hg). The chronic increase in workload led to a marked functional impairment of the heart in the PPAR{alpha}–/– mice as shown by the decline in EF and FS and the reduced LV systolic pressure and +dP/dtmax. The differences in contractility became even more pronounced when the TAC mice were subjected to a dobutamine stress test.

Interestingly, in sham-operated PPAR{alpha}–/– mice, CSA tended to be increased despite the fact that LV wall thickness is unchanged. Very recently, Guellich et al.30 also reported that CSA was enhanced in 9-month-old PPAR{alpha}–/– mice, whereas LV weight was not affected. They argued that this was because of loss of cardiomyocytes secondary to the increased oxidative stress to which the hearts of PPAR{alpha}–/– mice are exposed. Strikingly, it has also been reported that activation, instead of lack of PPAR{alpha} is associated with cardiomyocyte necrosis, secondary to enhanced oxidative stress.34 In the current study, we measured the expression of the putative PPAR-responsive gene heme oxygenase-1,35 but found no effect of genotype or TAC on the cardiac mRNA level of this anti-oxidant enzyme (data not shown). An alternative explanation could be that the number of muscle cells is lower because of the effect of PPAR{alpha} on the proliferation of cardiomyocytes during cardiac development and maturation. It is of note that the tendency to increased cardiomyocyte size is not associated with hallmarks of pathological cardiac hypertrophy, such as the shift in myosin heavy chain (MHC{alpha} -> MHCβ) protein isoform,30 or increased mRNA expression of ANF or {alpha}-SKA (Figure 4).

The chronic imposition of pressure overload for 4 weeks led to a more pronounced cardiac hypertrophy in mice lacking PPAR{alpha}, as shown by the substantially increased LV mass and the increase in LV wall thickness (echocardiography). After TAC, CSA also tended to be larger in PPAR{alpha}–/– mice than in wild-type mice (P = 0.081). It should be noted, however, that the relative increase in CSA in the PPAR{alpha}–/– and wild-type mice was comparable (~17%) because of the fact that, as discussed earlier, CSA was somewhat bigger in sham-operated PPAR{alpha}–/– mice compared with wild-type mice. Nonetheless, in line with the larger increase in cardiac mass after TAC, the rise in cardiac ANF and {alpha}-SKA expression was more pronounced in the PPAR{alpha}–/– mice, which also points to an enhanced hypertrophic response in the PPAR{alpha}–/– mice. The more pronounced hypertrophic response cannot be explained by differences in blood pressure. In fact, corroborating other studies,27,30 mean arterial pressure as measured under basal conditions in awake animals was even slightly lower (–10%) in PPAR{alpha}–/– mice than in wild-type animals (data not shown). Collectively, these findings indicate that the absence of PPAR{alpha} enhances pathological hypertrophy of the heart after imposition of pressure overload.

This conclusion is supported by earlier studies showing that activation of PPAR{alpha} via the administration of isoform-specific ligands blunts the hypertrophic response both of the heart in situ and in isolated cardiomyocytes. For example, fenofibrate was able to reduce endothelin-1-induced cardiomyocyte enlargement and protein synthesis.36,37 In the in vivo setting, treatment with fenofibrate normalized cardiac dimensions and FS in Dahl salt-sensitive rats and in mice subjected to TAC.15,38 Consistent with previous results in mildly hypertrophic hearts of rats subjected to abdominal aortic constriction,39 the TAC-induced cardiac hypertrophy in wild-type mice was not associated with a diminished expression of FA-handling genes. Likewise, in the myocardium of PPAR{alpha}–/– mice, the already reduced expression of these genes did not diminish further after TAC. It cannot be excluded that the chronic increase in afterload may gradually exhaust the already compromised energy-generating capacity of the cardiac muscle cells of the PPAR{alpha}–/– mice and, in this way, contribute to the observed cardiomyopathic changes.

The beneficial effect of PPAR{alpha} activation on LV remodelling has also been attributed to the interference of PPAR{alpha} with pro-inflammatory pathways converging on activator protein-1 (AP-1) and nuclear factor-{kappa}B (NF-{kappa}B), which are activated during hypertrophy.14,36 PPAR{alpha} activation was indeed associated with a decrease in both AP-1 and NF-{kappa}B DNA-binding activity in ET-1-treated neonatal rat cardiomyocytes and angiotensin II (AngII)-infused rats, respectively.14,36 By inference, this would imply that in hearts devoid of PPAR{alpha} inflammatory signalling pathways become more activated. Indeed, the current findings show that pro-inflammatory cytokines like IL-6, TNF-{alpha}, and the inducible form of cyclo-oxygenase (COX-2), the expression of which are under control of NF-{kappa}B, are more upregulated in the hearts of PPAR{alpha}–/– mice subjected to TAC than in wild-type mice. It has previously been demonstrated that activation of NF-{kappa}B is associated with an increased hypertrophic growth response.40,41 Moreover, targeted deletion or pharmacological inhibition of NF-{kappa}B, and hence the inflammatory response, attenuated cardiac hypertrophy in vivo.7,8 Furthermore, the expression of markers of extracellular matrix (ECM) remodelling, i.e. Col1 and MMP-2 was enhanced in PPAR{alpha}–/– mice. Sirius red staining, however, did not show changes in total collagen content in the affected hearts suggesting that, despite the enhanced ECM remodelling, collagen synthesis was still in balance with its degradation. The mRNA findings indicate that, in addition to interfering with the inflammatory pathway, PPAR{alpha} modulates ECM remodelling. This notion fits with the observation that the in vivo administration of synthetic PPAR{alpha} ligands to rats attenuates AngII-induced cardiac fibrosis.14


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
Funding
 References
 
The present findings show that the absence of PPAR{alpha} results in a more pronounced hypertrophic growth response and signs of functional deterioration, associated with an enhanced expression of markers of inflammation and extracellular matrix remodelling. By inference, this implies that in the normal heart, PPAR{alpha} attenuates pathological cardiac remodelling and, hence, mitigates the functional decline following pressure overload of the affected myocardium.

Conflict of interest: none declared.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Funding
References
 
Netherlands Organization for Scientific Research (912-04-017); EUGeneHeart (EU-FP6 grant LSHM-CT-2005-018833).


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

  1. Frey N, Olson EN. Cardiac hypertrophy: the good, the bad, and the ugly. Annu Rev Physiol (2003) 65:45–79.[CrossRef][Web of Science][Medline]
  2. Opie LH, Commerford PJ, Gersh BJ, Pfeffer MA. Controversies in ventricular remodelling. Lancet (2006) 367:356–367.[CrossRef][Web of Science][Medline]
  3. Porter KE, Turner NA, O’Regan DJ, Ball SG. Tumor necrosis factor alpha induces human atrial myofibroblast proliferation, invasion and MMP-9 secretion: inhibition by simvastatin. Cardiovasc Res (2004) 64:507–515.[Abstract/Free Full Text]
  4. Gurantz D, Cowling RT, Varki N, Frikovsky E, Moore CD, Greenberg BH. IL-1beta and TNF-alpha upregulate angiotensin II type 1 (AT1) receptors on cardiac fibroblasts and are associated with increased AT1 density in the post-MI heart. J Mol Cell Cardiol (2005) 38:505–515.[CrossRef][Web of Science][Medline]
  5. Thaik CM, Calderone A, Takahashi N, Colucci WS. Interleukin-1 beta modulates the growth and phenotype of neonatal rat cardiac myocytes. J Clin Invest (1995) 96:1093–1099.[Web of Science][Medline]
  6. Palmer JN, Hartogensis WE, Patten M, Fortuin FD, Long CS. Interleukin-1 beta induces cardiac myocyte growth but inhibits cardiac fibroblast proliferation in culture. J Clin Invest (1995) 95:2555–2564.[Web of Science][Medline]
  7. Gupta S, Young D, Sen S. Inhibition of NF-kappaB induces regression of cardiac hypertrophy, independent of blood pressure control, in spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol (2005) 289:H20–H29.[Abstract/Free Full Text]
  8. Higuchi Y, Chan TO, Brown MA, Zhang J, DeGeorge BR Jr, Funakoshi H, et al. Cardioprotection afforded by NF-kappaB ablation is associated with activation of Akt in mice overexpressing TNF-alpha. Am J Physiol Heart Circ Physiol (2006) 290:H590–H598.[Abstract/Free Full Text]
  9. Pascual G, Glass CK. Nuclear receptors versus inflammation: mechanisms of transrepression. Trends Endocrinol Metab (2006) 17:321–327.[CrossRef][Web of Science][Medline]
  10. Delerive P, De Bosscher K, Besnard S, Vanden Berghe W, Peters JM, Gonzalez FJ, et al. Peroxisome proliferator-activated receptor alpha negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-kappaB and AP-1. J Biol Chem (1999) 274:32048–32054.[Abstract/Free Full Text]
  11. Smeets PJ, Planavila A, Vusse Van der GJ, Van Bilsen M. Peroxisome proliferator-activated receptors and inflammation: take it to heart. Acta Physiol (2007) 191:171–188.[CrossRef]
  12. van Bilsen M, Vusse van der GJ, Gilde AJ, Lindhout M, Lee van der KA. Peroxisome proliferator-activated receptors: lipid binding proteins controling gene expression. Mol Cell Biochem (2002) 239:131–138.[CrossRef][Web of Science][Medline]
  13. Takano H, Nagai T, Asakawa M, Toyozaki T, Oka T, Komuro I, et al. Peroxisome proliferator-activated receptor activators inhibit lipopolysaccharide-induced tumor necrosis factor-alpha expression in neonatal rat cardiac myocytes. Circ Res (2000) 87:596–602.[Abstract/Free Full Text]
  14. Diep QN, Benkirane K, Amiri F, Cohn JS, Endemann D, Schiffrin EL. PPARalpha activator fenofibrate inhibits myocardial inflammation and fibrosis in angiotensin II-infused rats. J Mol Cell Cardiol (2004) 36:295–304.[CrossRef][Web of Science][Medline]
  15. Ichihara S, Obata K, Yamada Y, Nagata K, Noda A, Ichihara G, et al. Attenuation of cardiac dysfunction by a PPAR-alpha agonist is associated with down-regulation of redox-regulated transcription factors. J Mol Cell Cardiol (2006) 41:318–329.[CrossRef][Web of Science][Medline]
  16. Lee SS, Pineau T, Drago J, Lee EJ, Owens JW, Kroetz DL, et al. Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol Cell Biol (1995) 15:3012–3022.[Abstract]
  17. Rockman HA, Ross RS, Harris AN, Knowlton KU, Steinhelper ME, Field LJ, et al. Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy. Proc Natl Acad Sci USA (1991) 88:8277–8281.[Abstract/Free Full Text]
  18. De Celle T, Cleutjens JP, Blankesteijn WM, Debets JJ, Smits JF, Janssen BJ. Long-term structural and functional consequences of cardiac ischaemia-reperfusion injury in vivo in mice. Exp Physiol (2004) 89:605–615.[Abstract/Free Full Text]
  19. Creemers EE, Davis JN, Parkhurst AM, Leenders P, Dowdy KB, Hapke E, et al. Deficiency of TIMP-1 exacerbates LV remodeling after myocardial infarction in mice. Am J Physiol Heart Circ Physiol (2003) 284:H364–H371.[Abstract/Free Full Text]
  20. Lutgens E, Daemen MJ, de Muinck ED, Debets J, Leenders P, Smits JF. Chronic myocardial infarction in the mouse: cardiac structural and functional changes. Cardiovasc Res (1999) 41:586–593.[Abstract/Free Full Text]
  21. Teunissen BE, Smeets PJ, Willemsen PH, De Windt LJ, Vusse Van der GJ, Bilsen Van M. Activation of PPARdelta inhibits cardiac fibroblast proliferation and the transdifferentiation into myofibroblasts. Cardiovasc Res (2007) 75:519–529.[Abstract/Free Full Text]
  22. Vandesompele J, De Preter K, Pattyn F, Poppe B, Roy Van N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol (2002) 3:1–11.[Medline]
  23. Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J. Base relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol (2007) 8:R19.[CrossRef][Medline]
  24. Aoyama T, Peters JM, Iritani N, Nakajima T, Furihata K, Hashimoto T, et al. Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha). J Biol Chem (1998) 273:5678–5684.[Abstract/Free Full Text]
  25. Leone TC, Weinheimer CJ, Kelly DP. A critical role for the peroxisome proliferator-activated receptor alpha (PPARalpha) in the cellular fasting response: the PPARalpha-null mouse as a model of fatty acid oxidation disorders. Proc Natl Acad Sci USA (1999) 96:7473–7478.[Abstract/Free Full Text]
  26. Guerre-Millo M, Rouault C, Poulain P, Andre J, Poitout V, Peters JM, et al. PPAR-alpha-null mice are protected from high-fat diet-induced insulin resistance. Diabetes (2001) 50:2809–2814.[Abstract/Free Full Text]
  27. Watanabe K, Fujii H, Takahashi T, Kodama M, Aizawa Y, Ohta Y, et al. Constitutive regulation of cardiac fatty acid metabolism through peroxisome proliferator-activated receptor alpha associated with age-dependent cardiac toxicity. J Biol Chem (2000) 275:22293–22299.[Abstract/Free Full Text]
  28. Muoio DM, MacLean PS, Lang DB, Li S, Houmard JA, Way JM, et al. Fatty acid homeostasis and induction of lipid regulatory genes in skeletal muscles of peroxisome proliferator-activated receptor (PPAR) alpha knock-out mice. Evidence for compensatory regulation by PPAR delta. J Biol Chem (2002) 277:26089–26097.[Abstract/Free Full Text]
  29. Loichot C, Jesel L, Tesse A, Tabernero A, Schoonjans K, Roul G, et al. Deletion of peroxisome proliferator-activated receptor-alpha induces an alteration of cardiac functions. Am J Physiol Heart Circ Physiol (2006) 291:H161–H166.[Abstract/Free Full Text]
  30. Guellich A, Damy T, Lecarpentier Y, Conti M, Claes V, Samuel JL, et al. Role of oxidative stress in cardiac dysfunction of PPAR(alpha)–/– mice. Am J Physiol Heart Circ Physiol (2007) 293:H93–H102.[Abstract/Free Full Text]
  31. Luptak I, Balschi JA, Xing Y, Leone TC, Kelly DP, Tian R. Decreased contractile and metabolic reserve in peroxisome proliferator-activated receptor-alpha-null hearts can be rescued by increasing glucose transport and utilization. Circulation (2005) 112:2339–2346.[Abstract/Free Full Text]
  32. Panagia M, Gibbons GF, Radda GK, Clarke K. PPAR-alpha activation required for decreased glucose uptake and increased susceptibility to injury during ischemia. Am J Physiol Heart Circ Physiol (2005) 288:H2677–H2683.[Abstract/Free Full Text]
  33. Vusse van der GJ, Glatz JF, Stam HC, Reneman RS. Fatty acid homeostasis in the normoxic and ischemic heart. Physiol Rev (1992) 72:881–940.[Free Full Text]
  34. Pruimboom-Brees I, Haghpassand M, Royer L, Brees D, Aldinger C, Reagan W, et al. A critical role for peroxisomal proliferator-activated receptor-alpha nuclear receptors in the development of cardiomyocyte degeneration and necrosis. Am J Pathol (2006) 169:750–760.[Abstract/Free Full Text]
  35. Kronke G, Kadl A, Ikonomu E, Bluml S, Furnkranz A, Sarembock IJ, et al. Expression of heme oxygenase-1 in human vascular cells is regulated by peroxisome proliferator-activated receptors. Arterioscler Thromb Vasc Biol (2007) 27:1276–1282.[Abstract/Free Full Text]
  36. Irukayama-Tomobe Y, Miyauchi T, Sakai S, Kasuya Y, Ogata T, Takanashi M, et al. Endothelin-1-induced cardiac hypertrophy is inhibited by activation of peroxisome proliferator-activated receptor-alpha partly via blockade of c-Jun NH2-terminal kinase pathway. Circulation (2004) 109:904–910.[Abstract/Free Full Text]
  37. Liang F, Wang F, Zhang S, Gardner DG. Peroxisome proliferator activated receptor (PPAR)alpha agonists inhibit hypertrophy of neonatal rat cardiac myocytes. Endocrinology (2003) 144:4187–4194.[Abstract/Free Full Text]
  38. Duhaney TA, Cui L, Rude MK, Lebrasseur NK, Ngoy S, De Silva DS, et al. Peroxisome proliferator-activated receptor alpha-independent actions of fenofibrate exacerbates left ventricular dilation and fibrosis in chronic pressure overload. Hypertension (2007) 49:1084–1094.[Abstract/Free Full Text]
  39. Degens H, de Brouwer KF, Gilde AJ, Lindhout M, Willemsen PH, Janssen BJ, et al. Cardiac fatty acid metabolism is preserved in the compensated hypertrophic rat heart. Basic Res Cardiol (2006) 101:17–26.[CrossRef][Web of Science][Medline]
  40. Li Y, Ha T, Gao X, Kelley J, Williams DL, Browder IW, et al. NF-kappaB activation is required for the development of cardiac hypertrophy in vivo. Am J Physiol Heart Circ Physiol (2004) 287:H1712–H1720.[Abstract/Free Full Text]
  41. Purcell NH, Tang G, Yu C, Mercurio F, DiDonato JA, Lin A. Activation of NF-kappa B is required for hypertrophic growth of primary rat neonatal ventricular cardiomyocytes. Proc Natl Acad Sci USA (2001) 98:6668–6673.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
C. Pellieux, C. Montessuit, I. Papageorgiou, and R. Lerch
Angiotensin II downregulates the fatty acid oxidation pathway in adult rat cardiomyocytes via release of tumour necrosis factor-{alpha}
Cardiovasc Res, May 1, 2009; 82(2): 341 - 350.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
L. Makowski, R. C. Noland, T. R. Koves, W. Xing, O. R. Ilkayeva, M. J. Muehlbauer, R. D. Stevens, and D. M. Muoio
Metabolic profiling of PPAR{alpha}-/- mice reveals defects in carnitine and amino acid homeostasis that are partially reversed by oral carnitine supplementation
FASEB J, February 1, 2009; 23(2): 586 - 604.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
P. J. H. Smeets, H. M. de Vogel-van den Bosch, P. H. M. Willemsen, A. P. Stassen, T. Ayoubi, G. J. van der Vusse, and M. van Bilsen
Transcriptomic analysis of PPAR{alpha}-dependent alterations during cardiac hypertrophy
Physiol Genomics, December 12, 2008; 36(1): 15 - 23.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. J. H. Smeets, B. E. J. Teunissen, A. Planavila, H. de Vogel-van den Bosch, P. H. M. Willemsen, G. J. van der Vusse, and M. van Bilsen
Inflammatory Pathways Are Activated during Cardiomyocyte Hypertrophy and Attenuated by Peroxisome Proliferator-activated Receptors PPAR{alpha} and PPAR{delta}
J. Biol. Chem., October 24, 2008; 283(43): 29109 - 29118.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
M. Endoh
Novel signalling cascade for cardiac hypertrophy activation by uncoupling and internalization of {beta}1-adrenoceptors
Cardiovasc Res, April 1, 2008; 78(1): 5 - 7.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
78/1/79    most recent
cvn001v2
cvn001v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Smeets, P. J.H.
Right arrow Articles by van Bilsen, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smeets, P. J.H.
Right arrow Articles by van Bilsen, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?