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Cardiovascular Research Advance Access first published online on April 25, 2008
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Cardiovascular Research, doi:10.1093/cvr/cvn106
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Overexpression of human truncated peroxisome proliferator-activated receptor {alpha} induces apoptosis in HL-1 cardiomyocytes

Javier Beaumont1, Teresa Arias1, Susana Ravassa1 and Javier Díez1,2,*

1 Division of Cardiovascular Sciences, Centre for Applied Medical Research, University of Navarra, CIMA, Avda. Pío XII 55, 31008 Pamplona, Spain
2 Department of Cardiology and Cardiovascular Surgery, University Clinic, School of Medicine, University of Navarra, Pamplona, Spain

* Corresponding author. Tel: +34 948 194700 (ext. 3000); fax: +34 948 194716. E-mail address: jadimar{at}unav.es

Received 18 December 2007; revised 22 April 2008; accepted 22 April 2008

Time for primary review: 30 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: Our goal was to analyse whether truncated peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) overexpression induces apoptosis of cardiomyocytes.

Methods and results: We constructed a recombinant vector of human truncated PPAR{alpha} and a mammalian expression vector to transfect PPAR{alpha} into a line of murine cardiomyocytes designated HL-1. Four hallmarks of apoptosis were measured in these transfected cells: depolarization of mitochondrial membrane, activation of caspase-3, phosphatidylserine (PS) externalization, and DNA fragmentation. Co-transfection with human cyclic adenosine monophosphate response element-binding protein (CREB) and human CREB binding protein (CBP) and analysis of apoptosis regulatory proteins, Bcl-2 and Bax, were also performed in truncated PPAR{alpha}-transfected cells to determine the potential mechanisms by which truncated PPAR{alpha} may influence apoptosis. Progressive depolarization of mitochondrial membrane, activation of caspase-3, PS externalization, DNA fragmentation, and cell death were observed in HL-1 cells upon increasing levels of transfected truncated PPAR{alpha}. The expression of the antiapoptotic protein Bcl-2 decreased in transfected HL-1 cardiomyocytes, whereas no changes in the proapoptotic protein Bax were observed in these cells. Overexpression of CREB plus CBP abolished the inhibitory effect of truncated PPAR{alpha} on Bcl-2 protein.

Conclusion: These results demonstrate that human truncated PPAR{alpha} overexpression induces apoptosis in HL-1 cardiomyocytes. In addition, our findings suggest that truncated PPAR{alpha} may induce cardiomyocyte apoptosis through the inhibition of the antiapoptotic protein, Bcl-2. It is proposed that competition with CREB for coactivators like CBP could be involved in this inhibitory effect.

KEYWORDS Truncated PPAR{alpha}; Apoptosis; Cardiomyocytes


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Several studies have demonstrated the implication of cardiomyocyte apoptosis in the development of heart failure (HF). In fact, increased cardiomyocyte apoptosis has been shown in spontaneously hypertensive rats (SHR) with left ventricular hypertrophy (LVH) and HF compared with SHR with LVH,1 and in hypertensive patients with chronic HF and LVH compared with hypertensives with LVH and normal cardiac function.2,3

The peroxisome proliferator-activated repector {alpha} (PPAR{alpha}) is a transcription factor that has been shown to be involved in the development of LVH and the transition from LVH to HF.4 Two isoforms of PPAR{alpha} have been described in the human heart: a native isoform that gives rise to an active protein of 53 kDa and a truncated isoform that lacks the exon 6 and gives rise to a protein of 30 kDa.5,6 In vitro experiments performed in human hepatic cells and in cardiomyocytes demonstrated that truncated PPAR{alpha} has a repressive action on native PPAR{alpha} activity, probably through competition for essential coactivators.6,7 This repressive activity is not specific for native PPAR{alpha}, because truncated PPAR{alpha} is also a potent repressor of the activity of nuclear receptors like PPAR{gamma}, related orphan receptor-{alpha} (ROR{alpha}), and glucocorticoid receptor-{alpha} (GR{alpha}).6

Recently, our group demonstrated that, although a decrease in native PPAR{alpha} expression was associated with the development of LVH in hypertensive patients, overexpression of truncated PPAR{alpha} was associated with the transition from LVH to HF in hypertensives.2 The excess of truncated PPAR{alpha} protein was associated with a decrease in the ejection fraction and the dilatation of the left ventricular chamber in these patients. Moreover, we observed a direct association of truncated PPAR{alpha} protein with cardiomyocyte apoptosis in hypertensive patients.2 It is interesting to note that these associations were independent of the expression of native PPAR{alpha}.2

We thus have hypothesized that an increase of truncated PPAR{alpha} may induce cardiomyocyte apoptosis. To test this hypothesis, the present study was designed to analyse different hallmarks of cellular apoptosis in HL-1 cardiomyocytes transfected with human truncated PPAR{alpha}. In addition, the effects of transfection with truncated PPAR{alpha} on the expression of apoptosis regulatory proteins of the Bcl-2 family were investigated in HL-1 cells. As the cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)/CREB binding protein (CBP) complex may inhibit cardiomyocyte apoptosis through induction of Bcl-2,8,9 experiments were also performed to assess whether truncated PPAR{alpha} interacts with the above complex.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Construction of recombinant vectors
To obtain the cDNA of human truncated PPAR{alpha}, a reverse transcription polymerase chain reaction (RT-PCR) was performed. Reverse transcription was performed with 5 µg of total RNA from human library (Human Heart Total RNA, Ambion, TX, USA) using the Superscript III Reverse Transcriptase (Invitrogen, Barcelona, Spain). Two primers were designed to amplify the cDNA with the following sequences: (i) 5'-CACCATGGTGGACACGGAAAGCCCACT-3' corresponding to sequences 1–23 of the human truncated PPAR{alpha} cDNA and (ii) 5'-TCATGTATGACAAAAGGCGTTGTGTGACAT-3' corresponding to sequences 495–525 of the human truncated PPAR{alpha} cDNA. Taq Platinum High Fidelity DNA polymerase (Invitrogen, Barcelona, Spain) was used in the PCR to minimize the probability of DNA mutations. Amplified products were separated by electrophoresis, showing one band of about 530 bp, the expected size for truncated PPAR{alpha}. PCR products were purified, cloned into a pcDNA3.1/V5-His© TOPO® vector and transformed into TOP10 Escherichia coli cells. The recombinant vector obtained from these cells was sequenced using the dideoxy chain-termination method,10 showing the predicted sequence of human truncated PPAR{alpha}.6

To construct a recombinant vector for human CREB, an RT-PCR was performed as previously described for truncated PPAR{alpha}. The primers used for CREB amplification have the following sequences: (i) CREB forward: 5'-GACGGAGGAGCTTGTACCACCGGTAACTAA-3' and (ii) CREB reverse: 5'-TTCAGGTTGTGGCCAAGCCAGTCC-3'. CREB human cDNA was cloned, transformed, and sequenced as previously described.

2.2 Transfection of human truncated PPAR{alpha}
HL-1 murine cells were grown in Claycomb Medium supplemented with 10% foetal bovine serum, 1% penicillin/streptomycin, 1% norepinephrine, and 1% L-glutamine, in a 5% CO2-humidified atmosphere at 37°C.

All transfections were performed with a mixture of truncated PPAR{alpha} expression vector (60, 200, and 400 ng) and β-galactosidase expression vector as control for transfection efficiency (500 ng). All samples were complemented to an equal amount of transfected DNA using the pcDNA3.1/V5-His© TOPO® empty vector. Cells were transfected at 40–50% confluence using the Lipofectin reagent (Invitrogen, Barcelona, Spain) and OPTIMEM (GIBCO, Barcelona, Spain) serum-free medium. After 5 h of incubation with the transfection mixture, the medium was replaced with supplemented Claycomb Medium and cells were incubated for 24 h.

After the incubation for 24 h, HL-1-transfected cells were washed with phosphate-buffered saline (PBS) (Gibco, Barcelona, Spain), tripsinized, and collected by centrifugation at 200 g for 5 min at 4°C. HL-1 pellet was resuspended in PBS and two flasks of cells were counted in a Neubauer counting chamber, to obtain an approximation of the HL-1 cell number.

2.3 Detection of truncated PPAR{alpha} in HL-1-transfected cells
2.3.1 Detection of truncated PPAR{alpha} mRNA
The RNA of the transfected cells was isolated with TRIzol (Invitrogen, Barcelona, Spain) and subsequently purified using QIAGENs RNeasy Total RNA Isolation Kit according to the manufacturer's conditions. The presence of truncated PPAR{alpha} mRNA in transfected cells was demonstrated using RT-PCR. The reverse transcription was performed with 1 µg of total RNA and the cDNA obtained was amplified by real-time PCR using specific primers flanking exon 6 of human PPAR{alpha} cDNA with the following sequences: (i) 5'-TGTCGGGATGTCACACAACG-3' for the forward primer and (ii) 5'-GCCATACACAGTGTCTCCATATCAT-3' for the reverse primer.

2.3.2 Detection of truncated PPAR{alpha} protein
The truncated PPAR{alpha} protein expression was demonstrated in HL-1 transfected cells by western blot. Protein was extracted in lysis buffer (7 mol/L urea, 2 mol/L thiourea, 4% Chaps, 1% dithiothreitol) and aliquots containing 10 µg of total protein were diluted in 4x sample buffer (40% β-mercaptoethanol, 8% sodium dodecyl sulphate, 40% glycerol, 0.025% bromophenol blue, and 0.25 mmol/L Tris, pH 6.4), separated by electrophoresis on 10% polyacrylamide gel and transferred onto nitrocellulose membranes. Specific rabbit polyclonal antibody (Cayman Chemical, Ann Arbor, MI, USA.) against the amino-terminal portion of PPAR{alpha}, common to both isoforms of PPAR{alpha}, was incubated at a dilution of 1:500. Bands were detected by incubation with the peroxidase-conjugated anti-rabbit IgG (Amersham, Barcelona, Spain) at a dilution of 1:5000. Both PPAR{alpha} isoforms were visualized with the ECL-Plus chemiluminescence system (Amersham, Barcelona, Spain) and autoradiograms were analysed using an automatic densitometer (Quantity One, Bio-Rad, Barcelona, Spain).

2.4 Analysis of apoptotic hallmarks
To assess whether truncated PPAR{alpha} overexpression is associated with cardiomyocyte apoptosis, a number of apoptotic hallmarks were analysed in HL-1-transfected and in HL-1 non-transfected cells that served as controls. In each experiment, β-galactosidase activity was analysed in the same number of cells as control for transfection efficiency using fluorescein-β-D-galactopyranoside in a flow cytometer (FAC Scan, Becton Dickinson, Madrid, Spain) using the Cell Quest program. All analysed parameters were normalized to β-galactosidase activity.

2.4.1 Depolarization of mitochondrial membrane
To analyse the HL-1 mitochondrial membrane potential, we used the JC-1 dye (Molecular probes, Invitrogen, Barcelona, Spain) which shifts the fluorescence emission from red (approximately 590 nm) to green (approximately 527 nm) when the mitochondrial membrane is depolarized.11 Therefore, the ratio of the 527/590 nm absorbance, analysed in a flow cytometer, indicates the mitochondrial membrane depolarization.

Data are expressed as fold change in the number of cells with mitochondrial membrane depolarization with respect to non-transfected HL-1 cells.

2.4.2 Caspase-3 activation
To analyse the activation of caspase-3 in HL-1-transfected cells, an electrophoresis on 12% polyacrylamide gel was performed as previously described, and the proteins were transferred onto nitrocellulose membranes. The membranes were excised to detect the procaspase and the active caspase separately, and incubated overnight at 4°C with a caspase-3-specific rabbit polyclonal antibody (Cell Signaling, Danvers, MA, USA.) at a dilution of 1:1000 to analyse the inactive procaspase, and at 1:500 to analyse the 17–21 kDa active fragments. These fragments were detected by incubation for 1 h at room temperature with a peroxidase-conjugated anti-rabbit IgG (Amersham, Barcelona, Spain) at a dilution of 1:2500. The blots were reprobed with a monoclonal β-actin antibody (Sigma, Madrid, Spain) as a control for loading. The bands were visualized and analysed as previously described.

As the ratio between the active fragments and the inactive procaspase-3 is an indicator of caspase-3 activation,12 data are expressed as the ratio of 17–21 kDa active caspase-3/35 kDa procaspase-3.

2.4.3 Phosphatidylserine externalization
To analyse the cardiomyocyte phosphatidylserine (PS) exposition, the ‘Annexin A5-FITC’ Kit was employed (Pharmatarget, Maastricht, Netherlands). In this method, the annexin A5 conjugated with fluorescein isothiocyanate (FITC) fluorochrome binds to PS exposed on the plasma membrane. Cellular fluorescence was determined by flow cytometry analysis.13

Data are expressed as fold change in the number of annexin A5 positive cells with respect to non-transfected HL-1 cells.

2.4.4 DNA fragmentation
The Apo ssDNA Kit (Bachem, San Carlos, CA, USA.) can detect DNA damage in apoptotic cells using specific antibodies against single-stranded DNA (ssDNA).14

HL-1 cells were incubated for 1 h at room temperature with a mouse anti-ssDNA antibody at a dilution of 1:10 and 30 min at room temperature with an anti-mouse IgG antibody FITC-labelled (Amersham, Barcelona, Spain) at a dilution of 1:100. The fluorescence changes were analysed by flow cytometry.

Data are expressed as fold change in the number of HL-1 cells with apoptotic ssDNA fragmentation with respect to non-transfected HL-1 cells.

2.5 Viability assay in HL-1-transfected cells
To analyse the toxicity of truncated PPAR{alpha} in HL-1-transfected cells, propidium iodide viability staining assay was performed. The fluorescence of this reagent was analysed in a flow cytometer.

Data are expressed as fold change in the number of propidium iodide-positive cells with respect to non-transfected HL-1 cells.

2.6 Analysis of the apoptotic regulatory proteins Bcl-2, Bax, and cytochrome c
To analyse the expression of Bax and Bcl-2 in HL-1- transfected cells, an electrophoresis on 12% polyacrylamide gel was performed and proteins were transferred onto nitrocellulose membranes as previously described. To identify the presence of Bax and Bcl-2 proteins, a Bax-specific rabbit polyclonal antibody at a dilution of 1:500 and a Bcl-2-specific mouse polyclonal antibody at a dilution of 1:500 were used (Santa Cruz Biotechnology, Heidelberg, Germany). The membranes were incubated with both antibodies overnight at 4°C and 1 h at room temperature with a peroxidase-conjugated anti-rabbit IgG at a dilution of 1:2500 (Amersham, Barcelona, Spain) and with a peroxidase-conjugated anti-mouse IgG at a dilution of 1:2500 (Amersham, Barcelona, Spain) for Bax and Bcl-2 detection, respectively. The blots were reprobed with a monoclonal β-actin antibody (Sigma, Madrid, Spain) as a control for loading. The bands were visualized and analysed as previously described.

To analyse if the effect of truncated PPAR{alpha} on Bax and/or Bcl-2 protein expression could induce the release of cytochrome c from mitochondria to cytosol, subcellular fractionation of HL-1 lysates was realized, following the methodology employed by Nakagawa et al.,15 with some modifications. In these cellular fractions, the proteins were separated on 12% polyacrylamide gel and transferred onto nitrocellulose membranes as previously described. A polyclonal antibody against cytochrome c at a dilution of 1:1000 (Cell Signaling, Danvers, MA, USA.) and a conjugated anti-rabbit IgG at a dilution of 1:2500 (Amersham, Barcelona, Spain) were used to measure the cytochrome c protein expression. The bands were visualized and analysed as previously described.

To explore whether truncated PPAR{alpha} can modify the antiapoptotic pathway CREB-Bcl-2, additional experiments were performed measuring Bcl-2 expression in HL-1 cells co-transfected with 400 ng of truncated PPAR{alpha}, 1 µg of human CREB, and 400 ng of the human coactivator CBP. These co-transfections were performed in the same conditions previously described for truncated PPAR{alpha} transfections in HL-1 cardiomyocytes. In one group of HL-1 cells truncated PPAR{alpha} was co-transfected with CREB, and in other group of HL-1 cells truncated PPAR{alpha} was co-transfected with both CREB and CBP.

Data are expressed as arbitrary densitometric units (A.D.U.) relative to β-actin expression.

2.7 Statistical analysis
To analyse any tendency in the parameters measured, the linear test for trend was used. Differences between two samples were tested by a Student's t-test for unpaired data once normality was demonstrated (Shapiro–Wilks test); otherwise, a non-parametric test (Mann–Whitney U test) was used. The correlation between continuously distributed variables was tested by univariate regression analysis. The analysis was performed using the SPSS program (13.0 version). Values are expressed as mean±SEM. A value of P < 0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Expression of truncated PPAR{alpha} in transfected HL-1 cardiomyocytes
To analyse the presence of truncated PPAR{alpha} mRNA in HL-1-transfected cells, an RT-PCR was performed with specific primers flanking exon 6 of human PPAR{alpha} cDNA. PCR products were separated by electrophoresis, showing a single band of about 60 bp, the expected size for the truncated PPAR{alpha} fragment, only in HL-1-transfected cells (Figure 1A). As shown in Figure 1B, a single band of 53 kDa, which corresponds to endogenous native PPAR{alpha}, can be observed in both non-transfected and transfected HL-1 cells. However, a band of 30 kDa, specific for truncated PPAR{alpha}, can be detected only in transfected HL-1 cells. The expression of truncated PPAR{alpha} was thus demonstrated both at the mRNA level and at the protein level in transfected cells, these results demonstrating that the transfection experiments were successful. Transfection efficiency in HL-1 cardiomyocytes was about 15–20%.


Figure 1
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  		Figure 1 (A) Representative image of polymerase chain reaction products amplified with specific primers flanking exon 6 of human peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) gene and separated by electrophoresis in non-transfected and in truncated PPAR{alpha}-transfected HL-1 cells. (B) Representative image of a western blot for native PPAR{alpha} (53 kDa) and truncated PPAR{alpha} (30 kDa) in non-transfected and in truncated PPAR{alpha}-transfected HL-1 cells.

 
3.2 Induction of apoptosis by truncated PPAR{alpha} transfection in HL-1 cardiomyocytes
3.2.1 Depolarization of the mitochondrial membrane
We observed a progressive depolarization of the mitochondrial membrane (P for trend <0.05) in association with the progressive increase in the levels of truncated PPAR{alpha} transfected into HL-1 cells (Figure 2).


Figure 2
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  		Figure 2 The histogram shows the progressive depolarization of the mitochodrial membrane, analysed as the ratio JC1 monomers (527 nm)/JC1 aggregates (590 nm) in HL-1 cells transfected with increasing amounts of truncated peroxisome proliferator-activated receptor{alpha}. These results are expressed as fold change relative to non-transfected HL-1 cells (NT). Values are represented as mean + SEM.

 
3.2.2 Activation of caspase-3
A dose-dependent increment in caspase-3 activation (P for trend <0.05) was observed in HL-1-transfected cells (Figure 3).


Figure 3
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  		Figure 3 The histogram shows the progressive activation of caspase-3, analysed as the ratio between the active caspase-3 fragments (17–21 kDa) and the inactive procaspase-3 (35 kDa), in HL-1 cells transfected with increasing amounts of truncated peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}). These results are expressed as fold change relative to non-transfected HL-1 cells (NT) (upper panel). Representative image of caspase-3 active fragments (17–21 kDa) and inactive procaspase-3 (35 kDa) in non-transfected and truncated PPAR{alpha}-transfected HL-1 cells (lower panel). β-Actin was used as loading control. Values are represented as mean + SEM.

 
3.2.3 Phosphatidylserine externalization
Transfection of HL-1 with increasing amounts of truncated PPAR{alpha} induced the progressive increment (P for trend <0.05) in PS externalization in HL-1 cardiomyocytes (Figure 4).


Figure 4
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  		Figure 4 The histogram shows the progressive externalization of phosphatidylserine, analysed as HL-1 cells positive for Annexin A5, in HL-1 cells transfected with increasing amounts of truncated peroxisome proliferator-activated receptor {alpha}. These results are expressed as fold change relative to non-transfected (NT) HL-1 cells. Values are represented as mean + SEM.

 
3.2.4 DNA fragmentation
As shown in Figure 5, truncated PPAR{alpha} transfection induced DNA fragmentation in HL-1 cells (P for trend <0.05).


Figure 5
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  		Figure 5 The histogram shows the progressive increase in HL-1 cells positive for apoptotic single-stranded DNA fragmentation in HL-1 cells transfected with increasing amounts of truncated peroxisome proliferator-activated receptor {alpha}. These results are expressed as fold change relative to non-transfected (NT) HL-1 cells. Values are represented as mean + SEM.

 
3.3 Induction of cell death by truncated PPAR{alpha} transfection in HL-1 cardiomyocytes
Truncated PPAR{alpha} induced a dose-dependent increase (P for trend <0.01) in cell death in HL-1 cardiomyocytes (Figure 6).


Figure 6
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  		Figure 6 The histogram shows the progressive increase in cell death, analysed as HL-1 cells positive for propidium iodide, in HL-1 cells transfected with increasing amounts of truncated peroxisome proliferator-activated receptor {alpha}. These results are expressed as fold change relative to non-transfected (NT) HL-1 cells. Values are represented as mean + SEM.

 
3.4 Changes in Bcl-2 protein and cytochrome c release by truncated PPAR{alpha} transfection in HL-1 cardiomyocytes
The expression of the antiapoptotic protein Bcl-2 was reduced (P < 0.05) in HL-1 cells transfected with 400 ng of truncated PPAR{alpha} when compared with non-transfected cells (Figure 7). No differences were observed in Bax expression between non-transfected HL-1 cells and HL-1 cells transfected with 400 ng of truncated PPAR{alpha} (data not shown). Of interest, Bcl-2 protein expression was inversely correlated with mitochondrial membrane depolarization (r = –0.573, P < 0.001), caspase-3 activation (r = –0.543, P < 0.01), and PS externalization (r = –0.332, P < 0.05) in transfected HL-1 cells. Moreover, truncated PPAR{alpha} overexpression induced the release of cytochrome c from mitochondria to cytosol, increasing the ratio of cytosolic/mitochondrial cytochrome c (P < 0.05) in HL-1 transfected vs. non-transfected cells.


Figure 7
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  		Figure 7 The histogram shows Bcl-2 protein expression in HL-1 cells transfected with 400 ng of truncated peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) and/or 1 µg of cyclic adenosine monophosphate response element-binding protein (CREB) and/or 400 ng of human CREB-binding protein (CBP). These results are expressed as fold change relative to non-transfected (NT) HL-1 cells (upper panel). Representative image of Bcl-2 protein in non-transfected and transfected HL-1 cells. β-Actin was used as loading control (lower panel). A.D.U. denotes arbitrary densitometric units. Values are represented as mean ± SEM. *P < 0.05 vs. non-transfected cells. **P < 0.05 vs. HL-1 cells transfected with truncated PPAR{alpha} and HL-1 cells co-transfected with truncated PPAR{alpha} and CREB.

 
Interestingly, the ability of truncated PPAR{alpha} to inhibit Bcl-2 expression was abolished when HL-1 cells were co-transfected with CREB plus CBP (Figure 7).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
The main finding of this study is that human truncated PPAR{alpha} overexpression is associated with the induction of apoptosis in HL-1 cardiomyocytes. In fact, truncated PPAR{alpha} overexpression resulted in depolarization of mitochondrial membrane, activation of caspase-3, PS externalization, DNA fragmentation, and cardiomyocyte death.

Stimulation of cardiomyocyte apoptosis may compromise cardiac function through the decrease of the contractility mass because of the loss of cardiomyocytes and the dilatation of the left ventricle because of the side-to-side slippage of remaining cardiomyocytes.2,16,17 The stimulation of apoptosis may also impair the functionality of viable cardiomyocytes.18 In fact, caspase-3 activation has been shown to lead to cleavage of myofilaments, disruption of sarcomeric structure, and an alteration of contractile force in cardiomyocytes.19,20 Moreover, the release of cytochrome c from mitochondria to cytosol, a protein involved in ATP production, because of the depolarization of the mitochondrial membrane, may therefore interfere with energy production and lead to systolic dysfunction.21,22 Thus, truncated PPAR{alpha} emerges as a potential factor involved in the transition from LVH to HF in hypertension through cardiomyocyte apoptosis.

Another finding of this study is that truncated PPAR{alpha} overexpression is associated with Bcl-2 downregulation in HL-1 cardiomyocytes. As no changes in Bax expression were observed in transfected cells, it can be hypothesized that the relative deficiency of Bcl-2 may contribute to mitochondrial membrane depolarization and the release of apoptogenic molecules like cytochrome c from the mitochondria to the cytosol, inducing the activation of caspase-3 and, consequently, the externalization of PS and the fragmentation of DNA. In support of this possibility, we found that Bcl-2 downregulation was associated with the depolarization of the mitochondrial membrane, caspase-3 activation, and PS externalization in transfected cells, and that truncated PPAR{alpha} overexpression induces the release of cytochrome c from mitochondria to cytosol.

Our observation that the inhibitory effect of truncated PPAR{alpha} on Bcl-2 expression is reversed by overexpression of both CREB and CBP suggests that truncated PPAR{alpha} may downregulate the CREB-Bcl-2 pathway through sequestration of CBP. In this regard, it has been reported that truncated PPAR{alpha} inhibits the trancriptional activity of native PPAR{alpha} through competition for CBP.6

In summary, we have demonstrated for the first time that truncated PPAR{alpha} overexpression induces apoptosis in HL-1 cardiomyocytes, probably through downregulation of the antiapoptotic protein Bcl-2. It is tempting to speculate that sequestration of CBP could be a mechanism by which truncated PPAR{alpha} might inhibit the CREB-Bcl-2 pathway. Nevertheless, additional proapoptotic mechanisms cannot be excluded. Although in vivo experiments are necessary to confirm the proapoptotic effect of truncated PPAR{alpha} overexpression in the heart, findings here reported add mechanistic support to our previous report2 describing an independent association between truncated PPAR{alpha} overexpression and increased cardiomyocyte apoptosis in the myocardium of HF patients. Thus, truncated PPAR{alpha} may be a target for strategies aimed to inhibit apoptotic death of cardiomyocytes in the failing human heart.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
This work was funded through the agreement between the Foundation for Applied Medical Research (FIMA) and UTE project CIMA, the red Temática de Investigación Cooperativa en Enfermedades Cardiovasculares (RECAVA) from the Instituto de Salud Carlos III, Ministry of Health, Spain (grant RD06/0014/0008) and the European Union (InGenious HyperCare, grant LSHM-CT-2006-037093).


   Acknowledgements
 
HL-1 cells are a gift of Dr. W. Claycomb. Dr. Philippe Gervois is acknowledged for providing us the CBP clone. The authors also thank ‘La princesa Hospital’ (Inmunology Department) in Madrid, Spain, for HL-1 transfection knowledge and Xabier Aguirre for the help provided in PCR amplification methodology.

Conflict of interest: none declared.


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

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  3. Ravassa S, Gonzalez A, Lopez B, Beaumont J, Querejeta R, Larman, et al. Upregulation of myocardial Annexin A5 in hypertensive heart disease: association with systolic dysfunction. Eur Heart J (2007) 28:2785–2791.[Abstract/Free Full Text]
  4. Goikoetxea MJ, Beaumont J, Diez J. Peroxisome proliferator-activated receptor alpha and hypertensive heart disease. Drugs (2004) 64:9–18.[CrossRef][Web of Science][Medline]
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  6. Gervois P, Torra IP, Chinetti G, Grotzinger T, Dubois G, Fruchart JC, et al. A truncated human peroxisome proliferator-activated receptor alpha splice variant with dominant negative activity. Mol Endocrinol (1999) 13:1535–1549.[Abstract/Free Full Text]
  7. Gilde AJ, Van Bilsen M. Peroxisome proliferator-activated receptors (PPARS). regulators of gene expression in heart and skeletal muscle. Acta Physiol Scand (2003) 178:425–434.[CrossRef][Web of Science][Medline]
  8. Tokudome T, Horio T, Fukunaga M, Okumura H, Hino J, Mori K, et al. Ventricular nonmyocytes inhibit doxorubicin-induced myocyte apoptosis: involvement of endogenous endothelin-1 as a paracrine factor. Endocrinology (2004) 145:2458–2466.[Abstract/Free Full Text]
  9. Mehrhof FB, Muller FU, Bergmann MW, Li P, Wang Y, Schmitz W, et al. In cardiomyocyte hypoxia, insulin-like growth factor-I-induced antiapoptotic signaling requires phosphatidylinositol-3-OH-kinase-dependent and mitogen-activated protein kinase-dependent activation of the transcription factor cAMP response element-binding protein. Circulation (2001) 104:2088–2094.[Abstract/Free Full Text]
  10. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Biotechnology (1992) 24:104–108.[Medline]
  11. Smiley ST, Reers M, Mottola-Hartshorn C, Lin M, Chen A, Smith TW, et al. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci USA (1991) 88:3671–3675.[Abstract/Free Full Text]
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