p53-Mediated Upregulation of BAX Gene Transcription Is Not Involved in Bax-α Protein Overexpression in the Left Ventricle of Spontaneously Hypertensive Rats
Abstract—An association of increased apoptosis with overexpression of the proapoptotic protein Bax-α has been reported in the left ventricle of adult spontaneously hypertensive rats (SHR). Both alterations were corrected in SHR that received long-term treatment with the AT1 antagonist losartan. To gain insight into the regulation of cardiac Bax-α protein in genetic hypertension, we investigated the expression of the protein p53 (a BAX gene transcription factor) and BAX mRNA in the left ventricle of 30-week-old Wistar-Kyoto rats (WKY), SHR, and SHR treated with losartan (20 mg · kg−1 · d−1) during 14 weeks before death. The expression of p53 and Bax proteins was assessed by Western blot analysis. The expression of BAX mRNA was assessed by Northern blot analysis. The density of apoptotic cells was assessed by direct immunoperoxidase detection of biotin-labeled deoxyuridine nucleotides. Compared with WKY, untreated SHR exhibited increased apoptosis (P<0.05), increased Bax-α protein (P<0.05), and similar levels of p53 protein and BAX mRNA. Losartan given long term was associated with the normalization of apoptosis and Bax-α protein expression. The expression of BAX mRNA was decreased (P<0.05) in treated SHR compared with untreated SHR. No changes in the expression of p53 protein were observed in losartan-treated SHR. These results suggest that overexpression of the Bax-α protein seen in the left ventricle of adult SHR with increased apoptosis is not related to a p53-mediated upregulation of BAX gene transcription. Our data also suggest that normalization of Bax-α protein observed in SHR after long-term blockade of angiotensin II type 1 receptors may be due to the inhibition of BAX gene transcription.
Cardiomyocyte apoptosis is increasingly recognized as a contributing cause of cardiomyocyte loss with important pathophysiological consequences.1 2 For instance, increased apoptosis has been demonstrated recently in the hypertrophied left ventricle of young,3 adult,4 and aged5 spontaneously hypertensive rats (SHR). It has been suggested that apoptosis might be a mechanism involved in the reduction of cardiomyocyte mass that accompanies the development of interstitial fibrosis and the transition from stable compensation to heart failure in hypertensive heart disease.6
We have shown recently that an association exists between increased apoptosis and overexpression of the proapoptotic Bax-α protein in cardiomyocytes from the hypertrophied left ventricle of adult SHR.7 Long-term blockade of angiotensin II type 1 (AT1) receptors prevented Bax-α overexpression and normalized apoptosis in the left ventricle of SHR.7 These results suggested that the long-term effect of arterial hypertension in combination with local mechanisms (ie, the interaction of angiotensin II with the AT1 receptor) may facilitate cardiomyocyte apoptosis in the left ventricle of SHR by way of stimulation of Bax-α protein.
The BAX gene is a 6-exon, 4.5-kb gene, and a member of the Bcl-2 gene family that maps to chromosome 1q31.2. in the rat.8 It encodes different isoforms: Bax-α, Bax-β, Bax-γ, and Bax-δ. The heart has been shown to contain only the 21-kDa Bax-α protein.9 The expression of BAX has been found to be upregulated at the transcriptional level by p53.10 In fact, the BAX gene promoter was shown to contain 4 p53-binding sites that could be specifically transcriptionally transactivated by p53.11
This study was designed to determine whether the transcription of BAX gene is upregulated in the left ventricle of adult SHR and whether this is associated with changes in p53. In addition, we also determined whether blockade of AT1 receptors with losartan interferes with the transcriptional regulation of BAX in SHR.
All procedures were performed in accordance with European Community guidelines for ethical care and use of laboratory animals (Directive 86/609). The rats were provided by Harlan UK Limited (Bicester). The study followed the same design recently described by our team.7 Sixteen-week-old normotensive Wistar-Kyoto rats (WKY) (n=8) and 16-week-old untreated SHR (n=8) were observed in our laboratory for 14 weeks and killed at 30 weeks of age (groups WKY and SHR), and 16-week-old SHR (n=8) were treated with losartan (20 mg · kg−1 · d−1, PO) for 14 weeks and killed at 30 weeks (group SHR-L). Systolic blood pressure (SBP) was measured every 2 weeks by the standard tail-cuff method with the use of a LE 5007 Pressure Computer (Letica Scientific Instruments).
Preparation of Tissue Samples
All the animals were anesthetized with 30 mg/kg IP of sodium thiopental before death and were retrogradely perfused through the abdominal aorta as previously reported.7 After perfusion, the hearts were removed, the cardiac weight was measured, and the cardiac index was calculated by dividing the heart weight by the body weight for each animal. One portion of the left ventricle was fixed by immersion in 10% buffered formalin for 24 hours and embedded in paraffin. Coronal heart sections (5 μm thick) obtained from the equator of the heart were prepared for morphological studies. Another portion of the left ventricle was immediately stored at −70°C for Northern blot and Western blot analysis.
In Situ Detection of Apoptosis
The TUNEL methodology used for in situ end-labeling of DNA fragments was the same as recently described.7 Nuclei labeled with diaminobenzidine after TUNEL assay were quantified with an image analyzer. The presence of apoptotic cells was determined by means of the apoptotic density; the apoptotic density was calculated as the number of positive-staining nuclei per milimeter2 of myocardial surface area.
Northern Blot Analysis
Total RNA from frozen left ventricular tissue samples was prepared with the method of Chomczynski and Sacchi,12 with the use of Ultraspec RNA reagent (Biotecx Laboratories Inc). The purified RNA was quantified spectrometrically and run on ethidium bromide–stained agarose gels to check for its integrity. Total RNA (20 μg) was separated in a 1.3% denaturing formaldehyde agarose gel, blotted on nylon membranes by overnight capillary blotting, and fixed by UV irradiation. Blots were prehybridized in 5× SSC, 50% formamide, 5× Denhardt’s solution, 50 mmol/L sodium phosphate pH 6.5, 0.1% SDS, and 100 μg/mL salmon sperm DNA at 42°C. Hybridization was performed in 50% formamide solution at 42°C for 16 hours. Membranes were hybridized with a 505-bp fragment that encodes for rat BAX cDNA. The fragment was labeled with [α-32P]dCTP with the use of the Multiprime DNA labeling kit (Amersham Ibérica). The concentration of the labeled probe in the hybridization solution was 1×106 cpm/mL. After hybridization, membranes were successively washed twice in 2× SSC/0.1% SDS at room temperature for 20 minutes, twice in 1× SSC/0.1% SDS at 42°C for 20 minutes, and twice in 0.2× SSC/0.1% SDS at 65°C for 15 minutes. Standardization was performed by hybridization of the same membrane with a probe from rat GAPDH cDNA. After autoradiography, quantification of the signals was performed by densitometric analysis.
Western Blot Analysis
For immunoblot assay of Bax-α in the left ventricle, we used the procedure recently described.7 For the immunodetection of p53, 60 μg of proteins was separated in a 12% SDS–polyacrylamide gel and transferred to nitrocellulose membranes with the use of a Mini-Protean II Dual Stab Cell (BioRad). Membranes were blocked with 0.05% Tween and 10% dry skim milk in PBS (100 mmol/L sodium chloride, 80 mmol/L disodium phosphate, 25 mmol/L disodium monobasic phosphate, pH=7.5) overnight at 4°C and incubated with a mouse monoclonal anti-p53 (Pab240, Santa Cruz Biotechnology, Inc) at 1:400 in blocking solution for 1 hour at room temperature. After the proteins were washed, specific bound antibody was detected by a peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology, Inc) at 1:8000 in PBS and visualized by the ECL-Plus chemiluminescence detection system (Amersham). Values for p53 and Bax-α protein were expressed as arbitrary optical density units (AU).
Immunohistochemical Study of Bax-α Protein
For the immunohistochemical detection of Bax-α, avidin-biotin immunoperoxidase staining was performed as described previously,13 with some modifications. The deparaffinized and rehydrated sections were treated by microwave irradiation twice for 3 minutes at 550 W with 10 mmol/L citrate buffer (pH=6). After blocking with 4% normal goat serum was completed, sections were incubated for 1 hour at room temperature first with a rabbit polyclonal anti-mouse Bax antibody (Pharmingen) at a dilution of 1:100 in PBS , then with goat-biotinylated anti-rabbit IgG (Vector) at 1:100 in PBS, and finally with avidin-biotin complex that contained horseradish peroxidase (Vector). Immunostaining was detected with diaminobenzidine (Sigma), and tissues were counterstained with hematoxylin (Sigma). For all data presented, the specificity of the immunostaining results was confirmed by use of both preimmune serum, which produced entirely no background, and by preadsorption of anti-Bax-α antibody with competing peptide, which completely abrogated the immunostaining (not shown). To develop a semiquantitative scale, the amount of Bax-α was graded on a scale of 0 to 2+: 0 indicates the absence of Bax-α; 1+, mild deposits; and 2+, intense deposits.
Results are presented as mean±SEM computed from the average measurements obtained from each group of rats. Normal distribution of data was checked with the Shapiro-Wilk test. A Levene statistic test was performed to check the homogeneity of variances. Differences among the 3 groups of rats were tested by 1-way ANOVA. Subsequent analysis for significant differences between the 2 groups was performed with the use of the multiple comparison Student-Newman-Keuls test. When the normal distribution test was significant, the χ2 method (Kruskal-Wallis) was used to analyze the differences among the 3 groups of animals. For nonquantitative data, a χ2 method (Pearson) was used to analyze the differences among the 3 groups of animals. The significance level was assumed at P<0.05.
As shown in Table 1⇓, at 30 weeks of age, SBP was higher (P<0.05) in SHR than in WKY. Consistent with data previously reported,7 losartan diminished (P<0.05) SBP in SHR-L compared with SHR (Table 1⇓). Thus, no significant differences were found in SBP between SHR-L and WKY (Table 1⇓).
Both cardiac weight and index were greater (P<0.05) in SHR than in WKY (Table 1⇑). Thus, SHR were considered to have left ventricular hypertrophy (LVH). The values of the 2 parameters were not significantly different in SHR-L than in WKY (Table 1⇑), which indicates that losartan given long-term was associated with the regression of LVH already present in 16-week-old SHR.4
The apoptotic density was increased (P<0.05) in the left ventricle of SHR versus WKY (Table 1⇑). After treatment with losartan, the apoptotic density decreased (P<0.05) in SHR-L to values similar to those measured in WKY (Table 1⇑).
Expression of p53 Protein
As shown in Fig 1⇓, no significant differences were found in the expression of p53 among the 3 groups of animals. Optical density values for p53 were 0.89±0.15 AU for WKY, 1.14±0.16 AU for SHR, and 1.28±0.29 AU for SHR-L.
Expression of BAX mRNA and Bax-α Protein
A representative Northern blot of left ventricular BAX mRNA is shown in Fig 2⇓ (top). BAX mRNA levels were similar in the left ventricle of WKY and SHR (1.12±0.07 versus 1.02±0.05) (Fig 2⇓, bottom). Left ventricular BAX mRNA levels were decreased (P<0.05) in SHR-L (0.75±0.06) versus WKY and SHR (Fig 2⇓, bottom).
The amount of Bax-α protein was higher (P<0.05) in SHR (0.45±0.10 AU) than in WKY (0.19±0.03 AU) (Fig 3⇓). The expression of Bax-α protein (0.21±0.04 AU) was diminished (P<0.05) in SHR-L compared with SHR (Fig 3⇓). No significant differences in Bax-α protein were observed between SHR-L and WKY.
Left Ventricular Deposition of Bax-α Protein
The positive Bax-α immunoreactivity was primarily confined to the cardiomyocytes, which were easily distinguished from other nonmyocytes cells because of their morphology: well-shaped, cylindrical, and elongated cells that exhibit cross-striations and are branched. The photomicrographs in Fig 4⇓ show Bax-α deposition in the left ventricle of the 3 experimental rat groups. A semiquantitative analysis of Bax-α deposition was performed on all rats (Table 2⇓). Although more animals exhibited no deposition or a low grade of deposition of Bax-α in the WKY group, more animals exhibited high-grade deposition in the SHR group. After losartan was given, the distribution of SHR-L was displaced to a low grade of deposition of Bax-α. The analysis of the frequencies of distribution demonstrated differences among the 3 groups of animals (P<0.05).
The results of the current study provide the first indication that the expression of the BAX mRNA is unchanged in the left ventricle of adult SHR versus normotensive WKY. In addition, the reported data indicate that the expression of p53 protein is normal in the hypertrophied left ventricle of adult SHR that exhibit increased apoptosis of cardiomyocytes.
Previous observations suggest that BAX may function as a primary response gene in a p53-regulated pathway in some types of cells.10 11 Recently, Leri et al14 have shown that stretching of adult rat ventricular cardiomyocytes in vitro was coupled with enhanced p53 expression and binding to the promoter of BAX gene, which was followed by enhanced Bax-α protein expression and apoptosis. Thus, the authors suggest that activation of p53 results in the induction of BAX gene and increased susceptibility of cardiomyocytes to undergo apoptosis.14 Our results of normal levels of p53 and BAX mRNA in SHR do not support a role for the above mechanism of cardiomyocyte apoptosis in this in vivo model of genetic hypertension. This agrees with previous data that show that p53 transcripts are barely detectable in the adult rat myocardium and do not seem to increase with cardiac hypertrophy.15 Nevertheless, because upregulation in p53 binding activity may result from both increased expression of the protein and phosphorylation of its regulatory sites, without changes in its level of expression,16 caution is required to exclude definitively the participation of p53 in cardiac apoptosis in SHR.
Miyashita et al17 demonstrated that levels of Bax-α protein can be posttranslationally regulated by Bcl-2, a 25-kDa protein that blocks apoptosis. Gene transfer–mediated elevations in Bcl-2 protein resulted in increases in Bax-α by a reduction in the rate of Bax-α degradation in some types of cell lines.17 In a previous work, we have found that the concentration of Bcl-2 is increased by 53% in the left ventricle of SHR compared with WKY.7 Thus, a reasonable speculation is that the interaction of Bcl-2 with Bax-α, which leads to formation of heterodimers or complexes with other stoichiometry,18 somehow stabilizes the Bax-α, and thus leads to increases in the steady-state levels of this protein in the left ventricle of adult SHR. Nevertheless, alternative possibilities also deserve to be considered. These include differences in the levels or activity of proteases involved in proteolytic degradation, kinases and phosphatases that theoretically could control phosphorylation of Bax-α, and non-Bcl-2 family proteins that might bind to Bax-α.
A second finding of this study is that chronic treatment with losartan was associated with a parallel diminution of BAX mRNA and Bax-α protein levels in the left ventricle of SHR. Interestingly, no changes in p53 expression were observed in losartan-treated SHR. Together, these results suggest that long-term blockade of AT1 receptors in the left ventricle of SHR results in downregulation of BAX transcription through a p53-independent pathway. Some experimental observations support this possibility. First, stretch-mediated release of angiotensin II is coupled with stimulation of AT1 receptors and an increased level of Bax-α protein in adult rat ventricular cardiomyocytes.14 Thus, the interaction of angiotensin II with the AT1 receptor might participate in the regulation of BAX transcription in the left ventricle of SHR. Second, it has been shown that interleukin-6 downregulates BAX mRNA levels.19 However, because this cytokine also induces cardiac hypertrophy,20 it is unlikely that losartan treatment, which is associated with LVH regression in SHR, results in stimulation of interleukin-6. Third, we have reported previously that the blockade of AT1 receptors with losartan resulted in decreased expression of Bax-α in the absence of changes in the expression of Bcl-2 in left ventricular cells of SHR.7 Thus, it is unlikely that the ability of losartan to reduce the left ventricular levels of Bax-α protein can be the result of enhanced degradation of the protein in treated SHR.
Finally, in this study we found that losartan given long-term regressed LVH and prevented cardiomyocyte apoptosis in treated SHR. Thus, it seems that the treatment of SHR with losartan resulted in decreased cardiac mass despite a diminished loss of cardiomyocytes. This apparent discrepancy can be explained by the following considerations. First, it is unlikely that small changes in the density of cardiomyocytes, such as those observed in this work, may have a final significant effect on cardiac mass. Second, because cardiomyocytes may replicate in some conditions in the adult heart,21 further studies are required to assess whether losartan given long-term modifies the balance between cardiomyocyte apoptosis and replication in adult SHR. Third, it is possible that part of the cardiac weight decrease observed in losartan-treated SHR is due to a decrease in extracellular matrix. In experiments performed in our laboratory, we have found that the deposition of collagen fibers was significantly reduced in the left ventricle of adult SHR treated with losartan long-term versus untreated SHR.22
In summary, our results suggest that overexpression of the proapoptotic Bax-α protein present in the left ventricle of adult SHR is not due to p53-dependent upregulation of BAX gene transcription. Further studies are required to determine whether Bax-α overexpression can be the result of some kind of alteration in its posttranscriptional processing (ie, decreased degradation). In addition, our results suggest that the reduction in Bax-α levels observed in SHR that received long-term blockade of AT1 receptors can be related to inhibited transcription of the BAX gene. Whether this is the consequence of the interference with an angiotensin II–dependent pathway that regulates the transcription of BAX gene deserves further investigation.
- Received July 31, 1998.
- Revision received September 15, 1998.
- Accepted January 25, 1999.
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