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Cardiovascular Research 2006 71(1):118-128; doi:10.1016/j.cardiores.2006.03.014
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Copyright © 2006, European Society of Cardiology

Doxazosin induces activation of GADD153 and cleavage of focal adhesion kinase in cardiomyocytes en route to apoptosis

Sonia Eiras, Patricia Fernández, Roberto Piñeiro, María Jesús Iglesias, José Ramón González-Juanatey and Francisca Lago*

Unidad de Investigación del Servicio de Cardiología, Hospital Clínico Universitario, Santiago de Compostela, Spain

* Corresponding author. Laboratorio de Investigación Número 1, Área de Investigación y Docencia, Hospital Clínico Universitario de Santiago de Compostela, Travesía Choupana s/n, 15706 Santiago de Compostela, Spain. Tel.: +34 981 950902; fax: +34 981 950757. Email address: frlago{at}usc.es

Received 14 October 2005; revised 3 March 2006; accepted 17 March 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective The {alpha}1-adrenoreceptor blocker doxazosin, which in the ALLHAT trial was associated with a greater risk of heart failure than the diuretic chlorthalidone, induces the apoptosis of human and murine cardiomyocytes regardless of {alpha}1-adrenoreceptor blockade. We aimed to throw light on the mechanism of this process.

Methods Murine cardiomyocytes (HL-1) and primary cultures of human and neonatal rat cardiomyocytes were treated with 25 µmol/L doxazosin for between 0.5 and 48 h. cDNA microarray analysis, real-time RT-PCR, and Western blotting were performed to detect possible changes in gene expression and/or activation of proteins that could be involved in doxazosin-induced apoptosis.

Results Microarray analysis revealed changes in the expression of genes directly involved in the apoptotic end-stage of the cellular response to endoplasmic reticulum (ER) stress. Doxazosin considerably increased transcription and translation of gadd153, C/epbβ, and DOC-1 in cardiomyocytes as well as translocation of GADD153 to the nucleus, phosphorylation of p38 MAPK (a GADD153 activator), and the initial phosphorylation and subsequent cleavage of focal adhesion kinase (FAK). Experiments repeated following blockade of {alpha}1-adrenoreceptors showed no alteration of the above effects of doxazosin.

Conclusion Doxazosin induces the apoptosis of cardiomyocytes via the ER pathway, with increased production of C/EBPβ, GADD153 and DOC-1. Likewise it increases phosphorylation of the GADD153 activator p38 MAPK and induces first the phosphorylation, and then the cleavage, of FAK. These effects are not mediated by {alpha}1-adrenoreceptors.

KEYWORDS Apoptosis; Antihypertensive agents; Gene array analysis; Cell culture; Protein kinases


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Like other selective {alpha}1-adrenoceptor blockers, the well-known antihypertensive drugs doxazosin and prazosin lower blood pressure by dilating resistance and capacitance arteries. They also have a number of other cardiovascularly beneficial effects (improvement of lipid profile, fibrinolysis and insulin sensitivity, reduction of platelet aggregation, and regression of left ventricular hypertrophy) [1], and are extensively used to alleviate the symptoms of benign prostatic hyperplasia (BPH). However, in the Antihypertensive and Lipid-Lowering Treatment to prevent Heart Attack Trial (ALLHAT), the risk of congestive heart failure (CHF) was found to be twice as high with doxazosin as with the diuretic chlorthalidone, which forced the discontinuation of the doxazosin arm of the study and suggested that doxazosin, and possibly {alpha}-blockers in general, should no longer be used as first-line antihypertensive therapy [2]. A possible explanation for the ALLHAT results may be pointed to by our group's previous demonstration that doxazosin and prazosin induce apoptosis in cultured human and murine cardiomyocytes in a dose- and time-dependent fashion [3], since heart failure is known to involve the apoptosis of cardiomyocytes [4]. Doxazosin also induces the apoptosis of other cell types, including prostate cancer cells [5].

Proteins that have previously been described as possibly being actively or passively involved in mediating the apoptotic effects of doxazosin, include transforming growth factor β (TGF-β) [6], caspase 3 and focal adhesion kinase (FAK) [7], tumor necrosis factor {alpha} and reactive oxygen species [8], ether-a-go-go-gene-related potassium channels [9], and vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF-2) [10].

Apoptosis can occur through either of two types of pathway: "extrinsic" pathways initiated by specific extracellular "death ligands", and "intrinsic" pathways initiated by intracellular or nonspecific extracellular stressors (e.g., in the case of cardiomyocyte apoptosis, infectious pathogens, toxins, or morphological or functional cardiac abnormalities such as ischaemia, congenital malformations or contractile protein defects) [11,12]. Both types have been found to be involved in myocardial pathologies [11]. The early, stress-signalling stages of the intrinsic pathways converge on the induction of apoptogenic events in mitochondria and/or the endoplasmic reticulum (ER). Experimental and clinical evidence that cardiomyocytes of hypertrophic and failing hearts exhibit ER stress [13], suggests that an intrinsic pathway via the ER may play a significant role in the elevated rate of cardiomyocyte apoptosis known to take place in hearts with these conditions.

Our initial objective was to determine which type of pathway may be involved in doxazosin-induced cardiomyocyte apoptosis. To this end we used a cDNA microarray to screen for genes with altered expression in doxazosin-treated cardiomyocytes. This procedure detected increased expression of ten genes, five of which are known to be directly related to ER stress [14–17] and none to mitochondrion-mediated apoptosis. In the work described here we focused on two of the five genes related to ER stress: C/ebpβ, which encodes for the eponymous protein C/EBPβ (CCAAT/enhancer-binding protein β), and gadd153, which encodes for growth-arrest- and DNA-damage-inducible protein 153 [GADD153, also known as CHOP (C/EBP-homologous protein), DDIT3 (DNA-damage-inducible transcript 3) or C/EBP{zeta}]. We performed experiments to confirm the increased transcription and translation of these two genes, and we also explored the effects of doxazosin on events downstream of GADD153 and C/EBPβ. Specifically, we investigated its effects on the expression of downstream-of-CHOP gene 1 (DOC-1) [18]. DOC-1 encodes for a stress-inducible form of carbonic anhydrase VI that, by catalysing the hydration of CO2 to H2CO3, may increase stress and favour apoptosis by acidifying the intracellular medium [19]. We also investigated the possible involvement of the stress-signalling protein mitogen-activated protein kinase p38 (hereinafter "p38 MAPK" or simply "p38"), which has been reported to phosphorylate GADD153 [14]. Finally, the observation that doxazosin-treated cardiomyocytes underwent marked detachment from the culture plate during apoptosis drew our attention to FAK, since it is known that depression of FAK results in loss of cell adhesion and apoptosis [20]. Moreover, FAK is depressed by doxazosin in other cell types [7,10].

Our results suggest that doxazosin induces the apoptosis of cardiomyocytes via the ER pathway, with increased production of C/EBPβ, GADD153 and DOC-1; likewise increases phosphorylation of the GADD153 activator p38 MAPK; and induces first the phosphorylation, and then the cleavage, of FAK.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
All products (doxazosin included) were from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise stated.

2.1 Cells and doxazosin treatment
HL-1 adult murine cardiomyocytes (a gift of Dr. W.C. Claycomb, Louisiana State University Medical Center, USA) were cultured as previously described [3]. After 24 h (by which time confluence was approximately 80%), the medium was replaced with supplement-free ExCell 320, and 12 h later the cells were used in experiments in which non-control cells were treated with doxazosin at a concentration of 25 µM. This concentration is within the range in which the drug induces cardiomyocyte apoptosis in vitro (0.1–50 µM [3]), and is the least of the concentrations that have been used in other studies of the molecular mediators of its apoptotic effects [6–8]. A concentration of 1 µM in vitro is considered [5,21] to ensure intracellular concentrations similar to those achieved in vivo by therapeutic doses (in patients, serum doxazosin concentration reaches 0.122 µM with a single 8 mg dose, and 0.244 µM with a 16 mg dose [22]). It should be noted, moreover, that in our experiments doxazosin, that is usually employed for long-term treatment of hypertensive patients, caused apoptosis in vitro within just an hour.

Primary cultures of neonatal rat cardiomyocytes and human cardiomyocytes (the latter obtained from fragments of right atrial appendage following the informed consent of the patients) were cultured as described previously [3] and serodeprived for 12 h before being used in experiments as above. All procedures with animals were carried out in accordance with NIH guidelines (Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, revised 1996) and with the Declaration of Helsinki.

To note, in all the experiments, after the treatment with doxazosin, only cells adhering to the plate were collected and analysed.

2.2 Microarray analysis
Genes undergoing altered expression in doxazosin-treated murine cardiomyocytes were screened for using HL-1 cells. Doxazosin treatment lasted 3 h. Total RNA was extracted from the cells using an Rneasy Total RNA Extraction Kit (Quiagen K.K, London, UK), and its integrity was assessed with an AGILENT 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) using an RNA Nano LabChip (Agilent Technologies).

Double-stranded complementary DNA (cDNA) was synthesized from 8 µg of total RNA using a SuperScriptTM double-stranded cDNA synthesis kit (Invitrogen, San Diego, CA) with oligo(dT)24 primer linked to the T7 RNA polymerase promoter. Complementary RNA (cRNA) containing biotinylated cytidine triphosphate and uridine triphosphate was prepared therefrom using a Bioarray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY), and was then purified on an RNeasy column and fragmented by incubation at 94 °C for 35 min in 40 mmol/L Tris acetate (pH 8.1) containing 100 mmol/L potassium acetate and 30 mmol/L magnesium acetate. The integrity of cDNA, cRNA and fragmented cRNA was assessed by electrophoresis of samples on 1% agarose gels.

The expression of some 14,000 mouse genes was evaluated using Genechip MOE 430A mouse genome microarrays (Affymetrix Inc., Santa Clara, CA, USA). The scans were analyzed with the Affymetrix Microarray Suite 5.0 software. Intensities were scaled so that the average intensity in each array was 100, and each array was analyzed independently. Only genes with consistent mean intensities in three independent experiments were considered; those with low signal intensity, high background, and high variability among experiments were ignored. The change values were determined using the algorithm named "Signal Log Ratio" (Signal Log Ratio=Log2 Ratio). The Signal Log Ratio estimates the magnitude and direction of change of a transcript when two arrays are compared (experiment versus baseline). It is computed using a one-step Tukey's Biweight method by taking a mean of the log ratios of probe pair intensities across the two arrays. The Tukey's Biweight method gives an estimate of the amount of variation in the data, exactly as standard deviation measures the amount of variation for an average. A Signal Log Ratio of 1 indicates an increase of the transcript level by 2 fold, and – 1 indicates a decrease by 2 fold. In our microarray analysis, only fold changes of 2 or higher have been considered.

2.3 Real-time RT-PCR
Following two 2 h cycles of treatment with 10 IU/µL RNAase-free DNAase at 37 °C, reverse transcription of 1 µg of RNA was performed and the abundance of gadd153, Cebpβ or DOC-1 in the resulting cDNA was quantified relative to gapdh by real-time polymerase chain reaction using a SybrGreen kit (Roche Diagnostics, Barcelona, Spain) and the specific primers listed in Table 1. MgCl2 concentration was 4 mmol/L for gadd153 and Cebpβ, and 2 mmol/L for DOC-1. In each amplification cycle, denaturalization was performed for 10 min at 95 °C, annealing for 40 s at the temperatures listed in Table 1, and extension for 30 s at 72 °C; numbers of cycles are listed in Table 1. Baseline and threshold cycle (Ct) calculations were performed with Chromo 4 software (MJ Research, Inc, Reno, NV, USA); Ct was defined as the cycle in which the fluorescence reached 10 times the baseline signal, and this latter was defined as the fluorescence measured at the first minimum of the fluorescence curve (identified as the first maximum of its second derivative). The specificity of fluorescence was shown in each case by the presence of just a single peak in the melting curve. Fold change in gene expression relative to controls was calculated as 2-{delta}{delta}Ct, where {delta}Ct is [Ct(target gene) – Ct(gapdh)] and {delta}{delta}Ct is the {delta}Ct value for doxazosin-treated samples minus the {delta}Ct value for controls.


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  		Table 1 Primer sequences and real-time PCR conditions used to evaluate the expression of gadd153, C/ebpβ and DOC-1 relative to gapdh (m = mouse, r = rat, h = human)

 
2.4 Western blots of GADD153, C/EBPβ, p38 MAPK and FAK
Briefly, whole cell extracts of HL-1 cells treated with doxazosin for 0.5, 3, 6, 24 or 48 h were electrophoresed on 10–12% SDS-polyacrylamide gels, electrotransferred to PVDF membranes, reacted with rabbit polyclonal anti-GADD153, anti-C/EBPβ, anti-pp38-alpha, anti-p38-alpha, anti-pFAK and anti-FAK (all at dilutions of 1:1000; all from Santa Cruz Biotechnology, Delaware, CA, USA), and then exposed to peroxidase-conjugated goat anti-rabbit IgG or rabbit anti-goat IgG. Immunoreactive proteins were visualized using an enhanced chemiluminescence detection system (ECL, Amersham Pharmacia Ltd, London, UK) and quantified by densitometry using Typhoon (Amersham Pharmacia Ltd).

2.5 GADD153 translocation
HL-1 cardiomyocytes were treated with doxazosin for 0.5, 3 or 24 h and homogenized in a lysis buffer containing 0.5% NP-40, 10 µg/mL leupeptin, 2 µg/mL aprotinin and (mmol/L): HEPES buffer (pH 7.9), 10; EDTA, 1; EGTA, 1; KCl, 10; dithiothreitol, 1; and phenylmethylsulphonylfluoride, 1. Cytoplasmic and nuclear fractions were separated by centrifuging at 8000 x g for 15 min, and 40 µg of each fraction was subjected to Western blot with antibodies against GADD153 (1:1000) or histone H1 (as nuclear marker and gel loading control; 1:500) (Santa Cruz Biotechnology).

2.6 Statistical analysis
Data are presented as means±SEMs of at least three independent experiments. Differences between means were evaluated by Student's t test, a value of p<0.05 being considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Microarray analysis
As a first approximation to determine which among the apoptotic death pathways (death receptors, mitochondria, endoplasmic reticulum) might be most involved in doxazosin-induced cardiomyocyte apoptosis, we used microarray analysis. Table 2 shows the functional classification and fold change in expression of the mouse genes shown by microarray analysis to have altered expression in HL-1 cardiomyocytes after doxazosin treatment. Of the ten genes with increased expression, five are related to ER stress: gadd153, C/ebpβ, herpud1, atf3 and atf4. Those chosen for confirmatory RT-PCR experiments and further investigation were gadd153 and C/ebpβ. We could not detect with the microarray analysis changes in the gene expression of proteins involved in the mitochondrial death pathway after 3 h of doxazosin treatment of HL-1 cardiomyocytes.


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  		Table 2 HL-1 cardiomyocyte genes with significant microarray-evaluated change in expression (>2-fold) in response to 3 h exposure to 25 µmol/L doxazosin, grouped by gene product type or function (means of three independent experiments). Positive and negative values indicate increased and decreased expression, respectively

 
3.2 Induction of C/ebpβ and gadd153
To corroborate the doxazosin-induced increase in gadd153 and C/ebpβ expression indicated by the microarray analysis, real-time RT-PCR experiments were performed using HL-1 cells and primary cultures of cardiomyocytes.

Real-time PCR confirmed that 3 h treatment of HL-1 cardiomyocytes with 25 µmol/L doxazosin increased the expression of both C/ebpβ [(1.7±0.01)-fold; n=4, p<0.01] and gadd153 [(7.5±1.7)-fold; n=7, p<0.001] (Fig. 1A). Increased production of GADD153 and C/EBPβ was verified by quantitation of Western blots in arbitrary optical density units (Fig. 1C): GADD153 levels increased from 0.70±0.06 to 1.40±0.10 units, and C/EBPβ levels from 0.6±0.1 to 1.2±0.1 units (n=3, p<0.05).


Figure 1
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  		Fig. 1 Panel A: Fold changes in expression of gadd153 and C/ebpβ in HL-1 cells and primary cultures of neonatal rat cardiomyocytes (NRPC) treated with 25 µmol/L doxazosin for 3 h, as determined by real-time RT-PCR (means of at least 4 independent experiments; error bars indicate SEMs). Statistical significance with respect to controls: **, p<0.01; ***, p<0.001. Panel B: Expression of gadd153 in primary cultures of human cardiomyocytes treated with 25 µmol/L doxazosin for 3 h, as determined by RT-PCR. Panel C: Upper panel: Western blots showing increased levels of GADD153 and C/EBPβ in a typical experiment in which HL-1 cardiomyocytes were treated with 25 µmol/L doxazosin for 3 h. Lower panel: mean optical density (in arbitrary units) in three independent experiments of this kind; error bars indicate SEMs. *, p<0.05.

 
In real-time RT-PCR experiments, neonatal rat cells showed an (8.4±3.3)-fold increase in C/ebpβ expression and a (5.3±1.9)-fold increase in gadd153 expression (n=6, p<0.01 in both cases; Fig. 1A); while RT-PCR showed that doxazosin also induced a marked increase in gadd153 expression in human cardiomyocytes (Fig. 1B).

3.3 Phosphorylation of p38 MAPK
To determine if p38 MAPK activation was involved in doxazosin's proapoptotic effect on cardiomyocytes, we performed Western blot analysis. Doxazosin treatment of HL-1 cells significantly increased the level of phosphorylated p38 MAPK with respect to controls (1.3±0.1 vs. 0.5±0.1 after 0.5 h, 1.3±0.03 vs. 0.5±0.07 after 3 h, 1.2±0.10 vs. 0.7±0.07 after 6 h, 1.4±0.09 vs. 0.6±0.07 after 24 h; n=4, p<0.01 in all cases; Fig. 2).


Figure 2
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  		Fig. 2 A: Western blots showing the effect of 0.5–24 h 25 µmol/L doxazosin treatment on the levels of unphosphorylated p38{alpha} MAPK and phosphorylated p38{alpha} MAPK (pp38{alpha}) in HL-1 cardiomyocytes. B: Mean pp38{alpha} levels, in arbitrary optical density units (means of four independent experiments; error bars indicate SEMs; **, p<0.01).

 
3.4 GADD153 translocation
Western blot experiments were performed to investigate whether doxazosin might increase the ratio between nuclear and cytoplasmic levels of GADD153, which would be in keeping with its carrying the stress signal into the nucleus. GADD153 was significantly more abundant in the nucleus of doxazosin-treated HL-1 cells than in that of controls (values measured in arbitrary optical density units and corrected using nuclear Histone H1 as a standard or loading control) after 3 h (1.2±0.09 vs. 0.7±0.04) and after 24 h (1.6±0.4 vs. 0.6±0.2) (n=3, p<0.05 in both cases) (Fig. 3), thus showing that the amount of nuclear GADD153 increased progressively with the time of exposure to doxazosin.


Figure 3
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  		Fig. 3 A: Western blots of GADD153 in nuclear (n) and cytoplasmic (c) fractions of HL-1 cardiomyocytes treated with 25 µmol/L doxazosin for 0.5, 3 or 24 h, and in the corresponding controls (histone H1 was used as a nuclear marker and gel loading control). B: Dependence of nuclear GADD153 levels (in arbitrary optical density units) on time of exposure to doxazosin (means of three independent experiments; error bars indicate SEMs; *, p<0.05).

 
3.5 Induction of DOC-1 by doxazosin
We performed real-time RT-PCR to determine the possible GADD153 and CEBPβ-dependent induction of the novel carbonic anhydrase VI DOC-1 (recently identified as a novel stress-induced gene downstream of CHOP/GADD153 [18]) gene expression in HL-1 cardiomyocytes treated with doxazosin. In HL-1 cells, doxazosin increased the expression of DOC-1 (2.9±0.9)-fold after 6 h (n=4; p<0.05), (23.6±4.2)-fold after 24 h (n=4; p<0.001), and (80.5±28.3)-fold after 48 h (n=4; p<0.001) (Fig. 4). In primary cultures of neonatal rat cardiomyocytes, 24 h doxazosin treatment increased DOC-1 expression (8.5±3.2)-fold (n=3, p<0.05) (Fig. 4).


Figure 4
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  		Fig. 4 Fold change in expression of DOC-1 in HL-1 cells and primary cultures of neonatal rat cardiomyocytes (NRPC) exposed to 25 µmol/L doxazosin for 6, 24 or 48 h (HL-1) or 24 h (NRPC), as determined by real-time RT-PCR (means of at least 3 independent experiments). Statistical significance with respect to controls: *, p<0.05; ***, p<0.001.

 
3.6 Cleavage of FAK
To investigate the possible involvement of FAK in doxazosin-induced cardiomyocyte apoptosis, we analyzed, using Western blot, both FAK phosphorylation and FAK total protein expression. Both of them are relevant in order to determine the possible involvement of this kinase in doxazosin's proapoptotic effects on cardiomyocytes because: a) attenuation of FAK activation and function results in apoptosis [20], b) caspases cleave FAK during apoptosis [23] and c) doxazosin decreases FAK levels in other cell types [7,10].

Treating HL-1 cells with doxazosin for 0.5 or 3 h increased the abundance of phosphorylated FAK (pFAK) relative to controls (1.1±0.05 vs. 0.8±0.04 after 30 min, 1.1±0.06 vs. 0.8±0.06 after 3 h; n=4; p<0.01 in both cases), but after 6 h there was no difference between the pFAK levels of controls and doxazosin-treated cells (Fig. 5A and B). Total FAK levels at this time were unaltered by doxazosin treatment (Fig. 5A). After 24 h, however, the cleavage of FAK in doxazosin-treated cells was suggested by a fall in the levels of both FAK and phosphorylated FAK and the appearance of a fragment of lower molecular weight that is not present in control cells or cells treated with doxazosin for 3 h or less (Fig. 5A).


Figure 5
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  		Fig. 5 A: Western blots showing the effects of 0.5–48 h doxazosin treatment on phosphorylated FAK (pFAK) and total FAK levels in HL-1 cardiomyocytes. Duplicates confirm the cleavage of FAK after 24 or 48 h exposure. B: pFAK levels, in arbitrary optical density units (means of four independent experiments; error bars indicate SEMs; **, p<0.01). C: Photomicrographs (200 x) of HL-1 cardiomyocytes after 6, 24 or 48 h, with or without 25 µmol/L doxazosin treatment, showing marked detachment from the plate during doxazosin-induced apoptosis.

 
3.7 Independence of {alpha}1-adrenoceptor blockade
To confirm that the intracellular signalling changes described above were independent of {alpha}1-adrenoceptor blockade (like the overall apoptotic effect of doxazosin [3]), we repeated some experiments following 4 h exposure of the cells to the irreversible {alpha}-blocker phenoxybenzamine at a concentration of 1 µmol/L. In HL-1 cells, this exposure prevented neither the doxazosin-induced cleavage of FAK (Fig. 6A) nor the doxazosin-induced increase in the expression of DOC-1 [(19.2±4.1)-fold in the presence of both doxazosin and phenoxybenzamine, and (16.9±9.7)-fold in the presence of doxazosin alone (n=4, p>0.05, not significant) (Fig. 6B).


Figure 6
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  		Fig. 6 A: Western blots showing that 4 h prior exposure of HL-1 cardiomyocytes to the irreversible {alpha}1-adrenoceptor blocker phenoxybenzamine did not alter the effects of 24 h treatment with 25 µmol/L doxazosin on FAK cleavage. B: Fold change in expression of DOC-1 induced in HL-1 cardiomyocytes by 24 h treatment with 25 µmol/L doxazosin, with or without 4 h prior exposure to the irreversible {alpha}1-adrenoceptor blocker phenoxybenzamine, as determined by real-time RT-PCR (means of 4 independent experiments). Statistical significance with respect to controls: *, p<0.05; **, p<0.01.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In a previous paper we reported that doxazosin induces apoptosis in human and murine cultured cardiomyocytes by a mechanism that is independent of {alpha}-adrenoceptor blockade [3]. Since apoptosis is known to occur in the early stages of myocardial dysfunction and to contribute to progressive cardiomyocyte loss in heart failure [4], we suggested that this finding might help to explain why in ALLHAT [2] patients treated with doxazosin had more adverse cardiovascular events (particularly heart failure) than those treated with the diuretic chlorthalidone (which has no effect on the viability of isolated cardiomyocytes; results not shown). Doxazosin also induces apoptosis or cell cycle arrest in other cell types: through mechanisms that are independent of its {alpha}-adrenergic activity, it halts the cell cycle of cells of human smooth muscle [21,24] and bladder [25]; inhibits the adhesion and migration of, and invasion by, human vascular endothelial cells [10]; and induces the apoptosis of skin fibroblasts [8], normal and cancerous prostate cells [5,26], and non-prostatic cancer cell lines [8]. The unexpected adrenoreceptor-independent activities of doxazosin may be relevant to its long-term effects in patients treated with this drug.

There is evidence that the molecular mechanisms mediating doxazosin-induced apoptosis in prostate cancer cells involve TGF-β-1 signaling and I{kappa}B induction [6], whereas in normal and BPH-affected prostate stroma cells it seems that the mechanism involves, not this pathway, but an increase in reactive oxygen species associated with TNF-{alpha} signaling [8]. The objective of the work described in this paper was to gain insight into the proapoptotic mechanisms triggered by doxazosin in cultured cardiomyocytes. Although most of our experiments were, for convenience, performed on HL-1 cells (a cell line derived from murine atrial cardiomyocytes that maintains a heart-specific phenotype [27] and normally features all the cell-surface and intracellular components required for an {alpha}1-adrenergic response [28]), we also replicated some of our HL-1 results using primary cultures of neonatal rat or human cardiomyocytes.

As a first step, microarray analysis was used to identify those cardiomyocyte genes, the expression of which was significantly affected by doxazosin treatment (Table 2). In particular, we wished to determine whether the affected genes indicated that apoptosis proceeded via the extrinsic pathway, the intrinsic pathway, or both; and, if the intrinsic pathway were affected, whether the mitochondrial branch, the ER branch, or both were involved. We observed no alteration of directly apoptosis-related genes that were not related to ER stress. However, we cannot exclude the possibility that non-ER apoptosis paths might be activated by exposure to doxazosin for longer than the 3 h used in our microarray experiments.

Among the genes found to show depressed expression, many, such as clasp2 and elav14, are related to the cytoskeleton [29,30]. This is interesting, in that alterations of microtubules and other cytoskeletal structures have been observed in experimental studies of heart failure [31], and these gene suppression effects of doxazosin are accordingly being investigated in more detail in our laboratory. In the remainder of the present paper we discuss our results on processes associated with apoptosis-related genes that underwent increased expression following doxazosin treatment.

Five of the ten genes with a 2-fold or greater increase in expression following 3 h doxazosin treatment are directly involved in the ER stress response, during which they are upregulated. One, herpud1, encodes for a protein with an N-terminal ubiquitin-like domain located in the ER membrane, and upregulated during endoplasmic reticulum stress [17]. The other four, gadd153, C/ebpβ, atf3 and atf4, encode for bZip transcription factors: GADD153 dimerizes with C/EBPβ to alter the expression of numerous genes as part of the final, apoptotic stage of the ER stress response, and its expression is regulated by ATF3 (activating transcription factor 3) and by ATF4 [14] (to note, ATF4-C/EBPβ heterodimers induce the expression of gadd153 [16]). We focused our attention on the two that lie furthest downstream in the apoptotic process, and for which available information, though incomplete, is most abundant: gadd153 and C/ebpβ.

GADD153 is a small nuclear protein that is ubiquitous at very low levels in proliferating cells and is massively induced by growth arrest signals and toxic stress, especially ER stress. Specifically, GADD153 constitutes a major component of the final stage of the response to ER stress, in which cell cycle arrest or apoptosis is triggered [14]. It heterodimerizes with C/EBPβ and other members of the C/EBP family, and also with ATF4 and other bZip proteins, and these interactions modify the balance between proapoptotic and antiapoptotic effects [16]. In particular, the GADD153-C/EBP heterodimer does not act as a transactivator to increase the expression of classical C/EBP-activated genes, but does activate other target genes [14]. Importantly, GADD153 is induced in failing hearts [13], and it has been found that prolonged ER stress may contribute to cardiomyocyte apoptosis during the progression from cardiac hypertrophy to heart failure [13]. In this work, our microarray results showing increased expression of both gadd153 and C/ebpβ following doxazosin treatment were confirmed by real-time PCR analyses, and the corresponding increase in translation was confirmed by Western blots.

GADD153-mediated growth arrest and apoptosis has also been reported to be an effect of the antitumoral quinazoline gefitinib [32]. Since doxazosin and prazosin are also quinazoline derivatives, the present results, and the fact that prazosin, like doxazosin, induces apoptosis in cardiomyocytes, strongly suggest that the quinazoline structure is central to these activities.

Although the mechanisms by which GADD153 promotes cell death are far from fully elucidated, Wang et al. [18] found three target genes that they named DOCs (for downstream of CHOP). DOC-1 encodes for a stress-inducible form of carbonic anhydrase VI that reversibly catalyses the hydration of CO2 to H2CO3, and which may thereby favour apoptosis by increasing stress through acidification of the intracellular medium [19]. DOC-1 is upregulated by GADD153 (in keeping with which, a binding site for GADD153-C/EBP heterodimers has been identified in the DOC-1 promoter) [18], and its stress-induced expression is in fact dependent on both GADD153 and C/EBPβ [19]. Our finding in this work that doxazosin progressively increased DOC-1 expression in cardiomyocytes from 3-fold after 6 h exposure to 80-fold after 2 days is therefore consistent with the observed increases in GADD153 and C/EBPβ after 3 h exposure. It may be noted that increased expression of DOC-1 has been reported to be followed by the corresponding increase in translation [18].

GADD153 heterodimerizes when phosphorylated by activated p38 MAPK [14], which could therefore regulate apoptosis. In fact, there is evidence that p38 MAPK does regulate apoptosis in cardiomyocytes [12]. Activation of cardiomyocyte p38 MAPK is known to occur in response to mechanical stress [33] and to be increased in rabbits with pacing-induced heart failure [34], while inhibition of p38 MAPK improves cardiac function and attenuates left ventricular remodelling following myocardial infarction in rats [35]. In this work we found that 3 h doxazosin treatment increased both p38 MAPK phosphorylation and the translocation of GADD153 to the nucleus, suggesting that doxazosin-induced phosphorylation of p38 MAPK may be responsible for the activation of GADD153 and its translocation to the cell nucleus. However, it remains to be determined how doxazosin may induce the phosphorylation of p38.

As noted in the Introduction, we observed that cardiomyocytes treated with doxazosin underwent marked detachment from the plate during apoptosis (Fig. 5C). More specifically, exposure for more than 6 h induced progressive rounding and detachment from the fibronectin matrix. Such behaviour may be relevant to the apoptotic cardiac effects of doxazosin, because cardiomyocyte detachment from the extracellular matrix occurs early in left ventricular overload and contributes to the progression towards heart failure [36,37]. We accordingly investigated the effects of doxazosin on focal adhesion kinase (FAK), since it has been reported that in prostate cancer cells [7] and human vascular endothelial cells [10] doxazosin induces apoptosis by reducing FAK levels, and that cleavage of FAK accompanies cardiomyocyte apoptosis [38]. FAK is a non-receptor tyrosine kinase that plays a key role in the transmission of signals from the extracellular matrix to the cytoplasm, and hence in cell proliferation, migration and survival [20]. Phosphorylation of FAK forms part of the initial response of cardiomyocytes subjected to acute external stress [39,40], and it appears to be via the integrin-FAK-Src-Ras pathway that p38 MAPK is activated in the cardiomyocytes of hearts with stress-induced hypertrophy [33]. However, attenuation of FAK activation and function results in loss of cell adhesion and apoptosis [20], and, vice versa, caspases (which are generated during apoptosis) reduce functional FAK levels by cleaving FAK, which also generates FAK fragments that compete with FAK for phosphorylation [23].

The effects of doxazosin on FAK in the present study were in keeping with the above observations: short exposure (30 min to 3 h) induced the phosphorylation of FAK (possibly as a FAK-mediated recruitment of adaptive or anti-stress mechanisms) but prolonged exposure (longer than 6 h) resulted in FAK cleavage. A similar pattern has been reported in human glioblastoma cells under oxidative stress [41].

Finally, just as the induction of apoptosis by doxazosin in human and murine cultured cardiomyocytes is independent of {alpha}-adrenoreceptor blockade [3], we found in the present work that doxazosin-induced expression of DOC-1 and cleavage of FAK are also unaffected by prior irreversible blockade of {alpha}-adrenoreceptors by phenoxybenzamine. This shows that the mechanism by which doxazosin induces apoptosis in cardiomyocytes does not involve {alpha}-adrenoreceptors.

In conclusion, we have found that doxazosin treatment of cardiomyocytes leads to activation of the apoptotic stage of the ER stress response, and to initial phosphorylation and subsequent cleavage of FAK, and that both these effects are independent of {alpha}-adrenoreceptors. Additional research is necessary in order to clarify the possible link between the ER stress response and FAK cleavage in doxazosin-induced apoptosis. Our findings throw light on the cardiac effects of doxazosin, and suggest that the doxazosin-induced apoptosis of cardiomyocytes may have contributed to the incidence of heart failure in the ALLHAT study having been higher in patients treated with doxazosin than in those treated with chlorthalidone.


   Acknowledgements
 
This work was supported by the Spanish Ministry of Health through Health Research Fund (FIS) contract 03/0115. Francisca Lago's research is supported by the Spanish Ministry of Health and the Galician Health Service (SERGAS) under FIS contract 99/3040.


   Notes
 
Time for primary review 31 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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