Cardiovascular Research 71 (2006) 118 – 128 www.elsevier.com/locate/cardiores Doxazosin induces activation of GADD153 and cleavage of focal adhesion kinase in cardiomyocytes en route to apoptosis Downloaded from https://academic.oup.com/cardiovascres/article/71/1/118/269227 by guest on 01 October 2021 Sonia Eiras, Patricia Ferna´ndez, Roberto Pin˜eiro, Marı´a Jesu´s Iglesias, Jose´ Ramo´n Gonza´lez-Juanatey, Francisca Lago * Unidad de Investigacio´n del Servicio de Cardiologı´a, Hospital Clı´nico Universitario, Santiago de Compostela, Spain Received 14 October 2005; received in revised form 3 March 2006; accepted 17 March 2006 Available online 24 March 2006 Time for primary review 31 days Abstract Objective: The a1-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 a1-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 Amol/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/epbb, 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 a1-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/EBPh, 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 a1-adrenoreceptors. D 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved. Keywords: Apoptosis; Antihypertensive agents; Gene array analysis; Cell culture; Protein kinases 1. Introduction regression of left ventricular hypertrophy) [1], and are extensively used to alleviate the symptoms of benign Like other selective a1-adrenoceptor blockers, the well- prostatic hyperplasia (BPH). However, in the Antihyperten- known antihypertensive drugs doxazosin and prazosin lower sive and Lipid-Lowering Treatment to prevent Heart Attack blood pressure by dilating resistance and capacitance Trial (ALLHAT), the risk of congestive heart failure (CHF) arteries. They also have a number of other cardiovascularly was found to be twice as high with doxazosin as with the beneficial effects (improvement of lipid profile, fibrinolysis diuretic chlorthalidone, which forced the discontinuation of and insulin sensitivity, reduction of platelet aggregation, and the doxazosin arm of the study and suggested that doxazosin, and possibly a-blockers in general, should no longer be used as first-line antihypertensive therapy [2].A * Corresponding author. Laboratorio de Investigacio´n Nu´mero 1, A´ rea de possible explanation for the ALLHAT results may be Investigacio´n y Docencia, Hospital Clı´nico Universitario de Santiago de Compostela, Travesı´a Choupana s/n, 15706 Santiago de Compostela, Spain. pointed to by our group’s previous demonstration that Tel.: +34 981 950902; fax: +34 981 950757. doxazosin and prazosin induce apoptosis in cultured human E-mail address: [email protected] (F. Lago). and murine cardiomyocytes in a dose- and time-dependent 0008-6363/$ - see front matter D 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.cardiores.2006.03.014 S. Eiras et al. / Cardiovascular Research 71 (2006) 118–128 119 fashion [3], since heart failure is known to involve the to phosphorylate GADD153 [14]. Finally, the observation apoptosis of cardiomyocytes [4]. Doxazosin also induces the that doxazosin-treated cardiomyocytes underwent marked apoptosis of other cell types, including prostate cancer cells detachment from the culture plate during apoptosis drew [5]. our attention to FAK, since it is known that depression of Proteins that have previously been described as possibly FAK results in loss of cell adhesion and apoptosis [20]. being actively or passively involved in mediating the Moreover, FAK is depressed by doxazosin in other cell apoptotic effects of doxazosin, include transforming growth types [7,10]. factor h (TGF-h) [6], caspase 3 and focal adhesion kinase Our results suggest that doxazosin induces the apoptosis (FAK) [7], tumor necrosis factor a and reactive oxygen of cardiomyocytes via the ER pathway, with increased species [8], ether-a-go-go-gene-related potassium channels production of C/EBPh, GADD153 and DOC-1; likewise [9], and vascular endothelial growth factor (VEGF) and increases phosphorylation of the GADD153 activator p38 Downloaded from https://academic.oup.com/cardiovascres/article/71/1/118/269227 by guest on 01 October 2021 fibroblast growth factor 2 (FGF-2) [10]. MAPK; and induces first the phosphorylation, and then the Apoptosis can occur through either of two types of cleavage, of FAK. pathway: ‘‘extrinsic’’ pathways initiated by specific extra- cellular ‘‘death ligands’’, and ‘‘intrinsic’’ pathways initiated by intracellular or nonspecific extracellular stressors (e.g., in 2. Materials and methods the case of cardiomyocyte apoptosis, infectious pathogens, toxins, or morphological or functional cardiac abnormalities All products (doxazosin included) were from Sigma such as ischaemia, congenital malformations or contractile Chemical Co. (St. Louis, MO, USA) unless otherwise protein defects) [11,12]. Both types have been found to be stated. involved in myocardial pathologies [11]. The early, stress- signalling stages of the intrinsic pathways converge on the 2.1. Cells and doxazosin treatment induction of apoptogenic events in mitochondria and/or the endoplasmic reticulum (ER). Experimental and clinical HL-1 adult murine cardiomyocytes (a gift of Dr. W.C. evidence that cardiomyocytes of hypertrophic and failing Claycomb, Louisiana State University Medical Center, hearts exhibit ER stress [13], suggests that an intrinsic USA) were cultured as previously described [3]. After 24 h pathway via the ER may play a significant role in the (by which time confluence was approximately 80%), the elevated rate of cardiomyocyte apoptosis known to take medium was replaced with supplement-free ExCell 320, place in hearts with these conditions. and 12 h later the cells were used in experiments in which Our initial objective was to determine which type of non-control cells were treated with doxazosin at a pathway may be involved in doxazosin-induced cardio- concentration of 25 AM. This concentration is within the myocyte apoptosis. To this end we used a cDNA micro- range in which the drug induces cardiomyocyte apoptosis array to screen for genes with altered expression in in vitro (0.1–50 AM [3]), and is the least of the doxazosin-treated cardiomyocytes. This procedure detected concentrations that have been used in other studies of increased expression of ten genes, five of which are known the molecular mediators of its apoptotic effects [6–8].A to be directly related to ER stress [14–17] and none to concentration of 1 AM in vitro is considered [5,21] to mitochondrion-mediated apoptosis. In the work described ensure intracellular concentrations similar to those here we focused on two of the five genes related to ER achieved in vivo by therapeutic doses (in patients, serum stress: C/ebpb, which encodes for the eponymous protein doxazosin concentration reaches 0.122 AM with a single C/EBPh (CCAAT/enhancer-binding protein h), and 8 mg dose, and 0.244 AM with a 16 mg dose [22]). It gadd153, which encodes for growth-arrest- and DNA- should be noted, moreover, that in our experiments damage-inducible protein 153 [GADD153, also known as doxazosin, that is usually employed for long-term treat- CHOP (C/EBP-homologous protein), DDIT3 (DNA-dam- ment of hypertensive patients, caused apoptosis in vitro age-inducible transcript 3) or C/EBP~]. We performed within just an hour. experiments to confirm the increased transcription and Primary cultures of neonatal rat cardiomyocytes and translation of these two genes, and we also explored the human cardiomyocytes (the latter obtained from fragments effects of doxazosin on events downstream of GADD153 of right atrial appendage following the informed consent of and C/EBPh. Specifically, we investigated its effects on the the patients) were cultured as described previously [3] and expression of downstream-of-CHOP gene 1 (DOC-1) [18]. serodeprived for 12 h before being used in experiments as DOC-1 encodes for a stress-inducible form of carbonic above. All procedures with animals were carried out in anhydrase VI that, by catalysing the hydration of CO2 to accordance with NIH guidelines (Guide for the Care and H2CO3, may increase stress and favour apoptosis by Use of Laboratory Animals, NIH Publication No. 85-23, acidifying the intracellular medium [19]. We also investi-