Eur Respir J 2012; 40: 1420–1429 DOI: 10.1183/09031936.00011511 CopyrightßERS 2012 Dehydroepiandrosterone reverses chronic hypoxia/reoxygenation-induced right ventricular dysfunction in rats Eric Dumas de La Roque*,#,", Nade`ge Bellance#, Rodrigue Rossignol#, Hugues Begueret*,#,", Marie Billaud*,#, Pierre Dos Santos#,", Thomas Ducret*,#, Roger Marthan*,#,", Diana Dahan*,#, David Ramos-Barbo´n+,O´ scar Amor-Carro+, Jean-Pierre Savineau*,#," and Michael Fayon*,#," ABSTRACT: Dehydroepiandrosterone (DHEA) prevents chronic hypoxia-induced pulmonary AFFILIATIONS hypertension and associated right ventricle dysfunction in rats. In this animal model, *Univ-Bordeaux, Centre de Recherche Cardio-Thoracique U reoxygenation following hypoxia reverses pulmonary hypertension but not right ventricle 1045, dysfunction. We thus studied the effect of DHEA on the right ventricle after reoxygenation, i.e. #INSERM U 1045, U 688 and U after a normoxic recovery phase secondary to chronic hypoxia in rats. 1034, and " Right ventricle function was assessed in vivo by Doppler echocardiography and in vitro by the CHU de Bordeaux, Bordeaux, France. isolated perfused heart technique in three groups of animals: control, recovery (21 days of +Respiratory Dept, Hospital de la -1 hypoxia followed by 21 days of normoxia) and recovery DHEA (30 mg?kg every 2 days during Santa Creu i Sant Pau, Universitat the recovery phase). Right ventricle tissue was assessed by optical and electron microscopy. Auto`noma de Barcelona, Barcelona, DHEA abolished right ventricle diastolic dysfunction, as the echographic E wave remained Spain. -1 ¡SD ¡ ¡ close to that of controls (mean 76.5 2.4 and 79.7 1.7 cm?s , respectively), whereas it was CORRESPONDENCE diminished to 40.3¡3.7 in the recovery group. DHEA also abolished right ventricle systolic E. Dumas de la Roque dysfunction, as shown by the inhibition of the increase in the slope of the pressure–volume curve Hoˆpital Pellegrin Enfants in isolated heart. The DHEA effect was related to cardiac myocytes proliferation. CHU de Bordeaux Place Ame´lie Raba Le´on In conclusion, DHEA prevents right ventricle dysfunction in this animal model by preventing 33076 Bordeaux Cedex cardiomyocyte alteration. France E-mail: [email protected] KEYWORDS: Cardiac myocyte, chronic hypoxia, dehydroepiandrosterone, mitochondria, Received: pulmonary hypertension Jan 21 2011 Accepted after revision: March 26 2012 ypical chronic lung hypoxaemic diseases, We believe that such an experimental model may First published online: such as chronic obstructive pulmonary be relevant in patients suffering from COPD, as April 20 2012 T disease (COPD) [1], can lead to pulmonary they may alternate between successive severe hypertension (PH) and ultimately to right ven- hypoxic episodes related to exacerbations [10] tricular failure [2–4]. Rodents exposed to chronic and fewer hypoxic episodes related their oxygen hypoxia, either normo- or hyperbaric, consistute therapy [11]. a classical animal model used to investigate As mentioned, DHEA has been studied in animal mechanisms as well as therapeutic targets in this [7, 12] and human PH [13, 14]. More recently, it pulmonary vascular disease [5, 6]. In a rat model has been shown that DHEA can modulate cardiac of hypobaric chronic hypoxia, we have pre- function. DHEA reverses left ventricular stiffness viously demonstrated that dehydroepiandros- and fibrosis, which typically accompanies ageing terone (DHEA), an adrenal steroid, prevents in mice [15], and decreases the production of type I and decreases hypoxic PH and associated right collagen by cardiac fibroblasts [16]. DHEA can ventricle hypertrophy [7]. In the same animal also modulate oxidative stress in the rat heart [17]. model, reoxygenation following hypoxia, i.e. a normoxic recovery phase of 21 days of normoxia The purpose of this study was thus to evaluate secondary to a chronic hypoxic period of 21 days, the effect of DHEA on the right ventricle in this also reverses PH as it normalises pulmonary chronic hypoxia model followed by reoxygena- pressure and antagonises vascular remodelling tion, i.e. followed by a normoxic recovery phase European Respiratory Journal [8]. However, such normoxic recovery period in rats. We examined the additional effect of Print ISSN 0903-1936 does not correct right ventricular dysfunction [9]. DHEA on reoxygenation of the right ventricular Online ISSN 1399-3003 1420 VOLUME 40 NUMBER 6 EUROPEAN RESPIRATORY JOURNAL E. DUMAS DE LA ROQUE ET AL. PULMONARY VASCULAR DISEASE structure and function both in vivo and ex vivo. The signalling synthase were obtained from Mitoscience (Eugene, OR, USA). pathway activated by DHEA was also determined. The caspase 3, peroxisome proliferator-activated receptor c coactivator 1a (PGC1a), manganese superoxide dismutase METHODS (MnSOD) and endothelial nitric oxide synthase (eNOS) Animal model and DHEA treatment antibodies were obtained from Santa Cruz Biothechnology The investigation was carried out in agreement with the Guide Inc. (Santa Cruz, CA, USA). The antiphospho-cyclic adenosine for the Care and Use of Laboratory Animals [18] and European monophosphate response element binding (CREB) and anti- Directives [19]. Adult male Wistar rats (220–240 g) were proliferating cell nuclear antigen (PCNA) were obtained from randomised into three groups. The first group of rats was Abcam Biochemicals (Bristol, UK). kept in a hypobaric chamber for 21 days in order to induce a chronic hypoxic PH as previously described [7], followed by Cell proliferation and apoptosis normoxia for 21 days (recovery group); the second group of Tissues were fixed in phosphate-buffered 4% formaldehyde and rats was also kept in a hypobaric chamber for 21 days followed paraffin embedded. Myocyte cross-sectional areas were mea- by normoxia for 21 days while being treated with DHEA sured using an image-based quantitative analysis system (30 mg?kg-1 every 2 days) during the recovery period (recovery– (NanoZoomzer Digital Pathology Image software; Hamamatsu DHEA group). These two groups were compared with the Photonics France, Massy, France). The outlines of 50 myocytes control group, which was kept at normal atmospheric pressure. were traced in each animal studied. Cell proliferation and apoptosis detection were performed as previously described Assessment of heart tissue weight ratio and [24]. Proliferating cardiomyocytes were identified on the basis echocardiography of PCNA co-immunostaining with desmin. The anti-PCNA The right ventricle hypertrophy index was calculated as the monoclonal antibody was obtained from Calbiochem/Merck ratio of the weight of the right ventricle to that of the left (Darmstadt, Germany). Apoptotic cardiomyocytes were detected ventricle plus the septum; the right ventricle mass index was using the ApopTag peroxidase kit (Millipore, Billerica, MA, USA) calculated as the ratio of right ventricle weight to bodyweight according to the manufacturer’s instructions. Quantitative as previously described [20]. Echocardiography Doppler morphology was blindly performed on coded specimens. The imaging was performed as previously described [9]. The right tissue sections were scanned through systematic random field ventricle shortening fraction was then estimated as ((end- sampling using 0.143-mm2 calibrated fields captured with a diastolic diameter - end-systolic diameter)6100)/end-diastolic bright-field microscope and digital image acquisition system diameter. The right ventricle diastolic function was estimated by (Olympus, Tokyo, Japan). 11–24 (mean¡SEM 18.1¡1.2) fields per the E-wave and the E/A peak velocity ratio. Pulse-wave specimen were sampled through the complete tissue sections, Doppler (A and E wave) of the tricuspid valve was recorded and the numbers of PCNA+ cell nuclei were referenced to the in an apical four-chamber view. sampled surface area. Lung tissue sections from a murine asthma model published elsewhere [25] were entered in the staining Isolated and perfused heart technique batches as terminal deoxynucleotidyl transferase deoxyuridine The isolated and perfused heart technique was performed as triphosphate nick end labelling (TUNEL)+ control specimens. previously described [9]. Data analysis Histological measurements Data values are expressed as the mean¡SEM. Statistical analyses Optical and electronmicroscopy were performed as previously were performed using the NCSS 5.0 software (NCSS, Kaysville, described [7]. The right ventricular tissue was fixed in 4% (w/v) UT, USA), while intergroup differences were assessed by a formaldehyde and 3-mm thick sections were stained with Kruskal–Wallis ANOVA, as appropriate. In experiments with haematoxylin, eosin and saffron. The number (N) of myocytes comparison of two conditions, as unpaired Mann–Whitney test per ventricle was estimated according the following equation: was used. n refers to the animal sample size in the relevant N5(myocyte volume fraction6ventricular volume)/median experiment. Differences in the data were considered significant myocyte volume [21]. The myocyte volume fraction used was when p,0.05. 75%, and ventricular volume was calculated as the ventricular weight divided by the ventricular specific gravity (1.06 g?cm-3). RESULTS DHEA improves right ventricular dysfunction secondary to Reactive oxygen species measurement the normoxic recovery period Reactive oxygen species were measured by means of the In vivo, the right ventricular systolic (shortening fraction) and electron paramagnetic resonance technique on right ventricle diastolic (tricuspid