Pharmacological Research 95–96 (2015) 102–110 Contents lists available at ScienceDirect Pharmacological Research j ournal homepage: www.elsevier.com/locate/yphrs ␤ ␦ Activation of PPAR / protects cardiac myocytes from oxidative stress-induced apoptosis by suppressing generation of reactive oxygen/nitrogen species and expression of matrix metalloproteinases a b,d b b c,d Eleftheria Barlaka , Anikó Görbe , Renáta Gáspár , János Pálóczi , Péter Ferdinandy , a,∗ Antigone Lazou a Laboratory of Animal Physiology, School of Biology, Aristotle University of Thessaloniki, Greece b Cardiovascular Research Group, Department of Biochemistry, University of Szeged, Hungary c Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary d Pharmahungary Group, Szeged, Hungary a r a t i c l e i n f o b s t r a c t Article history: Heart failure still remains one of the leading causes of morbidity and mortality worldwide. A major Received 2 November 2014 contributing factor is reactive oxygen/nitrogen species (RONS) overproduction which is associated with Received in revised form 15 March 2015 cardiac remodeling partly through cardiomyocyte apoptosis. Peroxisome proliferator-activated receptors Accepted 15 March 2015 (PPARs) are ligand-activated transcription factors that belong to the nuclear receptor superfamily and Available online 28 March 2015 have been implicated in cardioprotection. However, the molecular mechanisms are largely unexplored. In this study we sought to investigate the potential beneficial effects evoked by activation of PPAR␤/␦ under Keywords: the setting of oxidative stress induced by H2O2 in adult rat cardiac myocytes. The selective PPAR␤/␦ ago- Cardiac myocytes nist GW0742 inhibited the H2O2-induced apoptosis and increased cell viability. In addition, generation Oxidative stress ␤ ␦ Apoptosis of RONS was attenuated in cardiac myocytes in the presence of PPAR / agonist. These effects were abol- ␤ ␦ ␤ ␦ Matrix metalloproteinases ished in the presence of the PPAR / antagonist indicating that the effect was through PPAR / receptor PPAR␤/␦ activation. Treatment with PPAR␤/␦ agonist was also associated with attenuation of caspase-3 and PARP Caspase 3 cleavage, upregulation of anti-apoptotic Bcl-2 and concomitant downregulation of pro-apoptotic Bax. Bcl-2 In addition, activation of PPAR␤/␦ inhibited the oxidative-stress-induced MMP-2 and MMP-9 mRNA upregulation. It is concluded that PPAR␤/␦ activation exerts a cytoprotective effect in adult rat cardiac Chemical compounds studied in this article: myocytes subjected to oxidative stress via inhibition of oxidative stress, MMP expression, and apopto- Hydrogen peroxide (PubChem CID: 784) sis. Our data suggest that the novel connection between PPAR signaling and MMP down-regulation in GW0742 (PubChem CID: 9934458) GSK0660 (PubChem CID: 46233311) cardiac myocytes might represent a new target for the management of oxidative stress-induced cardiac dysfunction. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction [1,2]. Excessive generation of reactive oxygen/nitrogen species (RONS) causes cellular dysfunction, protein and lipid peroxida- Cardiovascular diseases still remain one of the leading causes tion, DNA damage, activation of matrix metalloproteinases (MMPs) of mortality and morbidity worldwide. Over the past years, clinical and can lead to irreversible cell damage and cell death. The con- and experimental studies have provided substantial evidence that trol of myocyte loss through suppression of cell death pathways oxidative stress, defined as an imbalance between free radical pro- represents a promising strategy to prevent cardiac diseases. Ter- duction and their detoxification by antioxidants, is an important minally differentiated adult cardiac myocytes die primarily by contributing factor in the pathogenesis of a variety of cardiovas- apoptosis or necrosis [3]. Mitochondrial damage, cytochrome c cular diseases including ischemic heart disease and heart failure release and activation of cysteine proteases caspase 9 and 3 have been implicated in apoptotic pathways. The Bcl-2 family proteins, which contain both pro-apoptotic and anti-apoptotic members, ∗ have been recognized as important modulators of cardiac myocyte Corresponding author at: Laboratory of Animal Physiology, School of Biology, apoptosis as in other cells [4,5]. Bcl-2 family proteins act at the Aristotle University of Thessaloniki, Thessaloniki 54124, Greece. mitochondria either as heterodimers or as homodimers to regu- Tel.: +30 2310 998381; fax: +30 2310 998269. E-mail address: [email protected] (A. Lazou). late cytochrome c whereas the dynamic equilibrium between such http://dx.doi.org/10.1016/j.phrs.2015.03.008 1043-6618/© 2015 Elsevier Ltd. All rights reserved. E. Barlaka et al. / Pharmacological Research 95–96 (2015) 102–110 103 complexes appears to determine the predisposition to apoptosis [24]. Cells were finally resuspended in incubation medium, in 2+ [6]. which added Ca was gradually increased to 1 mM. Prepara- The zinc-dependent endopeptidases, matrix metalloproteinases tions were considered satisfactory only if the yield of rod-shaped (MMPs), which have been known for their ability to proteolyse cells was more than 70%. Experiments were performed 30 min extracellular matrix proteins, play an important role in tissue after dissociation. Cardiac myocytes were either left untreated, or remodeling associated with various physiological and pathological they were pre-treated with the PPAR␤/␦ agonist GW0742 (50 nM) processes including chronic heart failure [7]. It is now recognized (Sigma–Aldrich Chemie GmbH, Taufkirchen, Germany) for 10 min that MMPs can also have non-matrix related intracellular protein before exposure to H2O2 (0.1 mM). When the PPAR␤/␦ antago- targets and can regulate diverse cellular functions contributing to nist GSK0660 (0.5 ␮M) (Tocris Bioscience, Bristol, UK) was used, cardiac dysfunction, such as oxidative stress-induced cell death it was added 10 min before the addition of the agonist GW0742. [8–10]. Thus, MMPs are considered to be viable drug targets in the The concentration of H2O2 to induce apoptosis was chosen accord- therapy of cardiac pathologies. ing to our previous studies [24–26]. The optimal concentration of ␤ ␦ Peroxisome proliferator activated receptors (PPARs) are ligand PPAR / agonist and antagonist were chosen according to prelim- activated transcription factors that belong to the superfamily of inary experiments. Preliminary experiments also established that nuclear hormone receptors and include 3 members (␣, ␤/␦, ␥) with the concentrations of the agonist and antagonist used did not affect tissue specific but overlapping expression patterns. Upon activation the viability of cardiac myocytes. The experimental protocols are by their natural or pharmaceutical specific ligands, PPARs regulate summarized in Fig. 1. the expression of genes mostly involved in lipid and carbohydrate metabolism, energy production and inflammation [11]. It has been 2.3. Determination of cell viability also shown that PPARs may play a role in regulating transcriptional expression of antioxidants, such as Cu/Zn-superoxide dismutase Cell viability was determined using the 3-(4,5-dimethylthiazol- (SOD1), manganese superoxide dismutase (SOD2), and catalase 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay which [12,13]. Furthermore, emerging evidence indicates that activation assesses mitochondrial activity in living cells. Cardiac myocytes 5 of PPARs, in particular PPAR␣ or PPAR␥, may exert cardioprotec- (10 /sample) were either left untreated or they were pretreated ␤ ␦ tive effects beyond their metabolic effects, decreasing infarction with PPAR / agonist and/or antagonist (10 min), followed by after I/R and preventing cardiac remodeling and failure [14–17]. exposure to H2O2 for 3 h. Tetrazolium bromide at a concentration However, the mechanisms underlying these protective effects are 0.1 mg/ml was added 10 min before the end of incubation. At the largely unexplored, and it is still a matter of debate whether car- end of incubation, 1 ml of ethanol was added for 10 min to dissolve dioprotection is attributable to the modulation of cardiac energy formazan precipitate. Absorbance was measured at 570 nm using a metabolism or to antioxidative and anti-inflammatory effects of microplate autoreader. Cell viability was calculated as percentage PPARs [18,19]. Although PPAR␤/␦ is the predominant subtype in of control OD. Negative controls were used to ensure that the the heart, it is the least studied member of the PPAR family. Car- drugs used for the cell treatments did not interfere on their own diac PPAR␤/␦ deletion in mice resulted in depressed bioenergetics, with the formation of formazan. cardiac hypertrophy, and congestive heart failure [20] while it has been shown that PPAR␤/␦ is essential for the adult heart to maintain 2.4. Measurement of lactate dehydrogenase (LDH) mitochondrial capacity and oxidative metabolism [21]. Given the emerging role of PPAR␤/␦ in regulating mitochondrial LDH release into the cell culture medium was used as an biogenesis and metabolism, we hypothesized that PPAR␤/␦ acti- indicator of cell death. LDH activity was determined by spectro- vation may ameliorate oxidative stress-induced cardiac myocyte photometric analysis measuring the decrease in absorbance at damage. We used the highly selective and potent PPAR␤/␦ agonist 340 nm resulting from the oxidation of NADH. At the end of treat- GW0742 [22] and a specific PPAR␤/␦ antagonist, GSK0660