ARTICLE Modulation of mitochondrial respiratory function and ROS production by novel benzopyran analogues 1 Alexandra Petruş, Oana M. Duicu, Adrian Sturza, Lavinia Noveanu, Loránd Kiss, Maria Da ˘ nila ˘, István Baczkó, Danina M. Muntean, and Norbert Jost Abstract: A substantial body of evidence indicates that pharmacological activation of mitochondrial ATP-sensitive potassium channels (mK ATP ) in the heart is protective in conditions associated with ischemia/reperfusion injury. Several mechanisms have been postulated to be responsible for cardioprotection, including the modulation of mitochondrial respiratory function. The aim of the present study was to characterize the dose-dependent effects of novel synthetic benzopyran analogues, derived from a BMS-191095, a selective mK ATP opener, on mitochondrial respiration and reactive oxygen species (ROS) production in isolated rat heart mitochondria. Mitochondrial respiratory function was assessed by high-resolution respirometry, and H 2 O 2 production was measured by the Amplex Red fluorescence assay. Four compounds, namely KL-1487, KL-1492, KL-1495, and KL-1507, applied in increasing concentrations (50, 75, 100, and 150 mol/L, respectively) were investigated. When added in the last two concentrations, all compounds significantly increased State 2 and 4 respiratory rates, an effect that was not abolished by 5-hydroxydecanoate (5-HD, 100 mol/L), the classic mK ATP inhibitor. The highest concentration also elicited an important decrease of the oxidative phosphorylation in a K + independent manner. Both concentrations of 100 and 150 mol/L for KL-1487, KL-1492, and KL-1495, and the concentration of 150 mol/L for KL-1507, respectively, mitigated the mitochondrial H 2 O 2 release. In isolated rat heart mitochondria, the novel benzopyran analogues act as protonophoric uncouplers of oxidative phosphorylation and decrease the generation of reactive oxygen species in a dose-dependent manner. Key words: rat heart mitochondria, benzopyran analogues, protonophores, uncoupling, hydrogen peroxide. Résumé : De plus en plus de données indiquent que l’activation pharmacologique des canaux potassiques sensibles a ` l’ATP de la mitochondrie (mK ATP ) du cœur le protège lorsqu’il est exposé a ` des conditions associées au dommage d’ischémie/reperfusion. Plusieurs mécanismes pourraient être responsables de la cardioprotection incluant la modulation de la fonction respiratoire mitochondriale. Le but de l’étude présente était de caractériser les effets liés a ` la dose de nouveaux analogues synthétiques du benzopyrane, dérivés du BMS-191095, un composé qui ouvre sélectivement les mK ATP , sur la respiration mitochondriale et la production d’espèces réactives d’oxygène (ERO) dans les mitochondries isolées du cœur de rat et ce. La fonction respiratoire mitochondriale a été évaluée par respirométrie a ` haute résolution et la production de H 2 O 2 a été mesurée par un dosage en fluorescence a ` l’aide de la trousse Amplex Red. Quatre composés, a ` savoir KL-1487, KL-1492, KL-1495 et KL-1507, ajoutés en concentrations croissantes (50, 75, 100 et 150 mol/L, respectivement), ont été examinés. Lorsqu’ajoutés aux deux concentrations les plus élevées, tous les composés accroissaient significativement les taux respiratoires aux états 2 et 4, effet qui n’était pas aboli par le 5-hydroxydécanoate (5-HD, 100 mol/L), l’inhibiteur classique des mK ATP . La plus forte concentration induisait aussi une diminution importante de la phosphorylation oxydative de manière indépendante du K + . Les deux concentrations de 100 mol/L et 150 mol/L de KL-1487, KL-1492 et KL-1495, et la concentration de 150 mol/L de KL-1507 diminuaient respectivement la libération de H 2 O 2 de la mitochondrie. Les nouveaux analogues du benzopyrane agissent comme découplants protonophores de la phosphorylation oxydative dans les mitochondries isolées du cœur de rat, et diminuent la génération d’espèces réactives d’oxygène en fonction de leur concentration. [Traduit par la Rédaction] Mots-clés : mitochondries du cœur de rat, analogues du benzopyrane, protonophores, effet découplant, peroxyde d’hydrogène. Introduction In the past decades mitochondria have emerged as major con- tributors to the pathogenesis of myocardial ischemia/reperfusion (I/R) injury as well as important therapeutic targets in cardiopro- tection (Camara et al. 2011; Di Lisa et al. 2007). The mitochondrial ATP-sensitive potassium channel (mK ATP ) is one of the putative structures at the inner mitochondrial membrane that has been extensively studied in several experimental models as a major target that can be modulated by pharmacological agents and conditioning strategies to protect the heart against the deleteri- ous effects of reoxygenation/reperfusion (critically reviewed by Hanley and Daut (2005). The channel functions as potassium uni- porter, allowing the ions intake into the matrix and is inhibited by ATP, ADP, and fatty acids (Cardoso et al. 2010; Garlid and Received 26 January 2015. Accepted 24 June 2015. A. Petruş* and I. Baczkó. Department of Pathophysiology, “Victor Babeş” University of Medicine and Pharmacy of Timis ¸oara, 14, Tudor Vladimirescu st. 300173 Timisoara, Romania. O.M. Duicu,* A. Sturza, L. Noveanu, M. Da ˘ nila ˘ , D.M. Muntean, and N. Jost. Department of Pathophysiology, Center for Translational Research and Systems Medicine, “Victor Babeş” University of Medicine and Pharmacy of Timis ¸oara, Romania. L. Kiss. Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Szeged, Szeged, Hungary. Corresponding author: Danina Muntean (e-mail: daninamuntean@umft.ro). *These authors contributed equally to this work. 1 This article is part of a Special Issue entitled “Cardioprotection and Arrhythmias, Part 2.” 811 Can. J. Physiol. Pharmacol. 93: 811–818 (2015) dx.doi.org/10.1139/cjpp-2015-0041 Published at www.nrcresearchpress.com/cjpp on 30 June 2015. Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Dr Danina M. Muntean on 09/03/15 For personal use only.