Korean J Physiol Pharmacol 2016;20(2):201-211 http://dx.doi.org/10.4196/kjpp.2016.20.2.201 Original Article NecroX-5 protects mitochondrial oxidative phosphorylation capacity and preserves PGC1 expression levels during hypoxia/ reoxygenation injury Vu Thi Thu1,2,#, Hyoung Kyu Kim1,#, Le Thanh Long1, Bayalagmaa Nyamaa1, In-Sung Song1, To Thanh Thuy2, Nguyen Quang Huy2, Jubert Marquez1, Soon Ha Kim3, Nari Kim1, Kyung Soo Ko1, Byoung Doo Rhee1, and Jin Han1,* 1National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Cardiovascular and Metabolic Disease Center, Inje University, Busan 47392, Korea, 2Faculty of Biology, VNU University of Science, Hanoi 120036, Vietnam, 3Product Strategy and Development, LG Life Sciences Ltd, Seoul 03184, Korea ARTICLE INFO ABSTRACT Although the antioxidant and cardioprotective effects of NecroX-5 Received December 2, 2015 Revised January 11, 2016 on various in vitro and in vivo models have been demonstrated, the action of Accepted January 13, 2016 this compound on the mitochondrial oxidative phosphorylation system remains unclear. Here we verify the role of NecroX-5 in protecting mitochondrial oxidative *Correspondence phosphorylation capacity during hypoxia-reoxygenation (HR). Necrox-5 treatment Jin Han (10 M) and non-treatment were employed on isolated rat hearts during hypoxia/ E-mail: [email protected] reoxygenation treatment using an ex vivo Langendorff system. Proteomic analysis was performed using liquid chromatography-mass spectrometry (LC-MS) and Key Words non-labeling peptide count protein quantification. Real-time PCR, western blot, Hypoxia citrate synthases and mitochondrial complex activity assays were then performed Mitochondria to assess heart function. Treatment with NecroX-5 during hypoxia significantly NecroX preserved electron transport chain proteins involved in oxidative phosphorylation Oxidative phosphorylation and metabolic functions. NecroX-5 also improved mitochondrial complex I, II, and V PCG1 function. Additionally, markedly higher peroxisome proliferator-activated receptor- #These authors contributed equally to gamma coactivator-1 (PGC1) expression levels were observed in NecroX-5-treated this study. rat hearts. These novel results provide convincing evidence for the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity and in preserving PGC1α during cardiac HR injuries. INTRODUCTION further myocardial injury [1]. Potential mediators of ischemia- reperfusion (IR) injury play roles in oxidative stress, intracellular Myocardial ischemia (MI) results from reduced blood flow and mitochondrial Ca2+ overload, and the accumulation of to the heart. MI may damage the heart muscle and in severe inflammatory cells in the infarcted myocardial tissue [1-3]. A cases may cause heart failure and sudden death. Reperfusion- recent trend in the application of reperfusion therapy is targeted blood resupply to the cardiac muscle-may reduce myocardial drug delivery to the affected site [4,5]. damage and therefore is the first choice treatment for patients Emerging evidence has demonstrated that preservation of suffering from MI. Nevertheless, reperfusion itself results in mitochondrial function is important for limiting myocardial widespread oxidative modifications to lipids and proteins and damage in ischemic heart diseases [6-9]. Activation of mito- causes mitochondrial damage, which can eventually cause chondrial biogenesis, a complex process involving the coordi- This is an Open Access article distributed under the terms Author contributions: V.T.T. and H.K.K designed experiments and performed of the Creative Commons Attribution Non-Commercial animal experiments. L.T.L. performed langendorff heart experiments. B.N., I-S. License, which permits unrestricted non-commercial use, distribution, and S. and J.M. performed western blot analysis. T.T.T. and N.Q.H. contributed to reproduction in any medium, provided the original work is properly cited. proteome analysis. S.H.K. and N.K. contributed to chemical preparation. K.S.K. Copyright © Korean J Physiol Pharmacol, pISSN 1226-4512, eISSN 2093-3827 and B.D.R contributed to mitochondria complex assays. J.H. supervised and coordinated the study. V.T.T. and H.K.K. wrote the manuscript. www.kjpp.net 201 Korean J Physiol Pharmacol 2016;20(2):201-211 202 Thu VT et al nated expression of mitochondrial and nuclear genes, is a nature- Isolation of hearts adaptive reaction designed to restore mitochondrial function under ischemic conditions [10]. Alteration of mitochondrial Eight-week-old male Sprague–Dawley rats (200~250 g) were biogenesis is associated with changes in mitochondrial bioener- deeply anaesthetized with sodium pentobarbital (100 mg/kg) getics, including alterations in the oxidative phosphorylation administered intraperitoneally [19]. Successful anesthesia was (OXPHOS) system, mitochondrial membrane potential (ΔΨm), confirmed by the absence of nocifensive movement, such as the mitochondrial oxygen consumption, and ATP production. tail flick reflex [16]. Hearts were then removed from the chest Additionally, almost every functional aspect of OXPHOS cavity and quickly mounted and perfused with normal Tyrode’s strongly influences mitochondrial respiration [6]. Transcription (NT) solution to remove all blood. factors regulating the expression of major OXPHOS proteins play important roles in modifying cardiac energy metabolism Cardiac perfusion [11]. One of the most well-known of these transcription factors is peroxisome proliferator-activated receptor-gamma coactivator- In the HR experimental model, hearts were sequentially 1 (PGC1) [12]. Studies have demonstrated decreased PGC1 perfused with NT solution for 30 minutes and ischemic solution expression levels during myocardial infarction [13,14], and [20] for 30 minutes, followed by perfusion with NT solution treatment with metformin [14] or losartan [13] can reduce IR with (non-treated) or without 10 M NecroX-5 (treated) for 60 injuries with significant increases in PGC1 expression levels and minutes. In the control group, hearts were perfused with NT for enhancement of left ventricular function. 120 minutes. Recently, we developed NecroX compounds, cell-permeable necrosis inhibitors with antioxidant activity that localize mainly Isolation of mitochondria in the mitochondria [15]. One of these NecroX derivatives, NecroX-5 (C25H31N3O3S·2CH4O3S; molecular weight 453.61 Mitochondrial pellets were collected as described previously kDa), when administered during hypoxia, strongly protects [16,20,21]. Briefly, cardiac tissues were manually homogenized rat heart mitochondria against reperfusion injury by reducing and the homogenate clarified by centrifugation at 1,000 g for 5 mitochondrial oxidative stress, preserving the ΔΨm, improving min at 4oC. The supernatant was collected and centrifuged at mitochondrial oxygen consumption, and attenuating mito- 10,000 g for 10 minutes, and mitochondrial pellets were collected. chondrial Ca2+ accumulation by inhibiting the mitochondrial calcium uniporter [16]. NecroX-5 also suppresses sodium nit- RNA extraction and Real-time PCR roprusside-induced cardiac cell death by inhibiting c-Jun N-terminal kinases and caspase-3 activation [17]. Additionally, Total RNA was extracted using TRIZOL reagent (Invitrogen, NecroX-7, a NecroX series derivative functionally and chemically CA, USA) according to the manufacturer’s instructions. Frozen similar to NecroX-5, reduces hepatic necrosis and inflammation tissues (50~100 mg) were ground into powder in liquid nitrogen in IR injury [18]. These results together suggest that NecroX-5 and and then suspended in 1 mL TRIZOL Reagent. The aqueous other NecroX compounds may serve as drugs that can be used phase was used for RNA precipitation using an equal volume concurrently with reperfusion treatment to improve the overall of isopropanol. The RNA pellet was washed once with 1 mL efficiency of the treatment by reducing potentially harmful effects 75% ethanol, then air-dried and re-dissolved in an appropriate of reperfusion. Here, we further validate the role of NecroX-5 volume of RNase-free water. RNA was quantified using a in preventing mitochondrial dysfunction during IR injury by spectrophotometer (Nanodrop Technologies, Wilmington, DE, preserving mitochondrial oxidative phosphorylation capacity. USA). cDNA was synthesized using Taqman RT reagents (Applied Biosystems, NY, USA), following the manufacturer’s instructions. The cDNA synthesis thermal cycling program included the METHODS following steps: 65oC for 5 min, 4oC for 2 min, 37oC for 30 min, then 95oC for 5 min. Ethics statement Relative mRNA expression levels of Ndufv2, Idh3a, Ndufa1, Ndufb8, Idh1, Hyou1, Ak2, Lum, Cabc1 and Nexn genes This investigation conformed to the rules and protocols of the were performed in triplicate using SYBRⓇ Premix Ex TaqTM Guide for the Care and Use of Laboratory Animals published by II kit (Takara, Shiga, Japan) on CFX96. The mixture contains the United States National Institutes of Health (NIH Publication cDNA, SYBR green Taq polymerase mixture, and primers No. 85-23, revised 1996). All experimental procedures were (Table 1). According to the manufacturer's guidelines, relative reviewed and approved by the Institutional Review Board of quantification was analyzed using the ΔΔCT method for. The Animals at Inje University College of Medicine. Procedures were mRNA level was normalized by control group. performed according to the Institutional Review Board. Korean J Physiol