processes [6]. For instance, in the heart, hypoxia triggers CO production by inducing heme oxygenase-1 (HO-1) expression, which in turn may increase cGMP level leading to coronary artery vasodilatation as a cardiac protective response [7]. Studies using transgenic animal models also demonstrated that heme oxygenase overexpression was protective against ischemia/reperfusion injury [8]. Different approaches have been proposed to therapeutically deliver CO. Among them, CO-releasing molecules (CO-RMs) have been developed based on the chemical properties of transition metals [9]. CORM-3, ruthenium-based metal carbonyl, was the first water-soluble compound being characterized [10]. CORM-3 rapidly releases CO in biological fluids without a significant binding to haemoglobin in vivo [11]. We previously demonstrated that CORM-3 exerts cardioprotective effects by increasing mitochondrial function and biogenesis during experi- mental sepsis [12]. Upregulation of HO-1/CO pathway amelio- rates insulin sensitivity and glucose tolerance [13]. Therefore, CORMs may improve both cardiac mitochondria function and insulin sensitivity during metabolic syndrome. In this context, we first characterized the derangement of cardiac and mitochondrial function in a mouse model of metabolic syndrome. Then, we tested whether treatment of mice with CORM-3 would improve metabolic syndrome-induced cardiac dysfunction through changes in mitochondrial activities by primarily assessing dynamics, biogenesis and autophagy. Finally, we tested whether inhibition of autophagy was sufficient to abolish CORM-3-mediated cardioprotective effects in a model of isolated hearts. Materials and Methods Ethics Statement All experiments were carried out in accordance with national guidelines and approved by DDSV-NPDC-Lille (Permit Number 59-350206). Animal Care and Feeding Five-week-old C57BL/6 female mice were obtained from Charles River Laboratory (L’Arbresle, France). Mice were randomly divided in two groups: one had free access to a standard chow diet (normal diet ND); the other received a high-fat diet (HFD, D12492, SSNIFF, Soest, Germany) in which 60% of calories were from lard fat. Both groups had free access to water and were kept under a 12 h: 12 h light-dark cycle. Mice were weighed once a week and food intake was monitored daily. After 10–12 weeks of feeding, animals were injected with CORM-3 (10 mg.kg21) as described hereinafter (see ‘‘Treatments’’ para- graph of the current section) and studied 24 hours after the last injection. Analysis of Plasma Blood (100 ml) was withdrawn from tail after an overnight fasting period and glucose concentration was measured with a glucometer (Nova Biomedical, Les Ulis, France). Then, blood was collected into EDTA-coated tubes and centrifuged for 15 min at 1000 g at 4uC. The resultant plasma was aliquoted and stored at 280uC. Systemic insulin, adiponectin, leptin levels were deter- mined by the use of Lincoplex immunoassay kits (Millipore, Molsheim, France). True triglycerides were measured with the determination kit from Sigma Aldrich (St Quentin Fallavier, France). Total cholesterol was quantified with the cholesterol RTU and calibrator kits (Biome´rieux, Marcy-L’Etoile, France). CO Improves Mitochondrial Population Oral Glucose Tolerance Test (OGTT) Fasted animals received by gavage a glucose solution (2 mg.g21 total body weight). Glucose levels were measured on blood samples that were taken before glucose administration and 10, 20, 30, 60 and 120 min after glucose ingestion. Insulin Tolerance Test (ITT) Fasted animals received 0.5 mUI.g21 total body weight of insulin diluted in saline solution. Tail blood samples were taken before insulin injection and 15, 30 and 60 minutes later. Glucose concentration was measured using a glucose meter. Mice never presented any troubles or convulsions. In all cases, a glucose solution (200 mg.dl21) was ready for injection if the mice started exhibiting these symptoms. Exercise Stress Test First, mice performed a 10 m.min21 run, for 10 minutes, two days prior the exercise stress test to be acclimatized to the treadmill, as previously described [14]. Then, they were placed onto a single lane treadmill enclosed in a metabolic chamber connected to Oxymax oxygen (O2) and carbon dioxide (CO2) sensors (Columbus Instruments, Columbus, OH). After 30 min, basal whole body O2 uptake (VO2) and CO2 production (VCO2) were automatically calculated. The basal respiratory exchange ratio (RER), used to determine metabolism of the animal, was the ratio VCO2 to VO2. Mice started running on the treadmill at 10 m.min21 on a 0% incline. In order to evaluate maximal oxygen consumption increase, the treadmill speed was incremented every 3 min by 4 m.min21 until mice reached exhaustion, obtained when mice stayed on the electrical shocker plate for 5 sec without trying going back to the treadmill. Assessment of Myocardial Function After cervical dislocation, mouse heart was quickly mounted onto a Langendorff perfusion apparatus. Hearts were perfused at a constant coronary flow (2.5 ml.min21) with non-recirculating Krebs–Henseleit bicarbonate (KHB containing 11 mM glucose) buffer and paced at 9 Hz. Coronary perfusion pressure (CPP) was continuously monitored. When required, 3-methyladenine (10 mmol.l21) was added in the KHB perfusate 15 min before addition of CORM-3 (10 mmol.l21). The acute effects on heart mechanics and oxygen consumption were measured 60 min after CORM-3 addition. Mechanical activity was assessed through isovolumic contraction by inserting into the left ventricle a latex balloon connected to a pressure transducer. The balloon was filled with aqueous solution to achieve a left ventricular end-diastolic pressure of 6–8 mmHg. Pressure transducer was connected to a ML118 bridge amplifier that fed into a Powerlab 8 SP high- performance data acquisition system (ADInstruments Ltd. by Phymep, Paris, France). Contractile performance of the left ventricle was evaluated by developed pressure (LVDP) and its first derivatives. Oxygen partial pressure was measured in coronary perfusate and effluent using specific electrodes to calculate myocardial O2 uptake, MVO2 (mL.min21.g21). LVDP - heart rate (HR) product to MVO2 ratio was calculated as a surrogate of cardiac efficiency. Treatments In vivo treatment: CORM-3, tricarbonylchloroglycinato ruthe- nium(II) Ru(CO)3Cl(NH2CH2CO2), was synthesized as previously described [10]. The compound was solubilized in a saline solution (NaCl 0.9%). Mice received one injection of CORM-3 (10 mg.kg21 total body weight, intraperitoneally, a dose equivalent PLoS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e41836