to approximately 400 mM) at 6 pm and another one at 10 am the day after, as published before [12]. The experiments were then performed at 24, 48 or 72 hours after the second injection. In another set of experiments, mice were injected once a day for five consecutive days with CORM-3 (10 mg.kg21 total body weight) and cardiac function was evaluated the day after. In some experiments, to test the effect of the ruthenium compound, iCORM-3, the inactive form of CORM-3 prepared as previously described [12], was injected twice (10 mg.kg21 total body weight, intraperitoneally) and the effect on heart function was studied 24 hours after the last injection. Ex vivo treatment: in some experiments, isolated hearts from HFD mice were perfused onto a Langendorff apparatus. Then, CORM-3 (10 mmol.l21) was added to the KHB and acute effects on heart mechanics and oxygen consumption were measured 60 min after CORM-3 addition. To test whether autophagy was involved in the effects observed after ex vivo treatment with CORM-3, 3-methyladenine (10 mmol.l21) was added in the KHB perfusate 15 min before addition of CORM-3 in the perfusate. Determination of Mitochondrial Respiration Freshly excised heart was placed into ice-cold biopsy preserva- tion solution BIOPS [15]. Fiber bundles were separated and placed into a BIOPS solution containing 50 mg/mL saponin. After 30 min, permeabilized fibers were rinsed three times with mitochondrial respiration media Mitomed 2 [15]. Approximately 4 mg of wet fibers were placed in the oxygraph O2K (Oroboros Instruments, Innsbruck, Austria). First, 10 mmol.l21 glutamate +2 mmol.l21 malate were added into the chambers. The subsequent measured respiration, i.e. in absence of exogenous ADP, is referred to as state 2 [16]. Then, 2.5 mmol.l21 ADP were brought to obtain the ADP-coupled respiration called state 3. As in permeabilized fibers state 4 respiration cannot be achieved because of the presence of many intracellular ATPases that avoid total ADP depletion, the ratio state 3 to state 2 was used to evaluate the quality of the mitochondrial coupling between oxygen consumption and phosphorylation [17]. Measurements of Mitochondrial Membrane Potential and Calcium Retention Capacity Cardiac mitochondria were isolated as previously described [18]. 400 mg of mitochondria were placed into a multi-port chamber equipped with tetraphenylphosphonium (TPP+) and calcium -selective microelectrodes and reference electrodes (WPI, Sarasota, FL, USA). Mitochondria were gently stirred for 90 sec in the assay buffer [18] containing 1.5 mmol.l21 TPP+. Calcium pulses (10 mmol.l21) were added every 90 sec until a massive calcium release was observed. Calcium retention capacity was defined as the total amount of calcium added until permeability transition. Transmembrane potential DYm was calculated as 59log(v/V) - 59log(10DE/5921), where v is matrix volume (1.1 ml.mg21 mitochondrial protein), V is volume chamber (1 ml), and DE is voltage difference before and after calcium- induced permeability transition, expressed in millivolts. Purity and integrity of isolated mitochondria were assessed by measuring specific activities of nicotinamide adenine dinucleotide phosphate NADPH-cytochrome c reductase, as an endoplasmic reticulum marker enzyme, and cytochrome c oxidase, as an inner mitochondrial membrane marker enzyme. Quantitative RT-PCR Total RNAs from flash frozen heart tissue were extracted using TriZol reagent (Life Technologies SAS, Villebon sur Yvette, CO Improves Mitochondrial Population France) and purified with the PureLink RNA mini kit (Life Technologies SAS) according to manufacturer’s instructions. Total RNAs (1 mg) were reverse-transcribed using transcriptor first strand cDNA synthesis kit (Roche Applied Science, Meylan, France). Real-time RT-PCR was performed using an Eppendorf Realplex S2 (Eppendorf, Le Pecq, France) and Mesa Blue qPCR Master Mix Plus for SYBR green assay (Eurogentec, Angers, France). Primers used for the real-time RT-PCR are summarized in Table S1. b-actin was used as reference. Realplex software was used to quantify differences in gene expression. Results are expressed in fold expression compared to the ND group. Western-blotting Flash frozen hearts were homogenized at 4uC in RIPA buffer (in mmol.l21: Tris 10, NaCl 140, EDTA 5, PMSF 1, with Triton X- 100 1%, Deoxycholate 1%, SDS 0.1%, and in mg.ml21: aprotinin 10, leupeptin 10, pepstatin 10, at pH 7.4) using a glass tissue grinder. After a 15 min-centrifugation at 15,000 g at 4uC, supernatants were collected and stored at 280uC until use. Proteins (50 mg) were resolved by SDS-PAGE, transferred onto a nitrocellulose membrane and incubated with the following antibodies: anti-UCP3 rabbit polyclonal antibody (1/1000; Thermo Fisher Scientific, Brebieres, France), anti-GAPDH rabbit monoclonal antibody (1/5000; Cell Signaling Technology, Bev- erly, MA, USA), anti-PGC1-a rabbit polyclonal antibody (1/1000; Cell Signaling Technology), anti-LC3 rabbit polyclonal antibody (1/500; Abcam, Paris, France). Blots were developed with ECL Plus reagent (GE Healthcare, Templemars, France). Electron Microscopy Cardiac free wall was fixed in a formaldehyde-glutaraldehyde- picric acid solution. Once embedded, ultrathin sections were observed under a Zeiss EM 902 electron microscope, at a 7000X magnification. Mitochondrial morphometric analysis was per- formed on four different sections per heart. Each picture was analyzed by superimposing a grid of 144 mm2 made of lines spaced by 1 mm. The number of grid intersections superimposing mitochondria was scored and interpreted as mitochondrial volume density. In addition, surface of each individual mitochondrion was measured by the use of Gatan Digital Micrograph software (Gatan Inc, Pleasanton, CA, USA). Statistical Analysis Data are presented as means 6 S.E.M. Statistical analysis was carried out using two-tailed unpaired t-test when comparing two groups and ANOVA when comparing more than two groups. Analysis was performed on GraphPad Prism software 5.0 (San Diego, CA, USA). Differences were considered significant when p,0.05. Results High-fat Diet Led to Metabolic Syndrome and Cardiac Dysfunction At the onset of the regimen, mouse body weight was identical in both normal diet (ND) and high-fat diet (HFD) groups (Table S2). Over time, the body weight increased in both groups but after 2 months of feeding, the gain of weight was about 53% in the HFD compared to 15% in the ND mice (Table S2). Fasting glucose, insulin, cholesterol, triglycerides and leptin circulating levels increased significantly in the HFD mice compared to ND animals while blood adiponectin concentration was reduced (Table S2). Taken together, these data indicate that HFD mice develop a phenotype resembling to the human metabolic syndrome. PLoS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e41836