HFD and HFD + CORM-3 mice. Injection of CORM-3 in ND mice had no effects on all the above parameters (Figure 2). CORM-3 Ameliorated Cardiac Mitochondrial Function of HFD Mice Because of the positive effects of CORM-3 on (LVDP x HR) to MVO2 ratio (Figure 1C) and maximal VO2 increase (Figure 1D), we evaluated function of cardiac mitochondria. First, mitochon- drial respiration was evaluated on saponin-skinned cardiac fibers. Oxygen consumption stimulated by glutamate and malate in absence of exogenous ADP, so-called state 2 [16], was higher in HFD compared to ND mice (Figure 3A). On the contrary, ADP- coupled respiration, corresponding to state 3, was reduced in HFD compared to ND mice (Figure 3A). As a result, state 3 to state 2 ratio of HFD animals was reduced by 30% compared to ND littermates (Figure 3B). No differences were detected between ND and HFD + CORM-3 for both states 3 and 2 (Figure 3A), hence a better state 3/state 2 ratio for HFD + CORM-3 vs. HFD (Figure 3B). To test potential intrinsic mitochondrial defects, we isolated mitochondria to measure transmembrane potential DYm as well as calcium retention capacity. Compared to ND, mitochondria from HFD hearts displayed a reduced DYm (Figure 3C) as well as a two-fold decrease in calcium retention capacity (Figure 3D). CORM-3 ameliorated both parameters (Figure 3, C-D). CORM-3 -induced mitochondrial function improvement was not due to CO Improves Mitochondrial Population changes in uncoupling protein expression (Figure 4). Indeed, compared to ND mice, both Ucp2 and Ucp3 mRNA expressions were increased in HFD and remained elevated in the HFD + CORM-3 group (Figure 4A). In addition, UCP3 protein expression doubled in HFD and HFD + CORM-3 compared to ND (Figure 4, B-C). None of these mitochondrial parameters were affected by CORM-3 injection in ND mice (Figure 4). Because of the lack of effect of CORM-3 in ND mice on myocardial and mitochondrial functions, we focused our study on pathways regulating mitochondrial population quality in the ND, HFD and HFD + CORM-3 groups. CORM-3 Modulated Pathways Controlling Mitochondrial Quality Electron microscopy on cardiac sections revealed that HFD hearts had bigger mitochondria compared to ND hearts (Figure 5, A-B). Number of grid intersections superimposing mitochondria was higher in the HFD compared to ND group (Figure 5, A and C), indicating that mitochondria were either more numerous and/ or bigger. In addition, morphometric analysis revealed that HFD mice treated with CORM-3 displayed a mitochondrial population similar to ND hearts (Figure 5, A-C). We then explored mitochondrial dynamics, biogenesis and autophagy. Fission-related transcripts, Drp1 and Fis1, did not change in any groups (Figure 6A). While Mfn1 remained unchanged, fusion-related mRNAs, Mfn2 and Opa1, increased by Figure 3. CORM-3 improved mitochondrial function. (A) Oxygen consumption of permeabilized fibers in presence of glutamate and malate without exogenous ADP (state 2) or with 2.5 mM ADP (state 3) and (B) state 3 to state 2 ratio. Mitochondrial membrane potential (C) and calcium retention capacity (D) measured on isolated cardiac mitochondria. White bars = ND; vertically hatched bars = ND + CORM-3; black bars = HFD; crosshatched bars = CORM-3–treated HFD mice. Data are means 6 SEM. n = 6–8 in each group. * p,0.05 vs. ND, { p,0.05 vs. HFD. doi:10.1371/journal.pone.0041836.g003 PLoS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e41836