CO Improves Mitochondrial Population Figure 5. CORM-3 modified cardiac mitochondrial population. (A) Representative electron micrographs of ND, HFD and HFD + CORM-3 mouse hearts. Magnification x7000, scale bar = 2 mm. (B) Surface of every single mitochondrion from three randomized tissue sections prepared from four hearts in each group was measured. Black circles: ND; black squares: HFD; white diamonds: HFD + CORM-3. Results are expressed in percentage. (C) A grid of 144 mm2 was positioned onto each micrograph (3 sections per heart, 4 hearts per group) and the number of intersections with mitochondria was scored. White bars = ND; black bars = HFD; crosshatched bars = CORM-3–treated HFD mice. Data are means 6 SEM. * p,0.05 vs. ND. doi:10.1371/journal.pone.0041836.g005 Figure 6. Effects of CORM-3 on pathways controlling mitochondrial quality. RT-qPCR results of fusion/fission (A) and biogenesis (B) -related transcripts in cardiac tissue obtained from ND (white bars), HFD (black bars) or HFD + CORM-3 (crosshatched bars) mice. Data were normalized to aˆ- actin. (C) Representative western-blot of PGC-1a and GAPDH in ND, HFD and HFD + CORM-3 mouse hearts. Values are results of densitometric analysis normalized to GAPDH. (D) Representative western-blot of LC3 proteins in ND, HFD and HFD + CORM-3 mouse hearts. LC3-I represents the unactivated form; LC-II is the activated protein that has been lipidated. Numbers represent the pro-autophagy ratio LC3-II to LC3-I obtained after densitometric analysis. (E) Atg5 RT-qPCR results normalized to aˆ-actin. White bars = ND; black bars = HFD; crosshatched bars = CORM-3–treated HFD mice. Data are means 6 SEM. n = 5 in each group. * p,0.05 vs. ND, { p,0.05 vs. HFD. (F) Ratio (LVDP x Heart Rate)/MVO2 of isolated perfused heart from HFD mice. HFD hearts were pretreated or not with 10 mM 3-methyladenine (3-MA) for 15 min and then perfused with Krebs-Henseleit buffer containing 10 mM of CORM-3. Measurement was performed 60 min after CORM-3 perfusion. Black bars: isolated and perfused HFD hearts. Data are means 6 SEM. n = 4–5 in each group. { p,0.05 vs. untreated HFD hearts, # p,0.05 vs. CORM-3 treated HFD hearts. doi:10.1371/journal.pone.0041836.g006 PLoS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e41836