Figure 4. CORM-3 did not change UCP expression. (A) Results of quantitative RT-PCR detecting Ucp2 and Ucp3 mRNA expressions. Results were normalized to aˆ-actin. (B) Representative western-blot targeting UCP3 and GAPDH proteins and (C) densitometric analysis of UCP3 to GAPDH ratio. 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 = 5 in each group. * p,0.05 vs. ND. doi:10.1371/journal.pone.0041836.g004 50% in mice fed with high-fat regimen (Figure 6A). CORM-3 partially reduced high-fat-induced elevation in Mfn2 and Opa1 mRNA expression (Figure 6A). Pgc-1a mRNA and protein expressions were higher in the HFD group compared to ND (Figure 6, B-C). Downstream transcription factors Nrf1 and Tfam were upregulated in the HFD compared to CO Improves Mitochondrial Population ND (Figure 6B). CORM-3 abrogated HFD-induced increases in Pgc-1a, Nrf1 and Tfam expression (Figure 6, B-C). Autophagy also participates in the quality control of mitochon- drial population. While no differences were observed between ND and HFD, LC3-I protein lipidation into the active form LC3-II was increased in the HFD + CORM-3 group (Figure 6D). Consistently, expression of Atg5 was significantly increased after CORM-3 injection in HFD animals compared to ND and HFD mice (Figure 6E). CORM-3 injection, in vivo, in HFD mice was associated with both autophagy activation and improved cardiac function. We used 3-methyladenine, an autophagy inhibitor, on isolated HFD heart treated ex vivo by CORM-3 (see Materials and Methods) to test whether blocking defective mitochondria removal impeded improvement of cardiac function triggered by carbon monoxide. Addition of CORM-3 in the perfusate of HFD hearts led to an improvement of (LVDP 6 HR) to MVO2 ratio (Figure 6F). Importantly, pretreatment with 3-methyladenine abolished acute CORM-3 effects on myocardial function (Figure 6F). Discussion In the present study, we report that mice fed with high-fat diet developed glucose intolerance, insulin resistance, dyslipidemia, changes in adipocytokines levels and cardiac dysfunction. We further observed that myocardial contractile alterations were related to a poorer quality of the mitochondrial population. More importantly, we report for the first time that delivery of carbon monoxide by CORM-3 significantly attenuated HFD-induced cardiac dysfunction through improvement of mitochondrial function that may also involve elimination of defective mitochon- dria by autophagy activation. Consistently with published works [19], we found that three months after the onset of high-fat regimen, mice developed major abnormalities resembling to human metabolic syndrome such as glucose intolerance, insulin resistance, elevated glucose, insulin and leptin circulating levels and reduced adiponectin blood concentration. We also found that HFD led to intrinsic ventricular contractile dysfunction. Interestingly, carbon monoxide delivered by CORM-3 improved cardiac function but only transiently as improved function was only observed at 24 hours after the last injection. A possible explanation could be related to the half-life of carbon monoxide in the organism. Indeed, half-time of CO elimination is about four hours while breathing room-air [20]. On the other side, five consecutive injections may trigger a reduction in cardiac function. This is consistent with previous studies reporting that chronic CO exposure may induce cardiac hypertrophy [21] and arrhythmia [22]. Plus, five consecutive injections may result in elevated blood concentration of CO, mimicking the well-known poisonous effect on oxygen delivery to tissues or inhibition of mitochondrial cytochrome oxidase, leading to a reduced production of ATP [9]. As a consequence, although CORM-3 has striking beneficial effects when injected twice, it remains a molecule that has to be handle with care and that needs further investigation before its uses as a therapeutic agent. In association with improvement of cardiac function, the ratio (LVDP x HR) to MVO2, a surrogate of cardiac efficiency, and the increase in maximal oxygen consumption were higher in the CORM-3-injected group compared to untreated HFD mice. First, these results are in agreement with those obtained in genetically modified mice [5]. Second, as improvement of both cardiac function and animal oxygen uptake were not related to changes in metabolism, carbon monoxide may have changed the cellular redox state [23], reduced the pro-inflammatory state [24] or PLoS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e41836