exerted cytoprotective effects on the myocardium [25]. Here, we rather explored mitochondrial function as it may also contribute to the improvement of myocardial function. We used multiple approaches to characterize mitochondrial function. First, respiration measured from cardiac skinned fibers was lower when isolated from HFD animals. Second, mitochon- drial membrane potential and calcium retention capacity of isolated mitochondria were reduced in the HFD group. In all cases, CORM-3 exerted beneficial effects. Although carbon monoxide has been reported to exert beneficial effects on mitochondria through production of cGMP and reactive oxygen species and activation of Akt [26], a clear understanding of the underlying molecular mechanisms is still lacking and warrants additional studies. Here, we further assessed whether carbon monoxide improved mitochondrial function through changes in mitochondrial population (density and size of mitochondria) and intrinsic mitochondrial properties (oxidative capacity and uncou- pling level). Boudina et al. [5] suggested that cardiac UCP increase in diabetic hearts might participate to mitochondrial intrinsic dysfunction. In our model, we also detected increases in Ucp2 and Ucp3 but CORM-3 did not change their expression. Although we cannot rule out the fact that CORM-3, in a time-dependent manner, may directly modulate uncoupling and UCP activity in mitochondria [23], the ultimate effects of CORM-3 originating from the present study can be explained by an overall improvement of mitochondrial population quality. Electron microscopy revealed that HFD led to an increase in both mitochondrial size and number, two parameters modulated by CORM-3. Moreover, Mfn2 and Opa1 expressions increased in the HFD group. To our knowledge, this is the first time that such an observation has been made in cardiac tissue in the context of metabolic syndrome, diabetes or obesity. Physiological significance of such modifications in maintaining myocardial energy homeo- stasis remains largely unknown in the heart and requires further studies. In skeletal muscle, repression of Mfn2 reduces glucose oxidation, mitochondrial respiration and potential. Rhodes et al. [27] report that CO inhalation leads to increase in pro-fusion protein expression in biopsies of the vastus lateralis muscle. PGC-1a expression at both mRNA and protein levels was elevated in HFD mice. This observation is consistent with studies that reported mitochondrial proliferation in the db/db mice [5] and also in cardiac biopsies from diabetic patients [28]. CORM-3 blocked the HFD-induced elevations in Pgc-1a, Nrf1 and Tfam while other reports have found that CO activated mitochondrial biogenesis [29]. One hypothesis that could reconcile these apparent contradictory findings is that, because of a better mitochondrial function consecutive to CORM-3 treatment, mitochondrial biogenesis would not be required any longer. A balance between biogenesis and mitochondria autophagy, process known as ‘‘mitophagy’’, tightly controls the quality of mitochondrial population. An autophagy hallmark is the conju- gation of microtubule-associated light chain-3 (LC3-I) to phos- References 1. Boudina S, Abel ED (2010) Diabetic cardiomyopathy, causes and effects. Rev Endocr Metab Disord 11: 31–9. 2. Ong S-B, Subrayan S, Lim SY, Yellon DM, Davidson SM, et al. (2010) Inhibiting mitochondrial fission protects the heart against ischemia/reperfusion injury. Circulation 121: 2012–22. 3. Lehman JJ, Barger PM, Kovacs A, Saffitz JE, Medeiros DM, et al. (2000) Peroxisome proliferator-activated receptor gamma coactivator-1 promotes cardiac mitochondrial biogenesis. J Clin Invest 106: 847–56. 4. Nakai A, Yamaguchi O, Takeda T, Higuchi Y, Hikoso S, et al. (2007) The role of autophagy in cardiomyocytes in the basal state and in response to hemodynamic stress. Nat Med 13: 619–24. CO Improves Mitochondrial Population phatidylethanolamine (LC3-II), consecutively to the action of a complex constituted of several proteins including Atg5 [30]. Here, while there were no differences between ND and HFD groups, Atg5 expression and LC3-II to LC3-I ratio increased after treatment with CORM-3. Although additional studies are needed to understand the underlying mechanisms, consistent data from Lee et al. described that exposure of human alveolar adenocarci- noma epithelial cells and human bronchial epithelial cells to CO gas also triggered autophagy activation [31]. As a proof of concept, we performed ex vivo experiments in which 3-methyladenine, a pharmacological inhibitor of autophagy was administered. We found that 3-methyladenine prevented the positive effects of CORM-3 on cardiac efficiency within one hour, suggesting that CO may improve mitochondrial function through rapid and critical activation of autophagy. Although this striking effect may be questionable as changes in cardiac efficiency occurred within one hour of treatment, a 68% reduction in mitochondrial number consecutive to a 3.5-hour starvation has been observed in HL1 cardiac cell line [32]. In conclusion, improvement of mitochondrial population quality by CORM-3 is associated with a better mitochondrial efficiency, leading to amelioration of HFD-induced cardiac dysfunction. Although these results bring new therapeutic insight, further pharmacokinetic and toxicity studies on CORM-3 are required before administration in humans. Primers for RT-qPCR experiments. F: forward, R: Supporting Information Table S1 reverse. (DOC) Table S2 Model features obtained after ten weeks on specific diets. Plasmatic parameters were measured after a 12 hr-fasting period. n = 9–10 in each group. Left ventricular developed pressure (LVDP) and coronary perfusion pressure (CPP) were evaluated on five animals in each group. *P,0.05 vs. normal diet. (DOC) Acknowledgments We thank Thomas Comptdaer for his technical assistance. We thank Anne Loyens and Ce´cile Allet of the electron microscopy Core Facility of BiCeL- Campus Lille2 IFR114-IMPRT (Institut de Me´decine Pre´dictive et de Recherche The´rapeutique, Lille) for access to instruments and technical advices. We also thank Prof. Brian Mann for the synthesis of CORM-3. Author Contributions Conceived and designed the experiments: SL DM XM CM SMH BD C. Ballot. Performed the experiments: SL DM XM CM SMH BD C. Ballot C. Blazejewski DC BL. Analyzed the data: SL DM XM CM SMH BD RN. Contributed reagents/materials/analysis tools: RM. Wrote the paper: SL RM RN. 5. Boudina S, Sena S, Theobald H, Sheng X, Wright JJ, et al. (2007) Mitochondrial energetics in the heart in obesity-related diabetes: direct evidence for increased uncoupled respiration and activation of uncoupling proteins. Diabetes 56: 2457–66. 6. Wu L, Wang R (2005) Carbon monoxide: endogenous production, physiological functions, and pharmacological applications. Pharmacol Rev 57: 585–630. 7. Grilli A, De Lutiis MA, Patruno A, Speranza L, Gizzi F, et al. (2003) Inducible nitric oxide synthase and heme oxygenase-1 in rat heart: direct effect of chronic exposure to hypoxia. Ann Clin Lab Sci 33: 208–15. 8. Yet SF, Tian R, Layne MD, Wang ZY, Maemura K, et al. (2001) Cardiac- specific expression of heme oxygenase-1 protects against ischemia and reperfusion injury in transgenic mice. Circ Res 89: 168–73. PLoS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e41836