Mitochondrial dysfunction in skeletal muscle has been implicated in the development of type 2 diabetes. However, whether these changes are a cause or a consequence of insulin resistance is not clear. We investigated the structure and function of muscle mitochondria during the development of insulin resistance and progression to diabetes in mice fed a high-fat, high-sucrose diet. Although 1 month of high-fat, high-sucrose diet feeding was sufficient to induce glucose intolerance, mice showed no evidence of mitochondrial dysfunction at this stage. However, an extended diet intervention induced a diabetic state in which we observed altered mitochondrial biogenesis, structure, and function in muscle tissue. We assessed the role of oxidative stress in the develop-

Programme Hospitalier Recherche Clinique, Institut Pasteur, Inserm, French Public Health Agency.

M itochondria and the endoplasmic reticulum (ER) are separately considered key players in cell death signaling.1 Mitochondria and ER are interconnected organelles and form an endomembrane network. The contact points through which ER communicates with mitochondria are referred to as mitochondria-associated membranes (MAM).2 MAM are enriched in phospholipid-and glycosphingolipid-synthesis enzymes, as well as chaperone proteins, which transport lipids and exchange calcium between these 2 organelles.1 Several recent studies have identified new proteins enriched at the ER-mitochondria interface, highlighting the emerging understanding of the role of this region within the cell.3,4 One of them has identified a macromolecular complex composed of VDAC1, Grp75, and IP3R1 that regulates direct Ca 2+ transfer from ER to mitochondria. 5 Indeed, ER-mitochondria junctions are aligned with mitochondrial contact points where VDAC1 is abundantly present, thus creating a hot spot for the Ca 2+ transfer from the ER. 6 Although the role of this organelle cross talk is beginning to be understood in cell physiology, MAM involvement in cardiac pathologies remains unknown. Clinical Perspective on p 1565Calcium signaling is central for heart function through its physiological role in excitation-contraction coupling and the detrimental impact of Ca 2+ overload during heart failure and myocardial ischemia/reperfusion. During this latter condition, it is well accepted that the cytosolic accumulation of Ca 2+ subsequently results in mitochondrial Ca 2+ overload,Background-Under physiological conditions, Ca 2+ transfer from the endoplasmic reticulum (ER) to mitochondria might occur at least in part at contact points between the 2 organelles and involves the VDAC1/Grp75/IP3R1 complex. Accumulation of Ca 2+ into the mitochondrial matrix may activate the mitochondrial chaperone cyclophilin D (CypD) and trigger permeability transition pore opening, whose role in ischemia/reperfusion injury is well recognized. We questioned here whether the transfer of Ca 2+ from ER to mitochondria might play a role in cardiomyocyte death after hypoxia-reoxygenation. Methods and Results-We report that CypD interacts with the VDAC1/Grp75/IP3R1 complex in cardiomyocytes. Genetic or pharmacological inhibition of CypD in both H9c2 cardiomyoblasts and adult cardiomyocytes decreased the Ca 2+ transfer from ER to mitochondria through IP3R under normoxic conditions. During hypoxia-reoxygenation, the interaction between CypD and the IP3R1 Ca 2+ channeling complex increased concomitantly with mitochondrial Ca 2+ content. Inhibition of either CypD, IP3R1, or Grp75 decreased protein interaction within the complex, attenuated mitochondrial Ca 2+ overload, and protected cells from hypoxia-reoxygenation. Genetic or pharmacological inhibition of CypD provided a similar effect in adult mice cardiomyocytes. Disruption of ER-mitochondria interaction via the downregulation of Mfn2 similarly reduced the interaction between CypD and the IP3R1 complex and protected against hypoxia-reo...