Mitochondrial calcium has been postulated to regulate a wide range of processes from bioenergetics to cell death. Here, we characterize a mouse model that lacks expression of the recently discovered mitochondrial calcium uniporter (MCU). Mitochondria derived from MCU-/- mice have no apparent capacity to rapidly uptake calcium. While basal metabolism appears unaffected, the skeletal muscle of MCU-/- mice exhibited alterations in the phosphorylation and activity of pyruvate dehydrogenase. In addition, MCU-/- mice exhibited marked impairment in their ability to perform strenuous work. We further show that mitochondria from MCU-/- mice lacked evidence for calcium-induced permeability transition pore (PTP) opening. The lack of PTP opening does not appear to protect MCU-/- cells and tissues from cell death, although MCU-/- hearts fail to respond to the PTP inhibitor cyclosporin A (CsA). Taken together, these results clarify how acute alterations in mitochondrial matrix calcium can regulate mammalian physiology.

Abstract-Previous studies have revealed a novel interaction between deoxyhemoglobin and nitrite to generate nitric oxide (NO) in blood. It has been proposed that nitrite acts as an endocrine reservoir of NO and contributes to hypoxic vasodilation and signaling. Here, we characterize the nitrite reductase activity of deoxymyoglobin, which reduces nitrite approximately 36 times faster than deoxyhemoglobin because of its lower heme redox potential. We hypothesize that physiologically this reaction releases NO in proximity to mitochondria and regulates respiration through cytochrome c oxidase. Spectrophotometric and chemiluminescent measurements show that the deoxymyoglobin-nitrite reaction produces NO in a second order reaction that is dependent on deoxymyoglobin, nitrite and proton concentration, with a bimolecular rate constant of 12.4 mol/L -1 s -1 (pH 7.4, 37°C). Because the IC 50 for NO-dependent inhibition of mitochondrial respiration is approximately 100 nmol/L at physiological oxygen tensions (5 to 10 mol/L); we tested whether the myoglobin-dependent reduction of nitrite could inhibit respiration. Indeed, the addition of deoxymyoglobin and nitrite to isolated rat heart and liver mitochondria resulted in the inhibition of respiration, while myoglobin or nitrite alone had no effect. The addition of nitrite to rat heart homogenate containing both myoglobin and mitochondria resulted in NO generation and inhibition of respiration; these effects were blocked by myoglobin oxidation with ferricyanide but not by the xanthine oxidoreductase inhibitor allopurinol. These data expand on the paradigm that heme-globins conserve and generate NO via nitrite reduction along physiological oxygen gradients, and further demonstrate that NO generation from nitrite reduction can escape heme autocapture to regulate NO-dependent signaling. (Circ Res. 2007;100:654-661.)

Abstract-Nitric oxide has been shown to be an important signaling messenger in ischemic preconditioning (IPC).Accordingly, we investigated whether protein S-nitrosylation occurs in IPC hearts and whether S-nitrosoglutathione (GSNO) elicits similar effects on S-nitrosylation and cardioprotection. Preceding 20 minutes of no-flow ischemia and reperfusion, hearts from C57BL/6J mice were perfused in the Langendorff mode and subjected to the following conditions: (1) control perfusion; (2) IPC; or (3) 0.1 mmol/L GSNO treatment. Compared with control, IPC and GSNO significantly improved postischemic recovery of left ventricular developed pressure and reduced infarct size. IPC and GSNO both significantly increased S-nitrosothiol contents and S-nitrosylation levels of the L-type Ca 2ϩ channel ␣1 subunit in heart membrane fractions. We identified several candidate S-nitrosylated proteins by proteomic analysis following the biotin switch method, including the cardiac sarcoplasmic reticulum Ca 2ϩ -ATPase, ␣-ketoglutarate dehydrogenase, and the mitochondrial F1-ATPase ␣1 subunit. The activities of these enzymes were altered in a concentration-dependent manner by GSNO treatment. We further developed a 2D DyLight fluorescence difference gel electrophoresis proteomic method that used DyLight fluors and a modified biotin switch method to identify S-nitrosylated proteins. IPC and GSNO produced a similar pattern of S-nitrosylation modification and cardiac protection against ischemia/reperfusion injury, suggesting that protein S-nitrosylation may play an important cardioprotective role in heart. (Circ Res. 2007;101:1155-1163.) Key Words: preconditioning Ⅲ S-nitrosylation Ⅲ cardioprotection I schemic preconditioning (IPC) is a cellular adaptive phenomenon whereby brief episodes of myocardial ischemia and reperfusion (I/R) render the heart resistant to subsequent prolonged ischemic injury. 1 Through activation of a complex cascade of signaling events, IPC has been shown to reduce arrhythmias, infarct size, and postischemic contractile dysfunction. [2][3][4][5] Nitric oxide (NO) has been shown to be an important signal in cardioprotection. 6 -8 In acute IPC, NO has been shown to mediate protection at least in part by activation of guanylyl cyclase, resulting in the production of cyclic guanosine monophosphate (cGMP) and the activation of protein kinase G, which in turn leads to the opening of the mitochondrial K ATP channel. 6 Recently, it has also been shown that protein kinase G results in activation of an endogenous mitochondrial protein kinase C that is involved in activation of the mitochondrial K ATP channel. 9,10 The opening of the mitochondrial K ATP channel is reported to reduce mitochondrial Ca 2ϩ loading 11,12 and also to lead to generation of reactive oxygen species, which activate signaling cascades in a feed-forward manner to elicit cardioprotection. 13,14 In addition to activating cGMP/protein kinase G-dependent signaling pathways, NO can directly modify sulfhydryl residues of proteins through S-nitrosylation, which ha...