The central hypothesis of this competitive renewal is formulated on the basis of two key developments in the field of ischemia biology and cardioprotection: (i) the identification of multiprotein complex formation as a means for cardioprotective signaling and (ii) the establishment of the mitochondria as a critical organelle for cell death and survival. The functional link between these two developments is mitochondrial permeability transition (MPT) in ischemic injury. MPT involves impaired mitochondrial function leading to cell death and is carried out by a multiprotein complex, the MPT pore. Deciphering the proteomic basis of the MPT pore and elucidating its role in cardioprotection constitute the major goals of this application. The proposed studies will focus on nitric oxide (NO)-induced late preconditioning (PC), a well-characterized pharmacological means of cardioprotection with a poorly-defined proteomic basis for its underlying cellular mechanisms. To achieve our goals, the application directs its effort at two targets: the Oracle isozymes, a novel family of scaffolding proteins critical to NO protective signaling, and the subproteome of ANT1, a repertoire of ANT1-associating proteins that are essential to MPT pore function. We hypothesize that attenuated propensity for mitochondrial permeability transition is an essential signaling event in NO-mediated cardioprotection and that the Oracle family of proteins plays a critical role in this process by modulating the ANT1 subproteome and contributing to the protection of mitochondrial function. Three specific aims, with tightly related experimental protocols are proposed. Aim 1 will establish the essential role of Oracle, a molecular backbone of signaling complexes, in NO-induced preconditioning. Aim 2 will build upon knowledge gained in Aim 1 to elucidate the function of the MPT pore in NO-mediated cardioprotection and determine the role of Oracle in this process. Finally, Aim 3 will define a subproteome, composed of ANT1-associating proteins, which is critical to the regulation of MPT pore function in cardiac mitochondria. Importantly, for the first time, the proteomic basis of the participation of the MPT pore in NO protection will be determined and the role of Oracle in the assembly of this subproteome will be fully characterized. A comprehensive strategy encompassing pharmacological methods, transgenic models, and proteomic characterization is proposed. These studies will elucidate the proteomic basis of NO preconditioning, define mechanisms underlying ANT1 regulation, and identify novel molecular targets that are critical to the modulation of MPT pore function. Thus, the findings will have broad implications for future investigations concerning pharmacological cardioprotective interventions.