A central mechanism leading to necrosis and apoptosis during ischemia/reperfusion is believed to be the mitochondrial permeability transition (MPT), due to permeability transition pore (PTP) opening in the inner mitochondrial membrane. Based on our recent work, we hypothesize. In this revised application that two separable components predispose mitochondria to injury during anoxia/reoxygenation. The MPT threshold component is most relevant to the anoxic or ischemic period, and sets the threshold for MPT during reperfusion. It is manifested as progressive MPT-independent cytochrome c loss and inner membrane leakiness, which can be attributed to accumulation of long chain fatty acids (FA) and reactive oxygen species (ROS). The MPT trigger component is most relevant to reperfusion. Whether MPT occurs during reperfusion is determined by the interplay between MPT inducers/inhibitors present during rexoygenation (particularly matrix free Ca levels) and electron transport capacity for regenerating mitochondrial membrane potential (deltapsim),which in turn depends on cytochrome c content and inner membrane leak. Consistent with its known cardioprotective role, we find that mitoKATP channel agonist diazoxide protects against both the MPT threshold and MPT trigger components, and that this protection is blocked by mitoKATP antagonist 5-HD. In addition, PKC epsilon, a key signaling component in cardioprotection, protects against the MPT trigger component. The objective of this proposal is to further explore the signal transduction pathways protecting mitochondria from the MPT threshold and MPT trigger components under conditions generally relevant to ischemia/reperfusion. Our strategy is to integrate functional studies with proteomics analysis. Functional studies will use spectrofluorometric, imaging (fluorescent, confocal and high voltage electron microscopy), and adenoviral gene transfer techniques to study mitochondria and cardioprotection at three levels: isolated mitochondria, in situ mitochondria in permeabilized myocytes, and isolated myocytes. Proteomic analysis will dissect mitochondrial protein complexes associated with PKCepsilon and PTP components in protected and unprotected intact hearts. Using this integrated approach, we will 1) further characterize the mechanisms by which ischemic/reperfusion elements promote the MPT threshold and trigger components, and how mitoKATP channel agonists are protective; 2) define the roles of isoform-specific PKC signaling in protection against the MPT threshold and trigger components; 3) examine whether other signaling pathways implicated in cardioprotection modulate susceptibility to the MPT threshold and MPT trigger components. 4) identify, using functional proteomics, the proteins forming multiprotein signaling complexes with PKCepsilon and known PTP components in unprotected and protected hearts, and 5) characterize deltapsim depolarization waves induced by anoxia/reoxygenation to define their association with cytochrome c release and MPT and their responsive to mitoKATP activation and cardioprotective signaling pathways. ? ?
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