This proposal represents collaboration between investigators in the Sections of Pulmonary Medicine & Critical Care and Emergency Medicine under the newly formed Emergency Resuscitation Research Center at the University of Chicago. Studies are proposed to examine the mechanisms and consequences of ischemia/reperfusion (I/R)-induced apoptosis in cardiomyocytes and to examine the protective effects of hypoxic preconditioning on this apoptosis. The central hypotheses of this proposal are that preconditioning decreases apoptotic cell death through a number of mechanisms that block the initiation and/or propagation of apoptosis. To test these hypotheses, we propose three specific aims: (1) Test which reactive oxygen species (ROS) act as intracellular activators of apoptosis. Experiments will (a) Determine the sequence of apoptotic events including disruption of mitochondria membrane potential, release of cytochrome c into the cytosol, and activation of caspase-9 as """"""""initiator"""""""" caspase as well as downstream casases-3 and others; (b) Using specific inhibitory treatments, assess the effects of altered superoxide and/or H202 during (I/R) on these apoptotic events. (2) Determine whether preconditioning blocks certain apoptotic events via Bcl-2 family regulation through PKC-mediated phosphorylation. Experiments will (a) Determine pre- and post-ischemic levels of expression of Bcl-2, Bcl-x, and Bax in cardiomyocytes during I/R in preconditioned (PC) or non-PC cells; and determine the effects of PC on serine phopshorylation of Bcl-2 proteins (b) Assess the effects of over and underexpression/blockade of PKC, Bcl-2, and expression of a nonphosphorylatable Bcl-2 protein on oxidant stress, nitric oxide synthesis, NF-kappaB activation and apoptotic events. (3) Determine the relationship between NF-kappaB and nitric oxide (NO) synthesis in preventing apoptosis. (a) Determine the effect of PC on phosphorylation Of IkappaB and subsequent activation and nuclear translocation of NF-kappaB; (b) Determine the expression of immediate-early genes, Al and chIAP, and examine their effects on mitochondria events, ROS generation, oxidant stress, NO generation, caspase activity and apoptotic cell death through correlative and direct over expression and antisense studies. (c) Determine the levels of NO generation during reperfusion with or without PC and the direct effects of generated NO or specific blockade of NO generation on caspase activity and subsequent cell death in each condition. Results from these studies have the potential to improve post-resuscitation care following cardiac arrest and other ischemic diseases such as myocardial infarction and stroke.
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