Therapeutic hypothermia can significantly attenuate post-resuscitation injury and improve survival in some patients. In our cellular and animal models of global ischemia, there is significant post-resuscitation cardiac injury. The central hypothesis of this proposal is that hypothermia initiated during ischemia protects cardiomyocytes and preserves cardiac function through interrelated pathways, involving the production of nitric oxide (NO) and p53 down-regulation, which act at the mitochondria to prevent initiation of the apoptotic cascade. Using a combination of established normothermic and hypothermic cellular and animal models, we propose two major specific aims: 1) Determine the interacting roles of hypothermia-induced nitric oxide, modulation of reactive oxygen species (ROS) and the mitochondrion in protecting cardiomyocytes and the heart from ischemia/reperfusion (l/R)-induced injury. We hypothesize that intra-ischemic hypothermic protection involves the generation of an altered ROS and reactive nitrogen species (RNS) profile, allowing recovery of ischemia-reduced mitochondrial membrane potential and blocking the release of cytochrome c (cyt c) from the mitochondria. In these studies, we will examine the induction or modulation of specific oxidants, such as superoxide and nitric oxide, by hypothermia and these effects on the initiation of the mitochondrial pathway of apoptosis. 2) Determine the interactions between mitochondrial Bcl-2 family proteins, specifically Bax and Bak, and other """"""""mitochondria-targeted"""""""" proteins, specifically p53, through which hypothermia-induced NO generation provides protection and prevents release of pro-apoptotic mitochondrial proteins cyt c and activation of caspases. We hypothesize that activation of p53 during I/R leads to activation of Bax and/or Bak and hypothermic blockade of p53 activation, interactions with Bax/Bak, and/or translocation will inhibit these interactions, blocking the release of cyt c and protecting the cells from apoptosis. The proposed studies will elucidate the molecular mechanisms involved in the hypothermic induction of protection of cardiomyocytes, including details of the """"""""adaptive"""""""" or protective hypothermic oxidant profile. An understanding of mechanisms by which specific mitochondrial pathways of apoptosis are affected by hypothermic signaling will aid in developing optimal protective protocols in hypothermic, pharmacological and genetic therapeutic approaches.
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