The overall objective of this proposal is to elucidate the cellular mechanisms underlying the late phase of ischemic preconditioning (PC). The PI will attempt to develop a unifying pathogenetic paradigm applicable both to late PC against stunning and to late PC against infarction. The fundamental hypothesis is that, in both cases, NO plays a pivotal role, acting initially as the trigger and subsequently as the mediator of the protection (NO hypothesis of late PC). The PI proposes that NO formed during the initial PC stimulus activates a PKC-and NF-VB-dependent signal transduction cascade that culminates in increased expression of NOS, which then mediates the protection of late PC. Two different, but complementary, models (conscious rabbits and genetically-manipulated mice) will be used to arrive at conclusions that are definitive and, at the same time, physiologically relevant. A broad multidisciplinary approach will be used that will combine diverse techniques (integrative physiology, protein chemistry, cell biology, molecular biology, gene targeting and transgenesis) and will integrate genetic information at the molecular level with physiological information at the conscious animal level. The role of PKC as a critical effector of NO-initiated signaling in vivo will be directly investigated using isoform-selective assays of PKC in the presence or absence of NOS inhibitors or NO donors. The role of NF-VB as a key step in the signal transduction cascade responsible for late PC will be systematically evaluated by selective in vivo inhibition in conscious rabbits and selective gene manipulations in mice overexpressing a mutant lVBB that produces transdominant repression of NF-VB. The specific NOS isoforms responsible for initiating as well as mediating late PC will be identified both in conscious rabbits and in mice. The roles of iNOS, eNOS, and nNOS in rabbits will be interrogated by determining whether the changes in these isoforms after PC correlate with the time-course of protection and with the tissue levels of NOx and cGMP in the presence or absence of selective iNOS inhibitors. The posttranslational modulation of iNOS after PC will be explored by examining the effect of tyrosine kinase inhibitors on NOS activity and protein phosphorylation. The role of each individual NOS isoform in triggering vis-a-vis mediating late PC will be conclusively established by targeted disruption of the iNOS, eNOS, and nNOS gene in mice subjected to ischemic PC. A wide range of transgenic overexpressions of individual NOS isoforms will be examined to delineate in quantitative terms the relationship between NOS activity and cardioprotection. This proposal should produce important new insights into the molecular mechanisms of late PCA and into the role of NO in cardiovascular pathophysiology in general. Elucidation of the mechanism of late PC should facilitate the development of drugs that duplicate its powerful cardioprotective effects.
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