The overall objective of this proposal is to elucidate the signaling mechanisms responsible for the adaptive response of the heart to ischemic and pharmacologic stimuli. We will attempt to develop a unifying signal transduction paradigm applicable to three different forms of preconditioning (PC): the early phase of ischemic PC, the late phase of ischemic PC, and NO-induced (pharmacologic) PC. Our fundamental hypothesis is that, in all three cases, the activation of PKCepsilon and the subsequent recruitment of Src/Lck play an essential mechanistic role. We further propose that the PKCepsilon-Src/Lck signaling pathway induces late PC by upregulating iNOS at the transcriptional level on day 1 and at the post-translational level on day 2. A broad multidisciplinary approach will be used that will combine diverse techniques (integrative physiology, protein chemistry, mass spectrometry, cellular biology, molecular biology, gene targeting, and transgenesis) and will integrate genetic information at the molecular level with biochemical information at the protein structure level and physiologic information at the whole animal level. Unequivocal evidence for or against an essential role of PKCepsilon, Src, and Lck in PC will be provided by the use of two novel PKCepsilon transgenic mouse lines (constitutively active and dominant negative mutants of PKCepsilon) and Src and Lck knockout mice in a well-established murine model of PC. This will enable us to examine kinase- specific modulation of PKCepsilon, Src, and Lck in the intact animal. The role of PKCepsilon in ischemic PC and NO-induced PC will be conclusively established by determining the effects of specific transgenic activation or inhibition of this isozyme on the cardioprotection. The activity and protein content of all seven Src PTKs expressed in the mouse heart (Fyn, Fgr, Yes, Src, Lyn, Lck, and Blk) will be systematically measured at serial times after ischemic PC or NO donor administration. The role of Src PTKs in triggering versus mediating late PC will be discerned by comparing inhibition of these kinases on day 1 versus day 2. Targeted disruption of the Src and Lck gene will be employed to conclusively establish the specific function of individual PTKs in the PC protection. The hierarchical positions of PKCepsilon and Src/Lck in the signaling cascade of PC will be identified by determining whether specific transgenic activation of PKCepsilon leads to activation of Src/Lck and Src/Lck-dependent protection and, conversely, whether specific transgenic inhibition of PKCepsilon blocks the activation of Src/Lck and the concomitant protection induced by PC. The downstream targets of the PKCepsilon-Src/Lck pathway will be explored by analyzing the signaling elements known to govern NF-kappaB activity (NIK, MEKK1, IKKalpha, IKKbeta, IkappaB-alpha) in the absence or presence of targeted Src and Lck gene ablation. Finally, the role of Src PTKs in the posttranslational modulation of iNOS on day 2 will be elucidated by measuring iNOS activity and tyrosine phosphorylation in the absence and presence of Src PTK inhibitors. This proposal should provide important new insights into the molecular mechanisms of ischemic PC and pharmacologic PC and into the role of PKCepsilon and Src/Lck in cardiac signaling in general. Elucidation of the mechanism of PC should facilitate the development of novel therapeutic strategies that duplicate its powerful cardioprotective effects.
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