(Verbatim from the application): The overall objective of this proposal is to elucidate the cellular and molecular mechanisms underlying the late phase of ischemic preconditioning (PC). Recent studies have shown that the generation of nitric oxide (NO) and the activation of protein kinase C (PKC) and tyrosine kinases are associated with the development of late PC; however, the downstream signaling pathways that transduce the diverse signals elicited by ischemia into increased expression of cardioprotective gene(s) and protein(s) remain poorly understood. Our fundamental hypothesis is that activation of the Jak-Stati pathway after ischemic PC is necessary for the protective effect of late PC via the upregulation of iNOS. We further propose that ischemic PC activates this pathway through both tyrosine- (via NO generated by cNOS activation) and serine-phosphorylation (via the PKC-p44/42 MAPKs cascade) dependent mechanisms. All studies will be conducted in an established mouse model of late PC. This study will utilize a broad multidisciplinary approach that will combine diverse techniques, including integrative physiology, molecular biology, and gene targeting.
Aim 1. Activation of the Jak-Stati pathway after ischemic PC and its role in the cardioprotective effect of late PC will be systemically investigated. The requirement of tyrosine and serine phosphorylation of Stati for its nuclear translocation and DNA binding will be studied.
Aim 2. An obligatory role of Stati in iNOS expression and in late PC will be conclusively established by the use of Stati gene knockout mice and a novel Stati transgenic mouse line.
Aim 3. The role of the activation of specific cNOS isoforms (eNOS or nNOS) by ischemia in activating the Jak-Stati pathway leading to iNOS expression and cardioprotection will be established by the use of eNOS and nNOS gene knockout mice. To gain insights into the mechanism, NO donors will be used, in the absence of ischemia, to determine whether they activate the same Jak-Stat1 pathway as does ischemic PC leading to iNOS expression and cardioprotection.
Aim 4. Genetically-engineered mice via transgenesis of dominant negative mutants of the Stat1-Ser-727 residue and the Stat1-Tyr-701 residue will be used to determine whether pSer of Stat1 after ischemia is essential for transcriptional activation of iNOS gene and for the cardioprotection. This proposal will provide important new insights into the cellular and molecular mechanisms of the cardioprotection conferred by ischemia- and NO donor-induced late PC and into the role of the Jak-Stat1 pathway in iNOS expression and in cardiovascular pathophysiology in general. The information generated from this project may eventually lead to the development of novel pharmacological and/or gene therapeutic strategies to protect the heart in patients with coronary artery disease.
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