The overall objective of this proposal is to investigate the molecular mechanisms underlying the late phase of preconditioning (PC). We will attempt to develop a unifying pathogenic paradigm applicable both to ischemia-induced late PC and to NO donor-induced late PC. our fundamental hypothesis is that, in both cases, the central cellular adaptation is responsible for late PC is the transcriptional up- regulation of the iNOS gene. We propose that transcription of iNOS is mediated by the coordinated activation of NF-kappaB and other transcription factors, which is triggered by NC via a signal transduction cascade that includes PKC, tyrosine kinases, and IkappaB kinase (IKK). Two different forms of late PC (PC induced by ischemic stress and PC induced pharmacologically with NO releasing agents) will be systematically examined. Unequivocal evidence for or against the involvement of specific genes encoding transcription and signaling factors will be provided by the use of gene targeted and transgenic mice in a well-established murine model of late PC. A broad multi- disciplinary approach will be used that will combine diverse techniques (integrative physiology, molecular biology, protein chemistry, cell biology, gene targeting and transgenesis) and will integrate genetic information at the molecular level with physiological information at the whole animal level. The effects of PC upon NOS mRNA, protein, enzymatic activity, and cellular distribution will be systematically defined for all three isoforms (eNOS, iNOS, and nNOS), providing for the first time a thorough characterization of these changes in the mouse. The signaling mechanisms that control iNOS expression will be interrogated by determining the effect of pharmacologic inhibitors of NOS, PKC, tyrosine kinases, and NF-kappaB on infarct size and by correlating these effects with their effects on iNOS transcription. For the first time, the effect of PC on the phosphorylation activity of the IKK complex (which controls NF-kappaB) will be examined. The role of NF-kappaB in late PC will be conclusively established by targeted gene ablation of three specific NF- kappaB subunits (p50, Rel-B, c-rel) and by genetic blockade of the IKK/IkappaB/NF-kappaB signaling pathway. Two novel transdominant NF- kappaB mutants (IkappaBalpha/S32A, S36A and IKK-beta/k44A) will be used to determine the role of IKK and IkappaBalpha in iNOS up-regulation. The specific modulatory proteins that govern iNOS gene expression during late PC will be systematically identified by targeted genetic ablation of the transcription factors known to bind to the iNOS promoter (IRF-1, TNFalpha, STAT1, CREB, AP-1, IL-2, IL-6). This proposal should provide important new insights into the molecular mechanisms of late PC and into the role of NO and iNOS in cardiovascular pathophysiology in general Elucidation of the mechanism of late PC should facilitate the development of novel pharmacological and/or gene therapeutic strategies that duplicate its powerful cardioprotective effects.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL063034-01
Application #
2884177
Study Section
Cardiovascular and Renal Study Section (CVB)
Project Start
1999-07-01
Project End
2000-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Louisville
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Louisville
State
KY
Country
United States
Zip Code
40292
Tranter, Michael; Liu, Yemin; He, Suiwen et al. (2012) In vivo delivery of nucleic acids via glycopolymer vehicles affords therapeutic infarct size reduction in vivo. Mol Ther 20:601-8
Tranter, Michael; Helsley, Robert N; Paulding, Waltke R et al. (2011) Coordinated post-transcriptional regulation of Hsp70.3 gene expression by microRNA and alternative polyadenylation. J Biol Chem 286:29828-37
Wilhide, Michael E; Tranter, Michael; Ren, Xiaoping et al. (2011) Identification of a NF-?B cardioprotective gene program: NF-?B regulation of Hsp70.1 contributes to cardioprotection after permanent coronary occlusion. J Mol Cell Cardiol 51:82-9
Tranter, Michael; Ren, Xiaoping; Forde, Tiffany et al. (2010) NF-kappaB driven cardioprotective gene programs; Hsp70.3 and cardioprotection after late ischemic preconditioning. J Mol Cell Cardiol 49:664-72
Weintraub, Neal L; Jones, W Keith; Manka, David (2010) Understanding radiation-induced vascular disease. J Am Coll Cardiol 55:1237-9
Kwon, Ohwon; Tranter, Michael; Jones, W Keith et al. (2009) Differential translocation of nuclear factor-kappaB in a cardiac muscle cell line under gravitational changes. J Biomech Eng 131:064503
Jones, W Keith; Fan, Guo-Chang; Liao, Siyun et al. (2009) Peripheral nociception associated with surgical incision elicits remote nonischemic cardioprotection via neurogenic activation of protein kinase C signaling. Circulation 120:S1-9
Young, David; Popovic, Zoran B; Jones, W Keith et al. (2008) Blockade of NF-kappaB using IkappaB alpha dominant-negative mice ameliorates cardiac hypertrophy in myotrophin-overexpressed transgenic mice. J Mol Biol 381:559-68
Flaherty, Michael P; Brown, Maria; Grupp, Ingrid L et al. (2007) eNOS deficient mice develop progressive cardiac hypertrophy with altered cytokine and calcium handling protein expression. Cardiovasc Toxicol 7:165-77
Higuchi, Yoshihiro; Chan, Tung O; Brown, Maria A et al. (2006) Cardioprotection afforded by NF-kappaB ablation is associated with activation of Akt in mice overexpressing TNF-alpha. Am J Physiol Heart Circ Physiol 290:H590-8

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