Heart disease remains the leading cause of death worldwide. As a result of studies done by Barker and associates, our awareness of the significance of stress during gestation as a risk factor for heart diseases has expanded. We now know that events in utero can significantly alter gene expression patterns in heart tissue leading to increase susceptibility to ischemia reperfusion injury in adulthood. The focus of this project was to elucidate the role of chronic hypoxia in the programming of the cardio-protective gene, Protein Kinase C epsilon (PKCÎµ) in fetal rat heart. We used an animal, organ base, and cell culturing with the rat embryonic cell line H9c2 to determine the molecular events underpinning the heightened sensitivity to ischemia reperfusion injury of adult offspring exposed to chronic hypoxia in utero. We determined that chronic hypoxia directly represses PKCÎµ expression through increase methylation of CpG dinucleotides for two SP1 binding sites located at proximal region of the PKCÎµ promoter. Previous studies using reporter gene assays concluded the region encompassing both SP1 binding sites played a significant role in the activity of PKCÎµ promoter. Chromatin immunoprecipitation (ChIP) assays further verified the functional significance of methylation for both Sp1 sites in reducing SP1 protein binding. In the presence of the DNA methylation inhibitor, 5-aza-2-deoxycytidine, binding of SP1 to PKCÎµ promoter, promoter methylation, and PKCÎµ protein and mRNA were restored to control levels. Connecting epigenetics to chronic hypoxia in utero led us to further investigate the underlining mechanism of hypoxia-induced methylation of PKCÎµ promoter. The dominant pathway of cellular adaptation to hypoxic stress involves the stabilization of the Hypoxia-Inducible-Factor 1 alpha (HIF-1Î±). We found blockade of nuclear accumulation of HIF-1Î± did not restore PKCÎµ mRNA to control values. Next, we found the ROS Scavengers N-acetylcysteine and 4-hydroxy Tempo protect against hypoxia-induced repression of PKCÎµ gene activity, which linked oxidative stress to PKCÎµ repression in fetal hearts. This project has demonstrated that chronic hypoxia directly regulates PKCÎµ gene expression through ROS mediated epigenetic repression of PKCÎµ promoter, which leads to long term programming of the fetal heart.
School of Medicine
De Leon, Marino
Doctor of Philosophy (PhD)
Year Degree Awarded
Date (Title Page)
Library of Congress/MESH Subject Headings
Cardiovascular Diseases; Fetal Hypoxia -- physiopathology; Fetal Heart -- physiopathology; Fetal Development; Cardiovascular System -- physiopathology; Protein Kinase C; Endothelium, Vascular
Subject - Local
Heart disease; Chronic Hypoxia; Fetal rat hearts; Gene expression patterns; Cardioprotective gene; Vascular Endothelial Growth Factor
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This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.
Patterson, Andrew James Grant, "Chronic Hypoxia Induces Epigenetic Modifications in the Fetal Rat Heart" (2011). Loma Linda University Electronic Theses, Dissertations & Projects. 54.
Loma Linda University Electronic Theses & Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives