Title

Apoptotic signaling pathways in hypoxia-acidosis and hypoxia-reoxygenation of cardiac myocytes

Date of Award

2002

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Molecular and Cellular Pharmacology

First Committee Member

Keith A. Webster, Committee Chair

Abstract

Over 15 million people in the world die each year due to cardiovascular diseases. Many forms of these heart diseases are correlated with substantial loss of the myocardium. Apoptosis or programmed cell death is considered to play a major role in the cell death resulting from the two main components of heart disease: ischemia and reperfusion. As of yet, the signaling pathway resulting in apoptosis by ischemia alone is not well characterized, however it is likely that metabolite accumulation due to hindered blood flow is involved. Significantly more progress has been made on the ischemia-reperfusion pathway that results in apoptosis. After a brief ischemic event followed by reperfusion of the tissue, the newly oxygenated blood perfusing the previously ischemic tissue results in the generation of reactive oxygen species (ROS). By reacting with lipids, proteins, and DNA, ROS are capable of causing extensive damage to cells. These ROS, which also activate c-jun N-terminal kinase (JNK), are believed to initiate the signaling pathways required for the induction of apoptosis.The goal of this thesis was to further elucidate factors in the signaling pathways of both ischemia and ischemia-reperfusion-mediated apoptosis in a cell culture model. The first chapter of this dissertation serves as an introduction. The second chapter addresses the apoptosic signaling pathways in hypoxia/acidosis, with a focus on proapoptotic Bcl-2 family member BNIP3. Hypoxia induced the expression and accumulation of BNIP3 mRNA and protein in cardiac myocytes but acidosis was required to activate the death pathway. Acidosis stabilized BNIP3 protein and increased the association with mitochondria. Pretreatment with antisense BNIP3 oligonucleotides blocked cell death induced by hypoxia-acidosis. The pathway included extensive DNA fragmentation and opening of the mitochondria) permeability transition pore, but no caspase activation. Overexpression of wild type BNIP3, but not a translocation-defective mutant, activated cardiac myocyte death only when the myocytes were acidic.The third chapter is directed at hypoxia-reoxygenation-mediated apoptosis, specifically in the identification of the source of ROS responsible for activation of apoptosis as well as mitochondria) influences on reoxygenation-mediated JNK activation. Using mitochondria) complex inhibitors, JNK activation by reoxygenation was demonstrated to involve both ROS production via the electron transport chain as well as coupled electron transport through mitochondria) complexes II, III, and IV.Finally, as efficient delivery and conditional regulation of a therapeutic gene to the myocardium is one of the ultimate goals of the laboratory, Chapter four describes experiments investigating the protective effects of Dt-diaphorase, as well as the hypoxia regulation of a beta-galactosidase reporter gene in vitro and in vivo. DT-diaphorase transfection protected the myocytes from hypoxia-reoxygenation-mediated apoptosis. beta-galactosidase assay results confirmed the conditional regulation of the hypoxia-regulated adenovirus in vitro, but in vivo regulation of the reporter gene was not reproducible.

Keywords

Biology, Molecular

Link to Full Text

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