UBC Theses and Dissertations
Nitric oxide-induced cardiomyocyte cell death Klassen, Shaun Scott
Nitric oxide (NO), a regulator of diverse cardiovascular functions, modifies cardiac cell viability through mechanisms that remain uncertain. Several pathways were studied to understand these effects. The possibility that the protein p53 is involved in the cardiomyocyte response to the NO donor s-nitrosoglutathione (GSNO) or the peroxynitrite donor 3- morpholinosydnonimine (SIN-1) was explored. These donors induced a concentration-dependent increase of cell death in cultured embryonic chick cardiomyocytes. Expression of p53 protein was increased in response to GSNO, specifically in the nucleus. GSNO also caused DNA damage, but pifithrin, an inhibitor of p53 transactivation activity, did not alter the extent of this damage or cell death. Therefore, the role of increased nuclear p53 in response to NO and NO-induced DNA damage may not be specifically operative in NO-induced cell death. The action of GSNO also appears independent of mitochondrial pathways in cell death, as there was no association of p53 with the mitochondria. Neither GSNO- or SIN-1-induced cell death was altered by cyclosporin A, suggesting that permeability transition pore opening is not operative in these modes of induction of death. In contrast to SIN-1, GSNO did not reduce mitochondrial transmembrane potential, implying separate mechanisms of cell death. Immunocytochemistry demonstrated increased amounts of nitrotyrosine in response to GSNO or SIN-1, confirmed by Western blot following SIN-1. FeTPPS, an isomerase that converts peroxynitrite into the less toxic nitrate, produced a significant reduction of SIN-1-induced cell death and cellular protein nitration. FeTPPS did not reduce cell death from GSNO alone, but did from the combination of GSNO and hydrogen peroxide, a condition which promotes the generation of peroxynitrite. In summary, NO-induced cardiomyocyte cell death is due in part to the disruption of normal cellular functions by nitration of key proteins. Peroxynitrite decomposition reduces protein nitration and cell death, while p53 appears functions independent of the mitochondria or gene transactivation and may act in other pathways, such as cell repair.
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