Advisory Committee Chair
Richard B Marchase
Advisory Committee Members
John C Chatham
Victor M Darley-Usmar
Fang-Tsyr Lin
Bradley Yoder
Document Type
Dissertation
Date of Award
2008
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
Increased levels of protein O-linked-N-acetylglucosamine (O-GlcNAc) have been correlated with increased cell survival following stress. Therefore the goal of this study was to determine whether enhanced flux through the hexosamine biosynthesis pathway (HBP), which leads to elevated levels of O-GlcNAc, improved the tolerance of isolated neonatal rat ventricular myocytes (NRVMs) to ischemia/reperfusion (I/R) injury. NRVMs were exposed to hypoxic medium for 4 hours, followed by oxygenated complete medium for 16 hours. Acute treatment with 5 mM glucosamine showed an increase in viability and decreased both necrosis and apoptosis following I/R. Glucosamine increased O-GlcNAc levels maximally at 2 hours and went down at 16 hours of reperfusion. Pretreatment with 30 mM glucose promoted survival and decreased apoptosis following I/R while pretreatment with azaserine, an inhibitor of GFAT, which regulates glucose entry into HBP, abrogated this protection. In addition, reperfusion in the absence of glucose reduced O-GlcNAc levels and resulted in lower viability. The improved viability seen with glucosamine treatment was also associated with attenuation of ischemiainduced NFAT activation; suggesting that increased O-GlcNAc levels may reduce cellular Ca2+ during ischemia and reperfusion. Overexpression of O-GlcNAc transferase (OGT) increased O-GlcNAc levels and survival whereas blocking O-GlcNAc iii modification by OGT knockdown or pretreatment with alloxan, an inhibitor of OGT exacerbated cellular injury following I/R. Pretreatment with PUGNAc or NButGT, inhibitors of O-GlcNAcase was less protective against I/R, despite a 10 fold greater increase in overall O-GlcNAc levels. Interestingly, the protective effect of glucosamine was correlated with increasing levels of mitochondrial Bcl-2, protecting the loss of cytochrome C, which was also associated with attenuation of H2O2-induced loss of mitochondrial membrane potential. Therefore, the results from this study indicate at least two potential mechanisms contributing to the protection associated with elevated OGlcNAc: 1) attenuation of cellular calcium and 2) accumulation of mitochondrial Bcl-2, consequently protecting in mitochondria function which in final reduced cardiomyocyte necrosis and apoptosis. Taken together, these data support the notion that elevating OGlcNAc levels by increasing flux through OGT protects against ischemia-reperfusion injury in cardiomyocytes, suggesting that strategies designed to activate these pathways may represent novel interventions for inducing cardioprotection.
Recommended Citation
Champattanachai, Voraratt, "Effects Of Hexosamine Biosynthesis On An In Vitro Model Of Cardiacischemia" (2008). All ETDs from UAB. 3677.
https://digitalcommons.library.uab.edu/etd-collection/3677