Date on Master's Thesis/Doctoral Dissertation

12-2009

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Physiology and Biophysics

Committee Chair

Jones, Steven Paul

Author's Keywords

Myocardial ischemia; O-GlcNAc; Oxidative stress; Endoplasmic reticulum stress

Subject

Coronary heart disease

Abstract

O -linked ß-N-acetylglucosamine ( O -GlcNAc) is an inducible, dynamically cycling, and reversible post-translational modification of serine/threonine amino acid residues of nucleocytoplasmic and mitochondrial proteins. O -GlcNAc transferase (OGT) adds, while O -GlcNAcase (GCA) removes O -GlcNAc from proteins. Albeit being a recruitable stress-induced signal in other tissues, the role of O -GlcNAc in the heart is unknown. Therefore, we hypothesized that O -GlcNAc is recruited in the heart during acute stress, and enhanced O-GlcNAc is cardioprotective. Subjecting neonatal rat cardiac myocytes (NRCMs) to hypoxia, or mice to myocardial ischemia reduced O-GlcNAc signaling. Augmented O-GlcNAc signaling attenuated, while diminished O-GlcNAc signaling exacerbated post-hypoxic cardiomyocyte death. To determine how O-GlcNAc protects, we identified numerous proteins including voltage dependent anionic channel (VDAC) to be O-GlcNAc-modified via mass spectrometry and immunoprecipitation. Since VDAC is a putative member of the mitochondrial permeability transition pore (mPTP), we hypothesized that one mechanism of O-GlcNAc-mediated cardioprotection is by blocking mPTP formation. We ascertained if O-GlcNAc signaling affects key players in ischemic/hypoxic injury, Ca 2+ overload and oxidative stress, both inducers of mPTP. Enhanced O-GlcNAc significantly mitigated, while, reduced O-GlcNAc aggravated post-hypoxic Ca 2+ overload and ROS generation. Furthermore, augmented O-GlcNAc reduced, while, diminished O-GlcNAc sensitized mitochondria to mPTP formation according to Ca 2+ -induced swelling. Since mPTP formation induces loss of mitochondrial membrane potential (?? m ), we evaluated whether O-GlcNAc signaling affects post-hypoxic ?? m recovery. Enhanced O-GlcNAc significantly improved, while reduced O-GlcNAc minimized post-hypoxic ?? m recovery. Because ER stress contributes to ischemia-reperfusion injury, we evaluated whether inhibiting maladaptive ER stress reponse maybe another mechanism through which O-GlcNAc signaling cardioprotects. Indeed, augmented O-GlcNAc reduced maladaptive ER stress response according to diminished CHOP levels and PI positivity. To determine if such in vitro protection could be translated in vivo , we augmented O-GlcNAc levels (with PUGNAc) in adult, wild-type C57BL6 mice, subjected them to 40 minutes of left anterior descending coronary artery ligation, then reperfused for 24 hours, and assessed infarct size. Augmented O-GlcNAc levels significantly decreased infarct size. We conclude that O-GlcNAc mediates cardioprotection in vitro and in vivo via attenuating maladaptive ER stress response and recruitment of early events in the mitochondria! death pathway leading to mPTP formation.

Download

Share

COinS