Increased hexosamine biosynthesis and protein O-GLCNAC protect isolated rat heart from ischemia/reperfusion injury

by Ph.D. Liu, Jia

Increased levels of protein-associated O-linked N-acetylglucosamine (O-GlcNAc) have been correlated with increased tolerance to 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, increased the tolerance of the heart to ischemia and reperfusion (I/R). Hearts from male rats were isolated and perfused with Krebs-Henseliet buffer containing 5mM glucose, and global, no-flow ischemia was induced for 20min followed by 60 min reperfusion. 20 min pre-treatment with 10 mM glucosamine, which enters the HBP directly, significantly improved functional recovery and decreased cardiac Troponin I release during reperfusion. This protection was associated with a marked increase in the level of O-GlcNAc. Pre-treatment with 5mM alloxan, an inhibitor of O-GlcNAc transferase (OGT), the enzyme that catalyzes O-GlcNAcylation, completely reversed the protection seen with glucosamine and prevented the increase in O-GlcNAc. Glutamine: fructose-6-phosphate amidotransferase (GFAT) regulates the entry of glucose into the HBP, and glutamine is essential for GFAT activity. 30 min pre-treatment with 5 mM glutamine significantly improved functional recovery and decreased tissue injury during reperfusion. This protection was associated with a significant increase in the levels of UDP-N-acetylglucosamine (UDP-GlcNAc), protein O-GlcNAc and ATP. Pre-treatment with 80µM azaserine, an inhibitor of GFAT, completely reversed ii the protection seen with glutamine and prevented the increase in UDP-GlcNAc and protein O-GlcNAc. Inhibition of OGT with 5mM alloxan also reversed the protection associated with glutamine. Pre-ischemic treatment is frequently impractical in clinical practice; therefore, we also demonstrated that post-ischemic treatment with both glucosamime and PUGNAc, which is an effective inhibitor of O-GlcNAcase, improved functional recovery of hearts during reperfusion. This was associated with attenuation of calcium-mediated proteolysis of CaMKII and ?-fodrin. Taken together, these data support the notion that increasing cardiac O-GlcNAc levels may be a clinically relevant cardioprotective strategy and suggest that this protection could be due, at least in part, to inhibition of calcium-mediated stress responses. iii