Effect Of Ischemia-Reperfusion On O-Glcnacylation Of Specific Proteins In Isolated Rat Hearts

We have shown that increasing the level of O‐linked‐N‐acetylglucosamine (O‐GlcNAc) on proteins protects the heart against ischemia‐reperfusion injury; however, the specific proteins altered by O‐GlcNAc have yet to be determined. Isolated perfused hearts from male rats (n=5–9/groups) were subjected to 10 min no‐flow ischemia (ISC) or ISC followed by 60 min reperfusion (I/R). Overall protein O‐GlcNAc levels, determined by anti‐O‐GlcNAc immunoblots, were increased following ISC (p<0.05) compared to normoxic controls. Using MALDI‐TOF we identified proteins in two bands in which O‐GlcNAc levels significantly increased during ISC and subsequently decreased following I/R and one band that increased only following I/R. The proteins in the O‐GlcNAc positive bands that increased during ISC were identified as glycogen phosphorylase b and mitochondrial aconitase. The protein in the O‐GlcNAc band that increased with I/R was identified as the cytoskeletal protein vinculin. Vinculin and aconitase were subsequently immunoprecipitated and both were found to be O‐GlcNAc positive by immunoblot analysis. Further studies are needed to determine the effect of O‐GlcNAc on the function of these proteins and how this influences the response to I/R. NIH grants: HL067464 and HL079364.