Abstract 10267: Methylglyoxal Increases Cardiomyocyte Ischemia/Reperfusion Injury via Glycative Inhibition of Thioredoxin Activity
Diabetes (DM) causes cardiovascular morbidity and mortality, but the specific molecular mechanisms remain undefined. Methylglyoxal (MG), a glycative molecule, is increased in DM plasma, but has unclear role in cardiovascular pathology. Thioredoxin (Trx), an anti-apoptotic, cytoprotective molecule, is vulnerable to glycative inhibition, but whether MG may glycatively inhibit Trx (thereby causing increased cardiac injury) is unknown. Cultured H9c2 cardiomyocytes were treated with MG (200μM) or vehicle for 6 d followed by 8 h of simulated ischemia and 3 h of reoxygenation (SI/R). Cardiomyocyte death/apoptosis, Trx expression/activity, advanced glycation end-product (AGE) formation, Trx-apoptosis-regulating kinase-1 (Trx-ASK1) complex formation, and p38 mitogen-activated protein kinase (p38) phosphorylation and activity were determined. MG significantly increased SI/R-induced cardiomyocyte LDH release (17.1±0.78% of total cellular LDH vs. 10.9±0.58% in control cells), caspase-3 activity (88.9±2.09 vs. 68.5±4.54 nmolAFC/mg protein/h), and TUNEL positivity (18.7±1.33% of total nuclei vs. 10.4±1.20%), all P<0.01. Prior to SI/R, Trx activity was reduced (5.6±0.24 vs. 8.8±0.36 mUI/mg protein/min in vehicle-incubated cells, P<0.01), Trx-ASK1 complex formation was decreased, and both p38 activity and phosphorylation were increased in MG-treated cells. In addition, incubation of recombinant human Trx (hTrx) with MG increased CML (a glycation footprint) formation 2.8 fold over vehicle, and inhibited Trx activity (4.8±0.64 vs. 12.2±1.18 U/mg protein/min in vehicle-treated hTrx, P<0.01). Finally, glycation inhibitor aminoguanidine (AG) reduced AGE formation (57.4% reduction from MG alone), increased Trx activity (1.89-fold over MG alone), restored Trx-ASK1 interaction, inhibited p38 activation, decreased caspase-3 activation (68.5±2.29 vs. 83.2±2.55 nmol/mg protein/h in MG alone), and attenuated LDH release (12.3±0.69% of total cellular LDH vs. 16.3± 0.39% in MG alone), all P<0.01. We demonstrated for the first time that MG sensitized cardiomyocytes to SI/R injury by post-translational Trx glycative modification. Therapeutic interventions blocking protein glycation may attenuate I/R injury in the diabetic state.
- © 2010 by American Heart Association, Inc.