Methylglyoxal increases cardiomyocyte ischemia-reperfusion injury via glycative inhibition of thioredoxin activity

Abstract

Diabetes mellitus (DM) is closely related to cardiovascular morbidity and mortality, but the specific molecular basis linking DM with increased vulnerability to cardiovascular injury remains incompletely understood. Methylglyoxal (MG), a precursor to advanced glycation end products (AGEs), is increased in diabetic patient plasma, but its role in diabetic cardiovascular complications is unclear. Thioredoxin (Trx), a cytoprotective molecule with antiapoptotic function, has been demonstrated to be vulnerable to glycative inhibition, but whether Trx is glycatively inhibited by MG, thus contributing to increased cardiac injury, has never been investigated. Cultured H9c2 cardiomyocytes were treated with MG (200 muM) for 6 days. The following were determined pre- and post-simulated ischemia-reperfusion (SI-R; 8 h of hypoxia followed by 3 h of reoxygenation): cardiomyocyte death/apoptosis, Trx expression and activity, AGE formation, Trx-apoptosis-regulating kinase-1 (Trx-ASK1) complex formation, and p38 mitogen-activated protein kinase (MAPK) phosphorylation and activity. Compared with vehicle, MG significantly increased SI-R-induced cardiomyocyte LDH release and apoptosis (P < 0.01). Prior to SI-R, Trx activity was reduced in MG-treated cells, but Trx expression was increased moderately. Moreover, Trx-ASK1 complex formation was reduced, and both p38 MAPK activity and phosphorylation were increased. To investigate the effects of MG on Trx directly, recombinant human Trx (hTrx) was incubated with MG in vitro. Compared with vehicle, MG incubation markedly increased CML formation (a glycation footprint) and inhibited Trx activity. Finally, glycation inhibitor aminoguanidine administration during MG treatment of cultured cells reduced AGE formation, increased Trx activity, restored Trx-ASK1 interaction, and reduced p38 MAPK phosphorylation and activity, caspase-3 activation, and LDH release (P < 0.01). We demonstrated for the first time that methylglyoxal sensitized cultured cardiomyocytes to SI-R injury by posttranslational modification of Trx via glycation. Therapeutic interventions scavenging AGE precursors may attenuate ischemic-reperfusion injury in hyperglycemic state diseases such as diabetes.

Fig. 1.

Simulated ischemia-reperfusion (SI-R) injury is increased significantly in methylglyoxal (MG)-precultured cardiomyocytes, as measured by lactate dehydrogenase (LDH) release (A), apoptotic index by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive staining cells (B), and caspase-3 activity (C). D: MG preculture results in cardiomyocyte glycation prior to being subjected to SI-R, as measured by total advanced glycation end product (AGE) content; n = 6 independent experiments. **P < 0.01 vs. Sham SI-R; ##P < 0.01 vs. vehicle + SI-R. AFC, 7-amino-4-trifluoromethyl-coumarin.

Fig. 2.

Effect of aminoguanidine (AG) treatment on MG-enhanced LDH release (A) and caspase-3 activity (B) after SI-R; n = 5–6 independent experiments.

Fig. 3.

Effect of AG treatment on MG-enhanced apoptotic cell death determined by TUNEL-positive staining after SI-R; n = 5–6 independent experiments. DAPI, 4,6-diamino-2-phenylindole.

Fig. 4.

Effects of MG on N^ϵ-carboxymethyl lysine (CML) formation (A) and thioredoxin (Trx) activity of recombinant human Trx (hTrx) (B) in the absence and presence of AG, an AGE formation inhibitor; n = 6 independent experiments. Effect of AG treatment on MG-exposed Trx inactivation (C) and Trx expression (D) in cardiomyocytes prior to being subjected to SI-R. A and D, top: representative Western blots. Bar graphs represent density analysis (n = 5–8 independent experiments). AU, arbitrary units.

Fig. 5.

MG preculture decreases Trx-apoptosis-regulating kinase-1 (ASK1) binding (A) and increases p38 MAPK activation (B). A and B, top: representative Western blots. Bar graphs represent density analysis (n = 5–7 independent experiments). Effect of AG treatment on MG enhanced Trx-ASK1 dissociation (C) and p38 MAPK phosphorylation (D) in cardiomyocytes subjected to SI-R. C and D, top: representative Western blots. Bar graphs represent density analysis (n = 5–6 independent experiments).