Abstract 1373: Mice Lacking the Glutamate-Cysteine Ligase Modifier Subunit are Susceptible to Myocardial Ischemia-Reperfusion Injury Through Reduction of Mitochondrial Glutathione

Abstract

Reduced glutathione (GSH), tripeptide thiols, is a major anti-oxidant that protects cells from oxidative injury. We have previously reported that polymorphism of modifier subunit gene (GCLM) of glutamate-cysteine ligase, a rate-limiting enzyme for GSH synthesis, was an independent risk factor for myocardial infarction and coronary endothelial dysfunction. However, the molecular mechanisms underlying this association remain undefined. Thus, this study tested the hypothesis that chronic disruption of GSH synthesis may increase susceptibility to myocardial ischemia reperfusion (I/R) injury in GCLM knockout (GCLM ^−/−) mice. The GCLM ^−/− mice had no obvious abnormality in major tissues. Myocardial I/R injury was created by 1-hr ligation of left anterior descending coronary artery, followed by 24 hrs of reperfusion. The GCLM ^−/− mice had a 66% increase in myocardial infarct size, a 34% reduction of LV fractional shortening (by echocardiography), and a 86% increase of TUNEL positive cells and 30% increase of activated caspase-3 in the ischemic myocardium, as compared with GCLM ^+/+ mice. The mitochondrial GSH levels in non-ischemic myocardium were lower in GCLM ^−/− mice (39% of baseline GCLM ^+/+), and the mitochondrial GSH levels in ischemic myocardium decreased more in GCLM ^−/− mice (12% of baseline GCLM ^+/+) than GCLM ^+/+ mice (55% of baseline). GCLM ^−/− mice had a higher ESR signal intensity of DMPO-hydroxyl radicals adduct in the ischemic myocardium than GCLM ^+/+ mice. Hypoxia re-oxygenation induced greater mitochondrial damages in cultured GCLM ^−/− cardiomyocytes than GCLM ^+/+ cardiomyocytes, as evidenced by reduced membrane potential, increased protein carbonyl content, and increased GSSG/GSH rate in the isolated mitochondria, together with enhanced cytochrome c translocation to the cytosol and activation of caspase-3 in the cells. Furthermore, systemic administration of GSH ester reduced myocardial I/R injury by 50% as well as mitochondrial damage in association with restoration of mitochondrial GSH levels in GCLM ^−/− mice. GCLM ^−/− mice were more susceptible to myocardial I/R injury partly through an increased vulnerability of mitochondria to oxidative damage due to chronic reduction of mitochondrial GSH.