Abstract 1328: Selective Glutaredoxin (GRx1) Knockdown in H9c2 Cells Provides a Model for Age-Related Decreases in GRx1 in Rat Heart, and Increases Susceptibility to Oxidant Induced Apoptosis
Clinical and animal studies show that hearts of the elderly are more susceptible to irreversible damage after ischemia-reperfusion (I/R) than adult hearts. We reasoned this was due in part to changes in the enzymes that regulate sulfhydryl homeostasis which is perturbed by cardiac I/R, as indicated by increased protein mixed disulfides with glutathione (protein-SSG). In a rat model of aging (Fischer 344 rats), glutaredoxin (GRx1) content and activity were decreased (−40%) in hearts from elderly (24 m.o.) vs. adult (6 m.o.) animals, while the thioredoxin system was essentially unchanged. GRx1 is the major deglutathionylase in cells, regulating the steady-state S-glutathionylation and corresponding activity of many key proteins. Hence we hypothesized the greater oxidative heart injury in the elderly resulted from decreased GRx1 activity; and we used shRNA-mediated knockdown (KD) of GRx1 in H9c2 cells, a known cardiomyocyte model, to simulate the effect of decreased GRx1 in the aged heart. GRx activity was decreased by 40% in GRx1-KD cells compared to control, mimicking the diminution in elderly rat heart. Chromatin condensation (Hoescht staining) indicated a 3-fold increase in apoptosis induced by H2O2 (400μM, 5 min) in GRx1-KD cells. Likewise, simulated I/R (20 mM 2-deoxyglucose + 5 mM KCN for 1hr (“ischemia”), then control medium for 30 min (reperfusion”); or 95%N2/5% CO2, pH 6.2 for 3 hr (“ischemia”), 75%N2/20%O2/5%CO2, pH 7.4 for 2 hr (“reperfusion”)) revealed 16-fold and 5-fold increases in apoptosis, respectively, in GRx1-KD cells relative to like-treated control cells. NFκB promoter-dependent luciferase assays showed a 40% decrease in NFκB activity in GRx1-KD cells, implicating decreased anti-apoptotic activity of the NFκB pathway as a basis for increased sensitivity to oxidative stress by GRx1-KD cells. Also, the increase in intracellular calcium content in response to H2O2 treatment was 3.5-fold greater in the GRx1-KD cells, potentially contributing to the increased apoptotic susceptibility. Thus, selective knockdown of GRx1 in H9c2 cells (rat cardiomyocyte model), mimicking the effect of aging in the heart, predisposes the cells to oxidant-induced apoptosis, associated with a decrease in NFκB activity and altered intracellular calcium.