The proposed project will investigate potential mechanisms to explain why elderly patients experience increased morbidity and mortality following myocardial infarction (Ml) than their adult counterparts. We will test the hypothesis that an age-related decrease in the cytoprotective thiol disulfide oxidoreductase enzyme glutaredoxin (GRx) causes increased glutathionylation (i.e., protein-glutathione mixed disulfide formation) and functional modification of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), a protein that plays critical roles in cardiac function and maintenance of calcium homeostasis. Our experiments will be performed in 2 model systems: a cardiomyocyte cell line (H9c2) in which GRx content is genetically manipulated via overexpression or knock-down, and in heart tissue from adult (6 m.o.) and elderly (24 m.o.) Fisher 344 rats, an established model of human aging. We will investigate the effect of GRx on cytosolic free calcium ([Ca 2+]c) levels following an oxidative challenge using fluorescence microscopy, and on ? SERCA activity using selective radiolabel and fluorometric assays in both model systems. We will also ? determine the effect of changes in GRx content on the glutathionylation status of both calcium regulatory proteins using Western blot analysis with an anti-glutathione antibody, and confirming the specific protein modifications by mass spectrometry. By determining the effect of GRx content on SERCA activity and glutathionylation status, we expect to implicate GRx as an important cytoprotective enzyme (and potential therapeutic target) in recovery from Ml. This proposal investigates a specific molecular mechanism to explain why elderly patients do not recover after a heart attack as well as adult patients. Specifically, we will test the role of the enzyme glutaredoxin (GRx), which decreases with aging, in maintaining normal calcium balance in heart cells under oxidative stress conditions (i.e., simulated heart attack). Since increased cellular calcium levels can worsen heart function and damage heart tissue, identifying proteins that preserve normal calcium levels is important in understanding and treating heart attacks in elderly patients. ? ? ? ?
| Gallogly, Molly M; Starke, David W; Mieyal, John J (2009) Mechanistic and kinetic details of catalysis of thiol-disulfide exchange by glutaredoxins and potential mechanisms of regulation. Antioxid Redox Signal 11:1059-81 |
| Mieyal, John J; Gallogly, Molly M; Qanungo, Suparna et al. (2008) Molecular mechanisms and clinical implications of reversible protein S-glutathionylation. Antioxid Redox Signal 10:1941-88 |
| Gallogly, Molly M; Mieyal, John J (2007) Mechanisms of reversible protein glutathionylation in redox signaling and oxidative stress. Curr Opin Pharmacol 7:381-91 |
