Reactive Oxygen Species in Cardiac Pathophysiology: Modification of Sarcomeric Proteins
Avner, Benjamin S.
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The data described within this dissertation addresses the overall hypothesis that modification of essential sarcomeric proteins is a key route by which reactive oxygen species (ROS) trigger progression of heart disease. We isolated myofibrillar proteins from rat ventricular tissue before (“direct”) and after (“indirect”) treatment with hydrogen peroxide (H2O2), and measured in vitro ATP hydrolysis. Unlike direct application of H2O2, indirect H2O2 lowered the relative myofibrillar ATPase activity under low [Ca++] conditions. Furthermore, indirect H2O2 induced higher Ca++ sensitivity and rate of tension redevelopment (ktr) in detergent-skinned trabeculae, as well as higher phosphorylation of cardiac troponin I (cTnI) and myosin binding protein C due to a protein kinase C dependent pathway in isolated cardiomyocytes. These findings demonstrate that H2O2 redox signaling indirectly modifies sarcomeric protein phosphorylation and function. In order to analyze the aftermath of myocardial infarction (MI) and the contribution of ROS action on the sarcomere to the post-MI condition, we employed murine coronary ligation surgery as our model for acute MI. Our results demonstrated significant differences in sarcomere function 3-4 days post-MI in the surviving myocardium. Detergent-skinned papillary muscle fiber bundles displayed higher Ca++ sensitivity and lower cooperativity of activation compared to those from sham-operated animals. Net phosphorylation levels of cTnI and myosin light chain 2 were lower post-MI. Myosin ATPase activity was also lower in vitro in the sarcomeric fractions of infarcted ventricles. In addition to the above changes, protein oxidation by formation of disulfide products also occurred after MI. When analyzing these samples using specialized electrophoresis, we observed disulfide products containing tropomyosin. Western blot analysis of mixed glutathionylated protein disulfides in whole-ventricle sarcomere fractions revealed a greater amount post-MI. As disulfide products represent an experimental target to evaluate the functional consequences of ROS, we treated fiber bundles with a reducing agent and found that reduction reversed the effect of MI on sarcomeric calcium sensitivity. Overall, these findings demonstrate the effects of H2O2 redox signaling on cardiac sarcomeric protein structure and function, characterize the early pre-remodeling response to MI of the sarcomere, and demonstrate involvement of ROS-induced protein oxidation in the post-MI condition.
Subjectreactive oxygen species
cardiac sarcomeric protein