Numerous studies have shown that proteins are modified during myocardial ischemia. Earlier studies focused on the role of the Cadependent proteases to explain loss of contractile proteins during myocardial ischemia. Very little is known about the role that proteasome plays in removal of proteins damaged by oxidative stress produced by exposure of the heart to ischemia. Given the major role that proteasome plays in protein quality control and removal of oxidized/damaged proteins, it seems reasonable to suggest that one consequence of proteasome dysfunction during myocardial ischemia is failure to remove these damaged proteins resulting in cardiomyocyte dysfunction. A project is proposed to examine the hypothesis that removal of proteins damaged during myocardial ischemia is an integral function of proteasome and necessary for recovery of myocardial function.
Specific Aim 1 examines the hypothesis that proteasome plays an integral role in the turnover and removal of damaged/oxidized contractile proteins in cardiomyocytes. Using cultured rat neonatal ventricular myocytes (RNVCs) and adult cardiomyocytes (AC), turnover of actin and other oxidized proteins will be examined during exposure to an oxidizing environment. Studies using pharmacologic inhibitors or gene silencing techniques to affect loss of function will determine if loss of oxidized actin or other proteins is mediated by the proteasome. Still other experiments will transfect RNVCs and ACs with replication-deficient adenoviral constructs encoding for a mutated 25 subunit to decrease proteasome activity or with the PA281 subunit of the 11S activator ring to increase proteasome activity.
Specific Aim 2 examines the hypothesis that failure of dysfunctional proteasome to remove proteins damaged during myocardial ischemia leads to progressive contractile dysfunction. Experiments will use an in vivo rat LAD occlusion model to determine if a level of postischemic myocardial oxidized proteins correlates with activity of proteasome. Cause and effect between proteasome activity, removal of oxidized proteins, and recovery of function will be shown by using a transgenic approach to manipulate proteasome activity. Murine transgenic models expressing a mutated 25 subunit with 40% less constitutive chymotryptic activity, or overexpressing the PA281 subunit of the 11S activator ring will be used to assess the effect of diminished or increased proteasome activity, respectively. Additional studies will examine the hypothesis that a consequence of ischemic preconditioning is preservation of postischemic proteasome activity which facilitates removal of damaged proteins.
Specific Aim 3 examines the proteasome configuration responsible for clearing damaged proteins and will use RNAi to target specific subunits of the 20S proteasome, 11S activator ring, or 19S regulatory particle. These studies will yield new information on the role of the proteasome in removing proteins damaged during myocardial ischemia and aid in the development of new strategies or treatments to increase or preserve proteasome function to mitigate postischemic cardiac injury and prevent progression to failure.
AHA statistics indicate that over 70,000,000 Americans suffer from some form of cardiovascular disease and of these 13,000,000 have some form of coronary artery disease. These studies will yield new information on the role of the Ubiquitin Proteasome System in regulating processes that may determine cardiomyocyte death such as removal of critical proteins that may have been damaged during a heart attack. Failure to remove these damaged proteins may present a significant stress to the heart that may contribute to the development of heart failure. It is expected that these studies will aid in the development of new strategies or treatments for preservation of proteasome function, such as genetic manipulation to increase proteasome function, to prevent progression to heart failure.
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