Myocardial ischemia and reperfusion (I/R) results in increased glycolysis, changes in internal pH, inflammation, and elevated levels of reactive oxygen species (ROS) that contribute to I/R injury and its clinical outcomes. However, a common biochemical feature of myocardial I/R is increased formation of reactive carbonyls, which are generated in high abundance in the ischemic heart and their concentration is further increased upon reperfusion. Previous work has shown that increased enzymatic detoxification of these carbonyls decreases myocardial I/R injury. Nevertheless, it remains unclear which carbonyls are generated during I/R and how they contribute to tissue injury. As a result the general contribution of carbonyls to myocardial I/R injury remains unclear and no specific carbonyl quenching interventions have been developed to limit I/R injury. The current project is designed to understand the role of endogenous histidyl dipeptides, synthesized by the heart, in preventing carbonyl toxicity during I/R. Histidyl dipeptides such as carnosine and anserine, react avidly with structurally-diverse carbonyls, we propose that these peptides represent a general carbonyl detoxification pathway. To test this hypothesis, we will elucidate the role of histidyl dipeptides in myocardial I/R injury. Using both in situ and ex vivo models of myocardial I/R injury, we will examine whether increasing myocardial levels of histidyl dipeptides, either by overexpression of histidyl dipeptide synthase (HDS) in the heart or exogenous delivery will diminish tissue injury. To assess whether the cardioprotective effects of these peptides are due to increased carbonyl detoxification, we will determine whether increased myocardial levels of these peptides are associated with increased carbonyl removal and excretion Our studies will also delineate the mechanism by which histidyl dipeptides affect myocardial I/R injury. Specifically, we will examine whether histidyl dipeptides generated during I/R inhibit the proteasome and induce ER stress and that histidyl dipeptides decrease myocardial damage by preventing protein carbonylation and thereby attenuating proteasomal dysfunction and ER stress. Finally, we will examine the relative efficacy of endogenous and synthetic histidyl dipeptides against myocardial I/R injury. For this we will compare the relative efficacy of differet endogenous histidyl dipeptides and their synthetic analogs against carbonyl toxicity and investigate whether enhanced carbonyl quenching properties of these peptides result in greater protection against myocardial I/R injury. Completion of this project will lead to the identificatio and assessment of a novel antioxidant system present in the heart, and how components of this system (histidyl peptides, HDS) prevent critical features of ischemia injury (carbonyl toxicity). We expect that these studies will lead to the identification of specific carbonyls that mediate I/R injury, and how they contribute to myocardial damage. These findings might lead to the development of robust and effective interventions that could be used clinically to limit ischemic and oxidative injury in the heart as well as in other tissues.