Current evidence suggests that A3 adenosine receptors (A3ARs) are expressed in cardiac myocytes, where they regulate responses to ischemic stress. In addition, A3ARs are expressed in cells involved in the inflammatory response including neutrophils, macrophages, and endothelial cells. In these cells, they mediate anti-inflammatory actions. In preliminary studies, we have shown that newly developed A3AR agonists provide profound cardiac protection against tissue damage induced by myocardial ischemia/reperfusion (I/R) injury in mice, rabbits, and dogs whether they are given prior to ischemia or at the time of reperfusion. The goal of this research proposal is to understand the mechanisms responsible for this tissue protection. Our general hypothesis is that A3AR agonists act dually on cardiac myocytes to reduce myocardial injury during ischemia, and act on bone marrow-derived cells to reduce inflammation during reperfusion. The studies involve the use of genetic approaches to: 1) produce a congenic line of mice with the A3AR gene selectively deleted from cardiomyocytes using the Cre/LoxP strategy, and 2) to produce from congenic A3AR gene "knock-out" mice, chimeric mice lacking or expressing the A3AR in bone marrow-derived cells using standard transplantation techniques. An in vivo mouse model of infarction and an isolated mouse heart model of global ischemia and reperfusion will be used to determine the relative importance of A3ARs expressed in the myocardium versus inflammatory cells in regulating I/R injury. Additional studies are proposed to study cellular signaling responses of A3ARs in specific populations of inflammatory cells from "wild-type", conventional A2AAR gene "knock-out", and conventional A3AR gene knock-out" mice. Collectively, these studies will combine several state-of-the-art techniques to provide new definitive information on the cell-specific mechanisms by which A3AR activation provides protection from I/R injury.