Regulation of Protein Kinases by ATR Subtypes in Ischemia-Reperfusion

10. Project summary (must be completed on this page): ~ The goal of this research proposal is to determine if unopposed angiotensin II subtype 2 receptor (AT2R) stimulation contributes to the cardioprotective effects of angiotensin II subtype 1 receptor (AT1 R) blockade during myocardial ischemia-reperfusion injury. Studies from our lab demonstrated an increase in AT1R density following ischemia-reperfusion injury and an increase in AT2R density in hearts exposed to ischemic preconditioning and an AT1R blocker. In addition, the cardioprotective effect of an AT1 R blocker was no longer observed when combined with AT2R blockade during ischemia-reperfusion injury. The functional role of the AT2R is not fully understood but recent data suggest that stimulation of the AT2R leads to activation of various phosphatases that modulate MAP kinases. We hypothesize that endogenous angiotensin II contributes to myocardial ischemia-reperfusion injury and that unopposed AT2R stimulation contributes the cardioprotective effects of AT1R blockade. In addition, we hypothesize that co-administration of an AT2R antagonist will attenuate temporal changes in the activity of serine-threonine kinases (MAPKs and PKB) observed in the presence of an AT1 R antagonist following myocardial ischemia-reperfusion injury. To test this hypothesis we will determine the temporal effect of ischemic preconditioning, AT1R blockade, and AT2R blockade on cardiac angiotensin II receptor density and function in rat heart sections using receptor autoradiography and agonist-stimulated [35S]GTPgammaS autoradiography, respectively. In addition, we will elucidate the effect of ischemic preconditioning, AT1R blockade, and AT2R blockade on MAP kinase, PKB, and protein phosphatase-2A activity. The effects of each of these treatment strategies on cardiac function and infarct size will also be evaluated. These studies will provide novel information regarding myocardial angiotensin II receptor subtype density and their ability to couple to G-proteins and protein kinases following ischemia-reperfusion injury. In addition, these experiments will elucidate potential AT2R signaling pathways that contribute to the cardioprotective effects of AT1R blockade during myocardial ischemia-repefusion injury and lead to the development of cardioprotective strategies during reperfusion of the human ischemic heart following thrombolysis, percutaneous coronary intervention, coronary artery bypass, and cardiac transplantation. 11. Amount ReQuested:0r) $60,500 Year 1 $60,500 Year 2 $0 Year 3$0 Year 4 $0