The long-term objective of this application is to identify whether hyperglycemia with diabetes upregulates the myocardial renin-angiotensin system (RAS), resulting in the synthesis and secretion of Ang II Hyperglycemia-mediated enzymatic glycosylation of p53 may enhance transcription of angiotensinogen (Aogen) and AT1 receptor; Aogen is the limiting factor in the generation of Ang II and the AT1 effector pathway conditions cardiac cell death. Transmission of death signals by Ang II may involve an increase in cytosolic Ca2+ which, in turn, promotes the formation of reactive oxygen species (ROS), DNA damage and cell death. Ligand binding to AT1 receptor leads to phosphorylation of p53 by p38-MAP kinase, enhancing p53 function chronically and, thereby, the cellular production of Ang II, cytosolic Ca2+ and oxygen toxicity. ROS may induce DNA strand breaks and subsequently an interaction of injured DNA with p53. Autoproteolytic cleavage of p53 may occur and p53 fragments lacking the C-terminus, p50 (dC), or the N-terminus, p50 (dN), are formed. Since the C-terminus is required for cell death to take place, p50 (dC) may interact with single DNA strand breaks, upregulating p2l, inhibiting growth and inducing DNA repair. Conversely, p50 (dN) may bind to double DNA strand breaks, triggering apoptosis. These possibilities associated with the induction of diabetes by streptozotocin administration will be tested in a mouse model in which targeted mutation of the p66shc gene increases the resistance of cells to oxidative stress. In the p66shc-/- mouse, hyperglycemia may be characterized by an attenuated effect of ROS on DNA damage, leading to single DNA strand breaks, growth arrest and DNA repair. In the wild-type mouse, diabetes may result in extensive DNA injury, p50 (dN) activation and diffuse cell death. High levels of ROS are coupled with cell necrosis and lower levels with apoptosis. The initiation of cell death is largely dependent on the antioxidant defense mechanisms of cardiac cells. Because mitochondria comprise more than one third of the myocyte cytoplasm, large amounts of ROS may be generated in this cell population with hyperglycemia and its antioxidant enzymes may be less effective than in other cardiac cell types. Although, cells interact with each other in situ, making difficult to predict the cell population first injured by oxidative challenge with diabetes, myocytes constitute a reasonable initial target. To validate the primary role of hyperglycemia in the development of a decompensated diabetic myopathy, the experimental studies on insulin-dependent diabetes mellitus will be complemented and compared with studies of human hearts affected by non-insulin-dependent diabetes mellitus.
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