Project Summary/Abstract - Project 1 Increased cardiac Ang II activity-as a result of upregulated Ang II formation in the intracellular or interstitial compartment acting via autocrine or paracrine mechanisms- contributes to cardiac hypertrophic remodeling, arrhythmias, and fibrosis. RAS inhibitors are less effective in blocking Ang II-mediated adverse cardiac remodeling because Ang II production in cardiac myocytes follows a non-canonical pathway through the processing of angiotensinogen (Aogen) into the dodecapeptide intermediate substrate Ang-(1-12). We further showed that Ang-(1-12) metabolism between rodents and humans is that chymase but not ACE converts Ang- (1-12) directly into Ang II in human heart tissue and that this process may include an additional step in which human Aogen may be processed into a newly identified precursor Ang-(1-25) prior to conversion into Ang-(1- 12). The human form of cardiac chymase, either expressed in cardiomyocytes or incorporated into these cells from activated mast cells, is the enzyme producing Ang II from Ang-(1-12) and likely Ang-(1-25). Pilot studies suggest a role for kallikrein as the enzyme cleaving Ang-(1-12) from Aogen. On these bases, Project 1 poses the hypothesis that Ang II-mediated adverse cardiac remodeling results from the processing of intermediate forms of Aogen-derived peptides through the hydrolytic activity of intracellularly formed or incorporated chymase. Furthermore, we propose that these biotransformation steps are species-specific. To achieve these objectives we will determine: Aim 1, the compartmentalization and role of kallikrein or a kallikrein family member enzyme in the production of Ang-(1-12) from cardiac Aogen in WKY and SHR and its potential hydrolytic activity in cardiac Ang-(1-12) production in a transgenic rat model expressing the genes from human Aogen [TGR(hAGT)L1623]; Aim 2, the role of Ang-(1-12) and Ang-(1-25) as substrates for cardiomyocyte Ang II production in WKY and SHR versus TGR(hAGT)L1623 rats; Aim 3, the hemodynamic and RAS profile and the enzymes accounting for the expression of Ang-(1-25) and Ang-(1-12) in TGR(hAGT)L1623. We will also assess whether the chymase-driven cardiac Ang II pathway is influenced by species-specificity through the crossing of the human Aogen transgenic rat with a rat expressing the genes for human chymase. Our work will reveal new therapeutic approaches to hypertensive cardiac hypertrophy and fibrosis that are based on non- canonical cardiac Ang II formation.