Abnormal iron deposition causes liver and cardiac failure that is preceded by tissue fibrosis. We showed that the beta-blocker analog, d-propranolol (d-Pro), accumulates in endothelial lysosomes and reduces both cellular and cardiac injury caused by iron overload. This proposal is based upon the following hypotheses: Iron-loaded lysosomes constitute a major source of releasable iron capable of inducing in vivo oxidative stress and tissue (cardiac and hepatic) fibrosis, as well as enhanced myocardial susceptibility to imposed ischemia / reperfusion [I/R] stress in vitro);Angiotensin II (Ang II) may promote lysosomal iron accumulation and exacerbate oxidative stress, tissue fibrosis and myocardial intolerance to I/R;and d-Pro and its major metabolite, 4-HO-propranolol (4-OH-Pro), attenuate iron-mediated fibrosis and associated injury as a result of their potent lysosomotropic / antioxidant properties. By using both the whole rat, isolated heart and cultured cell models, we propose to: 1) Determine the extent to which tissue iron accumulation occurs prior to cardiac and hepatic oxidative stress and fibrosis during iron overload;2) Study how Ang II facilitates this pathogenesis in vivo and assess the benefits of in vivo intervention (Ang II receptor blockade, lysosomotropic agents and/or metal chelation) on tissue stress parameters and myocardial intolerance to I/R;3) Investigate the cellular mechanisms underlying Ang ll-mediated iron accumulation and oxidative responses (NADPH oxidase activation, fibrogenic activity, protein and lipid oxidation) using in vitro macrophage, endothelial cell, hepatocyte, and stellate cell models;and 4) Assess cell-specific protection of Ang II receptor blockade and d-Pro /4-OH-Pro on iron transport and associated oxidative responses. Various sophisticated biophysical (ESR spin trapping), immunochemical and molecular biology techniques (real-time PCR) will be used to assess the sequence of events leading to tissue oxidative stress, fibrogenesis and cellular toxicity. The involvement of NADPH oxidase during iron overload will also be assessed using gp91 phox knockout mice (Subcontract). Early indications of cardiac functional defects during iron overload will be determined in situ using sensitive echocardiologic techniques in the mouse (Subcontract), and the impact of the above treatment(s) determined. The proposed studies may reveal potential benefits of using Ang II receptor blockade and lysosomotropic/antioxidant agents as adjunct therapy for iron-overloaded patients.
