Extracellular matrix (ECM) contributes significantly to remodeling processes of heart following myocardial damage. My long term goal is to study the biochemical and physiological functions and regulation of osteopontin (OPN), an ECM protein. Our preliminary data show that OPN is markedly induced in the mouse heart following myocardial infraction (MI). We have also found that the inducible nitric oxide synthase (iNOS) gene is expressed in the mouse heart following MI, and may therefore be a source of oxidative stress by contributing reactive oxygen species (ROS). We previously demonstrated that OPN inhibits the induction of iNOS gene expression in cardiac myocytes in vitro, and others have provided evidence that OPN can reduce cellular oxidant levels. These observations, taken together, have led us to the hypothesis that, by attenuating nitric oxide (NO)-and ROS-mediated cell damage, OPN serves a protective function during the myocardial remodeling that occurs following MI. To test this hypothesis we will use a recently developed mouse model of chronic myocardial failure caused by coronary ligation, and OPN-knockout mice. We will study morphologic, physiologic and molecular parameters of myocardial remodeling and heart failure and compare them in wild type and OPN-knockout mice subjected to MI. In the first set of experiments, we will examine gross left ventricular (LV) chamber morphology, structure (apoptosis, fibrosis and hypertrophy) and LV function. To address the mechanism by which OPN modifies myocardial remodeling following MI, we will measure the production of NO (by measuring iNOS gene expression and iNOS activity) and superoxide (by lucigenin assay) in wild-type and OPN-knockout mice subjected to MI. Using in vitro cell culture, we will study the involvement of MAPK-pathway in OPN-mediated suppression of iNOS expression induced by cytokines. In the third set of experiments, using in situ hybridization and immunohistochemical analysis, we will identify the cell types involved in the expression of OPN in the myocardium following MI. Using cell adhesion, radio- iodination and immunoprecipitation assays, we will identify the receptors for OPN on two major cell types (microvascular endothelial cells and myocytes) of the heart. The identification of cell type(s) involved in the expression of OPN will guide future in vitro studies to determine the mechanism that regulates OPN expression in the myocardium. These studies will advance our understanding of the process of remodeling which occurs after MI and could lead to new therapeutic approaches to heart failure.
