The overall goal of the proposed research is to analyze the interaction of the cell surface of cardiac myocytes and fibroblasts with the components of the extracellular matrix (ECM) during neonatal development, normal adults and in certain types of myocardial hypertrophy. Specifically, investigations will be conducted to: 1) analyze the ability the cardiac myocytes and fibroblasts to interact with components of the ECM; 2) characterize the components on the cell surface that are responsible for recognition of ECM components; 3) make antibodies to the cell surface receptors of ECM components as well as to the ECM components themselves; and 4) analyze the distribution of ECM components and their receptor(s) both in vivo during neonatal development, normal adults and certain types of myocardial hypertrophy as well as in vitro cultures of isolated cardiac myocytes and fibroblasts. Cell adhesion assays will be conducted to analyze the ability of the myocytes and fibroblasts to recognize and attach to ECM components and the effect(s) of the ECM substrates on the subsequent progression of the cells into culture. Components on the cell surface of the myocytes responsible for the recognition of the ECM components will be isolated and antibodies produced against these antigens. Both polyclonal and monoclonal antibodies will be produced and assayed for their ability to inhibit the attachment of the myocytes and fibroblasts to ECM components. Antibodies will be raised against the cell surface components on hepatocytes that recognize ECM components and their effects will also be assayed on cardiac myocytes and fibroblasts to determine if similar receptor molecules are present. In addition, soluble cell surface components will be used to neutralize the effects of the antibodies to block attachment to the different substrates. Antibodies will also be used to determine the morphology of the ECM both in vitro and in vivo. Myocytes will be isolated and cultured from the different stages of development and hypertrophy. The ECM in vitro will be analyzed by using antibodies against ECM components as well as by SDSPAGE and other biochemical methods. This analysis of the pericellular matrix of myocytes and fibroblasts will be compared to similar stages in vivo. Antibodies against ECM receptors will be analyzed at different development stages to correlate the different physiological functions of the heart seen during neonatal development and in the hypertrophied myocardium. These studies will aid in our understanding of the role of the ECM in normal development and during the disease process as related to myocardial hypertrophy.
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