The long-term goal of this research project is to understand the molecular mechanisms involved during cardiac morphogenesis. The endocardial cushion structures, which contain activating, migrating and proliferating mesenchymal cells (or called heart fibroblasts) and their secreted matrix materials are important in their development for structures in the atrioventricular (AV) canal region of the heart. Actin cytoskeleton known to have a critical role in cell motility, adhesion and spreading, and transcription factors known to control specific gene expression at the specific stage of embryogenesis are likely involved in the formation of endocardial cushion structures and then valves and septa. Therefore, we will investigate the differences in the expression of cytoskeletal proteins and heparin-binding proteins (containing transcription factors) between heart and skin fibroblasts of human individuals with AV canal defects. We will prepare monoclonal antibodies against those proteins that are identified to have different expression levels between heart and skin cells. These monoclonal antibodies will be most useful in determining the temporal-and spatial appearances of these molecules during heart development. We will use another approach (differential display of mRNA species) to identify the differences in mRNA species between paired heart and skin fibroblasts of individuals with AV canal defects. Since the different RNA species-are displayed in their cDNA form, they are readily to be isolated and subcloned. These cDNA probes will be used not only in the study of normal endocardial cushion development in mouse model but also in the linkage studies proposed in the Project l. Since cardiomyocytes are found in association with some valves and septa, the understanding of mechanisms that control cardiac-specific gene expression is also important. Therefore, we will continue to use our rat cardiac troponin T gene system (funded by current SCOR) to isolate and characterize the cDNA clones encoding cardiac muscle-specific transcription factors. The characterization of both cardiac-derived fibroblasts and cardiomyocytes in terms of their cardiac-specific phenotype expression may provide valuable information about not only the formation of the heart valves and septa but also the etiology of AV canal defects in congenital heart diseases.
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