The structural underpinning of the essential physiological role of the heart resides in the synthesis and maintenance of those proteins that are responsible for the contractile mechanics, the polypeptides found in the sarcomere. The theme of this proposal is study both differentiation and development of the mammalian heart using gene targeting to produce defined mutations in four different genes, and to generate animal models in the mouse that will be used for molecular genetic, biochemical, cytological and physiological studies on fundamental mechanisms. In Project 1 the alpha-cardiac myosin heavy chain will be studied. Using gene targeting techniques, both ablation and site directed mutagenesis on structural and regulatory sequences will be carried out resulting in mice which carry the defined mutation in the correct chromosomal context. This Project will delineate the physiological importance of myosin isoform shifts in the myocardium, define transcriptional regulatory cassettes that are active in vivo, and will delineate structural sequences responsible for the unique ATPase activities of the different cardiac myosins. Project 2 uses the identical technological approach, but will analyze tropomyosin isoforms. This complements the analyses in Project 1, since the tropomyosin isoforms are generated at the post-transcriptional level, and interact with the myosins in the contractile apparatus. Similarly, Project 3 will analyze, again by gene targeting techniques, the structure/function relationships between the other major protein of the contractile apparatus, alpha-cardiac actin. Project 4 deals with a more basic facet of cardiomyogenesis, the basic fibroblast growth factor. While Projects 1, 2, and 3 focus on the proteins that are expressed during the terminal stages of cardiogenesis, basic fibroblast growth factor plays a role both in the differentiation of early mesoderm and in the later developmental stages. The factor's impact on the growth and development of both the myoblast as well as the other major cell type in the myocardium, the fibroblast, will be determined using gene targeting strategies. The entire group of projects is supported in their technological and analytical thrusts by 3 cores: the Embryonic Stem Cell Core, a Pathology Core and a Physiology Core which will use both whole hearts and isolated tissues to examine the cardiac mechanics of the resultant mutants. Taken together, these Projects should reveal specific and fundamental information on a mutation(s) effect(s) on cardiac differentiation, development and function.
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