This four year proposal is aimed at testing the general feasibility of using molecular approaches to alter cardiovascular physiology in the large animal heart. The long term objectives are to create animal models of human cardiovascular disease by transgenic remodeling of the cardiac compartments. A number of important data implicate the regulatory, or phosphorylatable light chain 2 (MLC2) as playing a role in cardiac function. Our objective is to test this hypothesis directly in an animal model whose heart, in terms of its contractile isoform content, closely resembles the human's. The first specific aim will test the hypothesis that the alpha myosin heavy chain (MyHC) and beta MyHC gene promoters are capable of driving cardiac-compartment specific expression in the rabbit heart. Like other larger mammals, including man, and unlike the mouse which is normally used for transgenic approaches, the rabbit expresses the two cardiac MyHC isoforms in a compartment-specific fashion in the adult animal. Therefore, each of the MyHC promoters will be linked to the reporter gene, cat, and used to drive cat expression at high levels in either the ventricle or atrium during different developmental stages. The second specific aim will use these defined reagents to address fundamental questions concerning MLC2's function in the rabbit heart by effectively remodeling the contractile apparatus. The promoters will be used to ectopically express the ventricular-specific form of the regulatory light chain (MLC2v) in the atrium, and in the ventricle where it is already being expressed.
This aim i s directed at testing the hypothesis that MLC2 has an important functional role and that structure/activity correlates are associated with the compartment- specific expression of the unique MLC isoforms. Expression of MLC2v in the ventricle will test the hypothesis that the simple act of over- expressing a transgene does not lead to a detectable phenotype. The hearts will be analyzed at the molecular, biochemical, cellular and whole organ levels. The third specific aim will test the hypothesis that MLC2v also plays a unique functional role in the ventricle by trangenically effecting an MLC2v->MLC2a transition. Again, the resultant phenotype will be analyzed at the molecular, cellular, biochemical and whole organ levels, both in terms of structure and function.
These aims are directed towards providing a large animal model of defined genetic etiology for the study of cardiovascular disease as well as an unambiguous assignation of basic function and the physiologic or pathophysiologic significance of differential MLC2 isoform content.
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