The changes in cardiovascular function that occur during development or in response to different physiological stimuli can depend upon contractile protein isoform diversity. This application proposes to use a combination of molecular, genetic and functional approaches to understand the significance of myosin (MyHC) diversity in the mammalian heart. The long term goal of this project is to investigate the functional correlates directly by establishing the different functional roles the unique cardiac MyHC's play in the working heart. Using the techniques of gene targeting coupled with transgenic overexpression, it is now possible to replace the MyHC isoforms specifically in the cardiac compartment and thereby establish the functional consequences of a directed isoform change.
SPECIFIC AIM 1 will test the ability of an alpha-MyHC transgene to restore wild type function in a mouse carrying either 1 or 2 copies of a null alpha-MyHC allele. Both the structural and functional consequences of the transgene's expression will be determined.
SPECIFIC AIM 2 will effect a replacement of the alpha-MyHC-encoded isoform (V1) with expression of the V3 MyHC that is normally only expressed by Beta-MyHC in the embryonic/fetal ventricle. Expression of a full length Beta-MyHC cDNA will be driven in the heart using the alpha- MyHC promoter in the wild-type, alpha-MyHC+/ and alpha-MyHC-/- backgrounds. This will yield a spectrum of animals displaying varying V1/V3 ratios in the absence of any other intrinsic or extrinsic stimuli. Breeding a Beta-MyHC transgenic overexpressor into the alpha-MyHC null background will yield an animal with a complete isoform replacement. The functional changes will be measured at the molecular, biochemical, cellular, structural and whole organ levels in order to directly establish the consequences of isoform replacement.
SPECIFIC AIM 3 will explore the structural underpinnings of the two different cardiac molecular motors by testing the hypothesis that the different biochemical behaviors can be ascribed to discrete regions in each gene transcript and that these behaviors are manifested at the functional level within the context of the intact animal. To establish the structure/function relationships between the two isoforms, two domains from V3 encoding surface loops, will be placed into the context of V1 and the chimeric MyHC expressed against the null background. Again, the consequences will be determined at the molecular, biochemical and whole organ levels, and the activity of the hybrid molecular motor in the in vitro motility assay measured.
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