TROPOMODULIN FUNCTION IN HEART DEVELOPMENT

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The formation of perfectly aligned myofibrils in cardiac muscle represents a dramatic examine of supramolecular assembly in eukaryotic cells; the mechanisms by which this occurs are still incompletely understood. The long term objective of this research is to identify the molecular components and mechanisms that regulate contractile protein interactions during myofibril assembly. The goal of this proposal is to define the function(s) of tropomodulin, and its specific molecular interactions, in regulation of thin filament assembly and organization using a novel model of de novo cardiac myofibril assembly that faithfully recapitulates myofibril assembly in vivo. My career research development plan, which encompasses the establishment of an interdisciplinary cardiac muscle research program, will include gaining expertise in molecular developmental biology and advanced microscopy techniques (including real-time fluorescence image analysis) by formal course work, interactions with colleagues and hands-on efforts. All the recourses needed will be available at the University of Arizona. This will allow for the investigations of cytoskeletal proteins in live cells and within the context of organisms. The Aims are: (1) To investigate myofibril assembly in differentiating cardiac myocytes. Explants from precardiac regions of avian embryos will be used to examine the temporal pattern of appearance and assembly of myofibril proteins. 2) To examine tropomodulin-tropomyosin interactions and to identify other molecular interactions required for actin filament capping by microinjecting function-blocking antibodies or overexpression of truncated tropomodulins that can act as dominant negative inhibitors. Thin filament length and organization will be examined using confocal (3D), polarization, deconvolution and two-photon microscopy. Contractile function will be studied using video microscopy; 3) To identify the domains of tropomodulin and of tropomyosin isoforms that are required for the assembly of tropomodulin using a novel permeabilized cell model and expression of green fluorescent- tagged proteins. 4) To identify the functional significance of the interaction of tropomodulin with a putative cardiac nebulin- like molecule. Understanding the mechanisms of thin filament assembly and determining the roles of key regulatory proteins such as tropomodulin in modulating actin filament dynamics are pivotal for future considerations of the molecular bases for myopathies seen in various types of heart disease, including familial hypertrophic cardiomyopathies.