Function of titin & a novel nebulin in heart development

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Despite major advances in understanding the molecular basis of active contraction, surprisingly little is known about mechanisms controlling myofibril formation in cardiac myocytes. It is likely that determination of the unique molecular properties of titin and nebulin, members of the "third and fourth filament systems" respectively, will provide a molecular explanation of these unsolved questions. The long term objective of this research is to identify the molecular components and mechanisms that regulate contractive protein interactions during myofibril assembly.. The goal of this proposal is to define the molecular role of titin, the titin binding protein T-cap, and a novel cardiac nebulin-related protein in the regulation of thick and thin filament assembly, length and organization using a model of de novo cardiac myofibril assembly that faithfully recapitulates myofibril assembly in vivo. The Aims are: (1) To complete the investigation of myofibril assembly in differentiation cardiac myocytes. Explants from pre-cardiac regions of avian embryos will be used to examine the temporal pattern of appearance and assembly of thick and thin filament proteins, and their relationship to titin, T-cap and nebulin-related protein. (2) To clone and characterize cDNAs encoding a novel nebulin-related protein, and to create molecular reagents (for aims 3 and 4) for investigating its function(s). (3) To determine the role(s) of titin, T-cap and the nebulin-related protein by over-expression of defined fragments that act as dominant negative inhibitors using adenoviral delivery. Also, specific regions of titin, T-cap and nebulin-related protein required for targeting their assembly into myofibrils will be identified. (4) To examine the effects of reducing functional expression of titin, T-cap and nebulin-related protein during myofibrillogenesis using antisense strategies and by targeting gene mutation in mouse embryonic stem cells. Establishment of an in vitro model for cardiac myofibril assembly and identifying the roles of titin, T-cap and nebulin of modulating thick and thin filament assembly and dynamics are pivotal for understanding the molecular bases of various types of cardiac myopathies, including familial hypertrophic cardiomyopathies.