Although cardiac muscle has been the focus of intense research, little is known about how myocytes regulate the assembly and organization of actin and associated proteins into thin filaments of strikingly precise lengths: properties that are critical for productive contraction. One molecule that has been proposed to function as a molecular ruler to specify thin filament lengths is nebulin, a giant (MW 500-900 kDa), modular protein that spans the entire length of the thin filaments. Although cardiac nebulin has been defined in some detail biochemically, studies addressing its functional properties are lacking. We have exciting preliminary data revealing that nebulin indeed is involved in thin filament length regulation and may also be multifunctional. The goal of this proposal is to delineate nebulin's functional properties and definitively determine whether it can indeed function as a molecular ruler to regulate cardiac thin filament lengths. First, we will directly test the hypothesis that nebulin determines thin filament length by expressing a novel mini-nebulin in living myocytes (containing modules from all of its unique regions but significantly fewer thin filament-binding super-repeats), in the presence or absence of endogenous nebulin. We propose that the lengths of the cardiac thin filaments will correspond to our molecular manipulation of mini-nebulin's size. Next, the molecular mechanisms by which nebulin functions in thin filament assembly will be deciphered by analyzing the significance of its interactions with the actin filament capping proteins, tropomodulin and capping protein (CapZ), using dominant-negative techniques combined with siRNA strategies during defined stages of development. Finally, we will begin to decipher nebulin's potential other functions (including Z-line assembly and contractile activity), by investigating its interactions with novel binding partners that we have identified by yeast two-hybrid screens. A combination of biochemical, molecular and cellular biological approaches including live-cell imaging will be used in conjunction with primary cultures of myocytes, a unique murine embryonic stem (ES) cell culture system to study de novo cardiac myofibrillogenesis, and analysis of hearts from nebulin -/- mice. We hypothesize that nebulin indeed acts as a multifunctional giant: it is a molecular ruler for thin filament length regulation, and its distinct regions have unique physiological roles critical for the proper functioning of striated muscle. The clinical relevance of these investigations is underscored by the identification of nebulin mutations resulting in various human myopathies, highlighting its pivotal role in normal muscle development and function. Additionally, studying mechanisms responsible for myofibril assembly is critical since mutations in >10 genes encoding sarcomeric proteins are responsible for familial hypertrophic cardiomyopathy, the most common heritable cardiovascular disease. These findings imply that our studies will provide valuable insight into the molecular bases of various muscle diseases. ? ? ? ? ? ?
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