The overall goal of the proposed experiments is to determine the molecular mechanisms involved in the regulation of cardiac muscle contraction by troponin and to determine its role in the genesis of familial hypertrophic cardiomyopathy (FHC). There are two major projects: In I., we will determine the fundamental role of cardiac troponin T (CTnT) in the regulation of cardiac muscle contraction. Traditionally, it has been thought that the primary role of TnT is to interact with and anchor the complex of Tnl and TnC to the actin- containing thin filaments through TnT's interactions with tropomyosin (Tm) and Tnl. Recent results from our lab, with skeletal muscle, additionally suggest that: 1) Ca/2+ binding to STnC causes an interaction between STnC and the C-terminus of STnT and causes an activation of the ATPase activity; and 2) the maximum level of ATPase activation is determined by the particular N-terminal TnT variant which is present. Due to the similarities in primary structure between CTnT and STnT, we hypothesize that CTnT may play a comparable or related role in cardiac muscle. Biochemical, molecular and physiological approaches will be used to test this hypothesis and to determine the fundamental role of HCTnT in the regulation of cardiac muscle contraction. Project II. FHC is an autosomal dominant disease which has been shown to be associated with mutations in HCTnT, and HCTnl. In this section we will attempt to learn how these mutations affect the biochemical and contractile properties of cardiac muscle and give rise to FHC. Our current working hypothesis is that mutations of CTnT and CTnI lead to changes in the interactions between the CTn subunits, or changes in their interactions with the other thin filament proteins, which in turn lead to changes in contractility and/or the Ca/2+ affinity of CTnC (which would change the intracellular [Ca/2+] transient), and that these changes could trigger the cellular mechanisms responsible for the hypertrophic process. To test this hypothesis, transgenic animal models, skinned fiber, myofibrillar, and actomyosin systems, combined with biochemical, molecular, biophysical and physiological techniques, will be utilized. The combined results from these two projects will yield important new information on the role of CTnT and CTnI in the regulation of cardiac contraction and in FHC.
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