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 familial hypertrophic cardiomyopathy (FHC).
SPECIFIC AIM 1. Cardiac TnT (CTnT) Isoforms and the Regulation of Contraction. CTnT isoforms are generated mainly by alternative splicing of two exons encoding the N-terminal variable region and are expressed in the human heart as four isoforms (HCTnT1 through HCTnT4). The expression of these isoforms at the protein level has previously been found to differ in the normal and failing adult and fetal human heart. Based on recent results from this laboratory we hypothesize that HCTnT N-terminal isoforms modulate Ca2+-sensitivity and the relaxation properties of cardiac muscle. In order to determine the fundamental role of HCTnT isoforms in the regulation of cardiac muscle contraction and to test this hypothesis, we will substitute the various CTnT isoforms into reconstituted skinned muscle and thin filament systems and study their individual properties.
SPECIFIC AIM 2. Functional Consequences of Tn FHC Mutations. Our working hypothesis is that mutations in Tn subunits lead to changes in the interactions between the CTn subunits and or changes in their interaction with other thin filament proteins, which in turn lead to changes in the Ca2+-affinity of CTnC and or changes in muscle mechanics eventually resulting in FHC. We propose to address the following issues: 1) to determine the in vitro properties of different Arg mutations in HCTnl (Arg 21, Arg 145 and Arg 204). Arg FHC mutations in HCTnT and HCTnl have been reported to be mutated to six different amino acids (Cys, Gly, Trp, Leu, Pro, and Gin); 2) to determine the in vitro properties of different Arg mutations in HCTnT (Arg 92, Arg 94, Arg 130 and Arg 278); 3) to characterize the first FHC mutant found in HCTnC; and 4) to determine the effect of different ratios of TnT, Tnl and TnC FHC mutants on the Ca 2+- sensitivity and level of actomyosin ATPase activity. ? All of the Specific Aims listed above focus on the effect of cardiac Tn, containing CTn subunits with FHC mutations, on the regulation of cardiac muscle contraction. Knowledge on these mutations will provide insights into the mechanisms by which these mutations cause cardiac disease as well as provide information about the importance of various regions in these CTn subunits. ? ?
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