Calcium regulation of cardiac thin filament activation

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DESCRIPTION (provided by applicant): Despite the facts that troponin T (TnT) is the only subunit of the troponin (Tn) complex that is clearly known to interact strongly with Tropomyosin (Tm) and that it has close proximity to half of the actin monomers in the functional unit and is closely associated with troponin I (TnI), how this pivotally positioned protein modulates cardiac function is not understood. The amino terminus (N) the carboxyl terminus (C) of rat fast skeletal TnT (fsTnT) are important for modulating kinetic rates of transition between 'on' and 'off' states of thin filaments, and for Ca 2+ regulation of Tm movement on the actin filament, respectively. The corresponding regions in rat cardiac TnT (cTnT) differ considerably, which indicates that Ca2+ regulation of thin filament activation in cardiac muscle is different. Our hypothesis is that the unique structural features of cTnT underlie the molecular mechanism(s) by which the Ca2+ activation is so exquisitely modulated in cardiac muscle. Specific Aims 1 and 4 will address the questions of how the N- and C- domains of cTnT control the dynamics of transitions from non-force bearing to force-beating myosin cross-bridge states in cardiac muscle, and how the special features of length-dependent activation of cardiac muscle are due to unique structural features of cTnT. Specific aims 2 and 3 will focus on how the cooperative activation of cardiac muscle differs from that in fast skeletal muscle because of unique structural features in both the N and C termini of cTnT. This proposal exploits differences in rat cTnT and rat fsTnT in order to provide novel insights about the mechanisms underlying the unique aspects Ca 2+ and length-dependent regulation of cardiac muscle. Furthermore, FHC-related cTnT mutants will be used to understand how modifications to the N and C termini of cTnT contribute to cardiac disease. Methods include measurement of Ca 2+ dependent force, ATPase activity, rate of force redevelopment (ktr), and myofiber dynamic stiffness in rat cardiac fiber bundles reconstituted with recombinant cTnT-fsTnT chimeras. Interactions between Tn subunits and Tm will be measured by using steady-state fluorescence assays. We will use mathematical model ofmyofilament mechano-dynamics to understand how changes in myofilament regulation by cTnT are expressed as changes in global myofilament mechano-dynamics. We will use a novel method for measuring Ca 2+ binding kinetics in a fully regulated thin filament system. This proposal aims to provide new and diverse insight into the molecular mechanism by which myofilament response to Ca 2+ is so exquisitely modulated in cardiac muscle.