Calcium regulation of cardiac thin filament activation

说明(申请人提供)：尽管肌钙蛋白T(TNT)是肌钙蛋白(TN)复合体中唯一已知的与原肌球蛋白(TM)相互作用强烈的亚单位，而且它与功能单位中一半的肌动蛋白单体非常接近，并与肌钙蛋白I(TnI)密切相关，但这种关键位置的蛋白质如何调节心脏功能尚不清楚。大鼠快速骨骼TnT(FsTnT)的氨基末端(N)和羧基末端(C)分别对调节细丝‘开’和‘关’状态转换的动力学速率和钙对肌动蛋白微丝上TM运动的调节起着重要作用。大鼠心肌TnT(CTnT)相应区域差异较大，提示钙对心肌细丝激活的调控不同。我们的假设是，cTnT独特的结构特征是心肌钙激活被如此精细地调控的分子机制(S)的基础。具体目标1和4将解决以下问题：cTnT的N-和C-结构域如何控制心肌从非承重状态到用力跳动的肌球蛋白跨桥状态的转变，以及cTnT独特的结构特征如何导致心肌依赖于长度的激活的特殊特征。具体目标2和3将集中于心肌的协同激活与快速骨骼肌的不同之处，因为cTnT的N和C末端都有独特的结构特征。这一建议利用大鼠cTnT和大鼠fsTnT的差异，以提供关于心肌独特方面的钙和长度依赖调节的机制的新见解。此外，与FHC相关的cTnT突变体将被用来了解cTnT N和C末端的修改如何导致心脏病。方法包括测定重组cTnT-fsTnT嵌合体构建的大鼠心肌纤维束的钙依赖力、ATPase活性、力再发展率(Ktr)和肌纤维动态硬度。TN亚基和TM之间的相互作用将通过稳态荧光分析来测量。我们将使用肌丝机械动力学的数学模型来理解cTnT对肌丝调节的变化是如何表达为全球肌丝机械动力学的变化的。我们将使用一种新的方法来测量完全调节的细丝系统中的钙结合动力学。这一建议旨在为心肌中肌丝对钙的反应如此精细地调节的分子机制提供新的和多样化的见解。

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.