Calcium regulation of cardiac thin filament activation

描述（由申请人提供）：尽管肌钙蛋白T（TnT）是肌钙蛋白（Tn）复合物中唯一已知与原肌球蛋白（Tm）强烈相互作用的亚基，并且其与功能单元中的一半肌动蛋白单体非常接近，并且与肌钙蛋白I（TnI）密切相关，但尚不清楚这种枢转定位蛋白如何调节心脏功能。 大鼠骨骼肌钙蛋白T（fsTnT）的氨基端（N）和羧基端（C）分别对调节微丝的“开”和“关”状态之间的动力学转换速率和Ca 2+对肌动蛋白丝上Tm运动的调节起重要作用。 大鼠心肌肌钙蛋白T（cTnT）的相应区域差异较大，表明Ca ~（2+）对心肌细纤维激活的调节是不同的。 我们的假设是，cTnT的独特结构特征是心肌中Ca 2+激活如此精细调节的分子机制的基础。 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中非受力肌球蛋白跨桥状态向受力肌球蛋白跨桥状态转变的动力学，以及心肌长度依赖性激活的特殊特征如何归因于cTnT的独特结构特征。 具体目标2和3将重点关注心肌的协同激活与快速骨骼肌的协同激活有何不同，因为cTnT的N端和C端具有独特的结构特征。 该建议利用大鼠cTnT和大鼠fsTnT的差异，以提供新的见解的独特方面的Ca 2+和心肌长度依赖性调节的机制。 此外，FHC相关cTnT突变体将用于了解cTnT的N和C末端的修饰如何导致心脏疾病。 方法测定重组cTnT-fsTnT嵌合体重建的大鼠心肌纤维束的Ca 2+依赖力、ATP酶活性、力重建率（ktr）和肌纤维动态刚度。 Tn亚基和Tm之间的相互作用将通过使用稳态荧光测定来测量。 我们将使用数学模型ofmyofilament mechano-dynamics了解肌钙蛋白T的肌丝调节的变化是如何表达为全球肌丝mechano-dynamics的变化。 我们将使用一种新的方法来测量钙离子结合动力学在一个完全调节细丝系统。 该建议旨在提供新的和不同的见解的分子机制，肌丝对Ca 2+的反应是如此精细地调节心肌。

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.