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）复合体中唯一已知与原肌球蛋白（Tm）强烈相互作用的亚基，并且它在功能单元中与一半的肌动蛋白单体非常接近，并且与肌钙蛋白I （TnI）密切相关，但这种关键位置的蛋白如何调节心脏功能尚不清楚。大鼠快速骨骼TnT （fsTnT）的氨基端(N)和羧基端(C)分别对调节细丝在“开”和“关”状态之间的转换动力学速率和ca2 +对肌动蛋白丝上Tm运动的调节具有重要意义。大鼠心肌中相应区域的TnT （cTnT）差异较大，说明Ca2+对心肌细丝活化的调控是不同的。我们的假设是，cTnT的独特结构特征是Ca2+激活在心肌中如此精细调节的分子机制的基础。具体目标1和4将解决cTnT的N-和C-结构域如何控制心肌从非力承受到力跳动的肌球蛋白过桥状态转变的动力学，以及cTnT独特的结构特征如何导致心肌长度依赖性激活的特殊特征。具体目标2和3将关注由于cTnT的N和C端独特的结构特征，心肌的协同激活与快速骨骼肌的协同激活有何不同。本研究利用大鼠cTnT和大鼠fsTnT的差异，为心肌ca2 +和长度依赖性调节的独特方面的机制提供新的见解。此外，fhc相关的cTnT突变体将用于了解cTnT的N和C端修饰如何导致心脏病。方法包括在重组cTnT-fsTnT嵌合体重建的大鼠心脏纤维束中测量ca2 +依赖性力、atp酶活性、力再开发率（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。我们将使用肌丝力学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整体肌丝力学动力学的变化。我们将使用一种新的方法来测量ca2 +在完全调节的细丝系统中的结合动力学。这一建议旨在为心肌肌丝对ca2 +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.