Role of Cardiac Myosin Binding Protein-C in the Regulation of Myocardial Contraction

摘要： 该项目的目标是了解心肌肌球蛋白结合蛋白-C(cMyBP-C)如何调节心脏 肌肉收缩以及cMyBP-C的失调如何导致收缩和舒张期功能障碍。工作地点： PI的实验室在过去的十年中坚定地证明了cMyBP-C与(肌动蛋白)细丝结合并激活 与钙离子相同的收缩方式和强结合的肌球蛋白跨越桥梁。这些发现 从根本上挑战了cMyBP-C仅通过抑制厚度影响收缩的先入之见 (肌球蛋白)细丝。CMyBP-C与细丝的直接相互作用也可以充分解释 CMyBP-C对心脏舒缩功能的调节作用然而，到目前为止，它仍在运行 CMyBP-C与肌动蛋白相互作用的影响纯粹是假设的，因为一直没有办法 在工作心脏中区分cMyBP-C与肌动蛋白或肌球蛋白结合的影响。另一个问题是 缺乏选择性修饰肌瘤中cMyBP-C的补充方法。如果没有这种组合 工具已经不可能在原位定位与cMyBP-C结合伙伴的特定相互作用。在这里我们 通过创建独特的资源来决定性地克服这些障碍，使我们能够从功能上剖析cMyBP-C 与细丝的相互作用。创新包括2个新的转基因小鼠模型，每个模型都有一个 一个高度保守的肌动蛋白结合序列的突变，我们在调控的M-结构域中发现了这个突变。这个 突变增加(L348P)或减少(E330K)cMyBP-C与细丝的结合。初步数据 提示cMyBP-C与肌动蛋白的相互作用控制着基本的收缩时间和 松弛，因为L348P突变增加了收缩射血持续时间，减慢了舒张期 E330K突变使收缩持续时间缩短。拟议实验的目标1将 使用L348P和E330K小鼠验证cMyBP-C与肌动蛋白结合保持细丝的假设 收缩末期的激活与钙离子或强结合交叉桥的活性下降无关。在……里面 目标2，我们创造了第三种独特的小鼠模型，称为“Spy-C”小鼠，它允许我们取代N‘- 肌瘤中cMyBP-C的末端结构域具有任何所需的修饰以探测功能。在AIM 2 我们将使用Spy-C系统来检验肌节长度动态调节cMyBP-C的假设 与肌动蛋白的结合作用，我们将进一步评估cMyBP的中间结构域(C3-C7)的影响- C，以及HCM错义突变热点在这些区域中的作用。我们将鉴定cMyBP-C 通过使用基于FRET的传感器标记cMyBP-C N‘末端结构域来与肌节中的伙伴相互作用。 这项工作的长期影响是，我们将能够选择性地定义cMyBP-C的影响 细丝与心脏收缩和舒张期功能的相互作用及识别新机制 CMyBP-C调控。

ABSTRACT: The goal of this project is to understand how cardiac myosin binding protein-C (cMyBP-C) regulates heart muscle contraction and how dysregulation of cMyBP-C causes systolic and diastolic dysfunction. Work from the PI's lab over the past decade firmly established that cMyBP-C binds to thin (actin) filaments and activates contraction in the same way as Ca2+ and strongly bound myosin cross-bridges. These discoveries fundamentally challenged the preconception that cMyBP-C affects contraction exclusively via inhibition of thick (myosin) filaments. Direct interactions of cMyBP-C with the thin filament can also adequately explain profound effects of cMyBP-C to modulate both diastolic and systolic cardiac function. However, until now functional effects due to cMyBP-C interactions with actin were purely hypothetical because there has been no way to distinguish between effects of cMyBP-C binding to actin or myosin in working hearts. An additional problem is a lack of complementary methods to selectively modify cMyBP-C in sarcomeres. Without this combination of tools it has been impossible to target specific interactions with cMyBP-C binding partners in situ. Here we decisively overcome these barriers by creating unique resources that allow us to functionally dissect cMyBP-C interactions with the thin filament. Innovations include 2 new transgenic mouse models, each with a single mutation in a highly conserved actin binding sequence that we identified in the regulatory M-domain. The mutations either increase (L348P) or decrease (E330K) cMyBP-C binding to the thin filament. Preliminary data from the mice suggest that cMyBP-C interactions with actin control fundamental timing of contraction and relaxation because the L348P mutation increased the duration of systolic ejection and slowed diastolic relaxation, while the E330K mutation decreased the duration of systole. Aim 1 of the proposed experiments will use the L348P and E330K mice test the hypothesis that cMyBP-C binding to actin maintains thin filament activation at the end of systole independent of declining activation by Ca2+ or strongly bound cross-bridges. In Aim 2, we created a third unique mouse model, referred to as “Spy-C” mice, that allows us to replace N'- terminal domains of cMyBP-C in sarcomeres in situ with any desired modification to probe function. In Aim 2 we will use the Spy-C system to test the hypothesis that sarcomere length dynamically regulates cMyBP-C binding interactions with actin and we will further assess the impact of the middle domains (C3-C7) of cMyBP- C and effects of HCM missense mutation hotspots in these domains for the first time. We will identify cMyBP-C interacting partners in the sarcomere by labeling cMyBP-C N'-terminal domains using FRET based sensors. The long-term impact of this work is that we will be able to selectively define the impact of cMyBP-C interactions with the thin filament on systolic and diastolic cardiac function and identify new mechanisms of cMyBP-C regulation.