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

心肌肌球蛋白结合蛋白-C 在心肌收缩调节中的作用

• 批准号: 10155578
• 负责人: Samantha P Harris
• 金额: $ 48.95万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10155578
• 关键词: Actins; Address; Affect; Affinity; Animal Model; Binding; Cardiac; Cardiac Myosins; Cells; Data; Diastole; Filament; Fluorescence Resonance Energy Transfer; Functional disorder; Funding; Generations; Genes; Goals; Head Movements; Heart; Heart Rate; Heart failure; Hypertrophic Cardiomyopathy; In Situ; Label; Lead; Length; Measures; Methods; Microfilaments; Missense Mutation; Modeling; Modification; Mus; Muscle Cells; Muscle Contraction; Muscle Proteins; Mutation; Myocardial Contraction; Myocardium; Myosin ATPase; N-terminal; Output; Phosphorylation; Point Mutation; Positioning Attribute; Proteins; Regulation; Relaxation; Research; Resources; Role; Sarcomeres; Stimulus; Stretching; System; Systole; Testing; Thick; Thick Filament; Thin Filament; Thinness; Time; Transgenic Mice; Work; base; experimental study; falls; fighting; heart function; in vivo; innovation; mechanical properties; mouse model; myosin-binding protein C; novel; response; sensor; tool; virtual

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.

摘要： 本研究的目的是了解心肌肌球蛋白结合蛋白C（cMyBP-C）如何调节心脏功能， 肌肉收缩以及cMyBP-C的失调如何导致收缩和舒张功能障碍。工作从 PI的实验室在过去的十年中坚定地建立了cMyBP-C与细（肌动蛋白）纤维结合并激活 以与Ca 2+相同的方式收缩，并强烈结合肌球蛋白跨桥。这些发现 从根本上挑战了cMyBP-C仅通过抑制厚膜细胞的收缩来影响收缩的偏见。 （肌球蛋白）丝。cMyBP-C与细丝的直接相互作用也可以充分解释深刻的 cMyBP-C调节舒张和收缩心脏功能的作用。然而，到目前为止， 由于cMyBP-C与肌动蛋白相互作用的影响纯粹是假设的，因为没有办法 区分cMyBP-C与肌动蛋白或肌球蛋白结合在工作心脏中的作用。另外一个问题是 缺乏选择性修饰肌节中cMyBP-C的补充方法。如果没有这种组合， 然而，由于cMyBP-C是一种新的靶向工具，因此不可能在原位靶向与cMyBP-C结合伴侣的特异性相互作用。这里我们 通过创造独特的资源，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 我们在调节M-结构域中鉴定的高度保守的肌动蛋白结合序列中的突变。的 突变增加（L348 P）或减少（E330 K）cMyBP-C与细丝的结合。初步数据 表明cMyBP-C与肌动蛋白的相互作用控制了收缩的基本时间， 因为L348 P突变增加了收缩期射血的持续时间，减慢了舒张期射血的持续时间， E330 K突变使收缩持续时间缩短。拟议实验的目标1将 使用L348 P和E330 K小鼠测试cMyBP-C与肌动蛋白结合维持细丝的假设 在收缩末期的激活独立于由Ca 2+或强结合的交叉桥引起的下降的激活。在 目的2，我们创建了第三种独特的小鼠模型，称为“Spy-C”小鼠，其允许我们替换N '- 原位肌节中cMyBP-C的末端结构域进行任何所需的修饰以探测功能。在目标2中 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 与肌动蛋白的结合相互作用，我们将进一步评估cMyBP的中间结构域（C3-C7）的影响。 C和HCM错义突变热点在这些领域的影响。我们将鉴定cMyBP-C 通过使用基于FRET的传感器标记cMyBP-C N '-末端结构域来检测肌节中的相互作用伴侣。 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。 与细细丝的相互作用对心脏收缩和舒张功能的影响，并确定新的机制， cMyBP-C调节。