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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

基本信息

批准号：
9913567
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
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-09-01 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9913567
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

项目摘要

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.

抽象的：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 失调如何导致收缩和舒张功能障碍。工作自 PI 的实验室在过去十年中坚定地证实 cMyBP-C 与细（肌动蛋白）丝结合并激活以与 Ca2+ 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，功能 cMyBP-C 与肌动蛋白相互作用产生的影响纯粹是假设的，因为没有办法区分工作心脏中 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节中的 cMyBP-C 的补充方法。如果没有这个组合工具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果断地克服这些障碍，使我们能够对 cMyBP-C 进行功能剖析与细丝的相互作用。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性 M 结构域中发现了高度保守的肌动蛋白结合序列的突变。这突变增加（L348P）或减少（E330K）cMyBP-C与细丝的结合。初步数据来自小鼠的研究表明，cMyBP-C 与肌动蛋白的相互作用控制着收缩和收缩的基本时间。松弛，因为 L348P 突变增加了收缩期射血的持续时间并减慢了舒张期舒张，而 E330K 突变则缩短了收缩期的持续时间。拟议实验的目标 1 将使用 L348P 和 E330K 小鼠测试 cMyBP-C 与肌动蛋白结合维持细丝的假设收缩末期的激活与 Ca2+ 或强结合跨桥的激活减弱无关。在目标 2，我们创建了第三种独特的小鼠模型，称为“Spy-C”小鼠，它允许我们替换 N'- 肌节中 cMyBP-C 的末端结构域原位，并对探针功能进行任何所需的修饰。目标 2 我们将使用 Spy-C 系统来测试肌节长度动态调节 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 以及首次在这些领域中 HCM 错义突变热点的影响。我们将识别 cMyBP-C 通过使用基于 FRET 的传感器标记 cMyBP-C N' 末端结构域，在肌节中相互作用。这项工作的长期影响是我们将能够选择性地定义 cMyBP-C 的影响与细丝对心脏收缩和舒张功能的相互作用，并确定新的机制 cMyBP-C 调节。