Blunting of the Myofilament Beta-Adrenergic Response in HCM: Structural-Dynamic Mechanisms

HCM 中肌丝 β 肾上腺素反应的钝化：结构动力学机制

基本信息

批准号：
10748921
负责人：
Romi L Castillo
金额：
$ 4.19万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-15 至 2025-08-14
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10748921
关键词：
ATP phosphohydrolase Actins Adrenergic Agents Affect Allosteric Regulation Behavior Binding Binding Sites Biological Assay Calcium Calcium Binding Cardiac Clinical Complex Computer Models Coupled Coupling Cryoelectron Microscopy Cyclic AMP-Dependent Protein Kinases Data Disease Dissociation Event Exercise Exhibits Experimental Designs Fluorescence Anisotropy Fluorescence Resonance Energy Transfer Free Energy Functional disorder Genetic Goals Health Heart Diseases Hypertrophic Cardiomyopathy Impairment In Vitro Individual Induced Mutation Kinetics Left Link Maps Measurement Mediating Methods Microfilaments Modeling Molecular Monitor Muscle Contraction Mutation Patients Peptides Performance Phenotype Phosphorylation Phosphorylation Site Play Point Mutation Process Proteins Regulation Relaxation Resolution Restrictive Cardiomyopathy Role Serine Severities Signal Transduction Site Striated Muscles Structure Surface System Thin Filament Time Tropomyosin Troponin Troponin C Troponin I Troponin T Variant Ventricular Work causal variant experimental study flexibility hemodynamics in silico intermolecular interaction molecular dynamics novel reconstitution response simulation stopped-flow fluorescence time resolved data time use

项目摘要

Project Summary: Hypertrophic cardiomyopathy (HCM) is a complex genetic cardiac disorder that affects ~1/300 – 1/500 individuals worldwide. A common clinical manifestation of patients with HCM is an impairment in left ventricular relaxation (diastolic dysfunction). Beta-adrenergic stimulation is a key regulator of diastolic performance. During beta- adrenergic stimulation protein kinase A (PKA) mediates phosphorylation of a variety of sarcomeric targets, including cardiac troponin I (cTnI) at serine 23/24 (Ser23/24). This phosphorylation event results in a significant increase in relaxation (or de-activation) rates at the myofilament level. Previous work has shown that this observation is due to increases in calcium dissociation rate from the cardiac thin filament. Additionally, some thin filament HCM mutations have been shown to exhibit an impaired response to phosphorylation of Ser23/24 in ATPase assays and force-pCa measurements. While extensive work by several groups has investigated the structural basis for this increase in calcium dissociation rate, all previous studies, to the best of our knowledge, lack the key thin filament binding partners actin and tropomyosin, crucial components for allosteric regulation of relaxation. In this proposal we will perform TR-FRET experiments to assess both intramolecular and intermolecular interactions between the N-terminus of cTnI and C-terminus of cTnI and the N-terminus of cTnI and Site II of cTnC in the presence and absence of phosphorylation at Ser23/24. The experimental design will provide distances that we will employ in our atomistic thin filament model. We will then use stopped flow fluorescence anisotropy in order to probe transitions in dynamic behavior in the C-terminus of cTnI when Ser23/24 is phosphorylated as calcium dissociates from the cardiac thin filament. We hypothesize that phosphorylation of Ser23/24 will alter the rate at which these transitions occur and that these mechanisms may be altered by HCM causative mutations. To explore the possibility that the degree of observed diastolic impairment (and potentially the severity of the end HCM phenotype) may be mutation-specific, we propose to investigate the molecular effects of 3 independent known cTnI mutations at residue R145 in cTnI. This mutational hotspot includes HCM- linked mutations R145G, R145Q and the restrictive cardiomyopathy (RCM) mutation R145W. We will couple structural data from TR-FRET experiments to changes in calcium dissociation rate to investigate how structural changes impact function and if the diastolic dysfunction is additive in the presence of Ser23/24 phosphorylation. We will employ metadynamics simulations to obtain free energy changes and identify specific changes in interactions that occur from these mutations and phosphorylation as well as in the two calcium states (on and off). The in-vitro—in-silico coupled approaches proposed in this application will provide atomic level resolution of the structural changes that occur upon Ser23/24 phosphorylation in the WT state and in the context of known cTnI-linked HCM/RCM mutations, with a long-term goal of identifying targetable disease mechanisms.

项目概要：肥厚型心肌病 (HCM) 是一种复杂的遗传性心脏病，影响 ~1/300 – 1/500 全世界 HCM 患者的一个常见临床表现是左心室受损。放松（舒张功能障碍）。β-肾上腺素能刺激是舒张功能的关键调节剂。在 β-肾上腺素能刺激过程中，蛋白激酶 A (PKA) 介导多种肌节的磷酸化靶标，包括丝氨酸 23/24 (Ser23/24) 处的心肌肌钙蛋白 I (cTnI)。先前的工作表明，肌丝水平的松弛（或失活）率显着增加。这一观察结果是由于心脏细丝中钙解离率的增加所致。一些细丝 HCM 突变已被证明对磷酸化反应受损 Ser23/24 在 ATP 酶测定和力 pCa 测量中的应用已由多个小组进行了大量工作。研究了钙解离率增加的结构基础，所有先前的研究都尽最大努力我们的知识，缺乏关键的细丝结合伙伴肌动蛋白和原肌球蛋白，它们是在本提案中，我们将进行 TR-FRET 实验来评估两者。 cTnI N 端和 cTnI C 端之间的分子内和分子间相互作用 Ser23/24 磷酸化存在和不存在时的 cTnI N 末端和 cTnC 位点 II。实验设计将提供我们将在原子细丝模型中使用的距离。使用停流荧光各向异性来探测 C 末端动态行为的转变当 Ser23/24 因钙从心脏细丝解离而被磷酸化时，cTnI。 Ser23/24 的磷酸化将改变这些转变发生的速率，并且这些机制可能会因 HCM 致病突变而改变探讨观察到的舒张程度的可能性。损伤（以及最终 HCM 表型的潜在严重程度）可能是突变特异性的，我们建议研究 cTnI 残基 R145 处 3 个独立的已知 cTnI 突变的分子效应。突变热点包括 HCM 相关突变 R145G、R145Q 和限制性心肌病 (RCM) 我们将 TR-FRET 实验的结构数据与钙解离的变化结合起来。调查结构变化如何影响功能以及舒张功能障碍是否会累加 Ser23/24 磷酸化的存在我们将采用元动力学模拟来获得自由能。变化并识别这些突变和磷酸化发生的相互作用的具体变化：以及两种钙状态（开和关）。应用程序将提供 Ser23/24 上发生的结构变化的原子级分辨率 WT 状态和已知 cTnI 相关的 HCM/RCM 突变背景下的磷酸化，具有长期确定可靶向疾病机制的目标。