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Integrative Approach to Divergent Remodeling in Thin Filament Cardiomyopathies

细丝心肌病发散性重构的综合方法

基本信息

批准号：
10391716
负责人：
Jil C Tardiff
金额：
$ 56.98万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-12-01 至 2025-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10391716
关键词：
Actins Actomyosin Address Alleles Animal Model Animals Basic Science Biological Markers Biology Biophysical Process Biophysics Cardiac Cardiomyopathies Clinical Clinical Research Collection Complex Computer Analysis Coupled Couples Cryoelectron Microscopy Data Development Dilated Cardiomyopathy Disease Disease Progression Dissociation Early treatment Exercise Fluorescence Resonance Energy Transfer Foundations Funding Future Gene Expression Profile Genes Genetic Transcription Genotype Goals Human Genetics Hypertrophic Cardiomyopathy Impairment In Vitro Individual Kinetics Knowledge Lead Link Measurement Methodology Microfilaments Modeling Molecular Mutation Myosin ATPase Organ Outcome Pathogenesis Pathogenicity Pathologic Patient-Focused Outcomes Patients Pattern Performance Phenotype Positioning Attribute Process Protein Isoforms Proteins Relaxation Resolution Rest Sarcomeres Side Signal Pathway Signal Transduction Site Spectrum Analysis Structure System Techniques Testing Therapeutic Thin Filament Time Transcriptional Activation Transgenic Animals Transgenic Mice Tropomyosin Ventricular Remodeling Work alpha helix base biomarker discovery clinically relevant drug discovery drug testing flexibility improved in vivo inherited cardiomyopathy inhibitor insight mouse model mutant novel novel strategies programs protein protein interaction reconstitution response stressor tool transcriptome sequencing transcriptomics

项目摘要

Project Summary: The cardiac thin filament is the essential regulator of cardiac contractility and relaxation at the molecular level. It is comprised of five discrete proteins: cTnC, cTnI, cTnT, actin and tropomyosin that have co-evolved to sustain efficient cardiac performance at rest, during exercise and, importantly, to respond to pathologic stressors. Mutations in genes encoding each of these proteins have been definitively linked to the development of a range of human genetic cardiomyopathies, including hypertrophic (HCM) and dilated (DCM) forms. Despite 25 years of study by many groups including ours, to define the direct link(s) between the biophysical insult and the resultant complex cardiomyopathy, many questions remain and significantly limit our ability to use genotype to prognosticate and eventually even treat individuals with genetic cardiomyopathies. The recent development of Mavacamten, a first-in-class, targeted myosin inhibitor is a game-changing advance that was predicated on decades of basic research into the fundamental biology of the sarcomere. Thus, the question is no longer “if” we can target the sarcomere, but for the thin filament the question is “what function to target” and eventually “when to treat”. The cardiac thin filament is a highly dynamic allosteric “machine” where most of the component proteins are comprised of a-helices connected by variably sized unstructured linkers, where dynamic flexibility is the rule, not the exception and this has limited the availability of high resolution structure for these regions. Most of the known pathogenic mutations in cTnI and cTnT are clustered within these highly flexible domains, where there is likely a “distribution” of tolerance, whereby mutations impair function (enough to cause disease) but do not break it. We thus propose that by examining the range of these dynamic perturbations within these domains we can identify new structural and dynamic disease mechanisms that can be functionally binned, studied and modulated. We provide proof-of-principle preliminary data in this proposal. Over the recent funding period we expanded our structural methodologies to include Time-Resolved FRET with a Single Donor – Dual Acceptor approach that allows us to use actin as an anchor to refine highly flexible structures. We will next use known, highly divergent (HCM vs DCM) mutations within each flexible domain to probe both structure and dynamics with the premise that these mutations will define the limits of “tolerability” in either direction and use spectroscopy and measurements of Ca2+ dissociation and association kinetics coupled to computation to define and test these hypotheses. Finally, we will “close the loop” by utilizing our existing extensively characterized transgenic animal models based on the same mutations used to set our limits and perform 3-timepoint RNA-Seq to discover unique early transcriptional signatures to help link these perturbations to the resultant early remodeling cascade that eventually leads to distinct patterns of ventricular remodeling. The long term goal is to use this coupled structural – dynamic – transcriptomic platform to identify new targets, both primary and secondary for future therapeutics and even biomarker discovery.

项目概要：心脏细丝是分子水平上心脏收缩和舒张的重要调节器。它由五种独立的蛋白质组成：cTnC、cTnI、cTnT、肌动蛋白和原肌球蛋白，它们共同进化为在休息时、运动期间维持有效的心脏功能，重要的是，对病理反应做出反应压力源。编码这些蛋白质的基因突变已明确与发育相关一系列人类遗传性心肌病，包括肥厚型心肌病 (HCM) 和扩张型心肌病 (DCM)。尽管包括我们在内的许多团体进行了 25 年的研究，以确定生物物理之间的直接联系侮辱和由此产生的复杂心肌病，许多问题仍然存在，并严重限制了我们的能力使用基因型来预测甚至最终治疗患有遗传性心肌病的个体。最近的 Mavacamten 是一种一流的靶向肌球蛋白抑制剂，其开发是一项改变游戏规则的进步基于数十年对肌节基础生物学的基础研究。因此，问题是不再是“如果”我们可以靶向肌节，而是对于细丝，问题是“靶向什么功能”和最终“何时治疗”。心脏细丝是一个高度动态的变构“机器”，其中大部分成分蛋白由通过不同大小的非结构化接头连接的 a 螺旋组成，其中动态灵活性是规则，而不是例外，这限制了高分辨率结构的可用性对于这些地区。大多数已知的 cTnI 和 cTnT 致病性突变都集中在这些高度灵活的域，其中可能存在耐受性的“分布”，由此突变会损害功能（足够导致疾病）但不要破坏它。因此，我们建议通过检查这些动态的范围通过这些领域内的扰动，我们可以识别新的结构和动态疾病机制，这些机制可以进行功能分类、研究和调制。我们在此提案中提供了原理验证的初步数据。在最近的资助期间，我们扩展了结构方法，将时间分辨 FRET 纳入其中采用单供体-双受体方法，使我们能够使用肌动蛋白作为锚点来精炼高度灵活的结构。接下来，我们将使用每个灵活域内已知的高度分歧（HCM 与 DCM）突变来探究结构和动力学，前提是这些突变将定义“耐受性”的极限任一方向并使用 Ca2+ 解离和缔合动力学耦合的光谱学和测量通过计算来定义和测试这些假设。最后，我们将利用现有的“闭环” 基于用于设定我们的极限的相同突变，广泛表征了转基因动物模型执行 3 个时间点 RNA 测序以发现独特的早期转录特征，以帮助将这些特征联系起来对由此产生的早期重塑级联的扰动最终导致心室的不同模式重塑。长期目标是利用这种耦合的结构-动态-转录组平台来识别新的目标，包括未来治疗甚至生物标志物发现的主要和次要目标。