Integrative Approach to Divergent Remodeling in Thin Filament Cardiomyopathies

项目摘要： 心脏细丝在分子水平上是心脏收缩和舒张的重要调节因子。 它由五种离散的蛋白质组成：cTnC，cTnI，cTnT，肌动蛋白和原肌球蛋白，它们共同进化， 在休息时、运动时维持有效心脏性能，重要的是， 压力源编码这些蛋白质的基因突变已被明确地与发展有关。 一系列人类遗传性心肌病，包括肥厚型（HCM）和扩张型（DCM）。 尽管包括我们在内的许多团体进行了25年的研究，但为了确定生物物理学之间的直接联系， 侮辱和由此产生的复杂心肌病，许多问题仍然存在，并大大限制了我们的能力， 利用基因型来诊断并最终治疗患有遗传性心肌病的个体。近期 Mavacamten是一种一流的靶向肌球蛋白抑制剂，它的开发是一个改变游戏规则的进步， 基于几十年来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"， 最后是“何时治疗”。心脏细丝是一个高度动态的变构"机器"， 组分蛋白质由α-螺旋组成，所述α-螺旋由尺寸较小的非结构化接头连接，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致病性突变都聚集在这些高度相关的突变中。 灵活的结构域，其中可能存在耐受性的"分布"，由此突变损害功能（足够 因此，我们建议，通过检查这些动态的范围， 在这些领域的扰动，我们可以确定新的结构和动态疾病机制， 被功能性地分类、研究和调节。我们在本提案中提供了初步的原理验证数据。 在最近的资助期间，我们扩展了我们的结构方法，包括时间分辨FRET 单供体-双受体方法，使我们能够使用肌动蛋白作为锚， 结构.接下来，我们将使用每个柔性结构域中已知的高度发散（HCM vs DCM）突变， 探索结构和动力学，前提是这些突变将定义“耐受性”的限制， 任一方向和使用光谱学和测量Ca2+解离和缔合动力学耦合 来定义和检验这些假设。最后，我们将通过利用现有的 广泛表征的转基因动物模型基于用于设定我们的限制的相同突变， 进行3个时间点的RNA-Seq，以发现独特的早期转录特征， 扰动所产生的早期重塑级联，最终导致不同的模式的心室 重塑长期目标是使用这种耦合的结构-动态-转录组平台来识别 新的目标，主要和次要的未来治疗，甚至生物标志物的发现。

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.