Elucidating Molecular Mechanisms of Hypertrophic Cardiomyopathy using Nano-Engineered Synthetic Myosin Thick Filaments

项目总结/摘要 肥厚型心肌病（HCM）是30岁以下人群心脏性猝死的主要原因。 临床上，肥厚型心肌病的特征是心肌收缩力亢进和心室壁增厚，严重程度直接影响心肌的功能。 取决于突变体与野生型蛋白质表达的比率，这一概念称为等位基因不平衡。HCM是 最常与编码肌节蛋白β-心肌肌球蛋白和 心肌肌球蛋白结合蛋白C（cMyBP-C）。β-心肌肌球蛋白是一种运动蛋白， 纤维和转换化学能从ATP到机械力产生杠杆臂摆动所需的 用于肌肉收缩。cMyBP-C是一种长的多模块结构蛋白，被认为可以抑制肌动蛋白， 肌球蛋白相互作用，减轻钙的影响，调节肌肉收缩。虽然很明显β- 心肌肌球蛋白和cMyBP-C对于正常的肌节功能至关重要，但尚不清楚这些基因突变如何影响肌节功能。 蛋白质产生HCM中所见的严重过度收缩表型。 本培训计划的中心目标是评估HCM突变对肌节相互作用的影响 和整体的集合表型。尽管在β-心肌肌球蛋白中发现了700多个HCM突变， 和cMyBP-C结合，在将基因型与疾病表型联系起来方面几乎没有成功。由于 由于机制的不确定性，不存在用于HCM的小分子疗法，并且治疗仍然是姑息性的。 表征HCM的困难可以部分归因于缺乏可用的技术来研究这些 高度组织化的蛋白质。单分子研究不能解释运动间 运动系综中的干扰和现有的体外测定受到以下因素的可变性和异质性的限制： 运动表面Sivaramakrishnan实验室因此开发并获得了初步数据 记录了DNA纳米管支架作为合成粗丝的实用性。肌球蛋白和cMyBP-C可以 以精确的间隔在DNA纳米管上形成图案，重现了天然的肌节相互作用。 我建议使用DNA纳米管技术通过两个目的来验证我的中心假设。首先我会 确定等位基因不平衡对整体整体表型的影响， HCM突变体和野生型的纳米管上。其次，我将使用合成粗细丝来解剖 cMyBP-C相互作用和改变的钙效应在HCM的过度收缩表型中的作用。 这些实验的发现将大大有助于我们理解基因型是如何 转化为HCM表型，并将有助于靶向药物的开发。

PROJECT SUMMARY/ABSTRACT Hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in people under 30. Clinically, HCM is characterized by hyper-contractility and a thickened ventricular wall with severity that directly depends on the ratio of mutant to wild type protein expression, a concept known as allelic imbalance. HCM is most commonly associated with mutations in genes encoding the sarcomeric proteins β-cardiac myosin and cardiac myosin-binding protein C (cMyBP-C). β-cardiac myosin is a motor protein that assembles into thick filaments and coverts chemical energy from ATP into a mechanical force-generating lever arm swing required for muscle contraction. cMyBP-C is a long multi-modular structural protein that is thought to inhibit the actin- myosin interaction and mitigate the effects of calcium, regulating muscle contraction. While it is clear that β- cardiac myosin and cMyBP-C are crucial for normal sarcomeric function, it is less clear how mutations in these proteins produce the severe hyper-contractile phenotype seen in HCM. The central goal of this training proposal is assess the impacts of HCM mutations on sarcomeric interactions and the overall ensemble phenotype. Despite the identification of over 700 HCM mutations in β-cardiac myosin and cMyBP-C combined, there has been little success in linking genotype to disease phenotype. Due to mechanistic uncertainty, no small molecule therapies for HCM exist and treatment remains palliative. The difficulty in characterizing HCM can be partially attributed to lack of available technologies to study these highly organized proteins on the sarcomeric level. Single-molecule studies do not account for inter-motor interference in the motor ensemble and existing in-vitro assays are limited by variability and heterogeneity of the motility surface. The Sivaramakrishnan lab has therefore developed and obtained preliminary data documenting the utility of a DNA nanotube scaffold as a synthetic thick filament. Myosin and cMyBP-C can be patterned onto the DNA nanotube at precise intervals, recapitulating the native sarcomeric interactions. I propose to use DNA nanotube technology to test my central hypothesis through two aims. First, I will determine the impact of allelic imbalance on the overall ensemble phenotype by patterning varying ratios of HCM mutant and wild type onto a nanotube. Second, I will use the synthetic thick filament to dissect the contributions of cMyBP-C interactions and altered calcium effects in the hypercontractile phenotype of HCM. The findings from these experiments will substantially contribute to our understanding of how genotype translates to HCM phenotype, and will aid in the development of targeted pharmaceuticals.