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

项目摘要/摘要 肥厚性心肌病(HCM)是30岁以下人群心源性猝死的主要原因。 临床上，肥厚性心肌病的特点是收缩过度和室壁增厚，其严重程度直接 取决于突变型和野生型蛋白表达的比例，这一概念被称为等位基因失衡。HCM是 最常见的与编码肌节蛋白β的基因突变有关-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，可以组装成厚的 细丝和转化化学能从三磷酸腺苷转化为机械力产生杠杆臂所需的摆动 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-C。 肌球蛋白相互作用，减轻钙离子的影响，调节肌肉收缩。虽然很明显，β- 心肌肌球蛋白和cMyBP-C对正常的肌节功能至关重要，目前尚不清楚这些基因的突变是如何 蛋白质产生在HCM中看到的严重的超收缩表型。 本培训计划的中心目标是评估HCM突变对肌瘤相互作用的影响 以及整体的合奏表型。尽管在β-心肌肌球蛋白中发现了700多个hCM突变 和cMyBP-C结合在一起，在将基因型与疾病表型联系起来方面取得的成功很少。由于 机制上的不确定性，不存在针对肥厚性心肌病的小分子疗法，治疗仍然是姑息性的。 描述HCM的困难可以部分归因于缺乏可用的技术来研究这些 肌节水平上高度组织化的蛋白质。单分子研究不能解释发动机间的相互作用 运动组和现有的体外检测中的干扰受到以下因素的影响 运动面。因此，Sivaramakrishnan实验室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用途。肌球蛋白和cMyBP-C可以 以精确的间隔排列在DNA纳米管上，重述了天然肌节的相互作用。 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 通过改变等位基因比例来确定等位基因不平衡对整体集合表型的影响 将HCM突变型和野生型转移到纳米管上。第二，我会用合成的粗丝来解剖 CMyBP-C相互作用和钙效应改变在肥厚性心肌细胞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.