Biomechanical analysis of sarcomeric mutations that cause early-onset cardiomyopa

描述(申请人提供)：肥厚型心肌病(HCM)是一种涉及心脏室壁增厚的疾病。在婴儿中，它的表现尤其严重，是儿科人群心源性猝死的主要原因。不幸的是，目前的治疗仅限于轻微病例的症状缓解和心脏移植。 对于严重的病例，这种突变引起的功能变化还不是很清楚。为了开发有针对性的治疗方法，我们必须首先更好地了解HCM的病因，据信大约75%的儿童HCM病例的本质是遗传的。据认为，β-心肌肌球蛋白的能量产生的改变会导致一系列的细胞变化，从而逐渐导致肥厚性心肌病的表型。到目前为止，几乎没有对突变进行严格的研究，以前的研究是使用非人类肌球蛋白进行的。这些研究产生了相互矛盾的结果，强调了表达人β-心肌肌球蛋白的必要性。一种新的基于小鼠成肌细胞的表达技术已经被开发出来，斯普迪奇实验室刚刚发表了第一个关于野生型和突变型人β-心肌肌球蛋白在整个人类系统中的生化特性研究。利用这个新的表达系统，将使用多种检测方法来评估5种儿科特异的hCM突变对人β-心肌肌球蛋白生物力学功能的影响：F-肌动蛋白激活的ATPase实验测量总周期时间，体外运动实验测量无负荷的最大收缩速度，以及双光束光学陷阱实验测量内力产生。我还将利用一种新开发的振动技术来施加不同的力，并测量作为力的函数的收缩速度。 通过对儿科特有的HCM突变进行第一次生物力学分析，以及对任何导致心肌病的突变进行第一次力-速度分析，我将确定这些突变在多大程度上改变肌球蛋白的移动和产生力的能力。通过强调突变可以改变分子马达功能的机制，这些儿科特有的突变可能为测试治疗HCM的潜在小分子疗法提供极好的模型。

DESCRIPTION (provided by applicant): Hypertrophic cardiomyopathy (HCM) is a disease involving the thickening of the ventricular walls of the heart. In infants, its presentation is particularly severe, and it is the leading cause of sudden cardiac death in pediatric populations. Unfortunately, current therapy is limited to symptomatic relief for mild cases and heart transplant for severe cases, and the functional changes caused by such mutations are not well understood. To develop targeted therapies, we must first better understand HCM's causes, which are believed to be genetic in nature for ~75% of pediatric HCM cases. It is believed that alteration of β-cardiac myosin's power generation by HCM mutations leads to a series of cellular changes that gradually cause the HCM disease phenotype. To date, few mutations have been rigorously investigated, and previous studies were performed using non-human myosins. These studies have produced conflicting results, underscoring the need for the expression of human β-cardiac myosin. A new murine myoblast-based expression technique has been developed, and the Spudich Lab has just published the first biochemical characterization study of wild type and mutant human β-cardiac myosin in an entirely human system. Using this new expression system will assess the effects of 5 pediatric-specific HCM mutations on the biomechanical function of human β-cardiac myosin using multiple assays: the F-actin activated ATPase assay to measure total cycle time, the in vitro motility assay to measure the unloaded maximum contractile velocity, and the dual beam optical trap assay to measure intrinsic force generation. I will also utilize a newly-developed oscillation technique to apply varying forces and measure velocity of contraction as a function of force. By performing the first biomechanical analysis of pediatric-specific HCM mutations and the first forcevelocity analysis of any cardiomyopathy-causing mutation, I will determine the extent to which these mutations alter myosin's ability to move and produce force. By highlighting the mechanisms by which mutations can alter molecular motor function, these pediatric-specific mutations may offer excellent models for testing potential small molecule therapeutics for treating HCM.