Biomechanical analysis of sarcomeric mutations that cause early-onset cardiomyopa

描述（由申请人提供）：肥厚型心肌病（HCM）是一种涉及心室壁增厚的疾病。在婴儿中，它的表现特别严重，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度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清楚。为了开发靶向治疗，我们必须首先更好地了解HCM的原因，据信75%的儿童HCM病例的原因是遗传性的。据信，HCM突变改变β-心肌肌球蛋白的能量生成导致一系列细胞变化，逐渐引起HCM疾病表型。迄今为止，很少有突变被严格研究，以前的研究是使用非人肌球蛋白进行的。这些研究产生了相互矛盾的结果，强调了表达人β-心脏肌球蛋白的必要性。一种新的基于鼠成肌细胞的表达技术已经开发出来，Spudich实验室刚刚发表了第一个在完全人类系统中野生型和突变型人类β-心肌肌球蛋白的生化表征研究。使用这种新的表达系统将评估5个儿科特异性HCM突变对人β-心肌肌球蛋白生物力学功能的影响，使用多种测定：F-肌动蛋白激活ATP酶测定以测量总循环时间，体外运动测定以测量无负荷最大收缩速度，以及双光束光阱测定以测量内力产生。我还将利用新开发的振荡技术来施加不同的力并测量收缩速度与力的函数关系。 通过对儿科特异性HCM突变进行第一次生物力学分析，并对任何引起心肌病的突变进行第一次力速度分析，我将确定这些突变改变肌球蛋白运动和产生力的能力的程度。通过强调突变可以改变分子运动功能的机制，这些儿科特异性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.