Molecular Biology And Pathophysiology Of Cardiomyopathy

心肌病的分子生物学和病理生理学

• 批准号: 8158030
• 负责人: NEAL DAVID EPSTEIN
• 金额: $ 32.41万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8158030
• 关键词: Cardiomyopathies; Functional disorder; Molecular Biology

Hypertrophic cardiomyopathy (HCM) is an inherited cardiac disease characterized by an increased left ventricular mass in the absence of another cause for hypertrophy. This disease provides a model to study the process of cardiac hypertrophy. In approximately 15% of affected families, the disease gene encodes a beta myosin heavy chain (BMHC) gene with a missense mutation. We previously identified 32 distinct mutations in the BMHC gene and mapped them onto the 3D structure of the head of skeletal muscle myosin. The families evaluated at NIH are referred to this tertiary care facility for severe disease. From a group of 320 such families more than 60 mapped to the Beta Myosin gene. The mutations cluster in 4 regions, suggesting different types of interference in the actomyosin cross-bridge kinetics as a function of mutation location. Subsequent large scale sequencing of many more patients by the Seidnman group has filled in the regions between these clusters. However, the patients that we studied had severe disease and perhaps this is why the clustering was so evident. We have studied the mechanical properties of extracted mutant myosins and muscle fibers expressing these myosins, to analyze the pathophysiology at a molecular level. One of the clusters of mutations has led us to the discovery of mutations in the myosin light chains which cause a variant of HCM characterized by an obstruction in the middle of the left ventricle. Through a series of arguments, the association of the myosin light chain mutations with the rare subtype of HCM led us to hypothesize the importance of the "stretch-activation response" to the function of the normal heart. The stretch-activation response in Drosophila flight muscle has been previously shown to be distorted by a mutation in the "regulatory" myosin light chain (RLC), resulting in flightless flies whose wings do not beat properly. We have developed transgenic mice expressing the mutant myosin "essential" light chain (ELC), from a patient with cardiac hypertrophy. The hearts from these mice also do not beat properly. That is, there is a shift of the frequency of maximum power output to a rate beyond the physiologic range, with consequent loss of oscillatory power. We have cloned a human enzyme that phosphorylates the RLC and and identified a genetic mutation in a small family. This mutant enzyme has been expressed and shown to have an increased maximum velocity compared to the normal enzyme. We have performed mechanical studies on isolated muscle fibers treated with this cloned enzyme and demonstrated a change in the stretch-activation response. We are continuing to use muscle fibers from normal and transgenic mice to study the basic biophysical response of muscle fibers to light chain phosphorylation. One of the observations from our recent temperature dependency studies evaluating cross-bridge kinetics of fast and slow muscle fibers has generated an intriquing hypothesis. That is, that cardiac muscle fibers may be able to drive the cross-bridge kinetics in reverse and produce the equivalent of new ATP. This is based on the observation of a Q10 of 2 for the force producing state of fast muscle myosin and a corresponding Q10 of 20 for cardiac myosin. This disparity suggests an isolated force producing state in fast myosin but a back linked force producing step in cardiac myosin. If elastic forces in the oscillating heart are timed correctly then portions of the heart that are stretched by contracting portions could conserve energy rather then produce heat with the net energy saving equivalent to new energy production. We are presently developing single molecule instrumentation that will allow the direct test of this hypothesis. We have published a manuscript (Biophys J. 2007 Apr 15;92(8):2865-74. Epub 2007 Jan 26.) that takes advantage of an observation we made in our basic single muscle fiber studies. Under rare conditions, a ploot of tension vs. temperature of fast skeletal muscle fibers is sigmoidal, with an inflection point. Therefore tension production can be represented as a simple 2 state reaction. This has allowed, for the first time, the calculation of the backwards and forward reaction rates of myosin force production as well as the enthalpy and entropy of the reaction. The forward and backward reactions are Arrhenious and anti-Arrhenious respectively, suggesting that force production involves a localized unfolding and the backwards reaction is inhibited by an entropic barrier (refolding). These findings are consistent with previous crystallographic findings of Myosin VI, published by the Houdusse laboratory. Our 2009 publication in PNAS compares the exponentials extracted from the temperature induced myosin force production (involving no movement) to movement induced force production. This highlights the buffering of tension by an ensemble of crossbridges that have released inorganic phosphate but not yet cycled through the strong attachment phase. Both of these papers add to our basic understanding of muscle contraction. Most recently, a review of our previous genetic screens has identified a naturally occurring mutation in a new cardiac kinase that phosphorylates both RLC and Myosin Binding Protein-C. This mutation causes severe disease in patients. The vector for a Knock-in mouse has been constructed and is now being sequenced before insertion into a stem cell for mouse production. The wild type and mutant form of this kinase is being expressed in a Baculoviral system to be used together with our previously expressed MLCK-2. Such studies will be carried out in single fiber studies, as we have done previously.

肥厚性心肌病（HCM）是一种遗传性心脏病，其特征是在没有另一个肥大原因的情况下左心室肿块增加。该疾病提供了研究心脏肥大过程的模型。在大约15％的受影响家庭中，该疾病基因编码具有错义突变的β肌球蛋白重链（BMHC）基因。我们先前鉴定了BMHC基因中的32个不同的突变，并将其映射到骨骼肌肌球蛋白头部的3D结构上。在NIH评估的家庭被转交给该三级护理机构，以解决严重疾病。从320个这样的家庭中，有60多个映射到β肌球蛋白基因。 突变簇在四个区域中，表明肌动菌素跨桥动力学中的不同类型的干扰是突变位置的函数。随后由Seidnman组对更多患者进行了更多患者的大规模测序，已经填补了这些簇之间的区域。 但是，我们研究的患者患有严重疾病，这也许就是为什么聚类如此明显的原因。 我们已经研究了表达这些肌球蛋白的提取的突变肌蛋白和肌肉纤维的机械性能，以分析分子水平的病理生理学。突变的簇之一使我们发现了肌球蛋白光链中突变的发现，这些突变引起了HCM的变体，其特征是左心室中间有阻塞。通过一系列参数，肌球蛋白轻链突变与HCM罕见的亚型的关联导致我们假设“拉伸激活响应”对正常心脏功能的重要性。果蝇飞行肌肉中的拉伸激活反应先前已被“调节”肌球蛋白轻链（RLC）突变扭曲，从而导致无飞的苍蝇的翅膀无法正常跳动。我们已经从心脏肥大的患者中开发出了表达突变肌球蛋白“必需”轻链（ELC）的转基因小鼠。这些老鼠的心也无法正常跳动。也就是说，最大功率输出的频率转移到了生理范围之外的速率，随之而来的振荡能力损失。我们已经克隆了一种人类酶，该酶磷酸化RLC并鉴定出一个小家族中的遗传突变。与正常酶相比，该突变酶已经表达并显示出最大速度的增加。我们已经对用这种克隆酶处理的分离的肌肉纤维进行了机械研究，并证明了拉伸激活反应的变化。我们继续使用正常小鼠和转基因小鼠的肌肉纤维来研究肌肉纤维对轻链磷酸化的基本生物物理反应。我们最近的温度依赖研究研究中的一种观察结果评估了快速和慢肌纤维的跨桥动力学，这产生了一个诱人的假设。也就是说，心肌纤维可能能够反向驱动跨桥动力学，并产生相当于新的ATP。这是基于对产生快速肌肉肌球蛋白的力状态的Q10的观察，而心脏肌球蛋白的相应Q10为20。这种差异表明，在快速肌球蛋白中产生孤立的力，但在心脏肌球蛋白中产生步骤的背部连接。如果正确定时振荡心脏中的弹性力，则通过收缩部分伸展的心脏的一部分可以节省能量，然后以等于新能量产生的净能量节省产生热量。我们目前正在开发单分子仪器，该仪器将允许直接检验该假设。 我们已经出版了一份手稿（Biophys J. 2007年4月15日； 92（8）：2865-74。Epub2007年1月26日），利用了我们在基本的单肌纤维研究中进行的观察。 在极少数情况下，张力与快速骨骼肌纤维的温度是sigmoidal，具有拐点。因此，张力产生可以表示为简单的2个状态反应。 这首先允许计算肌球蛋白力产生的后和正反应速率以及反应的焓和熵。 向前和向后的反应分别是狂热和反寄生的，这表明力产生涉及局部展开，而后退反应受到熵屏障（重折叠）的抑制。 这些发现与Houdusse实验室出版的肌球蛋白VI的先前晶体学发现是一致的。 我们2009年在PNA中的出版物比较了从温度诱导的肌球蛋白力产生（无运动）与运动引起的力产生的指数。 这突出了释放无机磷酸盐但尚未循环在强附着阶段的越野桥的集合来缓冲张力。 这两篇论文都增加了我们对肌肉收缩的基本理解。最近，对我们以前的遗传筛查的综述已经确定了新的心脏激酶中发生的自然突变，该突变磷酸化RLC和肌球蛋白结合蛋白-C。 这种突变会导致患者严重疾病。 敲入小鼠的载体已经构建，现在在插入干细胞之前对小鼠产生进行测序。 该激酶的野生型和突变形式正在杆状病毒系统中表达，与我们先前表达的MLCK-2一起使用。 像我们之前所做的那样，将在单纤维研究中进行此类研究。