Molecular Signaling in Hypertrophic Cardiomyopathy

描述（由申请人提供）：肌节蛋白基因突变导致家族性肥厚型心肌病（HCM）、散发性HCM、儿科HCM和老年人HCM，在美国约有100万人发生。这些突变产生LVH临床特征的分子机制仍不清楚。我们已经产生了携带选择性人类突变的小鼠模型，表征了组织病理学的发展，评估了触发肥大信号传导的候选分子，并在心室肌病理性重塑的早期和晚期进行了全面的（SAGE）转录谱分析。遗传上相同的HCM小鼠的表型表征表明，对肌节蛋白基因突变的反应是复杂的，在同一心脏内的不同肌细胞中激活不同的分子途径。这些不同的细胞通路必须由不同的环境因素激活。本申请的中心假设是，不同的肌细胞群体将通过不同的表达谱特征来区分，并且这些RNA特征的定义将有助于鉴定参与指导肥大反应的每个方面的关键分子。我们以前的努力，以确定转录签名的HCM涉及使用现有的技术来评估RNA表达在整个左心室的HCM小鼠。我们最初鉴定RNA谱特征的努力受到三个技术问题的困扰：1）现有的转录谱技术不允许评估低水平表达的RNA; 2）心脏组织被视为同质细胞群; 3）即使在遗传相同的小鼠之间，对肌节蛋白基因突变的反应也有很大差异。在这里，我们提出了两种方法来克服在表征肥大反应时遇到的技术困难。首先，我们将分离特定的细胞群，其中肥大反应的特定分子标志物已被激活。例如，我们将使用其中β-肌球蛋白重链（MHC）基因启动子驱动荧光黄蛋白的标记基因来分离其中该分子肥大标记被激活的细胞。其次，我们最近开发了一种改良的RNA分析方法，我们称之为PMAGE（polony multiplex analysis of gene expression），其灵敏度比现有技术高出约100倍。我们建议定义蛋白质的作用，其表达在不同的肌细胞亚群中发生改变。具体而言，我们提出：1）分离小鼠心肌细胞群体与共享的分子响应HCM突变。2)采用高度灵敏的RNA谱分析技术PMAGE来确定小鼠心肌细胞群体中的RNA谱。3)评估信号蛋白在肌节基因突变引发的肥大途径中的作用。4)评估RNA谱并筛选人类HCM样本中的突变候选基因。

DESCRIPTION (provided by applicant): Sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (HCM), sporadic HCM, pediatric HCM and HCM of the elderly and occur in approximately 1 million people in the US. The molecular mechanisms by which these mutations produce the clinical features of LVH remain largely unknown. We have produced mouse models that carry selective human mutations, characterized the development of histopathology, assessed candidate molecules for triggering hypertrophic signaling, and performed comprehensive (SAGE) transcriptional profiling early and late in pathologic remodeling of ventricular myocardium. Phenotypic characterization of genetically identical HCM mice demonstrated that responses to sarcomere protein gene mutations are complex, activating different molecular pathways in different myocytes within the same heart. These different cellular pathways must be activated by different environmental factors. The central hypotheses of this application is that different myocyte populations will be distinguished by different expression profile signatures and that definition of these RNA signatures will help to identify key molecules that are involved in directing each facet of the hypertrophic response. Our previous efforts to identify transcriptional signatures of HCM have involved using existing techniques to assess RNA expression in the entire left ventricle of HCM mice. Our initial efforts to identify RNA profile signatures were confounded by three technical problems: 1) Existing transcriptional profiling technologies did not allow assessment of RNAs that are expressed at low levels; 2) Cardiac tissue was treated as a homogenous cell population; 3) The response to sarcomere protein gene mutations varies considerably even between genetically identical mice. Here we propose two approaches to overcome the technical difficulties encountered in characterizing the hypertrophic response. First, we will isolate specific cell populations in which a particular molecular marker of a hypertrophic response has been activated. For example, we will use a marker gene in which the (3-myosin heavy chain (MHC) gene promoter drives a fluorescent yellow protein to isolate cells in which this molecular hypertrophy marker is activated. Second, we have recently developed a modified RNA profiling method, we have termed PMAGE (polony multiplex analysis of gene expression), which provides about 100 fold more sensitivity than existing techniques. We propose to define the role of proteins whose expression is altered in different myocyte subsets. Specifically we propose to: 1) Isolate mouse myocyte populations with shared molecular responses to HCM mutations. 2) Employ a highly sensitive RNA profiling technique PMAGE to define RNA profiles in mouse myocyte populations. 3) Assess roles of signaling proteins in hypertrophic pathways triggered by sarcomere gene mutations. 4) Assess RNA profiles and screen candidate genes for mutations in human HCM samples.