Molecular Signaling in Hypertrophic Cardiomyopathy

描述（由申请人提供）：肌瘤蛋白基因突变导致家族性肥厚性心肌病（HCM）、散发性HCM、儿童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在很大程度上仍然未知。我们制作了携带选择性人类突变的小鼠模型，描述了组织病理学的发展，评估了触发肥厚信号的候选分子，并在室性心肌病理重塑的早期和晚期进行了全面的（SAGE）转录谱分析。基因相同的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被不同的环境因素激活。本应用的中心假设是，不同的肌细胞群体将通过不同的表达谱特征来区分，这些RNA特征的定义将有助于识别参与指导肥厚反应各个方面的关键分子。我们之前鉴定HCM转录特征的工作涉及使用现有技术评估HCM小鼠整个左心室的RNA表达。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)心脏组织作为同质细胞群处理；3)即使在基因相同的小鼠之间，对肌节蛋白基因突变的反应也有很大差异。在这里，我们提出两种方法来克服表征肥厚反应所遇到的技术困难。首先，我们将分离特定的细胞群，其中一个特定的分子标记的肥厚反应已被激活。例如，我们将使用一种标记基因，其中(3-肌球蛋白重链（MHC）基因启动子驱动荧光黄色蛋白来分离这种分子肥大标记被激活的细胞。其次，我们最近开发了一种改进的RNA分析方法，我们称之为PMAGE（基因表达的多聚分析），它比现有技术的灵敏度高出约100倍。我们建议定义在不同肌细胞亚群中表达改变的蛋白的作用。具体来说，我们建议：1)分离对HCM突变有共同分子反应的小鼠肌细胞群。2)采用高灵敏度的RNA分析技术PMAGE来定义小鼠肌细胞群体中的RNA谱。3)评估信号蛋白在肌瘤基因突变引发的肥厚通路中的作用。4)评估RNA谱并筛选人类HCM样本中的候选突变基因。

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.