Molecular Signaling in Hypertrophic Cardiomyopathy

描述(申请人提供)：肌节蛋白基因突变导致家族性肥厚性心肌病(HCM)、散发性肥厚性心肌病、儿童肥厚性心肌病和老年肥厚性心肌病，在美国约有100万人发生。这些突变导致LVH临床特征的分子机制在很大程度上仍不清楚。我们已经建立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的候选分子，并在心室心肌病理重塑的早期和晚期进行了全面的(SAGE)转录图谱。基因完全相同的HCM小鼠的表型特征表明，对肌节蛋白基因突变的反应是复杂的，激活了同一心脏内不同心肌细胞中的不同分子通路。这些不同的细胞通路必须被不同的环境因素激活。这一应用的中心假设是，不同的心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大反应的各个方面的关键分子。我们以前的工作是识别HCM的转录特征，包括使用现有的技术来评估HCM小鼠整个左心室的RNA表达。我们最初识别RNA图谱特征的努力受到三个技术问题的干扰：1)现有的转录图谱技术不能对低水平表达的RNA进行评估；2)心脏组织被视为同质细胞群；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在描述肥大反应时遇到的技术困难。首先，我们将分离肥大反应的特定分子标记被激活的特定细胞群体。例如，我们将使用一个标记基因，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图谱分析方法，我们称之为PMAGE(Polony Multiex Analyst Of Gene Expression)，它提供的灵敏度大约是现有技术的100倍。我们建议定义在不同的心肌细胞亚群中表达改变的蛋白质的作用。具体地说，我们建议：1)分离对HCM突变有共同分子反应的小鼠心肌细胞群体。2)使用高灵敏的RNA图谱技术PMAGE来确定小鼠心肌细胞群体中的RNA图谱。3)评估信号蛋白在肌节基因突变引发的肥大通路中的作用。4)评估人类HCM样本中的RNA图谱并筛选候选基因的突变。

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.