"Connexin-Dependent Transcriptomic Networks in Controlling the Heart Rhythm"

描述（由申请人提供）：大量文献报告了主要间隙连接蛋白（连接蛋白，Cx）的表达改变和/或突变，该蛋白偶联心室（Cx43）和心房（Cx40）中的肌细胞，是心律失常的主要原因。我们已经发现，控制大量功能途径的许多基因在Cx43无效组织中受到调控，并且这些调控可以通过与野生型中的Cx43基因（Gja 1）的协调来准确预测。这些发现表明，这种疾病的另一个病因是与连接蛋白有关，但不一定与它们提供的细胞间偶联有关。我们的工作假设是，编码心脏节律决定因素（HRD）的基因是相互连接的连接蛋白依赖和连接蛋白独立的转录组网络，其拓扑结构可能会改变在发展过程中，表现出轻微的差异，两种性别之间。这种调控网络，其中连锁伙伴重新排列和强度修改的疾病，可以解释下游和平行的“涟漪”的表型变化。我们计划验证这一假设，并建立和表征心房和心室的基因编码的心脏节律决定因素的网络。此外，我们将确定和量化这些网络中的连接蛋白依赖的调控网络。特别感兴趣的是鉴定具有惊人相似或相反的配位特征的基因对，因为相似基因的上调或相反基因的下调预期会补偿对应基因的表达缺陷（如“转录组学跷跷板”）。为此，我们将在E19、1、2和4周的早期生命中分析野生型、Cx40缺失和Cx43条件性敲低雄性和雌性小鼠的心房和心室转录组。表达数据也将用于确定所有可量化的单基因的表达变异性和相互协调性，并研究HRD基因网的年龄和性别依赖性。我们已经发展了主基因分析，通过它来确定心脏节律决定因素（HRD），建立和表征其编码基因的网络。将通过比较培养的心肌细胞的转录组来测试“跷跷板”模型，其中Cx43或某些阳性“跷跷板”配偶体的表达通过siRNA处理被敲低。因此，这项研究有望揭示心脏转录组的新组织原则和间隙连接基因在该组织中的作用，长期目标是在心律失常的治疗中开辟新的治疗视野。公共卫生相关性：心肌细胞之间的缝隙连接提供了细胞间电流的通道，确保了整个心脏的收缩传播。越来越多的证据将许多心律失常归因于间隙连接及其组成蛋白的改变。我们假设间隙连接基因表达的这种作用部分是由于与影响心律的其他基因表达的联系，我们建议使用微阵列的基因表达谱来实现对心律失常的全面了解，并生成可通过更集中的方法进行检验的假设。我们计划分析雄性和雌性野生型小鼠和缺乏主要心脏间隙连接蛋白的小鼠在进化到成年状态期间的四个时间点的心房和心室中的基因表达，以在全基因组范围内鉴定和定量心律失常的连接蛋白相关转录组决定因素，分析其性别依赖性和成熟。一个主要的贡献将包括在确定候选基因的操作可能恢复正常的心脏节律的基础上，他们的相似或相反的表达协调与心脏连接蛋白在采样的转录组和其他基因的改变产生心律失常。该研究将揭示心脏转录组的新组织原理，长期目标是在心律失常的治疗中开辟新的治疗视野。我们的项目具有描述和量化心脏节律的Cx40和Cx43依赖性基因调控网络的独特功能，其长期目标是在心脏病学中开辟新的治疗视野。

DESCRIPTION (provided by applicant): Extensive literature has reported altered expression and/or mutation of the primary gap junction proteins (connexins, Cx) that couple myocytes in ventricles (Cx43) and atria (Cx40) among major causes of cardiac arrhythmias. We have found that numerous genes controlling a vast number of functional pathways are regulated in Cx43 null tissues and these regulations are accurately predicted from coordination with Cx43 gene (Gja1) in wildtypes. These findings suggest an additional etiology of the disease that is related to connexins but not necessarily to the intercellular coupling they provide. Our working hypothesis is that the genes encoding the heart rhythm determinants (HRD) are interconnected in connexin-dependent and connexin-independent transcriptomic networks whose topologies may change during development and exhibit slight differences between the two genders. Such regulatory networks, where linkage partners are rearranged and strength modified in disease, may explain downstream and parallel "ripples" of phenotypic change. We plan to verify this hypothesis, and build and characterize the atrial and ventricular webs of genes encoding hear rhythm determinants. In addition, we shall identify and quantify the connexin-dependent regulatory networks within these webs. Of particular interest will be to identify the gene pairs with strikingly similar or opposite coordination profiles because up-regulation of a similar one or down-regulation of an opposed are expected to compensate for the deficient expression of the counterpart (as "transcriptomic see-saws"). For these, we shall profile the atrial and ventricular transcriptomes of wildtype, Cx40 null and Cx43 conditional knockdown male and female mice at E19, 1, 2 and 4 weeks of their early life. The expression data will be also used to determine the expression variability and intercoordination of all quantifiable unigenes and study the age and gender dependence of the HRD gene webs. We have developed the Principal Gene Analysis by which to identify the heart rhythm determinants (HRD), build and characterize the webs of their encoding genes. The "see-saw" model will be tested by comparing the transcriptomes of cultured cardiomyocytes in which expression of either Cx43 or of certain positive "see-saw" partners are knocked-down through siRNA treatment. Thus, this study is expected to reveal new organizational principles of the heart transcriptome and the role of gap junction genes in this organization, with the long-term goal to open novel therapeutic horizons in the treatment of arrhythmia. PUBLIC HEALTH RELEVANCE: Gap junctions between cardiac muscle cells provide the channels for intercellular current flow that assures propagation of contraction throughout the heart. Increasing evidence attributes numerous cardiac arrhythmias to altered gap junctions and the proteins of which they are composed. We hypothesize that this role of gap junction gene expression is due in part to linkage to expression of other genes that affect the cardiac rhythm, and we propose to use gene expression profiling from microarrays to achieve a comprehensive understanding of arrhythmia and to generate hypotheses testable by more focused methods. We plan to profile the gene expressions in atria and ventricles of male and female wildtype mice and mice lacking the main cardiac gap junction proteins at four time- points during evolution to the adult state to identify and quantify on a genome-wide scale the connexin- related transcriptomic determinants of arrhythmia, analyzing their gender dependence and maturation. A major contribution will consist in identifying candidate genes whose manipulation might restore the normal cardiac rhythm based on their similar or opposed expression coordination with cardiac connexins in the sampled transcriptome and to that of other genes whose alteration generates arrhythmia. The study will reveal new organizational principles of the heart transcriptome, with the long- term goal to open novel therapeutic horizons in the treatment of arrhythmia. Our project has the unique feature of describing and quantifying the Cx40- and Cx43-dependent Gene Regulatory Networks of the heart rhythm, with the long term goal to open novel therapeutic horizons in cardiology.