Translation initiation in cardiac intercellular communication and stress-induced remodeling

总结 细胞间通讯的减少发生在几乎所有形式的心脏病中，并导致 心律失常导致心脏性猝死研究最深入的是椎间盘的组成部分， 工作的心肌细胞是连接蛋白43（Cx43）间隙连接。Cx43基因，GJA 1，编码一个多顺反子 经历内部翻译事件产生能够调节gap的截短多肽的mRNA 连接形成并在应激如局部缺血和肥大期间受到改变的调节。等 哺乳动物mRNA的蛋白质编码序列内的内部翻译只是最近才被鉴定， 关于细胞如何调节这一过程， 上游调节途径和相关蛋白质的操纵可以在治疗上被利用， 恢复患病心脏的正常细胞间通讯。本申请的目的是提供 改变翻译起始在病理性Cx43间隙连接调节中的作用的机制性见解， 重塑这项工作是有意义的，因为它将确定改变翻译启动作为一个共同的压力 心脏的反应，并通过以下方式提供对电通信调节的基本理解： 通过核糖体起始快速改变蛋白质组。我们的中心假设是， 细胞的翻译景观管理细胞间的通讯，是至关重要的维持适当的 心脏电耦合，并改变了病理性重塑过程中的综合应激反应， 心脏我们将测试我们的中心假设与以下具体目标：目的1：确定的作用， 改变间隙连接调节中的翻译起始。这一目标的工作假设是， 程序存在于翻译起始的水平，它调节从"连接“到”非连接“的过渡。 连接状态。目的2：阐明GJA 1 mRNA内部翻译的调控元件 入会仪式这一目标的工作假设是，GJA 1 mRNA具有基于序列和结构的功能。 通过募集特异性RNA结合蛋白进行内部翻译所必需的元件。目标3： 测试翻译起始的调节是否防止病理性心脏重塑。我们 为此目的的工作假设是整合的应激反应（ISR）改变了GJA 1的翻译起始， 在应激和肥厚心肌中减少GJA 1 - 20 k表达并限制缝隙连接形成。 本建议中概述的工作意义重大，因为它将（i）提供对角色的机械见解 在压力下调节间隙连接和细胞间通讯的内部翻译（ii）确定 这与肥厚和缺血性心脏的病理性重塑有何关系，以及（iii）测试是否调节 内部翻译是人类心脏病可行的治疗策略。

SUMMARY Reductions in intercellular communication occur in almost all forms of heart disease and lead to the arrhythmias of sudden cardiac death. The most intensely studied component of the intercalated disc of the working cardiomyocyte is the connexin 43 (Cx43) gap junction. The Cx43 gene, GJA1, encodes a polycistronic mRNA that undergoes internal translation events yielding truncated polypeptides capable of modulating gap junction formation and subject to altered regulation during stress such as ischemia and hypertrophy. Such internal translation within protein coding sequences of mammalian mRNAs has only recently been identified, and a substantial gap in the knowledge exists as to how this process is regulated by the cell and if manipulation of upstream regulatory pathways and associated proteins can be harnessed therapeutically to restore normal intercellular communication in diseased hearts. The objective of this application is to provide mechanistic insight into the role of altered translation initiation in pathological Cx43 gap junction regulation and remodeling. This work is significant because it will identify altered translation initiation as a common stress response in the heart and provide fundamental understanding of regulation of electrical communication through rapid alteration of the proteome by ribosomal initiation. Our central hypothesis is that dynamic regulation of the translational landscape of the cell governs intercellular communication, is vital in maintenance of proper cardiac electrical coupling, and is altered by the integrated stress response during pathological remodeling of the heart. We will test our central hypothesis with the following specific aims: AIM 1: Determine the role of altered translation initiation in gap junction regulation. The working hypothesis for this aim is that a cellular program exists at the level of translation initiation which regulates transition from a `junctional' to a `non- junctional' state. AIM 2: Delineate the regulatory elements of GJA1 mRNA critical for internal translation initiation. The working hypothesis for this aim is that GJA1 mRNA harbors sequence- and structure-based elements necessary for internal translation to occur via recruitment of specific RNA binding proteins. AIM 3: Test if modulation of translation initiation protects against pathological cardiac remodeling. Our working hypothesis for this aim is that the integrated stress response (ISR) alters translation initiation of GJA1, reducing GJA1-20k expression and limiting gap junction formation in stressed and hypertrophic myocardium. The work outlined in this proposal is significant because it will (i) provide mechanistic insight into the role of internal translation in regulation of gap junctions and intercellular communication during stress (ii) determine how this relates to pathological remodeling in hypertrophic and ischemic hearts, and (iii) test if modulation of internal translation is a viable therapeutic strategy for human heart disease.