Expression and function regulation of the sinoatrial ion channels

总体目标*本项目旨在基于功能基因组方法确定窦房结调节电功能的新的关键因素。这一知识可用于进一步了解窦房结功能的分子途径。*原理*窦房结(SAN)细胞由于多个离子通道及其相应的离子电流的协调激活而表现出自发的电活动。到目前为止，这些离子通道的转录和转录后分子调控及其对SAN功能的影响尚不清楚。为了应对这种复杂的调控，我们开发了一种基于系统生物学方法的跨学科策略。*目标*本项目的主要目标是寻找能够影响负责SAN电功能的离子通道表达的新的调控因子。考虑到起搏器(IF)和L型钙电流(ICAL)对窦房结自律性的重要贡献，本研究的目的将是寻找与离子通道表达相关的转录因子(TF)和微RNA(MiRNAs)来调节窦房结的活动。这些研究将利用基因组学、生物信息学、分子和细胞生物学以及电生理学技术相结合来实现，并将在老鼠和人类来源的细胞中进行。将进行以下具体目标：*实验计划*目的1.鉴定在SAN中特异表达的TF和miRNAs*将进行高通量差异表达研究，以比较和确定在小鼠SAN中唯一表达的TF和miRNAs。筛选出潜在的HCN和钙通道表达调控因子*生物信息学分析将分别预测鉴定出的TF和miRNAs是否能够分别结合DNA启动子或mRNA3‘非翻译区上的SAN离子通道基因。*目的3.确定所选择的TF和miRNAs在调节SAN离子通道表达和功能中的作用*考虑到所选择的调控因子可以与离子通道基因调控序列结合，我们还将研究它们的潜在贡献。首先，我们将研究它们对小鼠新生心肌细胞离子通道表达的影响，最后使用基因敲除策略(siRNA和antagomir)来研究它们对HiPSCs-CM离子电流调节的影响。*预期结果*本项目将有助于发现和识别新的调控SAN离子通道表达的TF和miRNAs。最终，这些发现可能有助于更好地理解控制SAN中电活动的基本机制。

Overall goal***This project aims to identify new key players in sinus node regulation of the electrical function based on a functional genomic approach. This knowledge could be used for further advancement in the understanding of molecular pathways underlying sinus node function.******Rationale***The sinoatrial node (SAN) cells exhibit spontaneous electrical activity due to a coordinated activation of a number of ion channels and their corresponding ionic currents. So far, transcriptional and post-transcriptional molecular regulation of these ion channels and their implication on SAN function remains unknown. To deal with this complex regulation, we developed an interdisciplinary strategy based on system biology approaches.******Objectives***The main goal of this project is to identify new regulators that can influence the ion channel expression responsible for SAN electrical function. Considering the significant contribution of the pacemaker (If) and L-type calcium (ICaL) currents to the automaticity of the SAN, the objectives of this study will be to identify transcription factors (TFs) and micro-RNAs (miRNAs) associated to ion channel expression that regulate SAN activity. These studies will be realized using a combination of genomic, bioinformatics, molecular and cell biology, as well as electrophysiological techniques and will be carried out in mouse- and human-derived cells. The following specific aims will be pursued.******Experimental plan***Aim 1. Identification of TFs and miRNAs specifically expressed in SAN***High-throughput differential expression studies will be performed to compare and determine the TFs and miRNAs that are expressed exclusively in the mouse SAN. A SAN preferential expression would support a role in the SAN electrical function.******Aim 2. Identification of the TFs and miRNAs that are potential regulators of HCN and calcium channels expression***Bioinformatics analysis will predict whether the identified TFs and miRNAs are able to bind SAN ion channel genes on the DNA promoter or mRNA 3'untranslated region, respectively.******Aim 3. Determine the contribution of the selected TFs and miRNAs in the regulation of SAN ion channel expression and function***Considering that the selected regulators can bind on ion channel genes regulatory sequences, we will also examine their potential contribution. First, we will study their effects on ion channel expressions in mouse neonatal cardiomyocytes and finally on ionic current regulation in hiPSCs-CM using knockdown strategies (siRNA and antagomir).******Expected Outcomes***This project will help uncover and identify new TFs and miRNAs that regulate SAN ion channel expression. Ultimately, these findings may help better understand the fundamental mechanisms that control the electrical activity of in the SAN.