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电功能调节的新关键参与者。这些知识可以用于进一步了解窦房结功能的分子途径。******原理***窦房结（SAN）细胞表现出自发的电活动，这是由于许多离子通道及其相应的离子电流的协调激活。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。为了处理这种复杂的调控，我们基于系统生物学的方法开发了一种跨学科的策略。******目标***该项目的主要目标是确定可以影响负责SAN电气功能的离子通道表达的新调节器。考虑到起搏器（If）和l型钙（ICaL）电流对SAN的自动性的重要贡献，本研究的目标将是确定与调节SAN活性的离子通道表达相关的转录因子（tf）和微rna （miRNAs）。这些研究将结合基因组学、生物信息学、分子和细胞生物学以及电生理技术来实现，并将在小鼠和人类来源的细胞中进行。将实现下列具体目标。******实验方案***目的鉴定在SAN中特异性表达的tf和mirna ***将进行高通量差异表达研究，以比较和确定仅在小鼠SAN中表达的tf和mirna。SAN优先表达式将支持SAN电气功能中的角色。* * * * * *的目标2。鉴定HCN和钙离子通道表达的潜在调控因子tf和mirna ***生物信息学分析将预测鉴定的tf和mirna是否能够分别结合DNA启动子或mRNA 3'非翻译区上的SAN离子通道基因。* * * * * *的目标3。确定所选择的tf和mirna对SAN离子通道表达和功能调控的贡献***考虑到所选择的调节剂可以结合到离子通道基因调控序列上，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，最后使用敲低策略（siRNA和安塔戈米尔）研究hiPSCs-CM中离子电流的调节。******预期结果***该项目将有助于发现和鉴定调节SAN离子通道表达的新tf和mirna。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.