Transcriptional Regulation of Ion Channels in Heart Failure and Arrhythmias

 描述(申请人提供)：心律失常是发达国家发病率和死亡率的主要原因。通常，这些心律失常与获得性心脏病有关，特别是心力衰竭(HF)，观察到许多离子转运体和通道的调节失调。特别是，观察到去极化的INA和重新极化的ITO两者之间的严重不平衡。我们以前的工作首次支持了这样一个新的想法，即Ina和Ito的表达可能共享共同的、尚未确定的涉及Ito的辅助亚基KChIP2的调控机制。KChIP2沉默导致InA和Ito来源的多个基因同时发生mRNA降解和潜在的翻译阻断，这可能是一种microRNA活性的机制，导致Ito和INA的显著丢失。这些结果表明，KChIP2可能具有额外的核功能，作为转录因子来调节其他关键的心脏电流。事实上，在神经元组织中发现的KChIP家族的成员KChIP3，也被称为DREAM，定位于细胞核，在那里它扮演着钙离子调节转录抑制因子的角色。鉴于KChIP2与DREAM有高度的序列同源性，并且已在核中发现，人们可以假设KChIP2在心脏设置中能够发挥类似的核作用。因此，我们假设KChIP2通过一种新的涉及microRNAs的转录后机制控制去极化Ina和复极化Ito的表达。事实上，我们的初步数据显示，有证据表明KChIP2在转录上调节一组被称为miR-34s的miRNAs，我们证明了参与产生inA和ito的靶基因。然而，该通路在病理条件下的意义尚不清楚，这是本提案的中心焦点。我们推测，病变心脏(肥大和/或心力衰竭)中KChIP2的丢失在基因表达水平上与心脏兴奋性功能障碍有关。鉴于KChIP2在心力衰竭中被显著抑制，我们的具体目标是：1.明确KChIP2 miRNA依赖的调控在心脏病理中的作用。2.评价恢复KChIP2表达对心律失常易感性和心力衰竭进展的影响。3.评价Anagomir阻断miRNA对心律失常易感性和心力衰竭进展的影响。为了验证这些目的，我们将使用大鼠和豚鼠的横动脉缩窄(TAC)心力衰竭模型和犬起搏诱导的心力衰竭模型。通过病毒载体传递KChIP2或注射miRNA抗体物阻断miR-34将被用来评估对INA和Ito表达的影响以及整体心律失常的易感性和心力衰竭的进展。阐明KChIP2调控的分子基础对于准确理解心脏去极化和复极化及其与致死性室性心律失常的关系至关重要。

 DESCRIPTION (provided by applicant): Cardiac arrhythmias are a leading cause of morbidity and mortality within developed nations. Often, these arrhythmias are associated with acquired heart diseases, notably cardiac heart failure (HF), where the dysregulation of a host of ion transporters and channels is observed. Particularly, a critical imbalance of both depolarizing INa and repolarizing Ito is observed. Our previous work was the first to support the nascent idea that expression of INa and Ito may share common, yet to be identified regulatory mechanisms involving KChIP2, an accessory subunit of Ito. KChIP2 silencing produced simultaneous mRNA degradation and potential translational block of multiple genes at the source of INa and Ito, suggesting a mechanism of microRNA activity, which led to significant loss of Ito and INa. These results suggested KChIP2 may have additional nuclear functions as a transcription factor to regulate other critical cardiac currents. Indeed, a member of the KChIP family found in neuronal tissues, KChIP3, also known as DREAM, is localized to the nucleus where it acts as a Ca2+-regulated transcriptional repressor. Given KChIP2 shares a high degree of sequence homology with DREAM and that it has been found in the nucleus, one can hypothesize that KChIP2 is capable of similar nuclear roles in cardiac settings. Therefore, we hypothesize that KChIP2 controls expression of depolarizing INa and repolarizing Ito through a novel posttranscriptional mechanism involving microRNAs. Indeed, our preliminary data show evidence demonstrating KChIP2 transcriptionally regulates a family of miRNAs known as miR-34s which we demonstrate targets genes involved in generating both INa and Ito. However the significance of this pathway under pathologic conditions is unknown and is the central focus of this proposal. We hypothesize that KChIP2 loss in the diseased heart (hypertrophy and/or HF) is responsible for dysfunction of cardiac excitability at the level of gene expression. Given that KChIP2 is significantly repressed in HF, our specific aims are to: 1. Define the role of KChIP2 miRNA-dependent regulation in cardiac pathology. 2. Evaluate the influence of restored KChIP2 expression on arrhythmia susceptibility and HF progression. 3. Evaluate the influence of miRNA blockade by antagomirs on arrhythmia susceptibility and heart failure progression. To test these aims rat and guinea pig heart failure models produced by transverse aortic constriction (TAC) and the canine pacing-induced HF model will be used. Delivery of either KChIP2 through viral vectors or miR-34 blockade by injection of miRNA antagomirs will be used to assess influence over INa and Ito expression as well as overall arrhythmia susceptibility and HF progression. The delineation of the molecular basis of KChIP2 regulation is essential for an accurate understanding of cardiac depolarization and repolarization and its implications with lethal ventricular arrhythmias.