Beta-arrestin signaling and microRNA biogenesis in cardioprotection

描述(由申请者提供)：心力衰竭(HF)是一个主要的公共健康问题，影响着500多万美国人。心力衰竭的总医疗费用每年超过300亿美元，预计随着人口老龄化而增加。因此，迫切需要制定预防和治疗心力衰竭的创新战略。1肾上腺素能受体(1AR，一种七种跨膜G蛋白偶联受体)信号对交感神经调节心功能至关重要，但在心力衰竭进展过程中，由于慢性儿茶酚胺刺激，该信号变得有害。最近，人们认识到1AR信号涉及多条途径，其中包括一条新的心肌保护信号(CPS)途径，在该途径中，1AR使用-arrestin(-ARR)在没有G蛋白激活的情况下促进心肌细胞(CM)的存活。然而，在下游的核过程中，ARR介导的1AR信号提供心脏保护的分子机制尚不清楚。基于越来越多的人认识到microRNAs(MiRs)是心血管功能的重要调节因子，我们推测miR可能代表着调节ARR介导的心脏保护的下游机制。我们的初步数据表明，1AR介导的-arr1的激活促进了miR的处理和几个已知调节细胞生存和凋亡途径的miR的表达。然而，增加的arr1调节的miRs(1-miRs)对心功能和组织损伤结局的重要性尚不清楚，也是当前研究的目标。本应用的目的是确定1-miR、miR-150及其两个CM靶基因(促凋亡的egr2和P2X7R)在1AR介导的arrestin1CPS中的重要性。中心假设是，-arr1介导的1AR调节机制通过miR-150处理和改变两个靶基因的表达，对心肌梗死(MI)和慢性儿茶酚胺刺激提供心脏保护。在大量初步数据的指导下，我们的假设将通过追求三个具体目标来检验：1)确定miR-150是否调节对冠状动脉结扎和慢性儿茶酚胺刺激的心功能反应。在活体内，对心肌梗塞和慢性儿茶酚胺治疗的反应将采用功能丧失和功能获得的方法。2)确定miR-150表达增加是否有助于卡维地洛(一种ARR偏向阻滞剂)介导的心脏保护作用。遗传和药理学方法都将用于CM和整个心脏。3)阐明miR-150的功能性CM靶点egr2和P2X7R在心力衰竭中的作用。将进行靶点功能丧失和功能恢复的研究，以显示它们在CM存活和心脏保护中的重要性。提出的工作是创新的，因为从来没有人研究过？-ARR信号对miR表达的调节。我们将通过-arrestin1偏向的1AR信号通路发现miR处理和功能的新调节机制，并识别参与CPS的潜在重要miR靶点对。提出的研究具有重要意义，因为偏向arr1的受体激动剂调节心肌保护性miR/促凋亡靶点对的能力为HF的药理学操作提供了希望。最终，这些知识有可能为各种心脏病预防和治疗的新方法的开发提供信息。

DESCRIPTION (provided by applicant): Heart failure (HF) is a major public health problem affecting more than 5 million Americans. The total healthcare costs of HF are over $30 billion annually, and are projected to increase as the population ages. Therefore, there is a critical need to develop innovative strategies for prevention and treatment of HF. ¿1-adrenergic receptor (¿1AR, a seven transmembrane G protein-coupled receptor) signaling is critical to sympathetic regulation of cardiac function, but becomes deleterious in response to chronic catecholamine stimulation during the progression of HF. More recently, it became appreciated that ¿1AR signaling involves multiple pathways including a novel cardioprotective signaling (CPS) pathway, in which ¿1AR uses ¿-arrestin (¿-arr) to promote cardiomyocyte (CM) survival in the absence of G protein activation. However, the molecular mechanisms in downstream nuclear processes by which ¿-arr-mediated ¿1AR signaling confers cardiac protection are not well understood. Based on increasing recognition of microRNAs (miRs) as important regulators of cardiovascular function, we postulated that miR may represent under-appreciated downstream mechanisms regulating the ¿-arr- mediated cardioprotection. Our preliminary data show that ¿1AR-mediated activation of ¿-arr1 enhanced miR processing and expression of several miRs that are known to regulate cell survival and apoptotic pathways. However, the importance of increased ¿-arr1-regulated miRs (¿1-miRs) to the outcome of cardiac function and tissue injury is unknown and a goal of the current study. The objective of this application is to define the importance of a ¿1-miR, miR-150 and two of its CM target genes (pro-apoptotic egr2 and p2x7r) in ¿1AR-mediated ¿-arrestin1 CPS. The central hypothesis is that ¿-arr1- mediated ¿1AR regulatory mechanisms confer cardioprotection against myocardial infarction (MI) and chronic catecholamine stimulation via miR-150 processing and altered expression of the two target genes. Guided by extensive preliminary data, our hypothesis will be tested by pursuing three specific aims: 1) Determine if miR-150 modulates cardiac functional responses to coronary ligation and chronic catecholamine stimulation. In vivo loss- and gain-of-function approaches in response to MI and chronic catecholamine treatment will be employed in mice. 2) Determine if increased expression of miR-150 contributes to carvedilol (a ¿-arr-biased ¿-blocker)-mediated cardioprotection. Both genetic and pharmacological approaches will be used in CM and whole hearts. 3) Elucidate the roles of egr2 and p2x7r, functional CM targets of miR- 150, in HF. Loss- and gain-of-function studies of targets will be performed to show their importance in CM survival and cardiac protection. The proposed work is innovative because the regulation of miR expression by ¿-arr signaling has never been studied. We will discover novel regulatory mechanisms of miR processing and function by ¿-arrestin1-biased ¿1AR signaling pathways and identify potentially important miR-target pairs that are involved in CPS. The proposed re- search is significant because the ability of ¿-arr1-biased agonism of ¿1AR to regulate cardioprotective miR/pro-apoptotic target pairs holds promise for pharmacological manipulation of HF. Ultimately, such knowledge has the potential to inform the development of novel approaches for the prevention and treatment of a variety of cardiac diseases.