Beta-arrestin signaling and microRNA biogenesis in cardioprotection

描述（由申请人提供）：心力衰竭（HF）是影响500多万美国人的主要公共卫生问题。HF的总医疗费用每年超过300亿美元，并且预计随着人口老龄化而增加。因此，迫切需要开发用于预防和治疗HF的创新策略。<$1-肾上腺素能受体（<$1AR，一种七跨膜G蛋白偶联受体）信号传导对心脏功能的交感神经调节至关重要，但在HF进展期间响应于慢性儿茶酚胺刺激而变得有害。最近，人们认识到，<$1AR信号转导涉及多个途径，包括一个新的心脏保护信号转导（CPS）途径，其中<$1AR使用<$-抑制蛋白（<$-arrestin）在G蛋白激活的情况下促进心肌细胞（CM）的存活。然而，<$arr介导的<$1AR信号传导赋予心脏保护作用的下游核过程中的分子机制还不清楚。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可能代表了调节心血管介导的心脏保护作用的未被充分认识的下游机制。我们的初步数据显示，<$1AR介导的<$-arr 1激活增强了miR加工和几种已知调节细胞存活和凋亡途径的miR的表达。然而，增加的<$-arr 1调节的miR（<$1-miR）对心脏功能和组织损伤的结果的重要性是未知的，并且是当前研究的目标。本申请的目的是确定Δ 1-miR、miR-150及其两个CM靶基因（促凋亡egr 2和p2 x7 r）在Δ 1 AR介导的Δ-arrestin 1 CPS中的重要性。核心假设是，<$-arr 1介导的<$1AR调节机制通过miR-150加工和两个靶基因的表达改变，赋予心脏保护作用，以对抗心肌梗死（MI）和慢性儿茶酚胺刺激。在广泛的初步数据的指导下，我们的假设将通过追求三个特定的目标进行测试：1）确定miR-150是否调节冠状动脉结扎和慢性儿茶酚胺刺激的心脏功能反应。将在小鼠中采用响应于MI和慢性儿茶酚胺治疗的体内功能丧失和获得方法。2)确定miR-150表达的增加是否有助于卡维地洛（一种有偏倚的受体阻滞剂）介导的心脏保护作用。遗传和药理学方法将用于CM和全心脏。3)阐明egr 2和p2 x7 r（miR- 150的功能性CM靶点）在HF中的作用。将对靶点进行功能丧失和获得研究，以显示其在CM生存和心脏保护中的重要性。这项工作具有创新性，因为从未研究过通过miR-1信号调节miR表达。我们将通过<$-arrestin 1-biased <$1AR信号通路发现miR加工和功能的新调控机制，并确定参与CPS的潜在重要miR-靶对。所提出的研究是重要的，因为<$1AR的<$-arr 1偏向激动调节心脏保护性miR/促凋亡靶对的能力为HF的药理学操作提供了希望。最终，这些知识有可能为预防和治疗各种心脏疾病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.