Roles for ER Stress Inducible Cardiomyokines in the heart

描述(由申请人提供)：概要：我们的长期目标是确定内质网应激在心脏中的作用。利用基因组学方法，我们确定了一组由内质网应激反应(ERSR)的ATF6分支诱导的基因，这些基因编码的蛋白预计对心脏具有辅助/自分泌作用。这些蛋白质，我们称之为内质网应激诱导的心肌细胞因子(ERS-CMK)，是独一无二的，因为它们是在应激(如缺血)时在心脏合成和分泌的，这种应激会损害大多数其他蛋白质的合成和释放。这项建议的重点是ERS-CMK，即中脑星形胶质细胞衍生神经营养因子(MANF)，这是一种新的功能，因为它在细胞内和细胞外发挥作用，影响心脏保护。在这个提议中，心肌细胞因子(CMK)被定义为心脏分泌的蛋白质，可以部分地通过直接与心脏细胞结合，以及与常驻的、也许是非心脏来源的干细胞结合来影响其功能，从而发挥旁/自分泌作用。大多数CMK是在粗大的内质网中合成和折叠的，被送到高尔基体，然后在分泌之前进入分泌小泡。在没有内质网胁迫的情况下，表达、折叠和分泌功能性CMK的条件是最佳的。然而，一些应激损伤了内质网中的蛋白质折叠，激活了内质网应激，使CMK的转录、翻译、折叠和分泌减少，从而导致CMK功能的丧失。内质网应激导致ATF6的激活，ATF6是内质网应激的一个节点传感器，它增加了许多已知ERSR基因的转录，这些基因编码内质网靶向蛋白，直接增强内质网蛋白的折叠。我们发现，ERSR的ATF6分支在缺血时被激活，在体外和体内都能保护心脏免受缺血和再灌注时的损伤。对小鼠心脏的微阵列分析揭示了许多ATF6诱导的基因，这些基因编码的蛋白预计是内质网靶向和分泌的(ERS-CMK)。一种ERS-CMK，MANF是不寻常的，因为它要么被保留，要么被分泌，这取决于压力。过表达MANF或在培养液中加入重组MANF(RMANF)可保护心肌细胞，而MANF基因敲除则增加模拟I&I/R介导的细胞死亡。假设：这一建议中提出的具体假设是，已知的激活内质网应激的缺血，以ATF6依赖的方式诱导ERS-CMK，MANF，并在细胞内和细胞外发挥保护心脏免受缺血性损伤的作用。针对这一假说的具体目的是：1.检测ATF6得失对缺血小鼠心脏MANF的表达，2.在体确定MANF得失对缺血心脏的影响，3.剖析细胞内和细胞外MANF的功能，描述其活动所需的MANF的结构特征，并确定与心肌细胞MANF功能有关的信号机制。 与公共卫生相关：心肌缺血经常导致心力衰竭，心力衰竭将在5年内导致目前被诊断为这种心血管疾病的500多万美国人中的50%死亡。内质网应激反应(ERSR)是治疗缺血性心脏病的一个有吸引力的治疗靶点，但其在这一重要应用中的潜力从未被探索过。这项应用提出了这样的探索，并将提供关于ERSR过程中诱导的新基因的新信息，这可能形成开发治疗缺血性心脏病的新疗法的基础。

DESCRIPTION (provided by applicant): Synopsis: Our long-term objective is to determine the roles of ER stress in the heart. Using a genomics approach we identified a group of genes induced by the ATF6 branch of the ER stress response (ERSR) that encode proteins predicted to have para/autocrine effects on the heart. These proteins, which we call ER stress-inducible cardiomyokines (ERS-CMKs), are unique, since they are synthesized in, and secreted from the heart during stresses, e.g. ischemia, that impair synthesis and release of most other proteins. The focus of this proposal is the ERS-CMK, mesencephalic astrocyte-derived neurotrophic factor (MANF), which is novel since it functions intra- and extracellularly to affect cardioprotection. In this proposal cardiomyokines (CMKs) are defined as proteins secreted by the heart that may exert para/autocrine effects, in part, by direct binding to heart cells, as well as binding to resident and, perhaps non-cardiac-derived stem cells to affect their function. Most CMKs are synthesized and folded in the rough ER, routed to the Golgi, then to secretory vesicles before secretion. In the absence of ER stress, conditions are optimal for expression, folding and, thus, secretion of functional CMKs. However, some stresses impair protein folding in the ER, activating ER stress, which decreases CMK transcription, translation, folding and secretion, thus leading to a loss of CMK function. ER stress leads to activation of ATF6, a nodal sensor of ER stress that increases transcription of many known ERSR genes that encode ER-targeted proteins that directly augment ER-protein folding. We showed that the ATF6 branch of the ERSR, which is activated during ischemia, protects the heart from damage during ischemia and reperfusion, ex vivo and in vivo. Microarray analyses of mouse hearts revealed numerous ATF6-inducible genes that encode proteins predicted to be ER-targeted and secreted (ERS-CMKs). One ERS-CMK, MANF, is unusual, since it is either retained or secreted, depending on the stress. Overexpression of MANF, or addition of recombinant MANF (rMANF) to culture medium, protected cardiomyocytes, while MANF knock-down increased simulated I & I/R-mediated cell death. Hypothesis: The specific hypothesis addressed in this proposal is that ischemia, which is known to activate ER stress, induces the ERS-CMK, MANF, in an ATF6-dependent manner, and acts intra- and extracellularly to protect the heart from ischemic damage. The Specific Aims that address this hypothesis are to: 1. examine expression of MANF in the ischemic mouse heart subjected to ATF6 gain- and loss-of-function, 2. determine the effects MANF gain- and loss-of-function in the ischemic heart, in vivo, and 3. dissect the functions of intra- and extracellular MANF, delineate the structural features of MANF required for its activities, and identify signaling mechanisms responsible for MANF function in cardiomyocytes. PUBLIC HEALTH RELEVANCE: Myocardial ischemia often precipitates the heart failure that will kill 50% of the over 5 million Americans currently diagnosed with this cardiovascular disease within 5 years. The ER stress response (ERSR) is an attractive therapeutic target for treating ischemic heart disease yet its potential for this important application has never been explored. This application proposes such an exploration and will provide new information about novel genes induced during the ERSR, which may form the basis of developing new therapies for ischemic heart disease.