Roles for ER Stress Inducible Cardiomyokines in the heart

描述（由申请人提供）： 我们的长期目标是确定ER应激在心脏中的作用。使用基因组学方法，我们确定了一组基因诱导的ATF 6分支的ER应激反应（ERSR），编码蛋白质预测有帕拉/自分泌作用的心脏。这些蛋白质，我们称之为ER应激诱导型心肌因子（ERS-CMK），是独特的，因为它们在应激（例如缺血）期间在心脏中合成和分泌，这损害了大多数其他蛋白质的合成和释放。这项建议的重点是ERS-CMK，中脑星形胶质细胞源性神经营养因子（MANF），这是新的，因为它的功能内和细胞外影响心脏保护。在该提案中，心肌因子（CMK）被定义为由心脏分泌的蛋白质，其可以部分地通过直接结合心脏细胞以及结合驻留的和可能非心脏来源的干细胞来影响其功能而发挥帕拉/自分泌效应。大多数CMK在粗糙的ER中合成和折叠，路由到高尔基体，然后在分泌之前到分泌囊泡。在不存在ER应激的情况下，条件对于功能性CMK的表达、折叠以及因此分泌是最佳的。然而，一些应激损害ER中的蛋白质折叠，激活ER应激，其降低CMK转录、翻译、折叠和分泌，从而导致CMK功能丧失。ER应激导致ATF 6的激活，ATF 6是ER应激的节点传感器，其增加许多已知ERSR基因的转录，所述ERSR基因编码直接增强ER蛋白折叠的ER靶向蛋白。我们发现，在缺血期间被激活的ERSR的ATF 6分支在离体和体内缺血和再灌注期间保护心脏免受损伤。对小鼠心脏的微阵列分析揭示了许多编码预测为ER靶向和分泌的蛋白质（ERS-CMKs）的ATF 6诱导基因。一种ERS-CMK，MANF，是不寻常的，因为它是保留或分泌，这取决于压力。MANF的过表达或向培养基中添加重组MANF（rMANF）可以保护心肌细胞，而MANF敲低则增加了模拟I和I/R介导的细胞死亡。假设：该提案中提出的具体假设是，已知激活ER应激的缺血以ATF 6依赖性方式诱导ERS-CMK、MANF，并在细胞内和细胞外起作用以保护心脏免受缺血性损伤。解决这一假设的具体目标是：1。检查在经受ATF 6功能获得和丧失的缺血小鼠心脏中MANF的表达，2.确定体内缺血性心脏中MANF功能获得和丧失的影响，以及3.剖析了心肌细胞内和细胞外MANF的功能，描述了MANF活性所需的结构特征，并确定了心肌细胞中MANF功能的信号传导机制。 公共卫生相关性： 心肌缺血通常会导致心力衰竭，目前诊断为心血管疾病的500多万美国人中有50%将在5年内死亡。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。本申请提出了这样的探索，并将提供新的信息，在ERSR过程中诱导的新基因，这可能形成开发新的治疗缺血性心脏病的基础。

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.