Roles for ER Stress Inducible Cardiomyokines in the heart

描述（由申请人提供）：简介：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质被预测对心脏有自分泌作用。这些蛋白质，我们称之为内质网应激诱导型心肌因子（ERS-CMKs），是独特的，因为它们是在心脏中合成的，并在缺血等压力下从心脏分泌，从而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，中脑星形细胞衍生的神经营养因子（MANF），它是新颖的，因为它在细胞内和细胞外都影响心脏保护。在这个建议中，心肌因子（CMKs）被定义为由心脏分泌的蛋白质，可以发挥自分泌作用，部分通过直接结合心脏细胞，以及与常驻和可能非心脏来源的干细胞结合来影响其功能。大多数CMKs在粗内质网中合成和折叠，然后进入高尔基体，然后在分泌前进入分泌囊泡。在没有内质网应激的情况下，条件是表达、折叠和分泌功能性cmk的最佳条件。然而，一些应激损害内质网中的蛋白质折叠，激活内质网应激，从而降低CMK的转录、翻译、折叠和分泌，从而导致CMK功能丧失。内质网应激导致ATF6的激活，ATF6是内质网应激的节点传感器，可增加许多已知ERSR基因的转录，这些基因编码内质网靶向蛋白，直接增加内质网蛋白折叠。我们发现ERSR的ATF6分支在缺血时被激活，保护心脏免受缺血和再灌注时的损伤，在体内和体外都是如此。小鼠心脏的微阵列分析揭示了许多atf6诱导基因，这些基因编码预测为er靶向和分泌的蛋白质（ERS-CMKs）。一种ERS-CMK，即MANF，是不寻常的，因为它是保留的还是分泌的，这取决于压力。过度表达MANF，或在培养基中加入重组MANF (rMANF)，可以保护心肌细胞，而MANF敲除会增加模拟I和I/ r介导的细胞死亡。假设：本提案提出的具体假设是缺血，已知可激活内质网应激，以atf6依赖的方式诱导ERS-CMK， MANF，并在细胞内和细胞外起作用，保护心脏免受缺血性损伤。解决这一假设的具体目标是：1。检测ATF6功能获得和功能丧失的缺血小鼠心脏中MANF的表达；2 .确定体内缺血心脏中MANF功能获得和功能丧失的影响；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，并确定心肌细胞中MANF功能的信号机制。

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.