Novel Mechanisms of ER Stress Regulation in Ageing and Diseased Heart

描述（由申请人提供）：心脏在衰老过程中不断受到压力的挑战。目前的研究表明，衰老伴随着氧化和内质网（ER）应激的增加。氧化损伤和ER应激通过保护性细胞内信号传导途径（包括来自ER的未折叠蛋白反应（UPR））来防止。普遍定期审议在与衰老有关的人类疾病中发挥着重要作用，其对心脏病的贡献刚刚开始被认识。大量的新发现导致了IRE 11，PERK和ATF 6的鉴定和表征，这些高度保守的UPR机制维持ER稳态。关于IRE 11在心脏病中的具体作用的信息很少。在初步研究中，IRE 11在培养的心肌细胞中的异位表达足以诱导瞬时下游活性，但不能持续下游ER应激信号传导。IRE 11下游活性的瞬时性质表明心肌细胞中存在IRE 11活性的有效负调节剂。蛋白磷酸酶2C（PP 2Ce）被鉴定为IRE 11信号传导复合物的组分。PP 2Ce在心脏中高度富集，并且专门靶向ER膜。它具有针对IRE 11的特异性磷酸酶活性，并作为IRE 11信号传导活性的有效负调节剂发挥作用。这些有趣的新发现导致PP 2Ce作为心脏中IRE 11的内源性负调节剂发挥作用并调节ER应激反应以响应衰老和其他病理应激的假设。具体目标1：表征IRE 11 PP 2Ce介导的信号传导在心肌细胞中的功能影响。新生大鼠心室肌细胞（NRVM）将用作心脏的体外模型系统。基因操作B腺病毒介导的NRVM基因转移将用于表征基础和氧化应激下UPR信号传导中IRE 11激活或PP 2 Ce灭活IRE 11的直接影响。将测量IRE 11 PP 2Ce途径对心肌细胞信号传导、肥大、形态、基因表达和活力的影响。具体目的2：研究PP 2Ce介导的IRE 11调控的分子机制。PP 2Ce介导的反馈机制的分子基础将在转录/蛋白质表达水平、蛋白质/蛋白质相互作用水平或PP 2Ce磷酸酶活性水平上确定。具体目标3：确定心脏IRE 11/PP 2Ce信号传导在体内的功能作用。已经开发了几种遗传模型，以研究IRE 11和PP 2Ce活性对心脏功能的功能作用以及响应于衰老和压力超负荷的病理过程。这项研究具有很大的潜力，可以揭示心脏病和衰竭的疾病机制的新见解，并在综合和多学科研究中提供全面和深入的培训。 公共卫生相关性：项目叙述心脏病影响着美国超过7900万人，并且患病率随着年龄的增长而增加。由于我们对衰老和心脏病的基本机制了解有限，心脏病的治疗仍然难以捉摸。这项研究旨在揭示保护机制，从氧化应激，这伴随着老化。重要的是，这项研究还旨在确定新的治疗策略。

DESCRIPTION (provided by applicant): The heart is constantly challenged by stresses during the ageing process. Current research suggests that ageing is accompanied by an increase in oxidative and Endoplasmic (ER) stress. Oxidative damage and ER stress is prevented by protective intracellular signaling pathways including the Unfolded Protein Response (UPR) from ER. UPR plays an important role in ageing-related human diseases and its contribution to heart disease has just begun to be recognized. A surge of new discoveries has led to the identification and characterization of IRE11, PERK, and ATF6, the highly conserved UPR machinery that maintains ER homeostasis. Little information is available about the specific roles of IRE11 in heart diseases. In preliminary studies, ectopic expression of IRE11 in cultured cardiomyocytes was sufficient to induce transient downstream activity but not sustained downstream ER stress signaling. The transient nature of IRE11 downstream activity indicates the existence of a potent negative regulator for IRE11 activity in cardiomyocytes. Protein Phosphatase 2C (PP2Ce) was identified as a component of an IRE11 signaling complex. PP2Ce is highly enriched in heart and is exclusively targeted to ER membrane. It possesses specific phosphatase activity against IRE11 and functions as a potent negative regulator of IRE11 signaling activity. These interesting new findings lead to the hypothesis that PP2Ce functions as an endogenous negative regulator of IRE11 in heart and modulates ER stress response in response to ageing and other pathological stresses. This hypothesis will be tested with biochemical, cellular and whole-heart studies Specific aim 1: Characterize the functional impact of IRE11 PP2Ce mediated signaling in cardiomyocytes. Neonatal Rat Ventricular Myocytes (NRVM) will be used as an in vitro model system for heart. Genetic manipulation b adenovirus-mediated gene transfer of NRVM will be used to characterize the direct impact of IRE11 activation, or IRE11 inactivation by PP2Ce, in UPR signaling under basal and oxidative stress. The impact of IRE11 PP2Ce pathway on cardiomyocytes signaling, hypertrophy, morphology, gene expression and viability will be measured. Specific Aim 2: Investigate the molecular mechanims of PP2Ce mediated IRE11 regulation. The molecular basis of PP2Ce mediated feedback mechanism will be determined at transcription/protein expression level, protein/protein interaction level or PP2Ce phosphatase activity level. Specific Aim 3: Determine the functional role of cardiac IRE11/PP2Ce signaling in vivo. Several genetic models have been developed in order to investigate the functional role if IRE11 and PP2Ce activity on cardiac function and the pathological process in response to ageing and pressure-overload. This study has great potential to uncover novel insights of disease mechanisms for heart diseases and failure and offers a comprehensive and in-depth training in an integrated and multi-disciplinary study. PUBLIC HEALTH RELEVANCE: Project Narrative Heart disease affects more than 79 million individuals in the U.S. and increases in prevalence with age. Treatments for heart disease remain elusive due to our limited understanding of fundamental mechanisms of both ageing and heart disease. This investigation seeks to uncover mechanisms of protection from oxidative stress, which accompanies ageing. Importantly, this study also aims to identify novel therapeutic strategies.