Novel Mechanisms of ER Stress Regulation in Ageing and Diseased Heart

描述(申请人提供)：心脏在老化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(ER)压力的增加。氧化损伤和内质网应激是通过保护细胞内信号通路，包括来自内质网的未折叠蛋白反应(UPR)来防止的。普遍定期审议在与老龄化有关的人类疾病中发挥着重要作用，其对心脏病的贡献才刚刚开始得到承认。大量的新发现导致了IRE11、PERK和ATF6的鉴定和鉴定，这是一种高度保守的UPR机制，维持内质网的动态平衡。有关IRE11在心脏病中的具体作用的信息很少。在初步研究中，IRE11在培养的心肌细胞中的异位表达足以诱导短暂的下游活动，但不能维持下游的内质网应激信号。IRE11下游活性的暂时性表明，心肌细胞中存在IRE11活性的有效负调节因子。蛋白磷酸酶2C(PP2Ce)是IRE11信号复合体的组成部分。PP2Ce在心脏中高度富含，并专门针对ER膜。它具有针对IRE11的特异性磷酸酶活性，并作为IRE11信号活性的有效负调节因子。这些有趣的新发现导致了一种假设，即PP2Ce在心脏中作为IRE11的内源性负调节因子，并调节内质网应激反应，以应对衰老和其他病理应激。这一假说将通过生化、细胞和全心脏研究来验证，具体目标1：表征IRE11 PP2Ce介导的信号对心肌细胞功能的影响。新生大鼠心室肌细胞(NRVM)将作为体外心脏模型系统。通过腺病毒介导的NRVM基因转移的基因操作将被用来表征基础和氧化应激下IRE11激活或PP2Ce失活在UPR信号中的直接影响。检测IRE11 PP2Ce通路对心肌细胞信号转导、肥大、形态、基因表达和活性的影响。特异性目的2：研究PP2Ce介导IRE11调控的分子机制。PP2Ce介导的反馈机制的分子基础将从转录/蛋白质表达水平、蛋白质/蛋白质相互作用水平或PP2Ce磷酸酶活性水平来确定。具体目的3：确定心脏IRE11/PP2Ce信号在体内的功能作用。为了研究IRE11和PP2Ce活性对心脏功能的作用，以及衰老和压力超负荷反应的病理过程，已经建立了几个遗传模型。这项研究具有揭示心脏病和心力衰竭疾病机制的新见解的巨大潜力，并在综合和多学科研究中提供全面和深入的培训。

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.