Novel Mechanisms of ER Stress Regulation in Ageing and Diseased Heart

描述（由申请人提供）：在衰老过程中，心脏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）应激的增加。氧化损伤和内质网应激可通过保护性细胞内信号通路，包括内质网的未折叠蛋白反应（UPR）来预防。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到。大量的新发现导致了IRE11、PERK和ATF6的鉴定和表征，它们是维持内质网稳态的高度保守的UPR机制。关于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性，但不能诱导持续的下游内质网应激信号。IRE11下游活性的短暂性表明心肌细胞中存在一种有效的IRE11活性负调节因子。蛋白磷酸酶2C （PP2Ce）被鉴定为IRE11信号复合体的一个组成部分。PP2Ce在心脏中高度富集，并且专门针对内质网膜。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控因子。这些有趣的新发现导致了PP2Ce作为心脏内源性IRE11负调节因子的假设，并调节内质网应激反应，以应对衰老和其他病理性应激。这一假设将通过生化、细胞和全心脏研究进行验证。具体目标1：表征IRE11 PP2Ce介导的心肌细胞信号传导的功能影响。将新生大鼠心室肌细胞（NRVM）作为心脏的体外模型系统。基因操作腺病毒介导的NRVM基因转移将用于表征IRE11激活或PP2Ce灭活对基础应激和氧化应激下UPR信号传导的直接影响。测量IRE11 PP2Ce通路对心肌细胞信号传导、肥厚、形态学、基因表达和活力的影响。具体目的2：探讨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在转录/蛋白表达水平、蛋白/蛋白相互作用水平或PP2Ce磷酸酶活性水平上确定。特异性目的3：确定心脏IRE11/PP2Ce信号在体内的功能作用。为了研究IRE11和PP2Ce活性在心脏功能和衰老和压力过载病理过程中的功能作用，已经建立了几个遗传模型。本研究具有揭示心脏病和心力衰竭发病机制的新见解的巨大潜力，并为综合和多学科研究提供了全面和深入的培训。

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. 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.