Reductive Stress Induces Proteotoxic Cardiac Disease

描述(由申请人提供)：蛋白毒性和蛋白病在衰竭心脏中的意义和致病后果最近已收到临床通知。在心肌细胞中，缺陷蛋白的积聚和聚集破坏了蛋白平衡，导致了病理后果。有缺陷的蛋白质的合成和降解之间的动态平衡(蛋白平衡)对动态活跃的心肌细胞至关重要。越来越多的证据表明，细胞骨架蛋白或伴侣蛋白突变引起的蛋白聚集性心肌病(PAC)大多涉及淀粉样蛋白前体聚集、氧化应激、线粒体功能障碍和心肌细胞的凋亡性死亡。我们现在证明，核红系样因子-2(NRF2)信号的结构性激活是这些致病过程中还原应激(RS)和PAC的潜在机制。我们的发现明确指出，RS是导致人类心脏病发病机制的代谢损伤。我们的长期目标是研究RS介导的蛋白毒性心脏病的分子机制，并探索相关的治疗干预措施。我们假设，细胞内还原能力的异常增加参与了RS，RS将通过受损的蛋白质质量控制机制导致蛋白毒性心脏重构和功能障碍。因此，我们提出以下目标：(1)确定慢性还原应激(CRS)是否足以导致心肌肥大和病理重构；(2)确定CRS是否损害内质网(ER)和泛素蛋白酶体功能以促进蛋白毒性和蛋白质聚集；(3)确定阻止RS或保留ER功能是否可以拯救CaNrf2-Tg小鼠免于蛋白毒性心脏重构和功能障碍。为了研究这些目标，我们通过在心脏中结构性激活Nrf2(CaNrf2)[CaNrf2-Tg或Keap1-/-：aMHC-CRE-Tg(心肌细胞特异性Nrf2信号的结构性激活)]来建立RS的小鼠模型。首先，我们将确定RS对经主动脉缩窄小鼠的心功能、结构重塑和应激性心肌肥厚的影响。接下来，我们将研究RS对患有蛋白毒性心脏病的CaNrf2-TG或Keap1-/-：aMHC-CreTG小鼠内质网氧化还原电位的影响，并探讨与内质网应激和未折叠蛋白反应通路相关的机制。然后，我们将使用药理学方法来预防RS或ER应激，以挽救蛋白毒性心脏病。或者，我们将使用遗传方法敲除Nrf2，以防止RS和由此产生的蛋白毒性。一个在心脏生理学、心脏成像、自由基化学、生物化学、分子生物学和基因治疗方面具有相关经验的科学家和医生团队将参与这个项目。该提案还包括一项强有力的计划，用尖端研究教育下一代(本科生和研究生)。这项研究的总体结果将产生关于RS和心脏蛋白毒性效应的新知识，这可能会在未来5-6年内加强对人类患者的治疗应用。

DESCRIPTION (provided by applicant): The significance and pathogenic consequences of proteotoxicity and proteinopathy in failing hearts have received recent clinical notice. Accumulation of defective proteins and their aggregation impair proteostasis and lead to pathological consequences in cardiomyocytes. A homeostatic balance (proteostasis) between synthesis and degradation of defective proteins is crucial for the dynamically active cardiomyocytes. Mounting evidence indicates that a majority of protein aggregation cardiomyopathies (PAC) caused by mutations in cytoskeletal proteins or chaperones involve pre-amyloid aggregates, oxidative stress, mitochondrial dysfunction and apoptotic death of cardiomyocytes. We now demonstrate that constitutive activation of nuclear erythroid-2 like factor-2 (Nrf2) signaling is a potential mechanism for Reductive Stress (RS) and PAC in these pathogenic processes. Our findings pinpoint RS as the metabolic insult responsible for pathogenesis in human cardiac disease. Our long-term goal is to investigate the molecular mechanisms for RS mediated proteotoxic cardiac disease and explore relevant therapeutic interventions. We hypothesize that abnormal increases in intracellular reducing power contributes to RS, which will cause proteotoxic cardiac remodeling and dysfunction through impaired protein quality control mechanisms. Accordingly, we propose the following aims: (1) To determine whether chronic reductive stress (CRS) is sufficient to cause cardiac hypertrophy and pathological remodeling, (2) To determine whether CRS impairs endoplasmic reticulum (ER) and ubiquitin- proteasome function to promote proteotoxicity and protein aggregation and (3) To determine whether preventing RS or preserving ER function rescues the CaNrf2-TG mice from proteotoxic cardiac remodeling and dysfunction. To study these aims, we have established mouse models for RS by constitutively activating Nrf2 (CaNrf2) in the heart [CaNrf2-TG or Keap1-/-:aMHC-Cre-TG (cardiomyocyte-specific constitutive activation of Nrf2 signaling)]. First, we will determine the effects of RS on cardiac function, structural remodeling and stress- induced cardiac hypertrophy in mice with trans-aortic constriction. Next, we will study the effect of RS on redox potential of ER and investigate the mechanisms associated with ER stress and unfolded protein response pathways in the CaNrf2-TG or Keap1-/-:aMHC-CreTG mice with proteotoxic cardiac disease. Then, we will use pharmacological approaches to prevent RS or ER stress to rescue the proteotoxic cardiac disease. Alternatively, we will use genetic approaches to knock down Nrf2 to prevent RS and resultant proteotoxicity. A team of scientists and physicians with relevant experience in cardiac physiology, cardiac imaging, free-radical chemistry, biochemistry, molecular biology and gene therapy will be involved in this project. The proposal also includes a strong plan for educating the next generation (undergraduates and postgraduates) with cutting-edge research. The overall outcome of this study will yield new knowledge on RS and proteotoxic effects in the heart, which will likely enhance therapeutic applications in human patients in the next 5-6 years.