Cardiac Sarcomere Protein Quality Control in Health and Disease

健康和疾病中的心脏肌节蛋白质量控制

• 批准号: 10445976
• 负责人: JONATHAN A KIRK
• 金额: $ 53.92万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445976
• 关键词: Actins; Address; Autophagosome; BAG3 gene; Binding; Biological Assay; Biophysics; Cardiac; Cardiac Myocytes; Cells; Client; Complex; Data; Depressed mood; Disease; Foundations; Functional disorder; Future; Generations; Grant; Half-Life; Health; Heart; Heart failure; Heat-Shock Proteins 70; Heat-Shock Response; Human; Impairment; In Vitro; Knockout Mice; Knowledge; Lasers; Lysosomes; Mechanical Stress; Mediating; Mental Depression; Methods; Modeling; Modification; Molecular Chaperones; Mus; Muscle Cells; Myocardial Infarction; Myosin ATPase; Nature; Neonatal; Pathway interactions; Patients; Phenotype; Process; Proteins; Publishing; Quality Control; Rattus; Regulation; Resolution; Role; Sarcomeres; Sedimentation process; Signal Transduction; Stimulus; Stress; Structure; System; Therapeutic; Transgenic Mice; Tropomyosin; Ubiquitin; Ubiquitination; Ventricular; Work; cell motility; cost; gene therapy; improved; in vivo; induced pluripotent stem cell; live cell imaging; misfolded protein; new therapeutic target; protein degradation; protein protein interaction; proteotoxicity; recruit; response; spatiotemporal; ubiquitin ligase

Cardiomyocytes are essentially non-renewing, so proteotoxicity from dysregulated Protein Quality Control (PQC) is intimately associated with heart failure. The proteins that comprise the cardiac sarcomere are responsible for force generation in the myocyte, and this constant mechanical stress uniquely predisposes them to misfolding. Despite PQC’s central role in heart failure, and the particular vulnerability of the sarcomere to misfolding, the PQC mechanisms that maintain the cardiac sarcomere are almost entirely unknown. This is a critical knowledge gap that we will address in this proposal. Our past work described a z-disc localized complex, anchored by the co- chaperone Bcl2-associated athanogene-3 (BAG3), that was essential for sarcomere PQC. Z-disc BAG3 levels were depressed in human heart failure (HF) and correlated with decreased sarcomeric force-generating capacity (Fmax). Similarly, cardio-myocyte specific inducible BAG3 KO mice had increased ubiquitination of sarcomeric proteins that remained integrated in the lattice, reducing force generation. Importantly, BAG3 gene therapy in a mouse HF model reversed this phenotype, indicating the potential of targeting sarcomere PQC to improve contractile function. In this renewal we will address the central hypothesis that sarcomere PQC occurs via sarcomere-localized pathways and depression of these systems in HF due to BAG3 instability results in accumulation of ubiquitinated proteins that induce dysfunction. In Aim 1 we will Explore the spatiotemporal organization of the key steps in sarcomere PQC. We will use super-resolution live cell imaging in neonatal rat ventricular myocytes (NRVMs) and human iPSC-CMs to visualize the spatiotemporal interplay between BAG3, autophagosomes, lysosomes, the z-disc, and BAG3-clients at baseline and in response to various stress and stimuli, such as heat shock, localized laser damage, hypertrophic signaling, and depressed BAG3 levels. In Aim 2 we will identify the functional consequences of sarcomeric protein ubiquitination. We will use in vivo and in vitro approaches to modulate sarcomere protein ubiquitination and assess the impact on sarcomere function with biophysical assays (force- Ca2+ relationship, tension cost, in vitro motility assay, super-relaxed state, co-sedimentation). In Aim 3 we will discover the regulation of BAG3 in the cardiomyocyte and how it is altered in heart failure. We will use several transgenic mouse lines and a myocardial infarction induced heart failure model, to discover the interplay between HSP70, BAG3, heart failure, and sarcomere PQC. We expect to identify new methods to stabilize BAG3 in the failing heart as a possible therapeutic strategy. These 3 aims establish a foundational understanding of sarcomere PQC, functional consequences of its misregulation, and how it can be modulated in vivo.

心肌细胞基本上是非更新性的，因此蛋白质失调引起的蛋白质毒性 质量控制（PQC）与心力衰竭密切相关。组成蛋白质的 心肌肌节负责在肌细胞中产生力，并且这种恒定的机械 压力使它们容易错误折叠。尽管PQC在心力衰竭中起着核心作用， 肌节对错误折叠的特殊脆弱性，维持肌节的PQC机制， 心脏肌节几乎完全未知。这是一个关键的知识差距，我们将 在这个提案中。我们过去的工作描述了一个z盘局部化的复合体，由co- Bcl 2-associated athanogene-3（BAG 3）是肌节PQC所必需的分子伴侣。Z盘 BAG 3水平在人心力衰竭（HF）中被抑制，并与肌节减少相关。 力生成能力（Fmax）。类似地，心肌细胞特异性诱导型BAG 3 KO小鼠具有 肌节蛋白的泛素化增加，这些蛋白仍然整合在晶格中， 一代重要的是，小鼠HF模型中的BAG 3基因治疗逆转了这种表型， 表明靶向肌节PQC改善收缩功能的潜力。在这次更新中，我们 将阐述肌节PQC通过肌节定位途径发生的中心假设 由于BAG 3的不稳定性，HF中这些系统的抑制导致 引起功能障碍的泛素化蛋白质。在目标1中，我们将探索时空 组织肌节PQC的关键步骤。我们将使用超分辨率活细胞成像技术， 新生大鼠心室肌细胞（NRVM）和人iPSC-CM的时空可视化 基线时BAG 3、自噬体、溶酶体、z盘和BAG 3-客户之间的相互作用， 响应于各种应激和刺激，例如热休克、局部激光损伤、肥大 信号传导和降低的BAG 3水平。在目标2中，我们将确定 肌节蛋白泛素化。我们将使用体内和体外的方法来调节肌节 蛋白质泛素化，并评估对肌节功能的影响与生物物理测定（力， Ca 2+关系、张力成本、体外运动性测定、超松弛状态、共沉降）。在Aim中 我们将发现心肌细胞中BAG 3的调节以及它在心力衰竭中是如何改变的。 我们将使用几种转基因小鼠品系和心肌梗死诱导的心力衰竭模型， 发现HSP 70、BAG 3、心力衰竭和肌节PQC之间的相互作用。我们期望 确定稳定衰竭心脏中BAG 3的新方法作为可能的治疗策略。这3 目的是建立对肌节PQC的基本理解，其功能后果， 以及它如何在体内被调节。