Role of the cardiac cytoskeleton in mRNA localization and hypertrophy

心脏细胞骨架在 mRNA 定位和肥大中的作用

• 批准号: 10230913
• 负责人: Emily A Scarborough
• 金额: $ 6.56万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10230913
• 关键词: Role; cardiac; cytoskeleton; mRNA; localization

PROJECT SUMMARY In the adult heart, remodeling in response to changing hemodynamic demands occurs primarily through hypertrophy, the addition of new sarcomeres to individual cardiomyocytes. Physiological hypertrophy, triggered by pregnancy or exercise, maintains normal organization of cardiac structure and can improve cardiac function. Pathological hypertrophy, however, often precedes heart failure. This can induce drastic changes to the cardiomyocyte cytoskeleton. However, with the exception of cellular mechanics, the role of the cardiomyocyte cytoskeleton in both health and disease has remained understudied. Recent data suggest that both the actinomyosin and microtubule network may play a role in mRNA and ribosomal localization in cardiomyocytes, though the mechanism remains unknown. In non-muscle cell types, actin-based and microtubule-based directed mRNA transport is well characterized. Additionally, myosin and other sarcomeric mRNAs, ribosomes, and protein degradation machinery appear to localize to the sarcomere in the cardiomyocyte, supporting a model of local translation for sarcomere maintenance and/or de novo formation. But how these new sarcomeres are formed remains particularly unclear, and the dependence of hypertrophy on proper mRNA transport and localization is unknown. The goal of my research proposal is to determine the relationship between mRNA localization and cardiac hypertrophy both in health and disease. My preliminary data establishes that the microtubule network is essential for mRNA localization in the rat cardiomyocyte in vivo and in vitro. To determine the specific mechanism of mRNA transport, I will utilize pharmacological reagents to destabilize the actin network and specifically inhibit motor proteins in isolated adult rat cardiomyocytes to test if mRNA localization changes. I will also use the MS2-MCP system to live track single mRNA transcripts to unambiguously define the mode of mRNA transport. I will leverage molecular biological tools to mislocalize specific sarcomeric transcripts and to visualize sites of new sarcomere deposition in growth-responsive neonatal rat cardiomyocytes to test if mRNA sublocalization is required for cardiac hypertrophy. Finally, I will evaluate if hypertrophy disrupts proper mRNA localization, and if so, dissect the relative contribution of pathophysiological microtubule network changes to this mislocalization using both in vitro and in vivo models of hypertrophy. Taken together, the proposed research will definitively establish the mechanism of mRNA transport and localization in the cardiomyocyte and its function in cardiac hypertrophy.

项目概要 在成人心脏中，响应变化的血流动力学需求而进行的重塑主要通过 肥大，向单个心肌细胞添加新的肌节。生理性肥大，引发 通过怀孕或运动，维持心脏组织的正常结构，并能改善心脏功能。 然而，病理性肥大通常先于心力衰竭。这可能会引起巨大的变化 心肌细胞的细胞骨架。然而，除了细胞力学之外，心肌细胞的作用 健康和疾病中的细胞骨架仍未得到充分研究。 最近的数据表明，放线肌球蛋白和微管网络可能在 mRNA 和 心肌细胞中的核糖体定位，但其机制仍不清楚。在非肌肉细胞类型中， 基于肌动蛋白和基于微管的定向 mRNA 运输已得到很好的表征。此外，肌球蛋白和 其他肌节 mRNA、核糖体和蛋白质降解机制似乎定位于肌节 在心肌细胞中，支持肌节维持和/或从头的局部翻译模型 形成。但这些新肌节是如何形成的仍不清楚，而且它们的依赖性 肥大对正确 mRNA 运输和定位的影响尚不清楚。我的研究计划的目标是 确定健康和疾病状态下 mRNA 定位与心脏肥大之间的关系。我的 初步数据表明微管网络对于大鼠 mRNA 定位至关重要 体内和体外的心肌细胞。为了确定 mRNA 转运的具体机制，我将利用 药理学试剂可破坏肌动蛋白网络的稳定性并特异性抑制分离的运动蛋白 成年大鼠心肌细胞测试 mRNA 定位是否发生变化。我也会使用MS2-MCP系统进行现场跟踪 单个 mRNA 转录本明确定义 mRNA 运输模式。我将利用分子 生物工具可对特定肌节转录本进行错误定位并可视化新肌节沉积位点 在生长反应性新生大鼠心肌细胞中测试心脏是否需要 mRNA 亚定位 肥大。最后，我将评估肥大是否破坏了正确的 mRNA 定位，如果是，则剖析 病理生理微管网络变化对这种错误定位的相对贡献 肥大的体外和体内模型。总而言之，拟议的研究将最终确定 心肌细胞中mRNA转运和定位的机制及其在心脏肥大中的作用。