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.

项目摘要 在成人心脏中，对血流动力学需求变化的反应主要通过以下方式发生重塑： 肥大，向个体心肌细胞添加新的肌节。生理性肥大，触发 通过怀孕或运动，维持心脏结构的正常组织，并能改善心脏功能。 然而，病理性肥大往往先于心力衰竭。这可能会导致 心肌细胞骨架然而，除了细胞力学，心肌细胞的作用 细胞骨架在健康和疾病中的作用仍然研究不足。 最近的数据表明，放线菌球蛋白和微管网络都可能在mRNA和 心肌细胞中的核糖体定位，尽管机制尚不清楚。在非肌肉细胞类型中， 基于肌动蛋白和基于微管的定向mRNA转运被很好地表征。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，支持用于肌节维持和/或从头开始的局部翻译模型 阵但是这些新的肌节是如何形成的仍然特别不清楚， 肥大对适当mRNA转运和定位的影响尚不清楚。我的研究计划的目标是 确定健康和疾病中mRNA定位与心脏肥大之间的关系。我 初步数据表明，微管网络对于大鼠的mRNA定位是必不可少的 在体内和体外心肌细胞中。为了确定mRNA转运的具体机制，我将利用 药理学试剂去稳定肌动蛋白网络和特异性抑制马达蛋白在分离的 成年大鼠心肌细胞，以测试mRNA定位是否改变。我还将使用MS 2-MCP系统实时跟踪 单个mRNA转录本以明确定义mRNA转运模式。我会利用分子 用于错误定位特定肌节转录物和可视化新肌节沉积位点的生物学工具 在生长反应性的新生大鼠心肌细胞中检测mRNA亚定位是否是心脏 肥厚最后，我将评估肥大是否破坏了正常的mRNA定位，如果是的话， 病理生理学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。综上所述，拟议的研究将明确建立 mRNA在心肌细胞中的转运和定位机制及其在心肌肥大中的作用。