An Investigation of Focal Adhesion Tension During Cardiomyocyte Contraction

心肌细胞收缩过程中局部粘附张力的研究

• 批准号: 10462935
• 负责人: Abigail Nagle
• 金额: $ 4.04万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10462935
• 关键词: Adhesions; Adhesives; Adrenergic Agents; Adrenergic beta-Agonists; Area; Calcium; Cardiac; Cardiac Myocytes; Cardiac Output; Cell Adhesion; Cell physiology; Cells; Cellular Structures; Characteristics; Complex; Contracts; Cues; Cytoskeleton; Data; Dependence; Distal; Energy Transfer; Engineering; Esthesia; Extracellular Matrix; Feedback; Fluorescence Resonance Energy Transfer; Focal Adhesions; Future; Generations; Geometry; Heart Diseases; Heterogeneity; Homeostasis; Image Analysis; Individual; Investigation; Isoproterenol; Knowledge; Location; Measurement; Measures; Mechanics; Mediating; Microfilaments; Modeling; Molecular; Morphologic artifacts; Muscle Cells; Myofibrils; Pathway interactions; Pattern; Physiological; Physiology; Positioning Attribute; Property; Proteins; RGD (sequence); Regulation; Relaxation; Role; Sarcomeres; Signal Transduction; Spatial Distribution; Stress; Structure; Techniques; Thin Filament; Time; Traction; Troponin C; VCL gene; Vinculin; Work; cell type; citrate carrier; data acquisition; experience; extracellular; fighting; force sensor; induced pluripotent stem cell; induced pluripotent stem cell derived cardiomyocytes; mechanical signal; mechanotransduction; microscopic imaging; mutant; overexpression; protein complex; response; sensor; sensor technology; stem cells; targeted treatment; tool

PROJECT SUMMARY Cardiomyocytes sense macro scale mechanical cues to adapt their structure and function, and disturbances of these mechanical signals can cause a negative feedback loop that leads to changes in cardiomyocyte structure and ultimately decreased cardiac output. Important to this mechanical sensing are focal adhesions, the mechanosensitive protein complexes that attach the cell cytoskeleton to the underlying extracellular matrix (ECM). Although focal adhesions have been shown to be necessary for myofilament maturation and are sensitive to external substrate characteristics, little is known about the specific forces sensed at these complexes during the physiological contraction cycle or how this adhesive tension is regulated by cardiomyocyte contractility. Furthermore, alterations of cardiomyocyte contractile tension initiate maladaptive cell remodeling, but this mechanism and the involvement of focal adhesions are poorly understood. Given that focal adhesions of other cell types have been shown to distribute focal adhesion tension unevenly within individual cells and microdomains and that there are regional heterogeneities in cardiomyocyte strain, it is important to understand the spatial distribution of mechanosensing in cardiomyocytes as the myofibrils contract against the ECM. This proposal aims to fill the crucial gap in knowledge of the role of focal adhesions and their interactions with both the myofibril structures and the ECM for the mechanical homeostasis of contracting cardiomyocytes. Previous studies of focal adhesion sensation during contraction have been limited by the inability to measure exact force across the focal adhesions in a time and spatially resolved manner. A recently developed tool that has been used to study mechanically driven cell processes is the FRET (Förster Resonant Energy Transfer) tension sensor, which I have engineered to express endogenously in induced pluripotent stem cells within the focal adhesion gene vinculin. Preliminary data from stem cell derived cardiomyocytes that express this sensor show myofibril contraction confers an increase in global focal adhesion tension sensation in static cells. However, the spatial and temporal generation of force in cardiomyocytes during contraction is not yet known and will be examined in this project using cutting edge microscopy and image analysis techniques. Importantly, this model overcomes previous limitations of overexpression artifacts or inconsistent sensor expression. Thus, I propose to first quantify spatial and temporal physiological focal adhesion tension during static and dynamic cardiomyocyte contraction. Second, I will modulate both the inherent contractility of cardiomyocytes and their connection to the ECM to determine the internal and external regulation of focal adhesion tension in cardiomyocytes. These aims will provide a complete understanding of focal adhesion tension generation during CM contraction. This understanding may inform future studies to develop better targeted therapies to maintain mechanical homeostasis in heart disease.

项目摘要 心肌细胞感受宏观尺度的机械线索，以适应其结构和功能， 这些机械信号可以引起负反馈回路， 最终导致心输出量下降对于这种机械感测重要的是局灶性粘连， 机械敏感蛋白复合物，其将细胞骨架附着于下层细胞外基质 （ECM）。虽然局灶性粘连已被证明是必要的肌丝成熟和敏感 到外部基底特性，很少有人知道在这些复合物中感测到的特定力， 生理收缩周期或这种粘附张力如何由心肌细胞收缩性调节。 此外，心肌细胞收缩张力的改变启动了适应不良的细胞重塑，但这一改变可能导致心肌细胞收缩力的降低。 机制和参与局灶性粘连知之甚少。考虑到其他组织的局灶性粘连 细胞类型已显示在单个细胞内不均匀地分布粘着斑张力， 微结构域和心肌细胞株中存在区域异质性，重要的是要了解 当肌原纤维收缩对抗ECM时，心肌细胞中机械感测的空间分布。这 该提案旨在填补对局灶性粘连的作用及其与两者相互作用的认识方面的关键空白 肌原纤维结构和ECM用于收缩心肌细胞的机械稳态。先前 由于不能测量精确的力 以时间和空间分辨的方式穿过粘着斑。最近开发的一种工具， 用于研究机械驱动细胞过程的是FRET（福斯特共振能量转移）张力 传感器，我已经设计了内源性表达的诱导多能干细胞内的病灶 粘着斑蛋白基因来自表达这种传感器的干细胞衍生的心肌细胞的初步数据显示， 肌原纤维收缩使静态细胞的全局局灶性粘附张力感觉增加。但 在收缩期间心肌细胞中力的空间和时间产生尚不清楚， 在这个项目中使用先进的显微镜和图像分析技术进行了检查。重要的是，这种模式 克服了先前过度表达伪像或不一致的传感器表达的限制。因此，我建议 第一次量化静态和动态心肌细胞过程中的空间和时间生理性局灶性粘附张力 收缩。第二，我将调节心肌细胞固有的收缩性及其与心肌细胞的连接。 ECM来确定心肌细胞中粘着斑张力的内部和外部调节。这些目标 将提供一个完整的理解，在CM收缩过程中的焦点粘附张力的产生。这 了解可能会为未来的研究提供信息，以开发更好的靶向治疗，以维持机械 心脏病的体内平衡