An Investigation of Focal Adhesion Tension During Cardiomyocyte Contraction

项目总结 心肌细胞感知大尺度机械信号以适应其结构和功能，以及 这些机械信号会导致负反馈环，从而导致心肌细胞结构的改变 最终降低了心输出量。对这种机械感觉很重要的是焦点粘连，即 将细胞骨架附着在细胞外基质上的机械敏感蛋白质复合体 (ECM)。尽管已经证明局部粘连是肌丝成熟所必需的，而且是敏感的 对于外部底物的特性，人们对在这些复合体中感受到的比力知之甚少 生理收缩周期或这种黏附张力是如何由心肌细胞收缩调节的。 此外，心肌细胞收缩张力的改变启动了适应性不良的细胞重建，但这 局灶性粘连的发生机制和参与机制尚不清楚。鉴于其他组织的灶性粘连 细胞类型已被证明在单个细胞内不均匀地分布焦点黏附张力，并且 微域和心肌细胞株存在区域异质性，了解这一点很重要 当肌原纤维收缩至细胞外基质时，心肌细胞机械感觉的空间分布。这 该提案旨在填补关于焦点粘连的作用及其与两者相互作用的知识的关键空白 收缩心肌细胞机械内稳态的肌原纤维结构和细胞外基质。上一首 由于无法测量精确的力，对收缩时局部粘连感觉的研究一直受到限制。 以时间和空间分辨的方式跨越焦点粘连。一个最近开发的工具，已经被 用于研究机械驱动电池过程的是FRET(Förster Resonant Energy Transfer)张力 传感器，我已经设计在病灶内的诱导多能干细胞中内源性表达 黏附基因纽蛋白。表达这种感受器的干细胞来源的心肌细胞的初步数据显示 肌原纤维收缩使静态细胞的局部粘连张力感增加。然而， 心肌细胞收缩过程中的空间和时间作用力尚不清楚，将来也将是未知的 在这个项目中使用尖端显微镜和图像分析技术进行了检查。重要的是，这种模式 克服了以往过度表达伪影或传感器表达不一致的限制。因此，我建议 首次量化静态和动态心肌细胞生理性粘着斑张力值 收缩。其次，我将调节心肌细胞固有的收缩能力及其与 ECM以确定心肌细胞局灶性黏附张力的内外调节。这些目标 将提供对CM收缩过程中焦点粘连张力产生的完整了解。这 了解这一点可能会为未来的研究提供信息，以开发更好的靶向疗法来维持机械 心脏病中的动态平衡。

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.