Novel Cell-in-Gel System for Mechanotransduction Study at the Single Cell Level

 描述(申请人提供)：心脏感知变化的机械负荷，并调整收缩强度，在节拍的基础上，以匹配负荷，以有效地泵入血液循环。高血压通常会导致心律失常和心脏病。结构蛋白缺陷，如肌营养不良，也可能导致心肌病。心肌细胞如何感知机械力并对其做出反应？什么分子起机械传感器的作用？在细胞内将机械压力转化为生化反应的信号通路是什么？所有这些重要的问题都需要通过在细胞和分子水平上研究机械力-化学转导(MCT)机制来回答。MCT机制研究的一个主要障碍是缺乏实现两个重要功能的技术：一是在模拟心肌的三维环境中在单个细胞水平上控制机械应力；二是在心肌细胞收缩过程中拉动特定的细胞表面机械传感器，以探讨它们在MCT中的作用。然而，目前所有可用的技术都不具备这两种能力。在这个项目中，Pi和她的跨学科团队将结合合成化学、肌肉力学以及细胞和分子生物学来实现两个主要目标：一个是生物工程目标，开发具有上述两种能力的创新的细胞-凝胶系统；另一个是科学目标，使用新工具研究机械负荷下心肌细胞收缩的MCT机制。与现有技术(使用碳纤维或玻璃棒拉伸细胞)相比，细胞凝胶系统有两个主要优势。(1)活的心肌细胞被包埋在三维水凝胶(由交联聚合物组成的弹性基质)中，因此它们在收缩过程中会受到三维机械应力(纵向拉伸、横向压缩、剪应力)，模拟体内环境。(2)凝胶化学允许将特定的细胞表面机械传感器(如营养不良多糖、整合素)系在凝胶基质上，以在细胞收缩期间对其施加机械应力。细胞凝胶系统将使科学家能够研究MCT复合体，它们的下游信号，以及活的心肌细胞和其他类型细胞的功能后果。我们将验证这一中心假设，即心肌细胞中两个主要的MCT复合体--dystrophin糖蛋白复合体(DGC)和纽蛋白-talin-整合素复合体(VTI)--以节拍为基础传递机械应力来调节钙信号系统，从而增强机械负荷下的钙瞬变和收缩能力，但同样的机制也可以在过度负荷下导致钙调节失调。解决这一MCT机制对于了解心脏如何对机械负荷做出反应以自动调节收缩能力，过度负荷如何导致心脏病，以及肌营养不良症的DGC突变如何导致钙调节失调和心功能障碍是至关重要的。

 DESCRIPTION (provided by applicant): The heart senses the changing mechanical load and adjusts the contractile strength, on a beat-to-beat basis, to match the load in order to effectivel pump blood into circulation. High blood pressure often leads to arrhythmias and heart diseases. Defects in structural proteins, such as in muscular dystrophy, can also lead to cardiomyopathy. How do the cardiomyocytes sense and respond to mechanical forces? What molecules serve as mechanosensors? What are the signaling pathways that transduce mechanical stress to biochemical reactions in the cell? All these important questions need to be answered by investigating the mechano-chemo- transduction (MCT) mechanisms at cellular and molecular levels. A major hindrance to studying MCT mechanisms is a lack of technology to achieve two important capabilities: one is to control mechanical stress at the single cell level in 3-D environment mimicking the myocardium; the other is to tug on specific cell-surface mechanosensors during myocyte contraction in order to interrogate their role in MCT. However, all currently available techniques come short of having both capabilities. In this project, the PI and her interdisciplinary team will combine synthetic chemistry, muscle mechanics, and cellular and molecular biology to achieve two major goals: one is the bioengineering goal to develop an innovative `Cell-in-Gel' system that have the above two capabilities; the other is the scientific goal of using the new tools to investigate the MCT mechanisms during cardiomyocyte contraction under mechanical load. The Cell-in-Gel system has two major advantages over existing techniques (stretching cells using carbon fibers or glass rods). (1) Live cardiomyocytes are embedded in a 3-D hydrogel (elastic matrix composed of crosslinking polymers) so they experience 3-D mechanical stresses (longitudinal tension, transverse compression, shear stress) during contraction, mimicking the in vivo environment. (2) The gel chemistry allows tethering specific cell-surface mechanosensors (e.g. dystroglycans, integrins) to the gel matrix to impose mechanical stress on them during cell contraction. The Cell-in-Gel system will enable scientists to study MCT complexes, their downstream signaling, and functional consequences in live cardiomyocytes and other cell types. We will test the central hypothesis that two major MCT complexes in cardiomyocytes-the dystrophin-glycoprotein complex (DGC) and the vinculin-talin-integrin complex (VTI)- transduce mechanical stress to modulate the Ca2+ signaling system on a beat-to-beat basis, which enhances Ca2+ transient and contractility in response to mechanical load, but this same mechanism can also cause Ca2+ dysregulation under excessive load. Resolving this MCT mechanism is fundamental to understanding how the heart responds to mechanical load to autoregulate contractility, how excessive loads cause heart diseases, and how DGC mutations in muscular dystrophy lead to Ca2+ dysregulation and cardiac dysfunction.