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

 描述（由申请人提供）：心脏感知不断变化的机械负荷，并在逐搏的基础上调整收缩强度，以匹配负荷，从而有效地将血液泵入循环。高血压常导致心律失常和心脏病。结构蛋白的缺陷，如肌营养不良症，也可能导致心肌病。心肌细胞如何感知和响应机械力？什么分子可以作为机械传感器？是什么信号通路将机械应力转化为细胞内的生化反应？所有这些重要的问题都需要从细胞和分子水平上研究机械-化学-转导（MCT）机制。研究MCT机制的一个主要障碍是缺乏实现两个重要能力的技术：一个是在模拟心肌的3-D环境中在单细胞水平上控制机械应力;另一个是在肌细胞收缩期间拖拽特定的细胞表面机械传感器，以询问它们在MCT中的作用。然而，所有目前可用的技术都不具备这两种能力。在这个项目中，主要研究者和她的跨学科团队将联合收割机结合合成化学，肌肉力学，细胞和分子生物学，以实现两个主要目标：一个是生物工程目标，开发一个创新的“Cell-in-Gel”系统，具有上述两种能力;另一个是科学目标，使用新的工具来研究机械负荷下心肌细胞收缩过程中的MCT机制。Cell-in-Gel系统相对于现有技术（使用碳纤维或玻璃棒拉伸细胞）具有两个主要优点。(1)活心肌细胞被嵌入3D水凝胶（由交联聚合物组成的弹性基质）中，因此它们在收缩期间经历3D机械应力（纵向张力、横向压缩、剪切应力），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（例如肌营养不良聚糖、整联蛋白）拴系到凝胶基质上，以在细胞收缩期间对其施加机械应力。Cell-in-Gel系统将使科学家能够研究MCT复合物，其下游信号传导以及活心肌细胞和其他细胞类型的功能后果。我们将检验中心假设，即心肌细胞中的两种主要MCT复合物-肌营养不良蛋白-糖蛋白复合物（DGC）和纽蛋白-talin-整合素复合物（VTI）-在逐搏的基础上抑制机械应力以调节Ca 2+信号传导系统，这增强了Ca 2+瞬态和对机械负荷的收缩性，但在过度负荷下，这种相同的机制也可能导致Ca 2+失调。解决这种MCT机制对于理解心脏如何响应机械负荷以自动调节收缩力，过度负荷如何导致心脏疾病以及肌营养不良症中的DGC突变如何导致Ca 2+失调和心功能障碍至关重要。

 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.