Adding Dimension:Mechanotransduction in mammalian endothelial Cells and Cardiomyocytes exposed to passive Stretchusing a novel multidirectional isotropic Cell-Stretch Technology

细胞通过粘着斑复合物（FAC）与其周围的细胞外基质（ECM）物理锚定。ECM向细胞膜传递的外力必须被感知并转导至细胞内信号传导途径，最终调节FAC模式和强度。机械感觉包括激活阳离子选择性机械敏感通道以允许Ca 2+内流。Ca 2+进入可能导致细胞粘附的变化，FAC蛋白的重新定向和重新调整，其信号传导回路尚未明确。对于内皮细胞（EC），牵张实验表明瞬时受体电位（TRP）通道参与调节机械转导。然而，生物力学测定通常涉及线性拉伸弹性体膜上涂覆的细胞的单轴拉伸装置。更可能反映细胞在体内经历的应变的拉伸制度，即等双轴或各向同性拉伸，没有得到很好的研究，因为目前的气动系统技术对高分辨率显微镜有限制。这成为学习时的一个主要限制，例如，在共聚焦显微镜下观察心肌细胞的多轴拉伸。此外，血管组织假设的内皮-肌肉串扰尚未在心脏准备中得到证实。在我们的项目中，我们的目标是研究EC和成人心肌细胞（CM）使用单轴和新颖的各向同性拉伸制度，以研究他们的特定响应机械应力，无论是循环或静态。为此，我们将应用我们的新型IsoStretcher技术进行自动拉伸协议和图像采集。定量fura-2 Ca 2+显微镜结合药理学来剖析离子通道靶点将揭示经典TRP通道（TRPC）对EC和CM机械转导的贡献。为此，我们将使用新型孔阻断TRPC抗体。在表征孤立的EC和CM机械敏感性，Ca 2+稳态及其与钙调神经磷酸酶/CaMKK介导的FAC蛋白调节的联系后，我们将在共培养中解决EC-CM串扰问题，并评估EC释放的旁分泌因子以调节CM反应。我们的项目提供了新的见解EC-CM机械转导和细胞生物力学研究的新工具。

Cells are physically anchored with their surrounding extracellular matrix (ECM) by focal adhesion complexes (FAC). External forces transmitted by ECM towards the cell membrane must be sensed and transduced to intracellular signalling pathways, eventually regulating FAC patterns and strength. Mechanosensation includes activation of cation-selective mechanosensitive channels to allow Ca2+-influx. Ca2+-entry may lead to changes in cellular adhesion, re-orientation and re-adjustment of FAC proteins, for which the signalling loop is not well defined. For endothelial cells (ECs), stretch experiments have shown that transient receptor potential (TRP) channels are involved in regulating mechanotransduction. However, biomechanical assays usually involve uniaxial stretch devices that linearly stretch cells coated on elastomer membranes. Stretch-regimes more likely reflecting the strain experienced by cells in vivo, i.e. equi-biaxial or isotropic stretch, are not well studied, as current technologies with pneumatic systems have limitations for high resolution microscopy. This becomes a major limitation when studying, e.g., single cardiomyocytes under multiaxial stretch with confocal microscopes. In addition, an endothelial-muscular cross-talk, as postulated for vascular tissue, has not been demonstrated in cardiac preparations. In our project, we aim to study both ECs and adult cardiomyocytes (CMs) using both uniaxial and novel isotropic stretch regimes to study their specific response to mechanical stress, either cyclic or static. For this, we will apply our novel IsoStretcher technology for automated stretch protocols and image acquisition. Quantitative fura-2 Ca2+ microscopy in conjunction with pharmacology to dissect ion channel targets will reveal the contribution of canonical TRP channels (TRPCs) to EC and CM mechanotransduction. For this, we will use novel pore-blocking TRPC antibodies. After characterizing isolated EC and CM mechanosensitivity, Ca2+-homeostasis and its link to calcineurin/CaMKK-mediated FAC protein regulation, we will address EC-CM crosstalk in co-cultures and evaluate paracrine factors released by ECs to modulate CM response. Our project provides new insights into EC-CM mechanotransduction and novel tools for cellular biomechanics research.