Third party funded individual grant
Start date : 01.10.2017
End date : 30.09.2020
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 signaling 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 signaling 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 cardiomy-ocytes 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.