Fluidization And Resolidification Of The Human Bladder Smooth Muscle Cell In Response To Transient Stretch

Cells in vivo are routinely subjected to mechanical stimuli, for instance, the human airway smooth muscle cells contract and relax at the breathing frequency of every other 6 seconds; the cells within the stomach wall and intestine are subjected to stimuli resulted from peristalsis; the endothelial cells are exposed to the shear stress resulted from blood flow; bladder smooth muscle cells contract and relax during the urine storage and emptying, and so on. Various studies show that these mechanical stimuli markedly influence the structure and the function of cells, therefore, if the in vitro experiments can accurately mimic the mechanical stimuli to which the cells in vivo are subjected, more breakthroughs could made in cell physiology and pathology.At present, the research of cell mechanics on cells in response to external forces mainly focuses on endothelial cells in response to shear stress and smooth muscle cells in response to mechanical stretch. For the latter, long-term stretch or cyclic stretch is usually used, results show it causes rapid actin polymerization and increased focal adhesion assembly, and thus the living cell stiffens and becomes more solid-like. In addition, long-term cell stretch triggers the activation of several biochemical pathways that cause cell stiffening in the course of seconds to minutes. Stretch–induced stiffening has been proposed to be a protective mechanism to preserve the structural integrity of cells and tissues. In contrast to this paradigm, some recent studies have shown that when subjected to a transient stretch, the cell acutely softens and becomes more fluid-like. Meantime, the rate of remodeling of the cytoskeleton is promptly accelerated. After cessation of the transient stretch, the cell slowly resolidifies and the rate of structural remodeling of the cytoskeleton progressively decays. This behavior has been observed at the subcellular level, at the single cell level, and at the tissue level. Fluidization constitutes an alternative mechanism for the cell to a dynamical environment. Stiffening and softening thus coexist in response to stretch, providing the cell with diverse mechanisms to maintain mechanical homeostasis.To further investigate the underlying mechanism of cell fluidization induced by transient mechanical stretch, in this study, we used human bladder smooth cells as experimental model. First, we applied a 10% transient stretch while measuring cell traction forces, elastic modulus, F-actin imaging and the F-actin/G-actin ratio; second, we pretreated the cells with a panel of signaling inhibitors which play important roles in sustained stretch to see if the fluidization would also be inhibited; finally, we applied a 10% transient compression to compare the different effects induced by transient stretch and transient compression. Our research results are as follows:?We used Optical Magnetic Twisting Cytometry to measure the elastic modulus of cells; we used Traction Force Microscopy to measure cell traction forces; we used fluorescent staining to measure F-actin intensity; and we used western blotting to measure the ratio of F-actin and G-actin. Immediately after a transient stretch, F-actin levels and cell stiffness were lower by about 50%, and traction forces were lower by about 70%, both indicative of prompt fluidization. Within 5 minutes, F-actin levels recovered completely, cell stiffness recovered by about 90%, and traction forces recovered by about 60%, all indicative of resolidification.?To investigate specificity of the fluidization response, we pretreated human bladder smooth muscle cells with a panel of signaling inhibitors whose effects on bladder smooth muscle cell physiology are well known, and then we applied a transient stretch. The result showed the extent of cell fluidization and the rate of resolidification didn't change compared with untreated cells, meaning that the fluidization response was not influenced by these signaling pathways tested.?We also applied a transient compression, and then we measured cell stiffness and F-actin levels. In contrary to a transient stretch, a transient compression couldn't change cell stiffness nor could it change F-actin levels. The result suggests that the cell can feel the difference of the external forces, only the tensile force generated by the transient stretch-unstretch disrupts and reassembles the F-actin cytoskeleton, which leads to the cell fluidization and resolidification.Taken together, these results implicate extremely rapid actin disassembly in the fluidization response, and slow actin reassembly in the resolidification response. In the bladder smooth muscle cell, the fluidization response to transient stretch occurs not through signaling pathways, but rather through release of increased tensile forces that drive acute disassociation of actin.