Contributions of the Active and Passive Components of the Cytoskeletal Prestress to Stiffening of Airway Smooth Muscle Cells

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Abstract
Airway smooth muscle cells exhibit stiffening during contractile activation. This stiffening may be interpreted as a result of the stabilizing influence of the mechanical prestress stored within the cytoskeleton (CSK). However, in vivo, airway smooth muscle cells contract while simultaneously experiencing breathing-induced stretching. Excessive stretching of cells could cause actin–myosin crosslinks, and possibly other cytoskeletal filaments, to break, thereby leading to dissipation of the prestress and inhibition of further cell stiffening. The aim of this study is to investigate the stiffening behavior of individual human airway smooth muscle (HASM) cells exposed to a combination of substrate stretching, contractile activation and relaxation. We treated cultured HASM cells with either contractile (histamine) or relaxing (DBcAMP) pharmacological agonists and used magnetic cytometry technique to investigate the stiffening behavior of these cells during uniform substrate stretching (0–30%). Cells that were not treated, as well as those treated with histamine, exhibited increasing stiffening during stretching up to 20% of substrate strain, with additional stiffening becoming inhibited for substrate strains of 20–30%. In contrast, in cells treated with DBcAMP, stretching produced moderate but continuous stiffening with increasing substrate strain. These results indicate that both active and passive components of the prestress contribute to cell stiffening. We also observed that cells permeabilized with saponin exhibited stiffening at low levels (<10%) of substrate stretching, similar to non-permeabilized cells, but not at high levels (10–30%) of stretching, where stiffening was inhibited. These data suggest that at low levels of substrate strains the relative contributions of ion channel activation as well as actin and focal adhesion remodeling are less important for stiffening than passive distension of the CSK. Taken together, our results suggest that both the active and passive components of the cytoskeletal prestress contribute to the stiffening behavior of HASM cells under physiological conditions, but that at high levels of cellular distensions there is a possible tradeoff between these two components with the contribution from the passive component becoming increasingly more important.