Atomic force microscopy as a method to study the responses of mechanosensitive Piezo1 channels of cardiac fibroblasts
DOI:
https://doi.org/10.33910/2687-1270-2024-5-1-50-59Keywords:
fibroblasts, organotypic tissue culture method, Piezo1 channels, Jedi2, atomic force microscopyAbstract
Jedi2, an activator of mechanosensitive Piezo1 channels, was found to affect the growth of embryonic heart tissue explants. The dependence of the change in the area index on the Jedi2 concentration is described by the Hill equation (Kd ≈ 20 μM, Hill coefficient — 1.6). A concentration of 10 μM was chosen to chemically activate mechanosensitive Piezo1 channels in the atomic force microscopy (AFM) study since it did not impact the growth of heart explants. Based on the obtained stimulus–response relationship, a force of 3 nN was chosen for mechanical action from the AFM probe when studying the effect of Jedi2 on fibroblasts, which does not lead to a change in cell stiffness in response to mechanical stimulation. In contrast to low forces (1–5 nN), high stimulation forces (6–7 nN) were associated with a sharp increase in the Young’s modulus of fibroblasts. An AFM study showed that Jedi2 augments fibroblast stiffness: the Young’s modulus of cells after exposure to the test substance (68 ± 7 kPa, n = 33) increases compared to the control (37 ± 4 kPa, n = 29). The effect of Jedi2 becomes stronger with time, with the maximum effect on fibroblast mechanical characteristics achieved after more than two hours of exposure to the substance. We assume that the increase in fibroblast stiffness observed under the influence of Jedi2 and at a stimulation force of 3 nN is associated with a shift in the threshold for triggering the cell response towards lower forces caused by the modulation of the Piezo1 channels.
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