Probing the viscoelastic behavior of cultured airway smooth muscle cells with atomic force microscopy: stiffening induced by contractile agonist

Abstract

Complex rheology of airway smooth muscle cells and its dynamic response during contractile stimulation involves many molecular processes, foremost of which are actomyosin cross-bridge cycling and actin polymerization. With an atomic force microscope, we tracked the spatial and temporal variations of the viscoelastic properties of cultured airway smooth muscle cells. Elasticity mapping identified stiff structural elements of the cytoskeletal network. Using a precisely positioned microscale probe, picoNewton forces and nanometer level indentation modulations were applied to cell surfaces at frequencies ranging from 0.5 to 100 Hz. The resulting elastic storage modulus (G') and dissipative modulus (G'') increased dramatically, with hysteresivity (eta = G''/G') showing a definitive decrease after stimulation with the contractile agonist 5-hydroxytryptamine. Frequency-dependent assays showed weak power-law structural damping behavior and universal scaling in support of the soft-glassy material description of cellular biophysics. Additionally, a high-frequency component of the loss modulus (attributed to cellular Newtonian viscosity) increased fourfold during the contractile process. The complex shear modulus showed a strong sensitivity to the degree of actin polymerization. Inhibitors of myosin light chain kinase activity had little effect on the stiffening response to contractile stimulation. Thus, our measurements appear to be particularly well suited for characterization of dynamic actin rheology during airway smooth muscle contraction.

FIGURE 1

(A) Typical force curve obtained from vertical scanning of a beaded AFM probe (at a rate of 0.317 Hz) such that it approaches, indents, retracts, and releases from a cell's surface. The inset is an illustration of a typical signal applied to the vertical scanner to perform indentation modulations. (B) A sample calibration of the hydrodynamic drag on a beaded AFM probe above a cell surface. The fit to the scaled spherical drag function (see text) and its extrapolation to surface indentation (⋆) are shown. The inset shows the linear dependence of the hydrodynamic drag on modulation frequency, and the purely viscous nature of this drag (H′ is the in-phase elastic component which is negligible compared to the out-of-phase viscous component, H″).

FIGURE 2

Force volume height (A, C) and elasticity (B, D) profiles of a confluent culture of rat tracheal smooth muscle cells using a constant maximum applied force of ∼0.4 nN (height color scale spans 1 μm in A and 0.3 μm in C; dark is stiff and light is soft in the elasticity maps). A and B covers an entire apical surface of a cell and portions of three neighboring cells. C and D are of the area within the square marked in A. These images reveal a stiff fibrous network that is highly nodal and spans the entire cell, although the nuclear region appears to provide a very soft background (large light region in the top left quarter of B). The higher resolution images in C and D clearly identify the nodes as well as their interconnecting fibers as the structures of mechanical rigidity within the cell.

FIGURE 3

Complex shear modulus (A and C) and hysteresivity (B and D) time-course measurements on two different cells, stimulated with the contractile agonist 5-HT (serotonin). The solid symbols in A and C are the elastic storage moduli and the open symbols are the dissipative loss moduli obtained from repetitive indentation modulation measurements (plotted without the coefficient, k₀ = k(1 − ν)/4(Rδ₀)^1/2). After an initial increase, likely a mechanosensitive response, the complex modulus reaches a relatively stable baseline before stimulation. 5-HT induces a dramatic stiffening response and reduced hysteresivity with variable kinetics. Two different modulation amplitudes were used in C and D (6.2 nm: squares for baseline and circles after 5-HT; 2.5 nm: triangles pointing up for baseline and pointing down for 5-HT) producing identical results, which indicates that cell rheology was probed in a linear deformation regime.

FIGURE 4

Example of the effects of the actin capping agent, cytochalasin-D, on the complex shear modulus and hysteresivity of an ASM cell after stimulation with 5-HT. The severe decrease in storage (G′) and loss (G″) modulus, to levels well below baseline, indicate that dynamic actin polymerization is largely responsible for maintenance of cellular rigidity in the contractile and resting states. The large increase in hysteresivity (η) characterizes the inhibition of actin polymerization as a transition toward fluidlike behavior of the cell.

FIGURE 5

Relative responses to stimulation (percent difference from baseline) calculated for each cell tested and averaged for control and MLCK inhibited conditions. Although mean values are reduced, there is no significant inhibition of the contractile responses (P > 0.05).

FIGURE 6

(A) Frequency dependence of the complex modulus of untreated ASM cells. Data are well fit by the weak power-law structural damping model with an additional Newtonian viscosity component (Eq. 2). The weak power-law behavior of the elastic modulus, spanning the entire frequency range tested, supports the hypothesis that cells behave as soft-glassy materials close to the glass transition (α = 0). (B) Frequency-dependent complex moduli under all treatment conditions, with a global fit to Eq. 2. The power-law exponent and Newtonian viscosity varied among treatments (fit parameters giving in Table 1). (C) Extrapolation of the storage moduli (G′) fits to the universal coordinate (f₀ = 10⁹ Hz and G₀ = 9.3 kPa).