Biochemical and Biophysical Research Communications
Rheological responses of cardiac fibroblasts to mechanical stretch
Highlights
► Microrheology was used to characterize cardiac fibroblast mechanical properties. ► Fibroblasts exhibited receptor-dependent changes in properties. ► Actin disruption softened the cells. ► Stretching reinforced the cells, even with actin disruption.
Introduction
Characterizing the physical properties of biological cells is crucial for understanding and modeling cellular and tissue response to mechanical stimuli. Since cells can be considered glass-like materials, rheological analysis characterizes cells based on their fluid- and solid-like characteristics. Most work on rheological characterization of cells uses external probes, such as AFM [1], [2], [3], magnetic twisting [4], [5], [6] or micromanipulation [7], [8], [9], [10], [11], or optical stretching [12], [13]. These techniques have various advantages, but the results can be overrepresentative of the cortical cytoskeleton and cell membrane and not of the cell interior. These methods also suffer the disadvantage that interpretation of mechanoresponses can be ambiguous because these techniques invariably impose mechanical stresses to the cells to measure their deformation.
Particle-tracking microrheology (PTM) is a technique developed to characterize mechanical properties of cell interiors, based on tracking small objects, such as beads. PTM yields the bead mean-squared displacement (MSD) over a range of lag times, and by fitting an appropriate model to the MSD, the complex modulus can be determined [14], [15], [16], [17], [18], [19], [20]. PTM is advantageous because after the beads are introduced, there are no further mechanical stimuli necessary for readout; thus this technique is ideally suited for examining the changes in the properties of living cells in response to mechanical stimuli.
PTM has been used in a variety of cell studies, including characterization of nuclear connections by the cytoskeleton [21], determination of prestress development [22], measurements of responses to cell–cell adhesion [23], characterization of 3D-matrix-embedded cells [14], and distinguishing primary from stem cells [24]. However, despite the importance of cell properties in response to physical forces, few studies have used PTM to characterize such responses. One study determined that cytoplasmic stiffening occurs in response to fluid shear in 3T3 cells [25], and another study established that alveolar epithelial cells exhibit diminished stiffness when stretched [26]. Many stretch sensitive cells, such as cardiac fibroblasts, have not been well-examined using PTM. While the molecular mechanotransduction of these cells is more extensively studied [27], [28], [29], the physical properties of the cells themselves are still not well-characterized. Cardiac fibroblast mechanotransduction is crucial for regulating heart properties, especially in remodeling in response to cardiovascular diseases. Thus, it is essential to understand how cell properties may be influenced by external perturbations.
Because actin is essential for regulating cellular rheological properties, we hypothesize that cardiac fibroblasts would exhibit diminished MSD in response to mechanical stretch application. Further, increases in MSD would accompany cytochalasin treatment due to disruption of the actin network. Finally, combining cytochalasin and stretch would be materially similar to cytochalasin treatment alone, given that a good deal of mechanoresponse is governed by intact actin networks. In this study, we quantitatively characterize rheological responses in primary cardiac fibroblasts to test these hypotheses and further determine whether there is receptor-dependence on such responses, since cardiac fibroblasts engage more than one type of adhesion molecule in vivo.
Section snippets
Cell culture
Cardiac fibroblasts were isolated from neonatal rat pups and maintained in high glucose Dulbecco’s Modified Eagle’s Medium (Sigma, St. Louis, MO) supplemented with 10% FBS and penicillin/streptomycin. Fibroblasts were maintained at 37 °C in a 5% CO2 environment. Cells were plated on either plastic cell culture dishes, or flexible silicone membranes, the latter coated with 5 ug/ml fibronectin (Life Technologies, Grand Island, NY) or 0.2 mg/ml collagen (Sigma). Cells were assayed at 70–80%
Effects of receptor-specific actin disruption
Incubating fibroblasts plated on cell-culture dishes in 1 μM cytochalasin D for an hour resulted in a higher bead MSD compared to that for cells incubated in DMSO, at all frequencies (p < 0.0001 at 1 Hz, Fig. 1A). G′ was zero at all analyzed frequencies for cytD-treated cells, but was 0.1 Pa (0.04, 0.13) for the DMSO-treated cells (at 1 Hz), indicating that actin disruption leads to a decrease in solid-like behavior of the cells. However, cell viscous properties was also altered by cytD treatment; G″
Discussion
The major conclusions of this study are that (1) cell rheological properties are receptor and mechanical-stimuli dependent, (2) both solid-like and fluid-like characteristics change in response to external stimuli, and (3) despite global actin disruption that clearly eliminates visible stress fibers, local cell properties at intermediate-to-long time frames are still sensitive to mechanical stimuli. Regarding the third point, the significance of these results is that mechanically conditioning
Acknowledgments
This work was supported in part by NSF CMMI-1130376.
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