1School of Biomedical Engineering, Dalhousie University, Halifax, Nova Scotia, Canada B3H 3J5; 2Physiology Program, Harvard School of Public Health, Boston, Massachusetts 02115; and 3Pediatrics Department, Case Western Reserve University, Cleveland, Ohio 44106
Submitted 2 September 2003 ; accepted in final form 2 April 2004
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ABSTRACT |
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mechanical stress; actin cytoskeleton; stiffness; airway smooth muscle cell; optical magnetic twisting cytometry; airway constriction and dilation; asthma
Our laboratory has previously reported that cultured ASM cells exposed to chronic (up to 12 days) cyclic mechanical strain exhibited increased stiffness, CSK reorganization, increased shortening capacity, and increased contractility and force production (32, 34, 36, 38). Mechanically strained cells also exhibit increased proliferation, increased velocity of shortening, and calcium sensitivity (35, 37, 38). These changes are consistent with bronchial hyperresponsiveness in vivo (6, 12, 24). Indeed, increased proliferation and altered contractile and biochemical characteristics have been observed in cells harvested from asthmatic subjects (5).
These mechanically induced changes in ASM cells have been documented for cells experiencing mechanical stimulation for 1012 days. On the other hand, it is known that CSK is a dynamic structure in a variety of cell types (11) and can respond to MS on a much shorter time scale through remodeling and typically causing increases in cell stiffness (10, 16, 17), thus approaching changes that have been observed when stimulated with contractile agonist. However, the effect of MS on ASM cells remains not well understood, especially during its acute application. Changes in CSK structure in ASM cells due to MS have only been qualitatively examined during chronically applied stress (34). Furthermore, although changes in CSK proteins induced by contractile activation have been studied (21, 26), structural changes were not examined, and it is not known how this compares with mechanically induced effects. Therefore, we investigated the response of the CSK structure in ASM cells to localized acute MS and its time course, as well as how the induced changes in CSK structure translate into altered mechanical function in terms of the CSK stiffness.
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MATERIALS AND METHODS |
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Magnetic twisting stimulator. We developed a device known as a magnetic twisting stimulator (MTS) (Fig. 1) to deliver localized MS to the CSK of cultured ASM cells by magnetically oscillating ferrimagnetic beads that were bound avidly to the CSK. Details of this technique have been published previously (8, 41). In brief, the beads were permanently magnetized and then twisted in a homogeneous magnetic field of varying amplitude and direction that was generated by a solenoid.
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The solenoid and the culture dishes were located in an incubator (Sanyo) so that the cells were maintained at 37°C in humidified air containing 5% CO2 while being mechanically stimulated. An electric fan was placed below the twisting coil to generate a moderate airflow for removing excess heat that resulted from resistive losses in the solenoid coil.
Fluorescence microscopy.
We used fluorescence microscopy to visualize the actin CSK of ASM cells of the cell-bead preparations on 12-mm glass coverslips. Filamentous actin was labeled with fluorescently conjugated phalloidin (Alexa 488; Molecular Probes, Eugene, OR). These preparations were either exposed to mechanical stimulation or used as time-matched controls. At different time points (0, 5, 15, 30, 60, and 120 min) after the initial 15 min of bead binding to the cells, the cells on the coverslip were washed with PBS and fixed in a solution containing 4% paraformaldehyde in PBS for 15 min. The cells were thoroughly washed in PBS and permeabilized with 0.3% Triton-X in PBS for 5 min. The cells were then rinsed twice and incubated in a blocking buffer (10% BSA in PBS) for 1 h at room temperature. Each of the coverslips was then submerged for 30 min in 200 µl of fluorescent phalloidin in PBS (6.6 µM) in a well of a 24-well cell culture plate. The cells were then thoroughly rinsed with PBS and mounted on a glass slide with mounting medium (Prolong Antifade kit; Molecular Probes). Fluorescently labeled actin CSK of ASM cells with surface-bound beads were examined under an epifluorescence microscope (Olympus IX70; Olympus Optical, Tokyo, Japan) and imaged by using a 1,280 x 1,024-pixel, 12-bit gray scale, and Peltier cooled monochrome charge-coupled device camera (SensiCam; Cooke, Auburn Hills, MI).
Image analysis and quantification of actin CSK remodeling.
For a given experimental condition and time point, three to six coverslips were prepared from repeated experiments. Coverslips with fluorescently labeled ASM cells were examined under the microscope at x100 magnification. On average, a field of view (0.18-mm diameter) covered one to three cells, containing approximately five attached beads in total. We examined the majority of the cells on each coverslip by scanning the field of view across the coverslip and recorded images from, on average, eight randomly chosen fields of view with no overlap where there were beads attached to the cells. For a given field of view, we examined the beads attached to the cells from below. Before taking images, we manually changed the focal plane, examining the three-dimensional structures for each bead-cell attachment. To fully capture any bead-associated actin structures for each field of view, we recorded between three and five images from different focal planes 12 µm apart.
Subsequent analysis of the recorded images revealed structures of a higher actin density on and/or near some beads, indicating changes of actin CSK structure due to bead-associated actin activity. The bead was counted as positive once actin was clearly associated with the bead in any of the recorded focal planes. We thus classified the beads with associated actin structures as positive beads against those as negative that were not associated with discernible actin structures (Fig. 2, BD). Because magnetic interference between beads can occur for beads that are clustered or that are within one bead diameter of each other, we excluded these from evaluation and examined only isolated beads, as exemplified in Fig. 2D. For each experimental condition, the total score of positive beads and negative beads was obtained. We then quantified actin CSK remodeling as the percentage of positive beads from the total number of evaluated beads, which ranged from over 100 to several hundred for each experiment condition.
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When a specific torque (), as defined in Eq. 1, is applied to the bead, the ratio of the torque to the resultant bead displacement
defines a complex stiffness
,
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Details of OMTC have been published elsewhere (14, 32). In brief, ASM cells cultured in 6.4-mm wells were prepared with beads, as described in MTS (20,000 beads per well). The cell-bead preparation was then placed in the twisting device fitted on the epifluorescence microscope by using a x20 objective (numerical aperture 0.40). A specific torque identical to that in MTS but with a frequency of 0.5 Hz (limited by the camera frame rate) was applied to the beads, while the charge-coupled device camera (SensiCam, described above) imaged
200 beads at 16 frames per twisting cycle, phase locked to the twisting field. Bead positions on the image were determined by using an intensity centroid algorithm (14). The resolution of the OMTC was 2.5 nm (root mean square) at x20 magnification. The displacement of beads in response to the applied torque was computed from the recorded bead positions after being digitally processed to eliminate drift and noise as well as beads with erratic, irreproducible motions (see Refs. 14 and 32 for details).
Experimental protocol. To assess the effects of MS on actin CSK remodeling and CSK stiffening in ASM cells, we subjected the cell-bead preparations to the mechanical stimulation of 56-Pa specific torque at 0.3 Hz, as defined in Magnetic twisting stimulator (referred to as MS hereafter) for up to 2 h. During that period, the beads were remagnetized every 15 min to maintain their magnetic moment and moment alignment. G' was measured after 15 min of bead binding (referred to as baseline, time t = 0) and after mechanical stimulation at time points of t = 5, 15, 30, 60, and 120 min. G' was also measured in time-matched controls, where beads were added but no mechanical stimulation was delivered, except during the brief measurement period of 32 s at each time point. The actin CSK of ASM cells was imaged at identical time points but with different cell-bead preparations, as described in Fluorescence microscopy. To compare actin CSK changes induced by MS to actin CSK changes induced by contractile stimulation (CS), we exposed both mechanically stimulated and unstimulated cells to contractile agonists KCl (80 mM) or ACh (104 M) at doses chosen to guarantee a maximum response in G' as measured by OMTC (1, 4, 22, 28). Cells stimulated with KCl or ACh alone are referred to as KCl or ACh, respectively, whereas cells with mechanical stimulation and KCl or mechanical stimulation and ACh are referred to as KCl + MS and ACh + MS, respectively. In experiments with CS, after 15 min of initial bead binding, the cell-bead preparations were placed in an isotonic 80 mM KCl solution or 104 M ACh (see Reagents), instead of the serum-free medium. Then the cell-bead preparations were either mechanically stimulated or not and fixed in 4% paraformaldehyde at t = 5, 15, 30, 60, and 120 min for KCl or at t = 5, 30, and 60 min for ACh. Cells were also fixed at corresponding time points without addition of KCl or ACh and without mechanical stimulation as baseline controls.
Data processing and statistics. The percentages of positive beads, as measured from image analysis, are given as means ± 1 SE, averaged over n repeated experiments (i.e., coverslip). For each data point (MS, controls, KCl, or MS + KCl at each time point), three to five repeated experiments were conducted (n = 35), except for MS at 60 min and KCl at 15 min, when n = 8 and 9, respectively. The number of beads (N) measured in each experiment ranged from 124 to 577 (mean of N = 299). For ACh or MS + ACh, experiments were repeated eight times at each time point, resulting in the number of beads from 118 to 763 and average of 430 beads. To test for significant differences between two experiment conditions, a Student's t-test with 95% confidence level was used (P < 0.05).
Because G' measured by OMTC was approximately log-normally distributed, results are given as median ± 1 SE. Student's t-tests were conducted on the logarithmically transformed data. We measured G' in four to six wells for each condition. Each well contained 300 beads, totaling roughly 1,000 beads for each condition.
Reagents. All chemicals were from Sigma Chemical (St. Louis, MO), unless indicated otherwise. Tissue culture reagents, including DMEM/F-12 medium and trypsin-EDTA solution, were purchased from GIBCO (Grand Island, NY). Isotonic potassium chloride solution (80 mM KCl) was prepared with 64.75 mM NaCl, 80 mM KCl, 1.2 mM Na2HPO4, 2 mM MOPS, 0.02 mM EDTA, 1.6 mM CaCl2, 1.2 mM MgSO4, and 5.6 mM glucose, dissolved in double-distilled and sterile water. ACh was diluted to 102 M in PBS as stock. We added 20 µl of stock ACh solution to the 2-ml serum free medium in 35-mm petri dishes containing the cells, giving a final concentration of 104 M.
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RESULTS |
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DISCUSSION |
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Magnetic beads for study of CSK. Various methods have been used to study cell responses to MS, including stretching cells cultured on a flexible membrane, shearing adherent cells in a flow chamber, cell poking with micropipettes, and manipulating microbeads attached to cells. Here we used magnetic beads to deliver localized MS to the CSK, as has been similarly used previously (10, 19, 25, 41). The level of mechanical stimulation that we applied is given as 56 Pa, which is the applied torque per bead volume and has a dimension of Pascal but does not represent the applied stress. Mijailovich et al. (27) have computed the stress delivered by an adherent microbead to a cell in a simulated condition similar to our technique and found the stress to be on the order of 1,000 Pa. The actual stress varies over the cell-bead contact area and depends greatly on the degree of embedment, and thus stress was heterogeneously applied in our study, necessitating assessment of a large number of beads. It is interesting to note that the level of applied MS is comparable to increases in stress found to be induced by contractile activation (42). Applying MS using adherent microbeads also allowed us to directly measure changes in CSK mechanics due to mechanical stimulation at the sites where mechanical stimulation was applied.
Fluorescent imaging of bead-associated actin structure. To quantify the actin CSK remodeling resulting from mechanical stimulation with magnetic beads, we imaged phalloidin-stained actin using fluorescence microscopy. A difficulty with this method is that out-of-focus light degrades the image. However, this light is diffuse, and, by adjusting the focal plane, distinct bead-associated actin features could easily be discerned, and images were recorded from several vertically spaced image planes chosen to capture these features. Furthermore, subconfluent ASM cells are very thin, which reduces the contribution from out-of-plane fluorescence. It might be that dim actin structures were overlooked by our technique, but this would most likely only affect early time points of mechanical stimulation or controls. In any case, with our method, we were able to discern clear differences in actin staining intensity near beads from the background intensity within the region of one bead diameter, as demonstrated in Fig. 6 and as others have also reported with epifluorescence imaging (16, 17).
Stiffness measurement using magnetic beads. Optical magnetic twisting cytometry has been previously used to measure CSK G' of cultured adherent cells, including ASM cells (15, 33). This technique probes the CSK mechanical properties by optically detecting the motion of individual beads in response to an externally applied force while the beads are connected to the CSK through ligand-receptor linkages. As reported previously, we found that G' was highly heterogeneous and distributed approximately log-normally (13, 14, 25, 32). This heterogeneity has been largely attributed to differences in bead attachment characteristics, such as the number of binding sites linking the bead to the focal adhesion, and the focal adhesion linkage to the CSK, but a significant portion of the heterogeneity is ascribed to variations in cell properties (14). This heterogeneity and the distribution of applied MS required that many beads (and cells) be probed to detect small G' differences (14). We measured over 300 beads for each experiment, usually repeated four to six times for each experimental condition, which provided more than a sufficient number of beads to observe significant differences.
Our results from cultured ASM cells are in general agreement with those findings from other cell types by using a variety of techniques from bead pulling to optical laser trapping. In particular, MS applied to fibroblasts and endothelial cells via surface-bound beads have been found to induce bead-associated local remodeling in CSK proteins, including actin filament accumulation and formation of focal adhesion complex containing actin, vinculin, and talin (9, 16, 17, 19). Mechanically induced stiffening in cells has also been reported to be associated with remodeling in the CSK (10, 16). Our results are consistent with these findings and extend them. We demonstrate quantitatively that localized MS induced actin remodeling in a time-dependent manner, correlated with the increasing G' of the CSK. Furthermore, MS induced a more than twofold increase in G', greater than that caused by contractile activation, as discussed below.
Contractile activation vs. MS. Contractile activation is known to stimulate actin polymerization in ASM (26) and increase the stiffness of ASM cells, due partly to increased actin polymerization and partly to enhanced actomyosin activity (1, 22). We used the contractile agonists KCl and ACh at doses chosen to evoke maximal changes in ASM cell G' and compared responses between contractile and mechanical stimulation. Both agonists increased bead-associated actin polymerization rapidly within 5 min, followed by a somewhat lower increase due to ACh by 30 min compared with KCl at 30 min (ACh 37.4 ± 5.1% vs. KCl 56.0 ± 1.5%; P < 0.05), and both reached comparable levels at 60 min (ACh 60.1 ± 1.9% vs. KCl 47.8 ± 1.9%; P > 0.05). Actin remodeling was comparable for both agonists and MS at early times. Although other contractile agonists may lead to different levels of actin polymerization, with the two agonists we employed, less bead-associated actin polymerization was found than with mechanical stimulation at times >30 min. Although the volume of agonist was very large compared with the cell number, such that dilution with time was unlikely, the lower response due to CS may be due, in part, to the effect of decreasing effectiveness of the contractile agonists. However, reports measuring stiffness changes by using magnetic beads found increases in stiffness in ASM cells, varying from 30 to 70% due to a variety of contractile agonists, including KCl and ACh (1, 22, 25, 42). Here we found that MS caused a greater increase in G' than in these studies, and that these changes in G' due to MS were well correlated with observed CSK remodeling in response to MS. That the increase we found with MS was greater than those reported with CS is also well correlated with the greater bead-associated CSK remodeling that we found with MS compared with CS, as we observed. Furthermore, the combination of MS and contractile agonist (KCl + MS or ACh + MS) did not produce any further increase in actin CSK remodeling than that induced by MS alone. Taken together, these data indicate that MS is a potent stimulus for CSK remodeling compared with contractile activation. Indeed, it may be that some of the actin polymerization that we and others observed (1, 22, 26) during CS could be a result of increased stress within the CSK that is brought about by actin-myosin activity. However, we cannot conclude that MS alone, in the absence of contractile activation, can induce increased actin polymerization. The contractile machinery of ASM cells in culture is partially activated (22, 25), and it may be that this contributed to the CSK stiffening and remodeling in ASM cells that we observed. Nevertheless, that MS is such a potent stimulus for increasing CSK stiffness in ASM has important physiological implications, as discussed below.
Actin structures. We found that the number of beads staining positive for bead-associated actin structures increased dramatically with the application of MS. However, we also observed that the intensity and extent of actin structures around the beads also appeared to change during exposure to MS. As shown in Fig. 6, markedly different structures formed near the beads. It was interesting to group them into four characteristic features that we termed ring, filament, spindle, and halo in the order of increasing relative actin staining intensity, which we further separated into two classes, larger actin structures, which we termed strong, and hairline ring actin structures, which we termed ring. These structures were not necessarily exclusive of each other, and a combination of different structures was occasionally present on a single positive bead. For example, some of the beads with ring features also showed strong features; of the positively staining beads during MS, <7% (30 min) and <16% (120 min) showed both ring features and strong features. We did this because measuring the positive beads alone did not account for changes of these structures or differences due to MS and contractile stimuli. We thus compared the changes of either strong features or ring features on positive beads in response to stimulation.
An interesting pattern emerged. Greater numbers of strong actin structures were caused by MS compared with KCl, which continued to increase with duration of MS (Fig. 7, left). On the other hand, ring features responded similarly to either MS or KCl (Fig. 7, middle) or ACh (data not shown). The large actin structures that appeared in MS-stimulated cells over time may have been partly due to growth of ring structures into strong features, as indicated by the changing percentage of ring and strong features among the positively stained beads (Fig. 7, right). Interestingly, the formation of strong actin structures caused by either KCl or ACh was rare, never exceeding 23% of the positive beads at any time examined.
Thus MS appeared to elicit the formation of larger, more intense, and more complex actin features, whereas CS involved a more modest response with a lack of development in those strong structures. This was also borne out in time-matched controls, where only 11% of the beads that stained positively for actin showed evidence of strong features. The increase in larger features due to MS compared with time-matched controls and, compared with CS, agreed with the greater increase in G' due to MS.
Mechanisms for stress-induced CSK remodeling and stiffening. The mechanisms by which MS induces and regulates changes in CSK structure and function are unclear. It has been proposed that MS applied to the cell via ligand-integrin linkage may result in geometric distortion in CSK components, changing their physiochemical properties locally, and create a milieu that is possibly preferable to actin assembly (23). On the other hand, it may be that MS may regulate actin assembly through protein-protein bond formation and kinetics (2). Possibly, MS may also activate stress-sensitive ion channels to trigger a transient increase of calcium influx, leading to a raised gradient of calcium near a bead (18, 30), and thus causing local actin polymerization (39). Stress also causes activation of focal adhesion proteins, such as FAK and paxillin (33), which could perhaps affect actin fiber polymerization. Each of these mechanisms may contribute to the increase of actin accumulation at the beads during mechanical stimulation.
Our aggregate data show that larger, more diverse actin structures appear to evolve from ring structures that formed early in either MS- or CS-stimulated cells, and larger structures occurred much more commonly in response to MS compared with CS. Whereas the mechanisms for the different behavior are unknown, CS involves the activation of contractile pathways throughout the cells, resulting in continuous increased cell stress, whereas MS involves cyclic loading localized at focal adhesions. Thus, although MS and CS both increase stress that resulted in increased bead-associated actin, the stimuli are quite different and likely differently transduced.
Here we have shown that actin CSK structure and G' changed progressively in response to up to 2 h of mechanical stimulation. Our laboratory (32, 34) has also shown previously that ASM cells undergo changes in CSK contents and functions, consistent with the present findings, with longer periods (212 days) of mechanical stimulation. Taking both observations together, we speculate that the early effects caused by MS, as we observed in this study, may subsequently lead to increased proliferation, enhanced contractility, and greater force production and shortening velocity, as well as ablated response to relaxant as the mechanical stimulation is prolonged (3538).
Physiological implications. The ability of the ASM CSK to rapidly remodel and stiffen in response to MS may have important implications to airway function and pathology, particularly in diseases such as asthma, where MS might be increased due to wheeze, increased inspiratory efforts, and exacerbations. Mechanically induced CSK remodeling may affect ASM function by altering force generation and normal regulation of the CSK structure. Indeed, the reorganization of the CSK is now strongly believed to be a normal feature of ASM function through several postulated mechanisms (15, 20, 31). Thus changes in the CSK due to mechanical stimulation may compromise or modulate these behaviors. Furthermore, enhanced stiffness may contribute to impaired dilation of constricted airways observed in asthma. Once constricted, airways of asthmatic subjects do not remain dilated after a deep inspiration, but rather narrow again (7). Enhanced stiffness could provide a plausible mechanism for maintained narrowed airways through increased elastic recoil.
In conclusion, we found that MS was a potent stimulus for promoting actin CSK remodeling in cultured ASM cells, thereby causing CSK stiffening. The capacity of the ASM cell to remodel its internal structures and increase its stiffness may have important consequences in asthma, where heightened MS are potentially prevalent. However, we do not imply that MS alone is sufficient for altered ASM behavior in asthma, as MS is inherent in the airways due to the action of breathing. Other factors must also contribute to ASM pathology with MS, such as inflammation, airway wall remodeling, and elevated bronchial tone, which are associated with asthmatic airways.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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