Biophysical basis for airway hyperresponsiveness

Airway hyperresponsiveness is the excessive narrowing of the airway lumen caused by stimuli that would cause little or no narrowing in the normal individual. It is one of the cardinal features of asthma, but its mechanisms remain unexplained. In asthma, the key end-effector of acute airway narrowing is contraction of the airway smooth muscle cell that is driven by myosin motors exerting their mechanical effects within an integrated cytoskeletal scaffolding. In just the past few years, however, our understanding of the rules that govern muscle biophysics has dramatically changed, as has their classical relationship to airway mechanics. It has become well established, for example, that muscle length is equilibrated dynamically rather than statically, and that in a dynamic setting nonclassical features of muscle biophysics come to the forefront, including unanticipated interactions between the muscle and its time-varying load, as well as the ability of the muscle cell to adapt (remodel) its internal microstructure rapidly in response to its ever-changing mechanical environment. Here, we consider some of these emerging concepts and, in particular, focus on structural remodeling of the airway smooth muscle cell as it relates to excessive airway narrowing in asthma.     

Modulation of host cell mechanics by Trypanosoma cruzi

To investigate the effects of Trypanosoma cruzi on the mechanical properties of infected host cells, cytoskeletal stiffness and remodeling dynamics were measured in parasite-infected fibroblasts. We find that cell stiffness decreases in a time-dependent fashion in T. cruzi-infected human foreskin fibroblasts without a significant change in the dynamics of cytoskeletal remodeling. In contrast, cells exposed to T. cruzi secreted/released components become significantly stiffer within 2 h of exposure and exhibit increased remodeling dynamics. These findings represent the first direct mechanical data to suggest a physical picture in which an intact, stiff, and rapidly remodeling cytoskeleton facilitates early stages of T. cruzi invasion and parasite retention, followed by subsequent softening and disassembly of the cytoskeleton to accommodate intracellular replication of parasites. We further suggest that these changes occur through protein kinase A and inhibition of the Rho/Rho kinase signaling pathway. In the context of tissue infection, changes in host cell mechanics could adversely affect the function of the infected organs, and may play an important role on the pathophysiology of Chagas' disease.     

Fluidization and Resolidification of the Human Bladder Smooth Muscle Cell in Response to Transient Stretch

Background

Cells resident in certain hollow organs are subjected routinely to large transient stretches, including every adherent cell resident in lungs, heart, great vessels, gut, and bladder. We have shown recently that in response to a transient stretch the adherent eukaryotic cell promptly fluidizes and then gradually resolidifies, but mechanism is not yet understood.

Principal Findings

In the isolated human bladder smooth muscle cell, here we applied a 10% transient stretch while measuring cell traction forces, elastic modulus, F-actin imaging and the F-actin/G-actin ratio. Immediately after a transient stretch, F-actin levels and cell stiffness were lower by about 50%, and traction forces were lower by about 70%, both indicative of prompt fluidization. Within 5min, F-actin levels recovered completely, cell stiffness recovered by about 90%, and traction forces recovered by about 60%, all indicative of resolidification. The extent of the fluidization response was uninfluenced by a variety of signaling inhibitors, and, surprisingly, was localized to the unstretch phase of the stretch-unstretch maneuver in a manner suggestive of cytoskeletal catch bonds. When we applied an “unstretch-restretch” (transient compression), rather than a “stretch-unstretch” (transient stretch), the cell did not fluidize and the actin network did not depolymerize.

Conclusions

Taken together, these results implicate extremely rapid actin disassembly in the fluidization response, and slow actin reassembly in the resolidification response. In the bladder smooth muscle cell, the fluidization response to transient stretch occurs not through signaling pathways, but rather through release of increased tensile forces that drive acute disassociation of actin.     

Alveolar pressure nonhomogeneity during small-amplitude high-frequency oscillation

In six excised canine lungs, regional alveolar pressures (PA) were measured during small-amplitude high-frequency oscillations applied at the airway opening. Both the regional distribution of PA's and their relationship to pressure excursions at the airway opening (Pao) were assessed in terms of amplitude and phase. PA was sampled in several capsules glued to the pleural surface and communicating with alveolar gas via pleural punctures. Pao and PA were measured over the frequency (f) range 1-60 Hz, at transpulmonary pressures (PL) of 5, 10, and 25 cmH2O. The amplitude of PA excursions substantially exceeded Pao excursions at frequencies near the resonant frequency. At resonance the ratio [PA/Pao] was 1.9, 2.9, and 4.8 at PL's of 5, 10, and 25 cmH2O, respectively. Both spatial homogeneity and temporal synchrony of PA's between sampled lung regions decreased with f and increased with PL. Interregional variability of airway impedance [(Pao - PA)/Vao] and tissue impedance (PA/Vao) tended to be larger than differences due to changing PL but not as large as between-dog variability. These data define the baseline nonhomogeneity of the normal canine lung and also suggest that there may be some advantage in applying high-frequency ventilation at frequencies at least as high as lung resonant frequency.     

In vivo estimation of tracheal distensibility and hysteresis in normal adults

We used the acoustic reflection technique to measure the cross-sectional area of tracheal and bronchial airway segments of eight healthy adults. We measured airway area during a slow continuous expiration from total lung capacity (TLC) to residual volume (RV) and during inspiration back to TLC. Lung volume and esophageal pressure were monitored continuously during this quasi-static, double vital capacity maneuver. We found that 1) the area of tracheal and bronchial segments increases with increasing lung volume and transpulmonary pressure, 2) the trachea and bronchi exhibit a variable degree of hysteresis, which may be greater or less than that of the lung parenchyma, 3) extrathoracic and intrathoracic tracheal segments behaved as if they were subjected to similar transmural pressure and had similar elastic properties, and 4) specific compliance (means +/- SE) for the intrathoracic and bronchial segments, calculated with the assumption that transmural pressure is equal to the transpulmonary pressure, was significantly (P less than 0.05) smaller for the intrathoracic segment than for the bronchial segment: (2.1 +/- 2.0) X 10(-3) cmH2O-1 vs. (9.1 +/- 2.1) X 10(-3) cmH2O-1. Direct measurements of airway area using acoustic reflections are in good agreement with previous estimates of airway distensibility in vivo, obtained by radiography or endoscopy.     

Selected contribution: time course and heterogeneity of contractile responses in cultured human airway smooth muscle cells.

We measured the time course and heterogeneity of responses to contractile and relaxing agonists in individual human airway smooth muscle (HASM) cells in culture. To this end, we developed a microrheometer based on magnetic twisting cytometry adapted with a novel optical detection system. Ferromagnetic beads (4.5 microm) coated with Arg-Gly-Asp peptide were bound to integrins on the cell surface. The beads were twisted in a sinusoidally varying magnetic field at 0.75 Hz. Oscillatory bead displacements were recorded using a phase-synchronized video camera. The storage modulus (cell stiffness; G'), loss modulus (friction; G"), and hysteresivity (eta; ratio of G" to G') could be determined with a time resolution of 1.3 s. Within 5 s after addition of histamine (100 microM), G' increased by 2.2-fold, G" increased by 3.0-fold, and eta increased transiently from 0.27 to 0.34. By 20 s, eta decreased to 0.25, whereas G' and G" remained above baseline. Comparable results were obtained with bradykinin (1 microM). These changes in G', G", and eta measured in cells were similar to but smaller than those reported for intact muscle strips. When we ablated baseline tone by adding the relaxing agonist dibutyryl cAMP (1 mM), G' decreased within 5 min by 3.3-fold. With relaxing and contracting agonists, G' could be manipulated through a contractile range of 7.3-fold. Cell populations exhibited a log-normal distribution of baseline stiffness (geometric SD = 2.8) and a heterogeneous response to both contractile and relaxing agonists, partly attributable to variability of baseline tone between cells. The total contractile range of the cells (from maximally relaxed to maximally stimulated), however, was independent of baseline stiffness. We conclude that HASM cells in culture exhibit a clear, although heterogeneous, response to contractile and relaxing agonists and express the essential mechanical features characteristic of the contractile response observed at the tissue level.     

Historical perspective on airway smooth muscle: the saga of a frustrated cell.

Despite the lack of a clearly defined physiological function, airway smooth muscle receives substantial attention because of its involvement in the pathogenesis of asthma. Recent investigations have turned to the ways in which the muscle is influenced by its dynamic microenvironment. Ordinarily, airway smooth muscle presents little problem, even when maximally activated, because unending mechanical perturbations provided by spontaneous tidal breathing put airway smooth muscle in a perpetual state of "limbo," keeping its contractile machinery off balance and unable to achieve its force-generating potential. The dynamic microenvironment affects airway smooth muscle in at least two ways: by acute changes associated with disruption of myosin binding and by chronic changes associated with plastic restructuring of contractile and cytoskeletal filament organization. Plastic restructuring can occur when dynamic length changes occur between sequential contractile events or within a single contractile event. Impairment of these normal responses of airway smooth muscle to its dynamic environment may be implicated in airway hyperresponsiveness in asthma.