An emerging tool in airway biology is the precision-cut lung slice (PCLS). Adoption of the PCLS as a model for assessing airway reactivity has been hampered by the limited time window within which tissues remain viable. Here we demonstrate that the PCLS can be frozen, stored long-term, and then thawed for later experimental use. Compared with the never-frozen murine PCLS, the frozenthawed PCLS shows metabolic activity that is decreased to an extent comparable to that observed in other cryopreserved tissues but shows no differences in cell viability or in airway caliber responses to the contractile agonist methacholine or the relaxing agonist chloroquine. These results indicate that freezing and long-term storage is a feasible solution to the problem of limited viability of the PCLS in culture.

Bronchospasm induced in non-asthmatic human subjects can be easily reversed by a deep inspiration (DI) whereas bronchospasm that occurs spontaneously in asthmatic subjects cannot. This physiological effect of a DI has been attributed to the manner in which a DI causes airway smooth muscle (ASM) cells to stretch, but underlying molecular mechanisms–and their failure in asthma–remain obscure. Using cells and tissues from wild type and zyxin-/- mice we report responses to a transient stretch of physiologic magnitude and duration. At the level of the cytoskeleton, zyxin facilitated repair at sites of stress fiber fragmentation. At the level of the isolated ASM cell, zyxin facilitated recovery of contractile force. Finally, at the level of the small airway embedded with a precision cut lung slice, zyxin slowed airway dilation. Thus, at each level zyxin stabilized ASM structure and contractile properties at current muscle length. Furthermore, when we examined tissue samples from humans who died as the result of an asthma attack, we found increased accumulation of zyxin compared with non-asthmatics and asthmatics who died of other causes. Together, these data suggest a biophysical role for zyxin in fatal asthma.

In response to a transient mechanical stretch, the airway smooth muscle (ASM) cell is found to promptly fluidize and then slowly resolidify. While fluidization is typified by prompt reduction of cytoskeletal stiffness and cell contractile forces, and a loss of actin stress fibers, resolidification is typified by a slow restoration of these same material properties. This resolidification response is ATP-dependent, moreover, and hence presumed to be driven by active molecular interactions. A likely candidate for regulation of these molecular interactions is the LIM protein zyxin, which is known to localize to actin stress fibers in response to stretch, and to stabilize actin stress -actinin fibers by recruiting VASP and α . Here we tested the hypothesis that zyxin mediates the resolidification response. Using unpassaged primary ASM cells isolated from the tracheas of WT and zyxin-/-mice, we measured changes in cell contractile force (represented by a scalar metric called the contractile moment) after transient stretch using Fourier transform traction microscopy. Following a transient isotropic stretch (5-10% strain), contractile force in WT ASM cells returned to baseline within 300 seconds, whereas contractile force in essential for zyxin-/-cells returned to only 80% of baseline within 500 seconds, (Figure 1). Based on this evidence, we suggest that zyxin is full recovery of contractile forces in a dynamic microenvironment and thereby provides a potential target for modulating contractile dynamics in ASM. Figure 1. Recovery of Contractile Force Following Transient Isotropic Stretch in WT and Zyxin-/-Primary ASM This abstract is funded by: None Am J Respir Crit Care Med 185;2012:A4130 Internet address: www.atsjournals.org Online Abstracts Issue

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This study examines the role of zyxin, a cytoskeletal regulatory protein, in the mechanics of airway smooth muscle (ASM) under both static and dynamic conditions. Primary tracheal ASM cells from WT and zyxin−/− mice were obtained and baseline contractile force and changes in contractile force before and after a series of 3 transient isotropic stretches (5–10% strain) were measured in single cells using Fourier transform traction microscopy. Additional contractile force measurements as well as other cell mechanics measurements were performed on zyxin−/− and GFP‐zyxin rescued murine embryonic fibroblasts (MEFs). Zyxin−/− primary ASM fail to resolidify to the same extent as the WT primary ASM following single transient stretches as well as a series of transient stretches, although there is no difference in their baseline contractility. Similar results were found in MEFs; additionally baseline cytoskeletal stiffness was found to be the same in zyxin−/− and GFP‐zyxin rescued MEFs. Based on these results, resolidification following transient stretch appears to be dependent on the presence of zyxin in ASM cells. This indicates that zyxin may be a potential target for modulating the contractile dynamics of ASM during bronchospasm in the dynamic mechanical environment of the asthmatic lung.