Airway smooth muscle cell tone amplifies contractile function in the presence of chronic cyclic strain

Abstract

Chronic contractile activation, or tone, in asthma coupled with continuous stretching due to breathing may be involved in altering the contractile function of airway smooth muscle (ASM). Previously, we (11) showed that cytoskeletal remodeling and stiffening responses to acute (2 h) localized stresses were modulated by the level of contractile activation of ASM. Here, we investigated if altered contractility in response to chronic mechanical strain was dependent on repeated modulation of contractile tone. Cultured human ASM cells received 5% cyclic (0.3 Hz), predominantly uniaxial strain for 5 days, with once-daily dosing of either sham, forskolin, carbachol, or histamine to alter tone. Stiffness, contractility (KCl), and "relaxability" (forskolin) were then measured as was cell alignment, myosin light-chain phosphorylation (pMLC), and myosin light-chain kinase (MLCK) content. Cells became aligned and baseline stiffness increased with strain, but repeated lowering of tone inhibited both effects (P < 0.05). Strain also reversed a negative tone-modulation dependence of MLCK, observed in static conditions in agreement with previous reports, with strain and tone together increasing both MLCK and pMLC. Furthermore, contractility increased 176% (SE 59) with repeated tone elevation. These findings indicate that with strain, and not without, repeated tone elevation promoted contractile function through changes in cytoskeletal organization and increased contractile protein. The ability of repeated contractile activation to increase contractility, but only with mechanical stretching, suggests a novel mechanism for increased ASM contractility in asthma and for the role of continuous bronchodilator and corticosteroid therapy in reversing airway hyperresponsiveness.

Acetylcholine beyond bronchoconstriction: roles in inflammation and remodeling

“…Of note, the effects of methacholine were similar to those of TGF-b, and shown to be mediated via enhanced release of TGF-b [42]. In vitro, it has been shown that muscarinic receptor stimulation, in combination with mechanical strain, can induce a-sm-actin and sm-myosin mRNA expression in bovine tracheal smooth muscle strips, and myosin light-chain kinase expression in human airway smooth muscle cells [43,44]. Moreover, contraction of airway smooth muscle cells induces TGF-b activation [45].…”

Development and characterization of a 3D multicell microtissue culture model of airway smooth muscle

“…Although not possible with the current iteration of microfabricated tissue gauges, it may also be important to determine whether applying dynamic mechanical strain alters the contractility of ASM in 3D microtissues. In traditional 2D cultures of ASM, dynamic mechanical strain can have potent effects, acutely eliciting cytoskeletal fluidization (40) and depending on the exact nature of the strain regime it can elicit chronic changes in ASM phenotype and function (22,44), but it is currently unclear whether 3D cell geometry may alter these responses.…”

“…Imaging of live and fixed microtissues was performed on a Leica DM IRB microscope with a ϫ10 or ϫ20 lens, and images were captured with a PCO Sensicam CCD camera with custom software [Beadtracker (22)]. Images were uniformly adjusted for brightness and contrast only, ensuring that no details were obscured.…”

“…Drug solutions were added to the dish, gently mixed, and incubated for 3 min, before beads were magnetized once and postdrug stiffness data were collected. Cytoskeletal stiffness (G=, Pa/nm) was calculated from the bead displacement and applied torque by use of custom MATLAB code (22), and the median value for each field was used to determine the percent change from baseline values. Stiffness changes measured by this method are directly proportional to the net contractile moment measured by traction force microscopy (60).…”

Regulation of airway inflammation and remodeling by muscarinic receptors: Perspectives on anticholinergic therapy in asthma and COPD

“…These pathways are also involved in the acquisition of contractile protein expression by TGF-β via transcriptional and translational processes Goldsmith et al, 2006;Zhou et al, 2005) and can be activated by muscarinic receptor stimulation (Halayko et al, 2008). Indeed, the expression of myosin light-chain kinase was augmented by carbachol in human airway smooth muscle cells exposed to cyclical mechanical strain (Fairbank et al, 2008). Additionally, we recently described that muscarinic receptor stimulation enhanced the TGF-β1-induced contractile protein expression in human airway smooth muscle cells (Oenema et al, 2010b).…”