Selected contribution: mechanical strain increases force production and calcium sensitivity in cultured airway smooth muscle cells.

Cultured airway smooth muscle cells subjected to cyclic deformational strain have increased cell content of myosin light chain kinase (MLCK) and myosin and increased formation of actin filaments. To determine how these changes may increase cell contractility, we measured isometric force production with changes in cytosolic calcium in individual permeabilized cells. The pCa for 50% maximal force production was 6.6+/-0.4 in the strain cells compared with 5.9+/-0.3 in control cells, signifying increased calcium sensitivity in strain cells. Maximal force production was also greater in strain cells (8.6+/-2.9 vs. 5.7+/-3.1 microN). The increased maximal force production in strain cells persisted after irreversible thiophosphorylation of myosin light chain, signifying that increased force could not be explained by differences in myosin light chain phosphorylation. Cells strained for brief periods sufficient to increase cytoskeletal organization but insufficient to increase contractile protein content also produced more force, suggesting that strain-induced cytoskeletal reorganization also increases force production.     

Chronic oscillatory strain induces MLCK associated rapid recovery from acute stretch in airway smooth muscle cells

A deep inspiration (DI) temporarily relaxes agonist-constricted airways in normal subjects, but in asthma airways are refractory and may rapidly renarrow, possibly due to changes in the structure and function of airway smooth muscle (ASM). Chronic largely uniaxial cyclic strain of ASM cells in culture causes several structural and functional changes in ASM similar to that in asthma, including increases in contractility, MLCK content, shortening velocity, and shortening capacity. However, changes in recovery from acute stretch similar to a DI have not been measured. We have therefore measured the response and recovery to large stretches of cells modified by chronic stretching and investigated the role of MLCK. Chronic, 10% uniaxial cyclic stretch, with or without a strain gradient, was administered for up to 11 days to cultured cells grown on Silastic membranes. Single cells were then removed from the membrane and subjected to 1 Hz oscillatory stretches up to 10% of the in situ cell length. These oscillations reduced stiffness by 66% in all groups (P < 0.05). Chronically strained cells recovered stiffness three times more rapidly than unstrained cells, while the strain gradient had no effect. The stiffness recovery in unstrained cells was completely inhibited by the MLCK inhibitor ML-7, but recovery in strained cells exhibiting increased MLCK was slightly inhibited. These data suggest that chronic strain leads to enhanced recovery from acute stretch, which may be attributable to the strain-induced increases in MLCK. This may also explain in part the more rapid renarrowing of activated airways following DI in asthma.     

Beneficial and harmful effects of oscillatory mechanical strain on airway smooth muscle

Airway smooth muscle (ASM) cells are constantly under mechanical strain as the lung cyclically expands and deflates, and this stretch is now known to modulate the contractile function of ASM. However, depending on the experimental conditions, stretch is either beneficial or harmful limiting or enhancing contractile force generation, respectively. Stretch caused by a deep inspiration is known to be beneficial in limiting or reversing airway constriction in healthy individuals, and oscillatory stretch lowers contractile force and stiffness or lengthens muscle in excised airway tissue strips. Stretch in ASM culture has generally been reported to cause increased contractile function through increases in proliferation, contractile protein content, and organization of the cell cytoskeleton. Recent evidence indicates the type of stretch is critically important. Growing cells on flexible membranes where stretch is non-uniform and anisotropic leads to pro-contractile changes, whereas uniform biaxial stretch causes the opposite effects. Furthermore, the role of contractile tone might be important in modulating the response to mechanical stretch in cultured cells. This report will review the contrasting evidence for modulation of contractile function of ASM, both in vivo and in vitro, and summarize the recent evidence that mechanical stress applied either acutely within 2 h or chronically over 11 d is a potent stimulus for cytoskeletal remodelling and stiffening. We will also point to new data suggesting that perhaps some of the difference in response to stretch might lie with one of the fundamental differences in the ASM environment in asthma and in culture--the presence of elevated contractile tone.     

Serum deprivation induces a unique hypercontractile phenotype of cultured smooth muscle cells

Chronic asthma is characterized by hypertrophyand hyperplasia of airway smooth muscle cells (SMC) that limit airflowby a geometric effect. Whether contractility of airway SMC is altered is not clear. Cultured cells were used as a model of hyperplasia. Phenotypic changes seen indicated conversion to a synthetic, weakly contractile type. At confluence, although limited reversal of proteinchanges was seen, no restoration in contractility occurred. Phenotypicmodulation of postconfluent cultured airway SMC under prolonged serumdeprivation (arrested cells) is reported here. Two phenotypicallydistinct groups of cells were identified in primary airway SMCcultures: 1) elongatedspindle-shaped cells, which expressed large amounts of smooth musclecontractile and regulatory proteins, and2) flat and stellate cells, whichexpressed very little. The first group showed a surprising shorteningcapacity and a velocity that was even greater than that of the freshly isolated cells, whereas the second group became spherical and noncontractile. Even more surprising was that the myosin heavy chain(MHC) isoform (SM-B) generally said to be associated with the highershortening velocity disappeared from the cell, while the content of thekey rate-limiting regulating enzyme, myosin light chain kinase (MLCK),increased 30-fold. We conclude that a functional, contractile phenotypeof airway SMC can be obtained by prolonged serum deprivation. Wespeculate that the increased contractility could be the result ofincreased phosphorylation of the 20-kDa myosin light chain resultingfrom increased content of smooth muscle MLCK rather than any increasein endogenous MHC ATPase activity. This model may be useful for studyof SMC differentiation and contraction.

     

Airway smooth muscle tone modulates mechanically induced cytoskeletal stiffening and remodeling.

The application of mechanical stresses to the airway smooth muscle (ASM) cell causes time-dependent cytoskeletal stiffening and remodeling (Deng L, Fairbank NJ, Fabry B, Smith PG, and Maksym GN. Am J Physiol Cell Physiol 287: C440-C448, 2004). We investigated here the extent to which these behaviors are modulated by the state of cell activation (tone). Localized mechanical stress was applied to the ASM cell in culture via oscillating beads (4.5 mum) that were tightly bound to the actin cytoskeleton (CSK). Tone was reduced from baseline level using a panel of relaxant agonists (10(-3) M dibutyryl cAMP, 10(-4) M forskolin, or 10(-6) M formoterol). To assess functional changes, we measured cell stiffness (G') using optical magnetic twisting cytometry, and to assess structural changes of the CSK we measured actin accumulation in the neighborhood of the bead. Applied mechanical stress caused a twofold increase in G' at 120 min. After cessation of applied stress, G' diminished only 24 +/- 6% (mean +/- SE) at 1 h, leaving substantial residual effects that were largely irreversible. However, applied stress-induced stiffening could be prevented by ablation of tone. Ablation of tone also inhibited the amount of actin accumulation induced by applied mechanical stress (P < 0.05). Thus the greater the contractile tone, the greater was applied stress-induced CSK stiffening and remodeling. As regards pathobiology of asthma, this suggests a maladaptive positive feedback in which tone potentiates ASM remodeling and stiffening that further increases stress and possibly leads to worsening airway function.     

Mechanical strain increases cell stiffness through cytoskeletal filament reorganization

We tested the hypothesis that cytoskeletal reorganization induced by cyclic strain increases cytoskeletal stiffness (G'). G' was measured by optical magnetic twisting cytometry in control cells and cells that had received mechanical strain for 10-12 days. G' was measured before and after both contractile and relaxant agonists, and in the strained cells both parallel (Para) and perpendicular (Perp) to the aligned cytoskeleton. Before activation, G' Para was 24 +/- 5% (+/- SE) greater compared with Perp (P < 0.05), and 35% +/- 6 greater compared with control (Cont, P < 0.01). The difference between strained and control cells was enhanced by KCl, increasing G' 171 +/- 7% Para compared with 125 +/- 6% Perp and 129 +/- 8% Cont (P < 10-5 both cases). The decrease in G' from baseline due to relaxant agonists isoproterenol and dibutyryl cAMP was similar in all groups. Long-term oscillatory loading of airway smooth muscle (ASM) cells caused stiffness to increase and become anisotropic. These findings are consistent with the hypothesis that cytoskeletal reorganization can enhance ASM stiffness and contractility. They imply, furthermore, that oscillatory loading of ASM may contribute to airway narrowing and failure of airway dilation in asthma.     

Insulin-induced myosin light-chain phosphorylation during receptor capping in IM-9 human B-lymphoblasts.

We have examined further the interaction between insulin surface receptors and the cytoskeleton of IM-9 human lymphoblasts. Using immunocytochemical techniques, we determined that actin, myosin, calmodulin and myosin light-chain kinase (MLCK) are all accumulated directly underneath insulin-receptor caps. In addition, we have now established that the concentration of intracellular Ca2+ (as measured by fura-2 fluorescence) increases just before insulin-induced receptor capping. Most importantly, we found that the binding of insulin to its receptor induces phosphorylation of myosin light chain in vivo. Furthermore, a number of drugs known to abolish the activation properties of calmodulin, such as trifluoperazine (TFP) or W-7, strongly inhibit insulin-receptor capping and myosin light-chain phosphorylation. These data imply that an actomyosin cytoskeletal contraction, regulated by Ca2+/calmodulin and MLCK, is involved in insulin-receptor capping. Biochemical analysis in vitro has revealed that IM-9 insulin receptors are physically associated with actin and myosin; and most interestingly, the binding of insulin-receptor/cytoskeletal complex significantly enhances the phosphorylation of the 20 kDa myosin light chain. This insulin-induced phosphorylation is inhibited by calmodulin antagonists (e.g. TFP and W-7), suggesting that the phosphorylation is catalysed by MLCK. Together, these results strongly suggest that MLCK-mediated myosin light-chain phosphorylation plays an important role in regulating the membrane-associated actomyosin contraction required for the collection of insulin receptors into caps.     

The laminin β1-competing peptide YIGSR induces a hypercontractile,hypoproliferative airway smooth muscle phenotype in an animal model of allergic asthma

Background

Fibroproliferative airway remodelling, including increased airway smooth muscle (ASM) mass and contractility, contributes to airway hyperresponsiveness in asthma. In vitro studies have shown that maturation of ASM cells to a (hyper)contractile phenotype is dependent on laminin, which can be inhibited by the laminin-competing peptide Tyr-Ile-Gly-Ser-Arg (YIGSR). The role of laminins in ASM remodelling in chronic asthma in vivo, however, has not yet been established.

Methods

Using an established guinea pig model of allergic asthma, we investigated the effects of topical treatment of the airways with YIGSR on features of airway remodelling induced by repeated allergen challenge, including ASM hyperplasia and hypercontractility, inflammation and fibrosis. Human ASM cells were used to investigate the direct effects of YIGSR on ASM proliferation in vitro.

Results

Topical administration of YIGSR attenuated allergen-induced ASM hyperplasia and pulmonary expression of the proliferative marker proliferating cell nuclear antigen (PCNA). Treatment with YIGSR also increased both the expression of sm-MHC and ASM contractility in saline- and allergen-challenged animals; this suggests that treatment with the laminin-competing peptide YIGSR mimics rather than inhibits laminin function in vivo. In addition, treatment with YIGSR increased allergen-induced fibrosis and submucosal eosinophilia. Immobilized YIGSR concentration-dependently reduced PDGF-induced proliferation of cultured ASM to a similar extent as laminin-coated culture plates. Notably, the effects of both immobilized YIGSR and laminin were antagonized by soluble YIGSR.

Conclusion

These results indicate that the laminin-competing peptide YIGSR promotes a contractile, hypoproliferative ASM phenotype in vivo, an effect that appears to be linked to the microenvironment in which the cells are exposed to the peptide.     

TNF-[alpha] modulates murine tracheal rings responsiveness to G-protein-coupled receptor agonists and KCl.

Although the mechanisms that underlie airway hyperresponsiveness in asthma are complex and involve a variety of factors, evidence now suggests that intrinsic abnormalities in airway smooth muscle (ASM) may play an important role. We previously reported that TNF-alpha, a cytokine involved in asthma, augments G-protein-coupled receptor (GPCR) agonist-evoked calcium responses in cultured ASM cells. Here we have extended our previous studies by investigating whether TNF-alpha also modulates the contractile and relaxant responses to GPCR activation using cultured murine tracheal rings. We found that in tracheal rings treated with 50 ng/ml TNF-alpha, carbachol-induced isometric force was significantly increased by 30% compared with those treated with diluent alone (P < 0.05). TNF-alpha also augmented KCl-induced force generation by 70% compared with rings treated with diluent alone (P < 0.01). The enhancing effect of TNF-alpha on carbachol-induced isometric force generation was completely abrogated in the tracheal rings obtained from TNF-alpha receptor (TNFR)1-deficient mice and in control rings treated with a TNF-alpha mutant that solely activates TNFR2. TNF-alpha also attenuated relaxation responsiveness to isoproterenol but not to PGE2 or forskolin. TNF-alpha modulatory effects on GPCR-induced ASM responsiveness were completely abrogated by pertussis toxin, an inhibitor of Gialpha proteins. Taken together, these data suggest that TNF-alpha may participate in the development of airway hyperresponsiveness in asthma via the modulation of ASM responsiveness to both contractile and beta-adrenoceptor GPCR agonists.     

Cyclic stretch enhances reorientation and differentiation of 3-D culture model of human airway smooth muscle

Activation of airway smooth muscle (ASM) cells plays a central role in the pathophysiology of asthma. Because ASM is an important therapeutic target in asthma, it is beneficial to develop bioengineered ASM models available for assessing physiological and biophysical properties of ASM cells. In the physiological condition in vivo, ASM cells are surrounded by extracellular matrix (ECM) and exposed to mechanical stresses such as cyclic stretch. We utilized a 3-D culture model of human ASM cells embedded in type-I collagen gel. We further examined the effects of cyclic mechanical stretch, which mimics tidal breathing, on cell orientation and expression of contractile proteins of ASM cells within the 3-D gel. ASM cells in type-I collagen exhibited a tissue-like structure with actin stress fiber formation and intracellular Ca²⁺ mobilization in response to methacholine. Uniaxial cyclic stretching enhanced alignment of nuclei and actin stress fibers of ASM cells. Moreover, expression of mRNAs for contractile proteins such as α-smooth muscle actin, calponin, myosin heavy chain 11, and transgelin of stretched ASM cells was significantly higher than that under the static condition. Our findings suggest that mechanical force and interaction with ECM affects development of the ASM tissue-like construct and differentiation to the contractile phenotype in a 3-D culture model.     

Localized mechanical stress induces time-dependent actin cytoskeletal remodeling and stiffening in cultured airway smooth muscle cells

Mechanical stress (MS) causes cytoskeletal (CSK) and phenotypic changes in cells. Such changes in airway smooth muscle (ASM) cells might contribute to the pathophysiology of asthma. We have shown that periodic mechanical strain applied to cultured ASM cells alters the structure and expression of CSK proteins and increases cell stiffness and contractility (Smith PG, Moreno R, and Ikebe M. Am J Physiol Lung Cell Mol Physiol 272: L20–L27, 1997; and Smith PG, Deng L, Fredberg JJ, and Maksym GN. Am J Physiol Lung Cell Mol Physiol 285: L456–L463, 2003). However, the mechanically induced CSK changes, altered cell function, and their time courses are not well understood. Here we applied MS to the CSK by magnetically oscillating ferrimagnetic beads bound to the CSK. We quantified CSK remodeling by measuring actin accumulation at the sites of applied MS using fluorescence microscopy. We also measured CSK stiffness using optical magnetic twisting cytometry. We found that, during MS of up to 120 min, the percentage of beads associated with actin structures increased with time. At 60 min, 68.1 ± 1.6% of the beads were associated with actin structures compared with only 6.7 ± 2.8% before MS and 38.4 ± 5.5% in time-matched controls (P < 0.05). Similarly, CSK stiffness increased more than twofold in response to the MS compared with time-matched controls. These changes were more pronounced than observed with contractile stimulation by 80 mM KCl or 10^–4 M acetylcholine. Together, these findings imply that MS is a potent stimulus to enhance stiffness and contractility of ASM cells through CSK remodeling, which may have important implications in airway narrowing and dilation in asthma. mechanical stress; actin cytoskeleton; stiffness; airway smooth muscle cell; optical magnetic twisting cytometry; airway constriction and dilation; asthma     

Heme oxygenase modulates oxidant-signaled airway smooth muscle contractility: role of bilirubin

Reactive oxygen species (ROS) increase the contractile response of airway smooth muscle (ASM). Heme oxygenase (HO) catabolizes heme to the powerful antioxidant bilirubin. Because HO is expressed in the airways, we investigated its effects on ASM contractility and ROS production in guinea pig trachea. HO expression was higher in the epithelium than in tracheal smooth muscle. Incubation of tracheal rings (TR) with the HO inhibitor tin protoporphyrin (SnPP IX) or the HO substrate hemin increased and decreased, respectively, ASM contractile response to carbamylcholine. The effect of hemin was reversed by SnPP and mimicked by the antioxidants superoxide dismutase (SOD) and catalase. Hemin significantly reduced the effect of carbamylcholine in rings treated with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), compared with ODQ-treated rings without hemin incubation, suggesting that the CO-guanosine 3',5'-cyclic monophosphate pathway was not involved in the control of tracheal reactivity. SnPP and hemin increased and decreased ROS production by TR by 18 and 38%, respectively. Bilirubin (100 pM) significantly decreased TR contractility and ROS production. Hemin, bilirubin, and SOD/catalase decreased phosphorylation of the contractile protein myosin light chain, whereas SnPP significantly augmented it. These data suggest that modulation of the redox status by HO and, moreover, by bilirubin modulates ASM contractility by modulating levels of phosphorylated myosin light chain.     

Myosin light-chain kinase regulates endothelial calcium entry and endothelium-dependent vasodilation.

Activation of smooth muscle myosin light-chain kinase (MLCK) causes contraction. Here we have proven that MLCK controls Ca2+ entry (CE) in endothelial cells (ECs): MLCK antisense oligonucleotides strongly prevented bradykinin (BK)- and thapsigargin (TG)-induced endothelial Ca2+ response, while MLCK sense did not. We also show that the relevant mechanism is not phosphorylation of myosin light-chain (MLC): MLC phosphorylation by BK required CE, but MLC phosphorylation caused by the phosphatase inhibitor calyculin A did not trigger Ca2+ response. Most important, we provide for the first time strong evidence that, in contrast to its role in smooth muscle cells, activation of MLCK in ECs stimulates the production of important endothelium-derived vascular relaxing factors: MLCK antisense and MLCK inhibitors abolished BK- and TG-induced nitric oxide production, and MLCK inhibitors substantially inhibited acetylcholine-stimulated hyperpolarization of smooth muscle cell membrane in rat mesenteric artery. These results indicate that MLCK controls endothelial CE, but not through MLC phosphorylation, and unveils a hitherto unknown physiological function of the enzyme: vasodilation through its action in endothelial cells. The study discovers a counter-balancing role of MLCK in the regulation of vascular tone.     

Effect of airway smooth muscle tone on airway distensibility measured by the forced oscillation technique in adults with asthma

Airway distensibility appears to be unaffected by airway smooth muscle (ASM) tone, despite the influence of ASM tone on the airway diameter-pressure relationship. This discrepancy may be because the greatest effect of ASM tone on airway diameter-pressure behavior occurs at low transpulmonary pressures, i.e., low lung volumes, which has not been investigated. Our study aimed to determine the contribution of ASM tone to airway distensibility, as assessed via the forced oscillation technique (FOT), across all lung volumes with a specific focus on low lung volumes. We also investigated the accompanying influence of ASM tone on peripheral airway closure and heterogeneity inferred from the reactance versus lung volume relationship. Respiratory system conductance and reactance were measured using FOT across the entire lung volume range in 22 asthma subjects and 19 healthy controls before and after bronchodilator. Airway distensibility (slope of conductance vs. lung volume) was calculated at residual volume (RV), functional residual capacity (FRC), and total lung capacity. At baseline, airway distensibility was significantly lower in subjects with asthma at all lung volumes. After bronchodilator, distensibility significantly increased at RV (64.8%, P < 0.001) and at FRC (61.8%, P < 0.01) in subjects with asthma but not in control subjects. The increased distensibility at RV and FRC in asthma were not associated with the accompanying changes in the reactance versus lung volume relationship. Our findings demonstrate that, at low lung volumes, ASM tone reduces airway distensibility in adults with asthma, independent of changes in airway closure and heterogeneity.     

Extracellular matrix proteins differentially regulate airway smooth muscle phenotype and function

Changes in the ECM and increased airway smooth muscle (ASM) mass are major contributors to airway remodeling in asthma and chronic obstructive pulmonary disease. It has recently been demonstrated that ECM proteins may differentially affect proliferation and expression of phenotypic markers of cultured ASM cells. In the present study, we investigated the functional relevance of ECM proteins in the modulation of ASM contractility using bovine tracheal smooth muscle (BTSM) preparations. The results demonstrate that culturing of BSTM strips for 4 days in the presence of fibronectin or collagen I depressed maximal contraction (E(max)) both for methacholine and KCl, which was associated with decreased contractile protein expression. By contrast, both fibronectin and collagen I increased proliferation of cultured BTSM cells. Similar effects were observed for PDGF. Moreover, PDGF augmented fibronectin- and collagen I-induced proliferation in an additive fashion, without an additional effect on contractility or contractile protein expression. The fibronectin-induced depression of contractility was blocked by the integrin antagonist Arg-Gly-Asp-Ser (RGDS) but not by its negative control Gly-Arg-Ala-Asp-Ser-Pro (GRADSP). Laminin, by itself, did not affect contractility or proliferation but reduced the effects of PDGF on these parameters. Strong relationships were found between the ECM-induced changes in E(max) in BTSM strips and their proliferative responses in BSTM cells and for E(max) and contractile protein expression. Our results indicate that ECM proteins differentially regulate both phenotype and function of intact ASM.     

A global, myosin light chain kinase-dependent increase in myosin II contractility accompanies the metaphase-anaphase transition in sea urchin eggs

Myosin II is the force-generating motor for cytokinesis, and although it is accepted that myosin contractility is greatest at the cell equator, the temporal and spatial cues that direct equatorial contractility are not known. Dividing sea urchin eggs were placed under compression to study myosin II-based contractile dynamics, and cells manipulated in this manner underwent an abrupt, global increase in cortical contractility concomitant with the metaphase-anaphase transition, followed by a brief relaxation and the onset of furrowing. Prefurrow cortical contractility both preceded and was independent of astral microtubule elongation, suggesting that the initial activation of myosin II preceded cleavage plane specification. The initial rise in contractility required myosin light chain kinase but not Rho-kinase, but both signaling pathways were required for successful cytokinesis. Last, mobilization of intracellular calcium during metaphase induced a contractile response, suggesting that calcium transients may be partially responsible for the timing of this initial contractile event. Together, these findings suggest that myosin II-based contractility is initiated at the metaphase-anaphase transition by Ca2+-dependent myosin light chain kinase (MLCK) activity and is maintained through cytokinesis by both MLCK- and Rho-dependent signaling. Moreover, the signals that initiate myosin II contractility respond to specific cell cycle transitions independently of the microtubule-dependent cleavage stimulus.     

Electron microscopic study of actin polymerization in airway smooth muscle

Actin polymerization as part of the normal smooth muscle response to various stimuli has been reported. The actin dynamics are believed to be necessary for cytoskeletal remodeling in smooth muscle in its adaptation to external stress and strain and for maintenance of optimal contractility. We have shown in our previous studies in airway smooth muscle that myosins polymerized in response to contractile activation as well as to adaptation at longer cell lengths. We postulated that the same response could be elicited from actins under the same conditions. In the present study, actin filament formation was quantified electron microscopically in cell cross sections. Nanometer resolution allowed us to examine regional distribution of filaments in a cell cross section. Airway smooth muscle bundles were fixed in relaxed and activated states at two lengths; muscle preparations were also fixed after a period of oscillatory strain, a condition known to cause depolymerization of myosin filaments. The results indicate that contractile activation and increased cell length nonsynergistically enhanced actin polymerization; the extent of actin polymerization was substantially less than that of myosin polymerization. Oscillatory strain increased thin filament formation. Although thin filament density was found higher in cytoplasmic areas near dense bodies, contractile activation did not preferentially enhance actin polymerization in these areas. It is concluded that actin thin filaments are dynamic structures whose length and number are regulated by the cell in response to changes in extracellular environment and that polymerization and depolymerization of thin filaments occur uniformly across the whole cell cross section.     

A fluorescent resonant energy transfer-based biosensor reveals transient and regional myosin light chain kinase activation in lamella and cleavage furrows

Approaches with high spatial and temporal resolution are required to understand the regulation of nonmuscle myosin II in vivo. Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its [Ca2+]4/calmodulin-binding state in living cells. We observe transient recruitment of diffuse MLCK to stress fibers and its in situ activation before contraction. MLCK is highly active in the lamella of migrating cells, but not at the retracting tail. This unexpected result highlights a potential role for MLCK-mediated myosin contractility in the lamella as a driving force for migration. During cytokinesis, MLCK was enriched at the spindle equator during late metaphase, and was maximally activated just before cleavage furrow constriction. As furrow contraction was completed, active MLCK was redistributed to the poles of the daughter cells. These results show MLCK is a myosin regulator in the lamella and contractile ring, and pinpoints sites where myosin function may be mediated by other kinases.     

A mathematical model of airway and pulmonary arteriole smooth muscle

Airway hyperresponsiveness is a major characteristic of asthma and is believed to result from the excessive contraction of airway smooth muscle cells (SMCs). However, the identification of the mechanisms responsible for airway hyperresponsiveness is hindered by our limited understanding of how calcium (Ca²⁺), myosin light chain kinase (MLCK), and myosin light chain phosphatase (MLCP) interact to regulate airway SMC contraction. In this work, we present a modified Hai-Murphy cross-bridge model of SMC contraction that incorporates Ca²⁺ regulation of MLCK and MLCP. A comparative fit of the model simulations to experimental data predicts 1), that airway and arteriole SMC contraction is initiated by fast activation by Ca²⁺ of MLCK; 2), that airway SMC, but not arteriole SMC, is inhibited by a slower activation by Ca²⁺ of MLCP; and 3), that the presence of a contractile agonist inhibits MLCP to enhance the Ca²⁺ sensitivity of airway and arteriole SMCs. The implication of these findings is that murine airway SMCs exploit a Ca²⁺-dependent mechanism to favor a default state of relaxation. The rate of SMC relaxation is determined principally by the rate of release of the latch-bridge state, which is predicted to be faster in airway than in arteriole. In addition, the model also predicts that oscillations in calcium concentration, commonly observed during agonist-induced smooth muscle contraction, cause a significantly greater contraction than an elevated steady calcium concentration.     

A theoretical model of inflammation- and mechanotransduction-driven asthmatic airway remodelling

Inflammation, airway hyper-responsiveness and airway remodelling are well-established hallmarks of asthma, but their inter-relationships remain elusive. In order to obtain a better understanding of their inter-dependence, we develop a mechanochemical morphoelastic model of the airway wall accounting for local volume changes in airway smooth muscle (ASM) and extracellular matrix in response to transient inflammatory or contractile agonist challenges. We use constrained mixture theory, together with a multiplicative decomposition of growth from the elastic deformation, to model the airway wall as a nonlinear fibre-reinforced elastic cylinder. Local contractile agonist drives ASM cell contraction, generating mechanical stresses in the tissue that drive further release of mitogenic mediators and contractile agonists via underlying mechanotransductive signalling pathways. Our model predictions are consistent with previously described inflammation-induced remodelling within an axisymmetric airway geometry. Additionally, our simulations reveal novel mechanotransductive feedback by which hyper-responsive airways exhibit increased remodelling, for example, via stress-induced release of pro-mitogenic and pro-contractile cytokines. Simulation results also reveal emergence of a persistent contractile tone observed in asthmatics, via either a pathological mechanotransductive feedback loop, a failure to clear agonists from the tissue, or a combination of both. Furthermore, we identify various parameter combinations that may contribute to the existence of different asthma phenotypes, and we illustrate a combination of factors which may predispose severe asthmatics to fatal bronchospasms.