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Passive stiffness of airway smooth muscle: The next target for improving airway distensibility and treatment for asthma?
Pulmonary Pharmacology & Therapeutics 26 (2013) 37e41
Contents lists available at SciVerse ScienceDirect
Pulmonary Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/ypupt
Passive stiffness of airway smooth muscle: The next target for improving airway distensibility and treatment for asthma?q Chun Y. Seow* Department of Pathology and Laboratory Medicine, The James Hogg Research Centre/St. Paul’s Hospital, University of British Columbia, 1081 Burrard Street, Rm. 166, Vancouver, BC V6Z 1Y6, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 March 2012 Received in revised form 25 June 2012 Accepted 27 June 2012
Reduced airway distensibility due to increased airway stiffness is a characteristic of asthma. Airway stiffness is determined by the property and structural organization of the various elements of the airway wall, and is often divided into active and passive components. Active stiffness is thought to be associated with activation of muscle cells in the airway wall. This component of stiffness can be inhibited when active force produced by the muscle is abolished. Passive stiffness, on the other hand, is thought to stem from non-muscle component of the airway wall, especially the collagen/elastin fibrous network of the extracellular matrix within which the muscle cells are embedded. In this brief review, the notion that passive stiffness is exclusively extracellular in origin is challenged. Recent evidence suggests that a substantial portion of the passive stiffness of an in vitro preparation of tracheal smooth muscle is calcium sensitive and is regulated by Rho-kinase, although the underlying mechanism and the details of regulation for the development of this intracellular passive stiffness are still largely unknown. To reduce airway stiffness different lines of attack must be tailored to different components of the stiffness. The regulatable passive stiffness is distinct from the relatively permanent stiffness of the extracellular matrix and the stiffness associated with active muscle contraction. To improve airway distensibility during asthma exacerbation, a comprehensive approach to reduce overall airway stiffness should therefore include a strategy for targeting the regulatable passive stiffness. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Regulatable airway stiffness Calcium-sensitive resistance to stretch Rho-kinase
It is known that airway distensibility is reduced in asthmatic patients and that multiple factors contribute to the stiffness of the airways [1e7]. Relaxing the smooth muscle in the airways increases airway distensibility in asthmatics as reflected in the improved pulmonary-conductance vs. recoil-pressure relationship, however, the asthmatic airways are still less distensible than normal even after the active muscle force is abolished [1,2]. A commonly held view is that remodeling of the asthmatic airways (more specifically, fibrotic thickening of the airway wall) as a result of chronic inflammation is responsible for the “permanent” increase in airway stiffness which is independent of smooth muscle tone. Airway distensibility is therefore thought to be largely determined by the structural and mechanical properties of the various components of the airway wall and these properties are usually classified as active (muscle tone dependent) or passive (muscle tone independent). Thus, those associated with activated airway smooth muscle (ASM) and regulated by calcium signaling pathways are referred to as q This work was supported by the Canadian Institutes of Health Research (CIHR). * Tel.: þ 1 604 806 9268; fax: þ 1 604 806 9274. E-mail address: [email protected]. 1094-5539/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pupt.2012.06.012
active properties and those exhibited by unstimulated ASM and non-ASM tissues are passive properties. However, tensile stiffness (i.e., the resistance to stretch) of unstimulated smooth muscle defies this simple classification. It is regarded as passive stiffness because it is a property associated with the muscle in the relaxed state, and yet it is calcium sensitive [8] and is regulated by enzymatic pathways associated with cell activation as revealed by recent studies [9,10]. The contribution of passive stiffness of ASM to airway distensibility could be mistakenly mixed up with those from non-muscle components of the airway, such as the collagen-elastin extracellular matrix whose properties can be altered in airway remodeling. One feature that differentiates passive ASM stiffness from the airway stiffness of non-muscle origin is that it can be altered rapidly through intervention of the signaling pathways [9]; airway remodeling (or its reversal), on the other hand, could change airway stiffness only over a prolonged process. Manipulating active tone in ASM for rapid modulation of airway stiffness is therefore not the only option we have. Passive ASM stiffness as a target for improving airway distensibility should be explored. Any cellular process or property regulated by enzymatic pathways is likely to serve some physiological function. This raises
1. The phenomenon of readily changeable passive tension and stiffness in ASM For the purpose of discussion in this review, passive muscle properties refer to those exhibited by muscles in the relaxed state and in the absence of detectable active tone. There are many ways with which muscle stiffness can be measured. Fig. 1 illustrates a simple protocol of estimating the stiffness. We have found that a value of 5e10% of resting muscle length for the length step (DL, Fig. 1) and a speed of stretch of 10 ms for the step change in length applied to a relaxed ASM preparation produces reliable measurements of muscle stiffness without altering the muscle’s ability to generate active force in subsequently induced contractions [9]. The clue that the development of passive stiffness in the relaxed smooth muscle may be activation-history dependent is evident in earlier studies showing a shift in the passive length-tension curve of smooth muscle adapted to different lengths [12e15]. Passive tension in unstimulated smooth muscle bundle increases approximately exponentially with muscle length (e.g., gray curve in Fig. 2).
ΔL
ΔF
Fig. 1. Illustration of time records of a step-change in length (upper trace) applied to a smooth muscle strip and force response (lower trace) from the strip. The ratio of DF/DL can be used as an estimate of muscle stiffness or resistance to stretch.
L ref
a question as to what physiological function may be served by passive stiffness of smooth muscle. In hollow organs smooth muscle modulates tension in the organ wall and maintains the organ shape. Active contraction and relaxation of smooth muscle is thought to be the driving force behind the changes in organ dimension. However, for maintaining a constant organ dimension or wall tension over a prolonged period of time, more efficient mechanisms may be preferred. In vascular smooth muscle it has been proposed that during a sustained contraction dephosphorylated myosin crossbridges may bind to actin filaments to form “latch” bridges that are able to maintain tension with low cycling rates and therefore low energetic cost [11]. In smooth muscles that possess little or no tone in the resting state, such as those found in airway and bladder [12,13], passive stiffness may be an alternative mechanism for maintaining wall tension in the relaxed state and modulating organ distensibility. In this brief review the effect of airway tensile stiffness (presumed to largely stem from the ASM layer) on airway distensibility is emphasized. This does not mean that airway stiffness exerts no effect on other mechanical properties of the airway or ASM itself. Passive compressive stiffness of the ASM could modulate shortening velocity of ASM, for example.
0.5 L ref
C.Y. Seow / Pulmonary Pharmacology & Therapeutics 26 (2013) 37e41
Tension
38
Length Fig. 2. Illustration of passive lengthetension relationships obtained from a smooth muscle strip adapted at its in situ length (used as a reference length, Lref) and half in situ length. The in situ passive length-tension curve (gray) is shifted horizontally to the left as the muscle is shortened and adapted at half of the in situ length.
Traditional explanation for this lengthetension property of resting smooth muscle is based on the assumption that most of the tension bearing structures reside in extracellular matrix in the form of elastin and collagen fibers. It is further assumed that the more compliant portion of the length-tension curve is mostly determined by elastin fibers and the stiffer portion of the curve is mostly determined by collagen fibers. Based on these principles Maksym and Bates [16] are able to demonstrate in a mathematical model of “exponential” stressestrain relationship typical of that seen in lung tissue. Small variations in the lengthetension relationship of resting smooth muscle could be explained by viscoelastic properties of the tissue. However, large shifts in the relationship, as that shown in Fig. 2, are not likely a manifestation of tissue viscoelasticity. (This argument will be made more compellingly when we discuss calcium-sensitive and enzyme-activity-dependent passive tissue properties in the following section). The shift in the lengthetension curve described in Fig. 2 involves adaptation of smooth muscle at different lengths. The gray curve is associated with the muscle adapted to its in situ or reference length (Lref). Adaptation of a muscle to a certain length often requires repeated cycles of contraction and relaxation under isometric conditions for a period of time during which maximal active force increases incrementally toward a plateau [17]. In the example shown in Fig. 2, the muscle is shortened to half of its initial reference length. The shift in the passive length-tension curve does not occur immediately. The gradual change in the muscle properties associated with length adaptation is better illustrated in Fig. 3, where the increase in passive stiffness over time is monitored. Immediately after a relaxed ASM is shortened by half, passive tension and stiffness are reduced to zero from a reference value determined before the length change (Fig. 3). During adaptation at the shortened length, passive stiffness increases toward a plateau. It should be pointed out that in adapting to a shortened length, unlike passive stiffness, passive tension often remains unchanged (at zero tension). Passive tension and passive stiffness therefore reflect different underlying mechanisms responsible for their evolution. For example, if slowly cycling cross-bridges exist in the resting state and are responsible for the development of stiffness, they will in time cause “passive” resting tension to increase. On the other hand, if muscle stiffness in the resting state stems from crosslinking of non-cycling cross-linkers (not necessarily myosin cross-bridges) then the development of stiffness will not have an effect on resting tension. The underlying mechanism for the development of passive stiffness is not clear (the cross-linker
Stiffness/Stiffness at L ref
C.Y. Seow / Pulmonary Pharmacology & Therapeutics 26 (2013) 37e41
39
3. Cell regulation of passive stiffness
Abrupt length change: Lref → 0.5 Lref
Reference stiffness
Time Fig. 3. Illustration of time course of recovery of passive stiffness (resistance to stretch) after an abrupt shortening of resting muscle from Lref to 0.5 Lref. Although the stiffness is measured in the relaxed state, in between measurements the muscle is activated briefly with electrical stimulation. The stiffness recovery normally reaches a plateau in about 30e40 min.
hypothesis is just one of the many possibilities), but the consequence is clear and drastic. As illustrated in Fig. 2, upon restretching the muscle that had been adapted to a short length back to its in situ length, the passive tension can reach a level much higher than the active force the muscle can generate at its in situ length. In the experiments by Wang et al. [12] and Naghshin et al. [15], it was found that the muscle strips could not be returned to their in situ length (after they had been adapted to shorter lengths) in a single step without causing permanent damage to the muscle tissue because of the high passive tension; the muscle could be returned to their in situ length only with multi-steps of incremental length increase followed by adaptation. 2. Calcium-sensitive vs. calcium-insensitive stiffness The smooth muscle layer in an airway is composed of ASM cells embedded in (mostly collagen-elastin) extracellular matrix. Mechanical properties of muscle preparations dissected from the muscle layer are therefore reflective of the properties of muscle cells and extracellular matrix. The most simplistic model regards the extracellular matrix as a parallel elastic component of the muscle preparation, with the matrix bearing all externally applied (passive) tension when the muscle cells are not activated. Because the properties of extracellular matrix are relatively inert, the relationship between passive tension and muscle length is considered to be static. The finding of changeable and regulatable passive stiffness in smooth muscle preparations (representative of the muscle layer in airways) indicates that not all passive tension is borne by extracellular matrix, and that smooth muscle cells are responsible for some of the passive tension. Removal of intracellular and extracellular calcium by incubating the muscle in calcium-free Krebs solution with EGTA causes a substantial decrease in the passive stiffness, and the loss of passive stiffness can be restored by reintroducing calcium back to the Krebs solution [9]. When extracellular calcium is prevented from entering the muscle cells by calcium-channel blockers active force is abolished over time, suggesting that intracellular calcium has been depleted. Under this condition, there is a loss of passive stiffness just the same as that found in calcium-free Krebs [9]. It is therefore tempting to conclude that the calcium-sensitive stiffness is of intracellular origin and the calcium-insensitive stiffness, extracellular. However, a definitive conclusion will require more direct evidence, possibly from single cell preparations in which muscle force, stiffness, and intracellular calcium can be monitored.
Calcium release into intracellular space triggers a cascade of reactions that characterizes cell activation. In smooth muscle cells the release of calcium leads to activation of actomyosin interaction and the development of active force and stiffening of the muscle. Re-sequestration of calcium returns the force to its resting level and abolishes the stiffness associated with active actomyosin binding, but retains the passive stiffness. As discussed in the previous section, part of the passive stiffness is likely intracellular in origin, because of its calcium sensitivity. Another characteristic of the passive stiffness is that it can be “softened” by large strains applied to the muscle cells [9,18,19]. In ASM strips, after strain softening, passive stiffness recovers completely in about 20 min [9]. In the experiments described by Raqeeb et al. [9], during the recovering period, the muscle was briefly and periodically activated (once every 5 min), and this appeared to aid the stiffness recovery. In rabbit urinary bladder smooth muscle, activation has also been shown to facilitate recovery of passive stiffness lost due to strain softening [20]. This is probably because the development of passive stiffness is Rho-kinase dependent, as demonstrated in both airway and bladder smooth muscles that Y27632 (Rho-kinase inhibitor) prevents redevelopment of a portion of the passive stiffness after strain softening [9,20]. The portion of passive stiffness sensitive to Y27632 is likely of intracellular origin, because the Rho-kinase inhibitor is not known to act extracellularly. Interestingly, the redevelopment of calcium-sensitive passive stiffness after strain softening is not affected by inhibition of the myosin light chain kinase (MLCK) [9,10]. With sufficiently high concentrations of ML-7 (an MLCK inhibitor), active force of ASM can be abolished but the ability of the muscle to redevelop passive stiffness after strain softening remains intact [9,10]. This suggests that the mechanism underlying active force generation in smooth muscle is separate from that responsible for stiffness development. This, however, does not preclude the possibility that the dephosphorylated, non-cycling (truly latch) myosin cross-bridges are the source of the passive stiffness. If this is the case, one would expect to see a positive correlation between the calcium-sensitive passive stiffness and the number of cross-bridges overlapping the actin filaments. Because the extent of cross-bridge overlap is proportional to the active force generated by the muscle, as indicated by the lengtheforce relationship [21], one would expect a positive correlation, over a certain length range, between calcium-sensitive passive stiffness and the ability of the muscle to generate force. However, by comparing the length vs. active force relationship and length vs. passive stiffness relationship, a negative correlation is found between active force and calcium-sensitive passive stiffness [9]. The results therefore do not support that notion that crosslinking of myosin cross-bridges with actin filaments is the source (or at least the main source) of the calcium-sensitive passive stiffness. This has raised an intriguing question of what is responsible for the calcium-sensitive passive stiffness. Part of the stiffness likely stems from ionic interactions among proteins and protein aggregates in the “crowded” intracellular environment [18,19]. At least some of these interactions must be calcium dependent for them to manifest as calcium-sensitive stiffness. Some of the interactions may only occur under activated condition but the consequence of the interaction (such as formation of cross-links) must be retained under resting condition for it to be considered as a “passive” property. Some interactions may occur at the basal level of calcium under the relaxed condition; it appears that at zero-calcium, the passive stiffness is all extracellular in origin [9]. If cross-linking of protein filaments is the mechanism underlying the intracellular passive stiffness, several players can be
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C.Y. Seow / Pulmonary Pharmacology & Therapeutics 26 (2013) 37e41
identified. On the filament side, actin filaments are probably the most important simply because they are the most abundant. Other filaments such as intermediate filaments, microtubules, and myosin filaments are possible players too. On the cross-linker side, there are many actin binding or bundling proteins, a-actinin and filamin for example. Adapter proteins that anchor actin filaments to adhesion plaques, such as vinculin, paxillin, and talin [22], may play a role as well. Proteins that bind both actin and intermediate filaments (such as a-actinin) could have a special role in the formation of dense-body “cables” that appear to be able to bear passive tension [23]. 3-D reconstruction reveals that the dense-body cables are made of dense bodies connected together in series by actin and intermediate filaments, and the cable length is adjustable according to the muscle cell length [23]. What regulates the cable length is not known, but the plastic cable could be a source of the regulatable passive stiffness. Another possible source of the passive stiffness could reside in the network of interaction among caldesmon, myosin, and actin filaments. We have found recently that caldesmon can be phosphorylated by Ca2þ-calmodulin-MLCK and that the time course of phosphorylation coincides with the development of “latch” bridges that is thought to be responsible for the maintenance of tension in the sustained phase of contraction [24]. In the presence of myosin, it is difficult to discern the exact contribution of caldesmon phosphorylation to the “latch” phenomenon, because caldesmon is able to bind both myosin and actin. It has been shown in vitro that caldesmon can reversibly cross-link actin filaments [25]. So there are two possible ways that caldesmon could contribute to the passive stiffness, one is through binding of myosin and actin, and the other is through binding of actin and actin. 4. Increasing airway distensibility as a strategy to improve lung function in asthmatics A recent study showed that asthmatic airways (from third to sixth generations) are narrower than those of normal controls [26]. To distend the airways back to normal diameters, asthmatics face several challenges: 1) If we approximate an airway as a thin-walled cylindrical vessel, LaPlace Law (governing the relationship between wall tension, distending pressure, and airway diameter) works against asthmatics, because it dictates that for countering the same wall tension a greater distending pressure is required in an airway with smaller diameter. 2) Many of the inflammatory mediators found in asthmatic airways are contractile agonists for ASM; chronic airway inflammation thus can lead to elevated ASM tone (or wall tension) and that the ASM may enter a “frozen” state [27] and become refractory to the relaxing effect of oscillatory stress that accompanies tidal breathing and deep inspirations. 3) Length adaptation [17] and force adaptation [28,29] of ASM at shorter lengths also work against asthmatics, because they allow the muscle to retain maximal contractility at short lengths. 4) The shift in the passive lengthetension relationship [12,15] and increase in passive stiffness associated with adaptation to short lengths [9,10] effectively augments airway wall tension under distending pressure. To overcome these challenges a comprehensive strategy that addresses all causes impeding airway distension (thus reducing airway resistance) need to be developed. It should be pointed out that in some airway diseases such as bronchiectasis and bronchomalacia an increase in airway distensibility is often associated with poor lung function and even collapsed airways. These should be distinguished from asthmatic airways where an increase in airway distensibility is often associated with improved lung function [1,2]. Relaxing ASM (with short and long acting b(2)-agonists) addresses only one cause of airway obstruction in asthma. The use of corticosteroids to suppress airway
inflammation can lead to reduced ASM tone and may prevent or reverse airway remodeling [30], but again the treatment does not address all the challenges asthmatics face. A recent innovative approach in treating airway reactivity in an allergen-induced rabbit model of asthma with continuous positive airway pressure (CPAP) [31] has proven to be effective. This is perhaps not surprising because its addresses the geometric and adaptive properties of asthmatic airways and ASM. However, none of the treatments of asthma (including animal models of asthma) so far has addressed all the above-described 4 challenges facing asthmatics. Highlighted in this review is the calcium-sensitive passive stiffness of the ASM layer that directly affects airway distensibility. This passive stiffness is “regulatable” because it is mechanically adjustable (e.g., through strain softening and length adaptation) and it can be modulated through intervention of the signaling pathways associated with ASM activation, thus making it an important component for a comprehensive approach to improve airway distensibility in asthmatics. References [1] Colebatch HJ, Finucane KE, Smith MM. Pulmonary conductance and elastic recoil relationships in asthma and emphysema. J Appl Physiol 1973;34: 143e53. [2] Colbatch HJ, Greave IA, Ng CKY. Pulmonary mechanics in diagnosis. In: De Kock MA, Nadel JA, Lewis CM, editors. Mechanisms of airway obstraction in human respiratory diseases. Rotterdam: Pub: Balkema; 1979. [3] Wilson JW, Li X, Pain MC. The lack of distensibility of asthmatic airways. Am Rev Respir Dis 1993;148(3):806e9. [4] Johns DP, Wilson J, Harding R, Walters EH. Airway distensibility in healthy and asthmatic subjects: effect of lung volume history. J Appl Physiol 2000;88(4): 1413e20. [5] Ward C, Johns DP, Bish R, Pais M, Reid DW, Ingram C, et al. Reduced airway distensibility, fixed airflow limitation, and airway wall remodeling in asthma. Am J Respir Crit Care Med 2001;164(9):1718e21. [6] Brown NJ, Salome CM, Berend N, Thorpe CW, King GG. Airway distensibility in adults with asthma and healthy adults, measured by forced oscillation technique. Am J Respir Crit Care Med 2007;176(2):129e37. [7] Pyrgos G, Scichilone N, Togias A, Brown RH. Bronchodilation response to deep inspirations in asthma is dependent on airway distensibility and air trapping. J Appl Physiol 2011;110(2):472e9. [8] Siegman MJ, Butler TM, Mooers SU, Davies RE. Calcium-dependent resistance to stretch and stress relaxation in the resting smooth muscle. Am J Physiol 1976;231(5):1051e8. [9] Raqeeb A, Jiao Y, Syyong HT, Pare PD, Seow CY. Regulatable stiffness in relaxed airway smooth muscle: a target for asthma treatment? J Appl Physiol 2011; 112(3):337e46. [10] Bossé Y, Solomon D, Chin LY, Lian K, Paré PD, Seow CY. Increase in passive stiffness at reduced airway smooth muscle length: potential impact on airway responsiveness. Am J Physiol Lung Cell Mol Physiol 2010;298(3): L277e87. [11] Dillon PF, Aksoy MO, Driska SP, Murphy RA. Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science 1981;211(4481): 495e7. [12] Wang L, Paré PD, Seow CY. Selected contribution: effect of chronic passive length change on airway smooth muscle length-tension relationship. J Appl Physiol 2001;90(2):734e40. [13] Speich JE, Dosier C, Borgsmiller L, Quintero K, Koo HP, Ratz PH. Adjustable passive length-tension curve in rabbit detrusor smooth muscle. J Appl Physiol 2007;102(5):1746e55. [14] Pratusevich VR, Seow CY, Ford LE. Plasticity in canine airway smooth muscle. J Gen Physiol 1995;105(1):73e94. [15] Naghshin J, Wang L, Pare PD, Seow CY. Adaptation to chronic length change in explanted airway smooth muscle. J Appl Physiol 2003;95(1):448e53. [16] Maksym GN, Bates JH. A distributed nonlinear model of lung tissue elasticity. J Appl Physiol 1997;82(1):32e41. [17] Bai TR, Bates JH, Brusasco V, Camoretti-Mercado B, Chitano P, Deng LH, et al. On the terminology for describing the length-force relationship and its changes in airway smooth muscle. J Appl Physiol 2004;97(6):2029e34. [18] Trepat X, Deng L, An SS, Navajas D, Tschumperlin DJ, Gerthoffer WT, et al. Universal physical responses to stretch in the living cell. Nature 2007; 447(7144):592e5. [19] Chen C, Krishnan R, Zhou E, Ramachandran A, Tambe D, Rajendran K, et al. Fluidization and resolidification of the human bladder smooth muscle cell in response to transient stretch. PLoS One 2010;5(8):e12035. [20] Speich JE, Borgsmiller L, Call C, Mohr R, Ratz PH. ROK-induced cross-link formation stiffens passive muscle: reversible strain-induced stress softening in rabbit detrusor. Am J Physiol Cell Physiol 2005;289(1):C12e21.
C.Y. Seow / Pulmonary Pharmacology & Therapeutics 26 (2013) 37e41 [21] Herrera AM, McParland BE, Bienkowska A, Tait R, Paré PD, Seow CY. ‘Sarcomeres’ of smooth muscle: functional characteristics and ultrastructural evidence. J Cell Sci 2005;118(Pt 11):2381e92. [22] Huang Y, Zhang W, Gunst SJ. Activation of vinculin induced by cholinergic stimulation regulates contraction of tracheal smooth muscle tissue. J Biol Chem 2011;286(5):3630e44. [23] Zhang J, Herrera AM, Paré PD, Seow CY. Dense-body aggregates as plastic structures supporting tension in smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2010;299(5):L631e8. [24] Sobieszek A, Sarg B, Lindner H, Seow CY. Phosphorylation of caldesmon by myosin light chain kinase increases its binding affinity for phosphorylated myosin filaments. Biol Chem 2012;391(9):1091e104. [25] Lynch WP, Riseman VM, Bretscher A. Smooth muscle caldesmon is an extended flexible monomeric protein in solution that can readily undergo reversible intra- and intermolecular sulfhydryl cross-linking. A mechanism for caldesmon’s F-actin bundling activity. J Biol Chem 1987;262(15): 7429e37.
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[26] Kurashima K, Hoshi T, Takayanagi N, Takaku Y, Kagiyama N, Ohta C, et al. Airway dimensions and pulmonary function in chronic obstructive pulmonary disease and bronchial asthma. Respirology 2012 Jan;17(1):79e86. [27] Gunst SJ, Fredberg JJ. The first three minutes: smooth muscle contraction, cytoskeletal events, and soft glasses. J Appl Physiol 2003;95(1):413e25. [28] Bossé Y, Chin LY, Paré PD, Seow CY. Adaptation of airway smooth muscle to basal tone: relevance to airway hyperresponsiveness. Am J Respir Cell Mol Biol 2009;40(1):13e8. [29] Bossé Y, Chin LY, Paré PD, Seow CY. Chronic activation in shortened airway smooth muscle: a synergistic combination underlying airway hyperresponsiveness? Am J Respir Cell Mol Biol 2010;42(3):341e8. [30] Durrani SR, Viswanathan RK, Busse WW. What effect does asthma treatment have on airway remodeling? Current perspectives. J Allergy Clin Immunol 2011;128(3):439e48. [31] Xue Z, Yu Y, Gao H, Gunst SJ, Tepper RS. Chronic continuous positive airway pressure (CPAP) reduces airway reactivity in vivo in an allergen-induced rabbit model of asthma. J Appl Physiol 2011;111(2):353e7.
Contents lists available at SciVerse ScienceDirect
Pulmonary Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/ypupt
Passive stiffness of airway smooth muscle: The next target for improving airway distensibility and treatment for asthma?q Chun Y. Seow* Department of Pathology and Laboratory Medicine, The James Hogg Research Centre/St. Paul’s Hospital, University of British Columbia, 1081 Burrard Street, Rm. 166, Vancouver, BC V6Z 1Y6, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 March 2012 Received in revised form 25 June 2012 Accepted 27 June 2012
Reduced airway distensibility due to increased airway stiffness is a characteristic of asthma. Airway stiffness is determined by the property and structural organization of the various elements of the airway wall, and is often divided into active and passive components. Active stiffness is thought to be associated with activation of muscle cells in the airway wall. This component of stiffness can be inhibited when active force produced by the muscle is abolished. Passive stiffness, on the other hand, is thought to stem from non-muscle component of the airway wall, especially the collagen/elastin fibrous network of the extracellular matrix within which the muscle cells are embedded. In this brief review, the notion that passive stiffness is exclusively extracellular in origin is challenged. Recent evidence suggests that a substantial portion of the passive stiffness of an in vitro preparation of tracheal smooth muscle is calcium sensitive and is regulated by Rho-kinase, although the underlying mechanism and the details of regulation for the development of this intracellular passive stiffness are still largely unknown. To reduce airway stiffness different lines of attack must be tailored to different components of the stiffness. The regulatable passive stiffness is distinct from the relatively permanent stiffness of the extracellular matrix and the stiffness associated with active muscle contraction. To improve airway distensibility during asthma exacerbation, a comprehensive approach to reduce overall airway stiffness should therefore include a strategy for targeting the regulatable passive stiffness. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Regulatable airway stiffness Calcium-sensitive resistance to stretch Rho-kinase
It is known that airway distensibility is reduced in asthmatic patients and that multiple factors contribute to the stiffness of the airways [1e7]. Relaxing the smooth muscle in the airways increases airway distensibility in asthmatics as reflected in the improved pulmonary-conductance vs. recoil-pressure relationship, however, the asthmatic airways are still less distensible than normal even after the active muscle force is abolished [1,2]. A commonly held view is that remodeling of the asthmatic airways (more specifically, fibrotic thickening of the airway wall) as a result of chronic inflammation is responsible for the “permanent” increase in airway stiffness which is independent of smooth muscle tone. Airway distensibility is therefore thought to be largely determined by the structural and mechanical properties of the various components of the airway wall and these properties are usually classified as active (muscle tone dependent) or passive (muscle tone independent). Thus, those associated with activated airway smooth muscle (ASM) and regulated by calcium signaling pathways are referred to as q This work was supported by the Canadian Institutes of Health Research (CIHR). * Tel.: þ 1 604 806 9268; fax: þ 1 604 806 9274. E-mail address: [email protected]. 1094-5539/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pupt.2012.06.012
active properties and those exhibited by unstimulated ASM and non-ASM tissues are passive properties. However, tensile stiffness (i.e., the resistance to stretch) of unstimulated smooth muscle defies this simple classification. It is regarded as passive stiffness because it is a property associated with the muscle in the relaxed state, and yet it is calcium sensitive [8] and is regulated by enzymatic pathways associated with cell activation as revealed by recent studies [9,10]. The contribution of passive stiffness of ASM to airway distensibility could be mistakenly mixed up with those from non-muscle components of the airway, such as the collagen-elastin extracellular matrix whose properties can be altered in airway remodeling. One feature that differentiates passive ASM stiffness from the airway stiffness of non-muscle origin is that it can be altered rapidly through intervention of the signaling pathways [9]; airway remodeling (or its reversal), on the other hand, could change airway stiffness only over a prolonged process. Manipulating active tone in ASM for rapid modulation of airway stiffness is therefore not the only option we have. Passive ASM stiffness as a target for improving airway distensibility should be explored. Any cellular process or property regulated by enzymatic pathways is likely to serve some physiological function. This raises
1. The phenomenon of readily changeable passive tension and stiffness in ASM For the purpose of discussion in this review, passive muscle properties refer to those exhibited by muscles in the relaxed state and in the absence of detectable active tone. There are many ways with which muscle stiffness can be measured. Fig. 1 illustrates a simple protocol of estimating the stiffness. We have found that a value of 5e10% of resting muscle length for the length step (DL, Fig. 1) and a speed of stretch of 10 ms for the step change in length applied to a relaxed ASM preparation produces reliable measurements of muscle stiffness without altering the muscle’s ability to generate active force in subsequently induced contractions [9]. The clue that the development of passive stiffness in the relaxed smooth muscle may be activation-history dependent is evident in earlier studies showing a shift in the passive length-tension curve of smooth muscle adapted to different lengths [12e15]. Passive tension in unstimulated smooth muscle bundle increases approximately exponentially with muscle length (e.g., gray curve in Fig. 2).
ΔL
ΔF
Fig. 1. Illustration of time records of a step-change in length (upper trace) applied to a smooth muscle strip and force response (lower trace) from the strip. The ratio of DF/DL can be used as an estimate of muscle stiffness or resistance to stretch.
L ref
a question as to what physiological function may be served by passive stiffness of smooth muscle. In hollow organs smooth muscle modulates tension in the organ wall and maintains the organ shape. Active contraction and relaxation of smooth muscle is thought to be the driving force behind the changes in organ dimension. However, for maintaining a constant organ dimension or wall tension over a prolonged period of time, more efficient mechanisms may be preferred. In vascular smooth muscle it has been proposed that during a sustained contraction dephosphorylated myosin crossbridges may bind to actin filaments to form “latch” bridges that are able to maintain tension with low cycling rates and therefore low energetic cost [11]. In smooth muscles that possess little or no tone in the resting state, such as those found in airway and bladder [12,13], passive stiffness may be an alternative mechanism for maintaining wall tension in the relaxed state and modulating organ distensibility. In this brief review the effect of airway tensile stiffness (presumed to largely stem from the ASM layer) on airway distensibility is emphasized. This does not mean that airway stiffness exerts no effect on other mechanical properties of the airway or ASM itself. Passive compressive stiffness of the ASM could modulate shortening velocity of ASM, for example.
0.5 L ref
C.Y. Seow / Pulmonary Pharmacology & Therapeutics 26 (2013) 37e41
Tension
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Length Fig. 2. Illustration of passive lengthetension relationships obtained from a smooth muscle strip adapted at its in situ length (used as a reference length, Lref) and half in situ length. The in situ passive length-tension curve (gray) is shifted horizontally to the left as the muscle is shortened and adapted at half of the in situ length.
Traditional explanation for this lengthetension property of resting smooth muscle is based on the assumption that most of the tension bearing structures reside in extracellular matrix in the form of elastin and collagen fibers. It is further assumed that the more compliant portion of the length-tension curve is mostly determined by elastin fibers and the stiffer portion of the curve is mostly determined by collagen fibers. Based on these principles Maksym and Bates [16] are able to demonstrate in a mathematical model of “exponential” stressestrain relationship typical of that seen in lung tissue. Small variations in the lengthetension relationship of resting smooth muscle could be explained by viscoelastic properties of the tissue. However, large shifts in the relationship, as that shown in Fig. 2, are not likely a manifestation of tissue viscoelasticity. (This argument will be made more compellingly when we discuss calcium-sensitive and enzyme-activity-dependent passive tissue properties in the following section). The shift in the lengthetension curve described in Fig. 2 involves adaptation of smooth muscle at different lengths. The gray curve is associated with the muscle adapted to its in situ or reference length (Lref). Adaptation of a muscle to a certain length often requires repeated cycles of contraction and relaxation under isometric conditions for a period of time during which maximal active force increases incrementally toward a plateau [17]. In the example shown in Fig. 2, the muscle is shortened to half of its initial reference length. The shift in the passive length-tension curve does not occur immediately. The gradual change in the muscle properties associated with length adaptation is better illustrated in Fig. 3, where the increase in passive stiffness over time is monitored. Immediately after a relaxed ASM is shortened by half, passive tension and stiffness are reduced to zero from a reference value determined before the length change (Fig. 3). During adaptation at the shortened length, passive stiffness increases toward a plateau. It should be pointed out that in adapting to a shortened length, unlike passive stiffness, passive tension often remains unchanged (at zero tension). Passive tension and passive stiffness therefore reflect different underlying mechanisms responsible for their evolution. For example, if slowly cycling cross-bridges exist in the resting state and are responsible for the development of stiffness, they will in time cause “passive” resting tension to increase. On the other hand, if muscle stiffness in the resting state stems from crosslinking of non-cycling cross-linkers (not necessarily myosin cross-bridges) then the development of stiffness will not have an effect on resting tension. The underlying mechanism for the development of passive stiffness is not clear (the cross-linker
Stiffness/Stiffness at L ref
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3. Cell regulation of passive stiffness
Abrupt length change: Lref → 0.5 Lref
Reference stiffness
Time Fig. 3. Illustration of time course of recovery of passive stiffness (resistance to stretch) after an abrupt shortening of resting muscle from Lref to 0.5 Lref. Although the stiffness is measured in the relaxed state, in between measurements the muscle is activated briefly with electrical stimulation. The stiffness recovery normally reaches a plateau in about 30e40 min.
hypothesis is just one of the many possibilities), but the consequence is clear and drastic. As illustrated in Fig. 2, upon restretching the muscle that had been adapted to a short length back to its in situ length, the passive tension can reach a level much higher than the active force the muscle can generate at its in situ length. In the experiments by Wang et al. [12] and Naghshin et al. [15], it was found that the muscle strips could not be returned to their in situ length (after they had been adapted to shorter lengths) in a single step without causing permanent damage to the muscle tissue because of the high passive tension; the muscle could be returned to their in situ length only with multi-steps of incremental length increase followed by adaptation. 2. Calcium-sensitive vs. calcium-insensitive stiffness The smooth muscle layer in an airway is composed of ASM cells embedded in (mostly collagen-elastin) extracellular matrix. Mechanical properties of muscle preparations dissected from the muscle layer are therefore reflective of the properties of muscle cells and extracellular matrix. The most simplistic model regards the extracellular matrix as a parallel elastic component of the muscle preparation, with the matrix bearing all externally applied (passive) tension when the muscle cells are not activated. Because the properties of extracellular matrix are relatively inert, the relationship between passive tension and muscle length is considered to be static. The finding of changeable and regulatable passive stiffness in smooth muscle preparations (representative of the muscle layer in airways) indicates that not all passive tension is borne by extracellular matrix, and that smooth muscle cells are responsible for some of the passive tension. Removal of intracellular and extracellular calcium by incubating the muscle in calcium-free Krebs solution with EGTA causes a substantial decrease in the passive stiffness, and the loss of passive stiffness can be restored by reintroducing calcium back to the Krebs solution [9]. When extracellular calcium is prevented from entering the muscle cells by calcium-channel blockers active force is abolished over time, suggesting that intracellular calcium has been depleted. Under this condition, there is a loss of passive stiffness just the same as that found in calcium-free Krebs [9]. It is therefore tempting to conclude that the calcium-sensitive stiffness is of intracellular origin and the calcium-insensitive stiffness, extracellular. However, a definitive conclusion will require more direct evidence, possibly from single cell preparations in which muscle force, stiffness, and intracellular calcium can be monitored.
Calcium release into intracellular space triggers a cascade of reactions that characterizes cell activation. In smooth muscle cells the release of calcium leads to activation of actomyosin interaction and the development of active force and stiffening of the muscle. Re-sequestration of calcium returns the force to its resting level and abolishes the stiffness associated with active actomyosin binding, but retains the passive stiffness. As discussed in the previous section, part of the passive stiffness is likely intracellular in origin, because of its calcium sensitivity. Another characteristic of the passive stiffness is that it can be “softened” by large strains applied to the muscle cells [9,18,19]. In ASM strips, after strain softening, passive stiffness recovers completely in about 20 min [9]. In the experiments described by Raqeeb et al. [9], during the recovering period, the muscle was briefly and periodically activated (once every 5 min), and this appeared to aid the stiffness recovery. In rabbit urinary bladder smooth muscle, activation has also been shown to facilitate recovery of passive stiffness lost due to strain softening [20]. This is probably because the development of passive stiffness is Rho-kinase dependent, as demonstrated in both airway and bladder smooth muscles that Y27632 (Rho-kinase inhibitor) prevents redevelopment of a portion of the passive stiffness after strain softening [9,20]. The portion of passive stiffness sensitive to Y27632 is likely of intracellular origin, because the Rho-kinase inhibitor is not known to act extracellularly. Interestingly, the redevelopment of calcium-sensitive passive stiffness after strain softening is not affected by inhibition of the myosin light chain kinase (MLCK) [9,10]. With sufficiently high concentrations of ML-7 (an MLCK inhibitor), active force of ASM can be abolished but the ability of the muscle to redevelop passive stiffness after strain softening remains intact [9,10]. This suggests that the mechanism underlying active force generation in smooth muscle is separate from that responsible for stiffness development. This, however, does not preclude the possibility that the dephosphorylated, non-cycling (truly latch) myosin cross-bridges are the source of the passive stiffness. If this is the case, one would expect to see a positive correlation between the calcium-sensitive passive stiffness and the number of cross-bridges overlapping the actin filaments. Because the extent of cross-bridge overlap is proportional to the active force generated by the muscle, as indicated by the lengtheforce relationship [21], one would expect a positive correlation, over a certain length range, between calcium-sensitive passive stiffness and the ability of the muscle to generate force. However, by comparing the length vs. active force relationship and length vs. passive stiffness relationship, a negative correlation is found between active force and calcium-sensitive passive stiffness [9]. The results therefore do not support that notion that crosslinking of myosin cross-bridges with actin filaments is the source (or at least the main source) of the calcium-sensitive passive stiffness. This has raised an intriguing question of what is responsible for the calcium-sensitive passive stiffness. Part of the stiffness likely stems from ionic interactions among proteins and protein aggregates in the “crowded” intracellular environment [18,19]. At least some of these interactions must be calcium dependent for them to manifest as calcium-sensitive stiffness. Some of the interactions may only occur under activated condition but the consequence of the interaction (such as formation of cross-links) must be retained under resting condition for it to be considered as a “passive” property. Some interactions may occur at the basal level of calcium under the relaxed condition; it appears that at zero-calcium, the passive stiffness is all extracellular in origin [9]. If cross-linking of protein filaments is the mechanism underlying the intracellular passive stiffness, several players can be
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identified. On the filament side, actin filaments are probably the most important simply because they are the most abundant. Other filaments such as intermediate filaments, microtubules, and myosin filaments are possible players too. On the cross-linker side, there are many actin binding or bundling proteins, a-actinin and filamin for example. Adapter proteins that anchor actin filaments to adhesion plaques, such as vinculin, paxillin, and talin [22], may play a role as well. Proteins that bind both actin and intermediate filaments (such as a-actinin) could have a special role in the formation of dense-body “cables” that appear to be able to bear passive tension [23]. 3-D reconstruction reveals that the dense-body cables are made of dense bodies connected together in series by actin and intermediate filaments, and the cable length is adjustable according to the muscle cell length [23]. What regulates the cable length is not known, but the plastic cable could be a source of the regulatable passive stiffness. Another possible source of the passive stiffness could reside in the network of interaction among caldesmon, myosin, and actin filaments. We have found recently that caldesmon can be phosphorylated by Ca2þ-calmodulin-MLCK and that the time course of phosphorylation coincides with the development of “latch” bridges that is thought to be responsible for the maintenance of tension in the sustained phase of contraction [24]. In the presence of myosin, it is difficult to discern the exact contribution of caldesmon phosphorylation to the “latch” phenomenon, because caldesmon is able to bind both myosin and actin. It has been shown in vitro that caldesmon can reversibly cross-link actin filaments [25]. So there are two possible ways that caldesmon could contribute to the passive stiffness, one is through binding of myosin and actin, and the other is through binding of actin and actin. 4. Increasing airway distensibility as a strategy to improve lung function in asthmatics A recent study showed that asthmatic airways (from third to sixth generations) are narrower than those of normal controls [26]. To distend the airways back to normal diameters, asthmatics face several challenges: 1) If we approximate an airway as a thin-walled cylindrical vessel, LaPlace Law (governing the relationship between wall tension, distending pressure, and airway diameter) works against asthmatics, because it dictates that for countering the same wall tension a greater distending pressure is required in an airway with smaller diameter. 2) Many of the inflammatory mediators found in asthmatic airways are contractile agonists for ASM; chronic airway inflammation thus can lead to elevated ASM tone (or wall tension) and that the ASM may enter a “frozen” state [27] and become refractory to the relaxing effect of oscillatory stress that accompanies tidal breathing and deep inspirations. 3) Length adaptation [17] and force adaptation [28,29] of ASM at shorter lengths also work against asthmatics, because they allow the muscle to retain maximal contractility at short lengths. 4) The shift in the passive lengthetension relationship [12,15] and increase in passive stiffness associated with adaptation to short lengths [9,10] effectively augments airway wall tension under distending pressure. To overcome these challenges a comprehensive strategy that addresses all causes impeding airway distension (thus reducing airway resistance) need to be developed. It should be pointed out that in some airway diseases such as bronchiectasis and bronchomalacia an increase in airway distensibility is often associated with poor lung function and even collapsed airways. These should be distinguished from asthmatic airways where an increase in airway distensibility is often associated with improved lung function [1,2]. Relaxing ASM (with short and long acting b(2)-agonists) addresses only one cause of airway obstruction in asthma. The use of corticosteroids to suppress airway
inflammation can lead to reduced ASM tone and may prevent or reverse airway remodeling [30], but again the treatment does not address all the challenges asthmatics face. A recent innovative approach in treating airway reactivity in an allergen-induced rabbit model of asthma with continuous positive airway pressure (CPAP) [31] has proven to be effective. This is perhaps not surprising because its addresses the geometric and adaptive properties of asthmatic airways and ASM. However, none of the treatments of asthma (including animal models of asthma) so far has addressed all the above-described 4 challenges facing asthmatics. Highlighted in this review is the calcium-sensitive passive stiffness of the ASM layer that directly affects airway distensibility. This passive stiffness is “regulatable” because it is mechanically adjustable (e.g., through strain softening and length adaptation) and it can be modulated through intervention of the signaling pathways associated with ASM activation, thus making it an important component for a comprehensive approach to improve airway distensibility in asthmatics. References [1] Colebatch HJ, Finucane KE, Smith MM. Pulmonary conductance and elastic recoil relationships in asthma and emphysema. J Appl Physiol 1973;34: 143e53. [2] Colbatch HJ, Greave IA, Ng CKY. Pulmonary mechanics in diagnosis. In: De Kock MA, Nadel JA, Lewis CM, editors. Mechanisms of airway obstraction in human respiratory diseases. Rotterdam: Pub: Balkema; 1979. [3] Wilson JW, Li X, Pain MC. The lack of distensibility of asthmatic airways. Am Rev Respir Dis 1993;148(3):806e9. [4] Johns DP, Wilson J, Harding R, Walters EH. Airway distensibility in healthy and asthmatic subjects: effect of lung volume history. J Appl Physiol 2000;88(4): 1413e20. [5] Ward C, Johns DP, Bish R, Pais M, Reid DW, Ingram C, et al. Reduced airway distensibility, fixed airflow limitation, and airway wall remodeling in asthma. Am J Respir Crit Care Med 2001;164(9):1718e21. [6] Brown NJ, Salome CM, Berend N, Thorpe CW, King GG. Airway distensibility in adults with asthma and healthy adults, measured by forced oscillation technique. Am J Respir Crit Care Med 2007;176(2):129e37. [7] Pyrgos G, Scichilone N, Togias A, Brown RH. Bronchodilation response to deep inspirations in asthma is dependent on airway distensibility and air trapping. J Appl Physiol 2011;110(2):472e9. [8] Siegman MJ, Butler TM, Mooers SU, Davies RE. Calcium-dependent resistance to stretch and stress relaxation in the resting smooth muscle. Am J Physiol 1976;231(5):1051e8. [9] Raqeeb A, Jiao Y, Syyong HT, Pare PD, Seow CY. Regulatable stiffness in relaxed airway smooth muscle: a target for asthma treatment? J Appl Physiol 2011; 112(3):337e46. [10] Bossé Y, Solomon D, Chin LY, Lian K, Paré PD, Seow CY. Increase in passive stiffness at reduced airway smooth muscle length: potential impact on airway responsiveness. Am J Physiol Lung Cell Mol Physiol 2010;298(3): L277e87. [11] Dillon PF, Aksoy MO, Driska SP, Murphy RA. Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science 1981;211(4481): 495e7. [12] Wang L, Paré PD, Seow CY. Selected contribution: effect of chronic passive length change on airway smooth muscle length-tension relationship. J Appl Physiol 2001;90(2):734e40. [13] Speich JE, Dosier C, Borgsmiller L, Quintero K, Koo HP, Ratz PH. Adjustable passive length-tension curve in rabbit detrusor smooth muscle. J Appl Physiol 2007;102(5):1746e55. [14] Pratusevich VR, Seow CY, Ford LE. Plasticity in canine airway smooth muscle. J Gen Physiol 1995;105(1):73e94. [15] Naghshin J, Wang L, Pare PD, Seow CY. Adaptation to chronic length change in explanted airway smooth muscle. J Appl Physiol 2003;95(1):448e53. [16] Maksym GN, Bates JH. A distributed nonlinear model of lung tissue elasticity. J Appl Physiol 1997;82(1):32e41. [17] Bai TR, Bates JH, Brusasco V, Camoretti-Mercado B, Chitano P, Deng LH, et al. On the terminology for describing the length-force relationship and its changes in airway smooth muscle. J Appl Physiol 2004;97(6):2029e34. [18] Trepat X, Deng L, An SS, Navajas D, Tschumperlin DJ, Gerthoffer WT, et al. Universal physical responses to stretch in the living cell. Nature 2007; 447(7144):592e5. [19] Chen C, Krishnan R, Zhou E, Ramachandran A, Tambe D, Rajendran K, et al. Fluidization and resolidification of the human bladder smooth muscle cell in response to transient stretch. PLoS One 2010;5(8):e12035. [20] Speich JE, Borgsmiller L, Call C, Mohr R, Ratz PH. ROK-induced cross-link formation stiffens passive muscle: reversible strain-induced stress softening in rabbit detrusor. Am J Physiol Cell Physiol 2005;289(1):C12e21.
C.Y. Seow / Pulmonary Pharmacology & Therapeutics 26 (2013) 37e41 [21] Herrera AM, McParland BE, Bienkowska A, Tait R, Paré PD, Seow CY. ‘Sarcomeres’ of smooth muscle: functional characteristics and ultrastructural evidence. J Cell Sci 2005;118(Pt 11):2381e92. [22] Huang Y, Zhang W, Gunst SJ. Activation of vinculin induced by cholinergic stimulation regulates contraction of tracheal smooth muscle tissue. J Biol Chem 2011;286(5):3630e44. [23] Zhang J, Herrera AM, Paré PD, Seow CY. Dense-body aggregates as plastic structures supporting tension in smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2010;299(5):L631e8. [24] Sobieszek A, Sarg B, Lindner H, Seow CY. Phosphorylation of caldesmon by myosin light chain kinase increases its binding affinity for phosphorylated myosin filaments. Biol Chem 2012;391(9):1091e104. [25] Lynch WP, Riseman VM, Bretscher A. Smooth muscle caldesmon is an extended flexible monomeric protein in solution that can readily undergo reversible intra- and intermolecular sulfhydryl cross-linking. A mechanism for caldesmon’s F-actin bundling activity. J Biol Chem 1987;262(15): 7429e37.
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[26] Kurashima K, Hoshi T, Takayanagi N, Takaku Y, Kagiyama N, Ohta C, et al. Airway dimensions and pulmonary function in chronic obstructive pulmonary disease and bronchial asthma. Respirology 2012 Jan;17(1):79e86. [27] Gunst SJ, Fredberg JJ. The first three minutes: smooth muscle contraction, cytoskeletal events, and soft glasses. J Appl Physiol 2003;95(1):413e25. [28] Bossé Y, Chin LY, Paré PD, Seow CY. Adaptation of airway smooth muscle to basal tone: relevance to airway hyperresponsiveness. Am J Respir Cell Mol Biol 2009;40(1):13e8. [29] Bossé Y, Chin LY, Paré PD, Seow CY. Chronic activation in shortened airway smooth muscle: a synergistic combination underlying airway hyperresponsiveness? Am J Respir Cell Mol Biol 2010;42(3):341e8. [30] Durrani SR, Viswanathan RK, Busse WW. What effect does asthma treatment have on airway remodeling? Current perspectives. J Allergy Clin Immunol 2011;128(3):439e48. [31] Xue Z, Yu Y, Gao H, Gunst SJ, Tepper RS. Chronic continuous positive airway pressure (CPAP) reduces airway reactivity in vivo in an allergen-induced rabbit model of asthma. J Appl Physiol 2011;111(2):353e7.