Critical roles of airway smooth muscle in mediating deep-inspiration-induced bronchodilation: a big stretch?

BACKGROUND: Deep inspiration (DI) has been shown to induce bronchodilation and bronchoprotection in bronchochallenged healthy subjects, but not in asthmatics. Strain-induced relaxation of airway smooth muscle (ASM) is considered one of the factors responsible for these effects. Other factors include the release or redistribution of pulmonary surfactant, alteration in mucus plugs, and changes in airway heterogeneity. MAIN BODY: The present review is focused on the DI effect on ASM function, based on recent findings from ex vivo sheep lung experiments showing a large change in airway diameter during a DI. The amount of stretch on the airways, when applied to isolated airway rings in vitro, caused a substantial decrease in ASM contractility that takes many minutes to recover. When challenged with a bronchoconstrictor, the increase in pulmonary resistance in the ex vivo ovine lungs is mostly due to the increase in airway resistance. CONCLUSIONS: Although non-ASM related factors cannot be excluded, the large strain on the airways associated with a DI substantially reduces ASM contractility and thus can account for most of the bronchodilatory and bronchoprotective effects of DI.

Airway and parenchymal tissue resistance and elastance in ex vivo sheep lungs: effects of bronchochallenge and deep inspiration.

Lung resistance (RL) is determined by airway and parenchymal tissue resistance, as well as the degree of heterogeneity in airway constriction. Deep inspirations (DIs) are known to reverse experimentally induced increase in RL, but the mechanism is not entirely clear. The first step toward understanding the effect of DI is to determine how each of the resistance components is affected by DI. In the present study, we measured RL and apparent airway resistance (RAW, which combines the effects of airway resistance and airway heterogeneity) simultaneously before and after a DI in acetylcholine (ACh)-challenged ex vivo sheep lungs. We found that at normal breathing frequency (0.25 Hz) ACh-challenge led to a doubling of RL, 80.3% of that increase was caused by an increase in RAW; the increase in apparent tissue resistance (RT) was insignificant. 57.7% of the increase in RAW was abolished by a single DI. After subtracting RAW from RL, the remaining RT was mostly independent of ACh-challenge and its reduction after a DI came mostly from the change in the mechanical properties of lung parenchyma. We conclude that at normal breathing frequency, RL in an unchallenged lung is mostly composed of RT, and the increase in RL due to ACh-challenge stems mostly from the increase in RAW and that both RAW and RT can be greatly reduced by a DI, likely due to a reduction in true airway resistance and heterogeneity, as well as parenchymal tissue hysteresis post DI.

Lung resistance and elastance are different in ex vivo sheep lungs ventilated by positive and negative pressures.

Lung resistance (RL) and elastance (EL) can be measured during positive or negative pressure ventilation. Whether the different modes of ventilation produce different RL and EL is still being debated. Although negative pressure ventilation (NPV) is more physiological, positive pressure ventilation (PPV) is more commonly used for treating respiratory failure. In the present study, we measured lung volume, airway diameter, and airway volume, as well as RL and EL with PPV and NPV in explanted sheep lungs. We found that lung volume under a static pressure, either positive or negative, was not different. However, RL and EL were significantly higher in NPV at high inflation pressures. Interestingly, diameters of smaller airways (diameters <3.5 mm) and total airway volume were significantly greater at high negative inflation pressures compared with those at high positive inflation pressures. This suggests that NPV is more effective in distending the peripheral airways, likely due to the fact that negative pressure is applied through the pleural membrane and reaches the central airways via the peripheral airways, whereas positive pressure is applied in the opposite direction. More distension of lung periphery could explain why RL is higher in NPV (vs. PPV), because the peripheral parenchyma is a major source of tissue resistance, which is a part of the RL that increases with pressure. This explanation is consistent with the finding that during high frequency ventilation (>1 Hz, where RL reflects airway resistance more than tissue resistance), the difference in RL between NPV and PPV disappeared.

Airway diameter at different transpulmonary pressures in ex vivo sheep lungs: implications for deep inspiration-induced bronchodilation and bronchoprotection.

Deep inspiration (DI)-induced bronchodilation is the first line of defense against bronchoconstriction in healthy subjects. A hallmark of asthma is the lack of this beneficial effect of DI. The mechanism underlying the bronchodilatory effect of DI is not clear. Understanding the mechanism will help us unravel the mystery of asthma pathophysiology. It has been postulated that straining airway smooth muscle (ASM) during a DI could lead to bronchodilation and bronchoprotection. The hypothesis is currently under debate, and a central question is whether ASM is sufficiently stretched during a DI for its contractility to be compromised. Besides bronchoconstriction, another contributor to lung resistance is airway heterogeneity. The present study examines changes in airway diameter and heterogeneity at different lung volumes. Freshly explanted sheep lungs were used in plethysmographic measurements of lung resistance and elastance at different lung volumes, whereas the airway dimensions were measured by computed tomography (CT). The change in airway diameter informed by CT measurements was applied to isolated airway ring preparations to determine the strain-induced loss of ASM contractility. We found that changing the transpulmonary pressure from 5 to 30 cmH2O led to a 51% increase in lung volume, accompanied by a 46% increase in the airway diameter with no change in airway heterogeneity. When comparable airway strains measured in the whole lung were applied to isolated airway rings in either relaxed or contracted state, a significant loss of ASM contractility was observed, suggesting that DI-induced bronchodilation and bronchoprotection can result from strain-induced loss of ASM contractility.

p116Rip promotes myosin phosphatase activity in airway smooth muscle cells.

Myosin phosphatase-Rho interacting protein (p116Rip ) was originally found as a RhoA-binding protein. Subsequent studies by us and others revealed that p116Rip facilitates myosin light chain phosphatase (MLCP) activity through direct and indirect manners. However, it is unclear how p116Rip regulates myosin phosphatase activity in cells. To elucidate the role of p116Rip in cellular contractile processes, we suppressed the expression of p116Rip by RNA interference in human airway smooth muscle cells (HASMCs). We found that knockdown of p116Rip in HASMCs led to increased di-phosphorylated MLC (pMLC), that is phosphorylation at both Ser19 and Thr18. This was because of a change in the interaction between MLCP and myosin, but not an alteration of RhoA/ROCK signaling. Attenuation of Zipper-interacting protein kinase (ZIPK) abolished the increase in di-pMLC, suggesting that ZIPK is involved in this process. Moreover, suppression of p116Rip expression in HASMCs substantially increased the histamine-induced collagen gel contraction. We also found that expression of the p116Rip was decreased in the airway smooth muscle tissue from asthmatic patients compared with that from non-asthmatic patients, suggesting a potential role of p116Rip expression in asthma pathogenesis.

Mechanopharmacology of Rho-kinase antagonism in airway smooth muscle and potential new therapy for asthma.

The principle of mechanopharmacology of airway smooth muscle (ASM) is based on the premise that physical agitation, such as pressure oscillation applied to an airway, is able to induce bronchodilation by reducing contractility and softening the cytoskeleton of ASM. Although the underlying mechanism is not entirely clear, there is evidence to suggest that large-amplitude stretches are able to disrupt the actomyosin interaction in the crossbridge cycle and weaken the cytoskeleton in ASM cells. Rho-kinase is known to enhance force generation and strengthen structural integrity of the cytoskeleton during smooth muscle activation and plays a key role in the maintenance of force during prolonged muscle contractions. Synergy in relaxation has been observed when the muscle is subject to oscillatory length change while Rho-kinase is pharmacologically inhibited. In this review, inhibition of Rho-kinase coupled to therapeutic pressure oscillation applied to the airways is explored as a combination treatment for asthma.

The Huxley crossbridge model as the basic mechanism for airway smooth muscle contraction.

The cyclic interaction between myosin crossbridges and actin filaments underlies smooth muscle contraction. Phosphorylation of the 20-kDa myosin light chain (MLC20) is a crucial step in activating the crossbridge cycle. Our current understanding of smooth muscle contraction is based on observed correlations among MLC20 phosphorylation, maximal shortening velocity (Vmax), and isometric force over the time course of contraction. However, during contraction there are changes in the extent of phosphorylation of many additional proteins as well as changes in activation of enzymes associated with the signaling pathways. As a consequence, the mechanical manifestation of muscle contraction is likely to change with time. To simplify the study of these relationships, we measured the mechanical properties of airway smooth muscle at different levels of MLC20 phosphorylation at a fixed time during contraction. A simple correlation emerged when time-dependent variables were fixed. MLC20 phosphorylation was found to be directly and linearly correlated with the active stress, stiffness, and power of the muscle; the observed weak dependence of Vmax on MLC20 phosphorylation could be explained by the presence of an internal load in the muscle preparation. These results can be entirely explained by the Huxley crossbridge model. We conclude that when the influence of time-dependent events during contraction is held constant, the basic crossbridge mechanism in smooth muscle is the same as that in striated muscle.

Smooth muscle function and myosin polymerization.

Smooth muscle is able to function over a much broader length range than striated muscle. The ability to maintain contractility after a large length change is thought to be due to an adaptive process involving restructuring of the contractile apparatus to maximize overlap between the contractile filaments. The molecular mechanism for the length-adaptive behavior is largely unknown. In smooth muscle adapted to different lengths we quantified myosin monomers, basal and activation-induced myosin light chain (MLC) phosphorylation, shortening velocity, power output and active force. The muscle was able to generate a constant maximal force over a two fold length range when it was allowed to go through isometric contraction/relaxation cycles after each length change (length adaptation). In the relaxed state, myosin monomer concentration and basal MLC phosphorylation decreased linearly, while in the activated state activation-induced MLC phosphorylation and shortening velocity/power output increased linearly with muscle length. The results suggest that recruitment of myosin monomers and oligomers into the actin filament lattice (where they form force-generating filaments) occurs during muscle adaptation to longer length, with the opposite occurring during adaptation to shorter length.

Hyperresponsiveness: Relating the Intact Airway to the Whole Lung.

We relate changes of the airway wall to the response of the intact airway and the whole lung. We address how mechanical conditions and specific structural changes for an airway contribute to hyperresponsiveness resistant to deep inspiration. This review conveys that the origins of hyperresponsiveness do not devolve into an abnormality at single structural level but require examination of the complex interplay of all the parts.

Bronchodilatory effect of deep inspiration in freshly isolated sheep lungs.

Taking a big breath is known to reverse bronchoconstriction induced by bronchochallenge in healthy subjects; this bronchodilatory effect of deep inspiration (DI) is diminished in asthmatics. The mechanism underlying the DI effect is not clear. Observations from experiments using isolated airway smooth muscle (ASM) preparations and airway segments suggest that straining of ASM due to DI could lead to bronchodilation, possibly due to strain-induced reduction in ASM contractility. However, factors external to the lung cannot be excluded as potential causes for the DI effect. Neural reflex initiated by stretch receptors in the lung are known to inhibit the broncho-motor tone and enhance vasodilatation; the former directly reduces airway resistance, and the latter facilitates removal of contractile agonists through the bronchial circulation. If the DI effect is solely mediated by factors extrinsic to the lung, the DI effect would be absent in isolated, nonperfused lungs. Here we examined the DI effect in freshly isolated, nonperfused sheep lungs. We found that imposition of DI on isolated lungs resulted in significant bronchodilation, that this DI effect was present only after the lungs were challenged with a contractile agonist (acetylcholine or histamine), and that the effect was independent of the difference in lung volume observed pre- and post-DI. We conclude that a significant portion of the bronchodilatory DI effect stems from factors internal to the lung related to the activation of ASM.

Heterogeneity of airway wall dimensions in humans: a critical determinant of lung function in asthmatics and nonasthmatics.

Airway remodeling, a key feature of asthma, alters every layer of the airway wall but most strikingly the airway smooth muscle (ASM) layer. Airway remodeling in asthmatics contributes to fixed airflow obstruction and can amplify airway narrowing caused by ASM activation. Previous modeling studies have shown that the increase in ASM mass has the largest effect on increasing maximal airway narrowing. Simulated heterogeneity in the dimensions and properties of the airway wall can further amplify airway narrowing. Using measurements made on histological sections from donor lungs, we show for the first time that there is profound heterogeneity of ASM area and wall area in both nonasthmatics and asthmatics. Using a mathematical model, we found that this heterogeneity, together with changes in the mean values, contributes to an increased baseline resistance and elastance in asthmatics as well as a leftward shift in the responsiveness of the airways to a simulated agonist in both nonasthmatics and asthmatics. The ability of heterogeneous wall dimensions to shift the dose-response curve is largely due to an increased susceptibility for the small airways to close. This research confirms that heterogeneity of airway wall dimensions can contribute to exaggerated airway narrowing and provides an actual assessment of the magnitude of these effects.

Airway smooth muscle tone increases airway responsiveness in healthy young adults.

Force adaptation, a process whereby sustained spasmogenic activation (viz., tone) of airway smooth muscle (ASM) increases its contractile capacity, has been reported in isolated ASM tissues in vitro, as well as in mice in vivo. The objective of the present study was to assess the effect of tone on airway responsiveness in humans. Ten healthy volunteers underwent methacholine challenge on two occasions. One challenge consisted of six serial doses of saline followed by a single high dose of methacholine. The other consisted of six low doses of methacholine 5 min apart followed by a higher dose. The cumulative dose was identical for both challenges. After both methacholine challenges, subjects took a deep inspiration (DI) to total lung capacity as another way to probe ASM mechanics. Responses to methacholine and the DI were measured using a multifrequency forced oscillation technique. Compared with a single high dose, the challenge preceded by tone led to an elevated response measured by respiratory system resistance (Rrs) and reactance at 5 Hz. However, there was no difference in the increase in Rrs at 19 Hz, suggesting a predominant effect on smaller airways. Increased tone also reduced the efficacy of DI, measured by an attenuated maximal dilation during the DI and an increased renarrowing post-DI. We conclude that ASM tone increases small airway responsiveness to inhaled methacholine and reduces the effectiveness of DI in healthy humans. This suggests that force adaptation may contribute to airway hyperresponsiveness and the reduced bronchodilatory effect of DI in asthma.

Cystic Fibrosis Transmembrane Conductance Regulator in Sarcoplasmic Reticulum of Airway Smooth Muscle. Implications for Airway Contractility.

RATIONALE: An asthma-like airway phenotype has been described in people with cystic fibrosis (CF). Whether these findings are directly caused by loss of CF transmembrane conductance regulator (CFTR) function or secondary to chronic airway infection and/or inflammation has been difficult to determine. OBJECTIVES: Airway contractility is primarily determined by airway smooth muscle. We tested the hypothesis that CFTR is expressed in airway smooth muscle and directly affects airway smooth muscle contractility. METHODS: Newborn pigs, both wild type and with CF (before the onset of airway infection and inflammation), were used in this study. High-resolution immunofluorescence was used to identify the subcellular localization of CFTR in airway smooth muscle. Airway smooth muscle function was determined with tissue myography, intracellular calcium measurements, and regulatory myosin light chain phosphorylation status. Precision-cut lung slices were used to investigate the therapeutic potential of CFTR modulation on airway reactivity. MEASUREMENTS AND MAIN RESULTS: We found that CFTR localizes to the sarcoplasmic reticulum compartment of airway smooth muscle and regulates airway smooth muscle tone. Loss of CFTR function led to delayed calcium reuptake following cholinergic stimulation and increased myosin light chain phosphorylation. CFTR potentiation with ivacaftor decreased airway reactivity in precision-cut lung slices following cholinergic stimulation. CONCLUSIONS: Loss of CFTR alters porcine airway smooth muscle function and may contribute to the airflow obstruction phenotype observed in human CF. Airway smooth muscle CFTR may represent a therapeutic target in CF and other diseases of airway narrowing.

Gene expression analysis in asthma using a targeted multiplex array.

BACKGROUND: Gene expression changes in the structural cells of the airways are thought to play a role in the development of asthma and airway hyperresponsiveness. This includes changes to smooth muscle contractile machinery and epithelial barrier integrity genes. We used a targeted gene expression arrays to identify changes in the expression and co-expression of genes important in asthma pathology. METHODS: RNA was isolated from the airways of donor lungs from 12 patients with asthma (8 fatal) and 12 non-asthmatics controls and analyzed using a multiplexed, hypothesis-directed platform to detect differences in gene expression. Genes were grouped according to their role in airway dysfunction: airway smooth muscle contraction, cytoskeleton structure and regulation, epithelial barrier function, innate and adaptive immunity, fibrosis and remodeling, and epigenetics. RESULTS: Differential gene expression and gene co-expression analyses were used to identify disease associated changes in the airways of asthmatics. There was significantly decreased abundance of integrin beta 6 and Ras-Related C3 Botulinum Toxin Substrate 1 (RAC1) in the airways of asthmatics, genes which are known to play an important role in barrier function. Significantly elevated levels of Collagen Type 1 Alpha 1 (COL1A1) and COL3A1 which have been shown to modulate cell proliferation and inflammation, were found in asthmatic airways. Additionally, we identified patterns of differentially co-expressed genes related to pathways involved in virus recognition and regulation of interferon production. 7 of 8 pairs of differentially co-expressed genes were found to contain CCCTC-binding factor (CTCF) motifs in their upstream promoters. CONCLUSIONS: Changes in the abundance of genes involved in cell-cell and cell-matrix interactions could play an important role in regulating inflammation and remodeling in asthma. Additionally, our results suggest that alterations to the binding site of the transcriptional regulator CTCF could drive changes in gene expression in asthmatic airways. Several asthma susceptibility loci are known to contain CTCF motifs and so understanding the role of this transcription factor may expand our understanding of asthma pathophysiology and therapeutic options.

Ultrastructure of human tracheal smooth muscle from subjects with asthma and nonasthmatic subjects. Standardized methods for comparison.

A characteristic feature of asthma is exaggerated airway narrowing, termed airway hyper-responsiveness (AHR) due to contraction of airway smooth muscle (ASM). Although smooth muscle (SM)-specific asthma susceptibility genes have been identified, it is not known whether asthmatic ASM is phenotypically different from nonasthmatic ASM in terms of subcellular structure or mechanical function. The present study is the first to systematically quantify, using electron microscopy, the ultrastructure of tracheal SM from subjects with asthma and nonasthmatic subjects. Methodological details concerning tissue sample preparation, ultrastructural quantification, and normalization of isometric force by appropriate morphometric parameters are described. We reasoned that genetic and/or acquired differences in the ultrastructure of asthmatic ASM could be associated with functional changes. We recently reported that asthmatic ASM is better able to maintain and recover active force generation after length oscillations simulating deep inspirations. The present study was designed to seek structural evidence to account for this observation. Contrary to our hypotheses, no significant qualitative or quantitative differences were found in the subcellular structure of asthmatic versus nonasthmatic tracheal SM. Specifically, there were no differences in average SM cell cross-sectional area; fraction of the cell area occupied by nonfilamentous area; amounts of mitochondria, dense bodies, and dense plaques; myosin and actin filament densities; basal lamina thickness; and the number of microtubules. These results indicate that functional differences in ASM do not necessarily translate into observable structural changes.

Biphasic force response to iso-velocity stretch in airway smooth muscle.

Airway smooth muscle (ASM) in vivo is constantly subjected to oscillatory strain due to tidal breathing and deep inspirations. ASM contractility is known to be adversely affected by strains, especially those of large amplitudes. Based on the cross-bridge model of contraction, it is likely that strain impairs force generation by disrupting actomyosin cross-bridge interaction. There is also evidence that strain modulates muscle stiffness and force through induction of cytoskeletal remodeling. However, the molecular mechanism by which strain alters smooth muscle function is not entirely clear. Here, we examine the response of ASM to iso-velocity stretches to probe the components within the muscle preparation that give rise to different features in the force response. We found in ASM that force response to a ramp stretch showed a biphasic feature, with the initial phase associated with greater muscle stiffness compared with that in the later phase, and that the transition between the phases occurred at a critical strain of ∼3.3%. Only strains with amplitudes greater than the critical strain could lead to reduction in force and stiffness of the muscle in the subsequent stretches. The initial-phase stiffness was found to be linearly related to the degree of muscle activation, suggesting that the stiffness stems mainly from attached cross bridges. Both phases were affected by the degree of muscle activation and by inhibitors of myosin light-chain kinase, PKC, and Rho-kinase. Different responses due to different interventions suggest that cross-bridge and cytoskeletal stiffness is regulated differently by the kinases.

Force maintenance and myosin filament assembly regulated by Rho-kinase in airway smooth muscle.

Smooth muscle contraction can be divided into two phases: the initial contraction determines the amount of developed force and the second phase determines how well the force is maintained. The initial phase is primarily due to activation of actomyosin interaction and is relatively well understood, whereas the second phase remains poorly understood. Force maintenance in the sustained phase can be disrupted by strains applied to the muscle; the strain causes actomyosin cross-bridges to detach and also the cytoskeletal structure to disassemble in a process known as fluidization, for which the underlying mechanism is largely unknown. In the present study we investigated the ability of airway smooth muscle to maintain force after the initial phase of contraction. Specifically, we examined the roles of Rho-kinase and protein kinase C (PKC) in force maintenance. We found that for the same degree of initial force inhibition, Rho-kinase substantially reduced the muscle's ability to sustain force under static conditions, whereas inhibition of PKC had a minimal effect on sustaining force. Under oscillatory strain, Rho-kinase inhibition caused further decline in force, but again, PKC inhibition had a minimal effect. We also found that Rho-kinase inhibition led to a decrease in the myosin filament mass in the muscle cells, suggesting that one of the functions of Rho-kinase is to stabilize myosin filaments. The results also suggest that dissolution of myosin filaments may be one of the mechanisms underlying the phenomenon of fluidization. These findings can shed light on the mechanism underlying deep inspiration induced bronchodilation.