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Human trachealis and main bronchi smooth muscle are normoresponsive in asthma.

RATIONALE Airway smooth muscle (ASM) plays a key role in airway hyperresponsiveness (AHR) but it is unclear whether its contractility is intrinsically changed in asthma. OBJECTIVES To investigate whether key parameters of ASM contractility are altered in subjects with asthma. METHODS Human trachea and main bronchi were dissected free of epithelium and connective tissues and suspended in a force-length measurement set-up. After equilibration each tissue underwent a series of protocols to assess its methacholine dose-response relationship, shortening velocity, and response to length oscillations equivalent to tidal breathing and deep inspirations. MEASUREMENTS AND MAIN RESULTS Main bronchi and tracheal ASM were significantly hyposensitive in subjects with asthma compared with control subjects. Trachea and main bronchi did not show significant differences in reactivity to methacholine and unloaded tissue shortening velocity (Vmax) compared with control subjects. There were no significant differences in responses to deep inspiration, with or without superimposed tidal breathing oscillations. No significant correlations were found between age, body mass index, or sex and sensitivity, reactivity, or Vmax. CONCLUSIONS Our data show that, in contrast to some animal models of AHR, human tracheal and main bronchial smooth muscle contractility is not increased in asthma. Specifically, our results indicate that it is highly unlikely that ASM half-maximum effective concentration (EC50) or Vmax contribute to AHR in asthma, but, because of high variability, we cannot conclude whether or not asthmatic ASM is hyperreactive.

Unphosphorylated calponin enhances the binding force of unphosphorylated myosin to actin.

BACKGROUND Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin. METHODS To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (Funb) in the absence and presence of calponin or phosphorylated calponin. RESULTS Funb from F-actin alone (0.12±0.01pN; mean±SE) was significantly increased in the presence of calponin (0.20±0.02pN). This enhancement was lost when calponin was phosphorylated (0.12±0.01pN). To further verify that this enhancement of Funb was due to the cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the Funb obtained at a [KCl] of 25mM (0.21±0.02pN; mean±SE) was significantly decreased at a [KCl] of 150mM, (0.13±0.01pN). CONCLUSIONS This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state. GENERAL SIGNIFICANCE This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.

Peripheral Airway Smooth Muscle, but Not the Trachealis, Is Hypercontractile in an Equine Model of Asthma

Heaves is a naturally occurring equine disease that shares many similarities with human asthma, including reversible antigen-induced bronchoconstriction, airway inflammation, and remodeling. The purpose of this study was to determine whether the trachealis muscle is mechanically representative of the peripheral airway smooth muscle (ASM) in an equine model of asthma. Tracheal and peripheral ASM of heaves-affected horses under exacerbation, or under clinical remission of the disease, and control horses were dissected and freed of epithelium to measure unloaded shortening velocity (Vmax), stress (force/cross-sectional area), methacholine effective concentration at which 50% of the maximum response is obtained, and stiffness. Myofibrillar Mg(2+)-ATPase activity, actomyosin in vitro motility, and contractile protein expression were also measured. Horses with heaves had significantly greater Vmax and Mg(2+)-ATPase activity in peripheral airway but not in tracheal smooth muscle. In addition, a significant correlation was found between Vmax and the time elapsed since the end of the corticosteroid treatment for the peripheral airways in horses with heaves. Maximal stress and stiffness were greater in the peripheral airways of the horses under remission compared with controls and the horses under exacerbation, potentially due to remodeling. Actomyosin in vitro motility was not different between controls and horses with heaves. These data demonstrate that peripheral ASM is mechanically and biochemically altered in heaves, whereas the trachealis behaves as in control horses. It is therefore conceivable that the trachealis muscle may not be representative of the peripheral ASM in human asthma either, but this will require further investigation.

Accumulating Evidence for Increased Velocity of Airway Smooth Muscle Shortening in Asthmatic Airway Hyperresponsiveness

It remains unclear whether airway smooth muscle (ASM) mechanics is altered in asthma. While efforts have originally focussed on contractile force, some evidence points to an increased velocity of shortening. A greater rate of airway renarrowing after a deep inspiration has been reported in asthmatics compared to controls, which could result from a shortening velocity increase. In addition, we have recently shown in rats that increased shortening velocity correlates with increased muscle shortening, without increasing muscle force. Nonetheless, establishing whether or not asthmatic ASM shortens faster than that of normal subjects remains problematic. Endobronchial biopsies provide excellent tissue samples because the patients are well characterized, but the size of the samples allows only cell level experiments. Whole human lungs from transplant programs suffer primarily from poor patient characterization, leading to high variability. ASM from several animal models of asthma has shown increased shortening velocity, but it is unclear whether this is representative of human asthma. Several candidates have been suggested as responsible for increased shortening velocity in asthma, such as alterations in contractile protein expression or changes in the contractile apparatus structure. There is no doubt that more remains to be learned about the role of shortening velocity in asthma.

Epithelial Cells Induce a Cyclo-Oxygenase-1–Dependent Endogenous Reduction in Airway Smooth Muscle Contractile Phenotype

&NA; Airway smooth muscle cells (ASMCs) are phenotypically regulated to exist in either a proliferative or a contractile state. However, the influence of other airway structural cell types on ASMC phenotype is largely unknown. Although epithelial cells are known to drive ASM proliferation, their effects on the contractile phenotype are uncertain. In the current study, we tested the hypothesis that epithelial cells reduce the contractile phenotype of ASMCs. To do so, we measured force production by traction microscopy, gene and protein expression, as well as calcium release by Fura‐2 ratiometric imaging. ASMCs incubated with epithelial‐derived medium produced less force after histamine stimulation. We observed reduced expression of myocardin, &agr;‐smooth muscle actin, and calponin within ASMCs after coculture with epithelial cells. Peak calcium release in response to histamine was diminished, and depended on the synthesis of cyclo‐oxygenase‐1 products by ASM and on prostaglandin E receptors 2 and 4. Together, these in vitro results demonstrate that epithelial cells have the capacity to coordinately reduce ASM contraction by functional antagonism and by reduction of the expression of certain contractile proteins.

Rapid Time Resolved Analysis of Filament Motility

In vitro motility assays are a valuable tool in understanding and characterizing motor protein mechanics, but existing algorithms are not optimized for time resolved analyses. We propose an algorithm that combines trace detection with time resolved analysis. By tracking filament ends, we double the number of data points and minimize data loss from crossing filaments. A movement trace formed by each filament end is stored such that the frame number in which a filament either first (filament tip) or last (filament tail) occupies a certain pixel, is stored. A frame number vs distance curve is generated from this trace, which is segmented into regions by slope to detect stop-and-go movement. We generated mock motility videos with known velocities, motile fraction profiles and varying filament lengths. Our algorithm accurately detected velocity and motile fraction changes for velocities less than 0.05 pixels per frame, without manual trace dropping and regardless of filament crossings. Two established algorithms, with manual trace dropping of unrealistic or not analyzable traces, showed much greater error in average and individual filament velocity as well as motile fraction. To demonstrate the utility of the algorithm we tested two actual motility experiments: 1) Adenosine triphosphate (ATP) added to skeletal myosin in rigor state; 2) myosin light chain phosphatase (MLCP) added to phasic smooth muscle myosin. Our algorithm revealed features not previously detectable: 1) a near instantaneous increase in motile fraction paralleled by a slow increase in velocity as the ATP concentration increases and 2) simultaneous reductions in velocity and motile fraction as MLCP diffuses into the motility chamber at very low velocities. Our tests show that the developed algorithm surpasses existing algorithms in time resolved measurements of motile fraction and velocity at a wide range of filament velocities and lengths, with minimal user input and CPU time.

Maintenance of Contractile function of isolated airway smooth muscle after cryopreservation.

Isolated human airway smooth muscle (ASM) tissue contractility studies are essential for understanding the role of ASM in respiratory disease, but limited availability and cost render storage options necessary for optimal use. However, to our knowledge, no comprehensive study of cryopreservation protocols for isolated ASM has been performed to date. We tested several cryostorage protocols on equine trachealis ASM using different cryostorage media [1.8 M dimethyl sulfoxide and fetal bovine serum (FBS) or Krebs-Henseleit (KH)] and different degrees of dissection (with or without epithelium and connective tissues attached) before storage. We measured methacholine (MCh), histamine, and isoproterenol (Iso) dose-responses and electrical field stimulation (EFS) and MCh force-velocity curves. We confirmed our findings in human trachealis ASM stored undissected in FBS. Maximal stress response to MCh was decreased more in dissected than undissected equine tissues. EFS force was decreased in all equine but not in human cryostored tissues. Furthermore, in human cryostored tissues, EFS maximal shortening velocity was decreased, and Iso response was potentiated after cryostorage. Overnight incubation with 0.5 or 10% FBS did not recover contractility in the equine tissues but potentiated Iso response. Overnight incubation with 10% FBS in human tissues showed maximal stress recovery and maintenance of other contractile parameters. ASM tissues can be cryostored while maintaining most contractile function. We propose an optimal protocol for cryostorage of ASM as undissected tissues in FBS or KH solution followed by dissection of the ASM bundles and a 24-h incubation with 10% FBS before mechanics measurements.