A method for isolating contractile smooth muscle cells from cryopreserved tissue.

Multiple techniques have been developed to isolate contractile smooth muscle cells (SMCs) from tissues with varying degrees of success. However, most of these approaches rely on obtaining fresh tissue, which poses logistical challenges. In the present study, we introduce a novel protocol for isolating contractile SMCs from cryopreserved smooth muscle (SM) tissue, thereby enhancing experimental efficiency. This protocol yields abundant viable, spindle-shaped, contractile SMCs that closely resemble those obtained from fresh samples. By analyzing the expression of contractile proteins, we demonstrate that both the isolated SMCs from cryopreserved tissue represent more accurately fresh SM tissue compared with cultured SMCs. Moreover, we demonstrate the importance of a brief incubation step of the tissue in culture medium before cell dissociation to achieve contractile SMCs. Finally, we provide a concise overview of our protocol optimization efforts, along with a summary of previously published methods, which could be valuable for the development of similar protocols for other species.NEW & NOTEWORTHY We report a successful protocol development for isolating contractile smooth muscle cells (SMCs) from cryopreserved tissue reducing the reliance on fresh tissues and providing a readily available source of contractile SMCs. Our findings suggest that SMCs isolated using our protocol maintain their phenotype better compared with cultured SMCs. This preservation of the cellular characteristics, including the expression of key contractile proteins, makes these cells more representative of fresh SM tissue.

Automated, high temporal resolution mechanics measurements during incubation of contractile tissues.

Muscle tissue mechanics and contractility measurements have a great advantage over cultured cell level experiments as their mechanical and contractile properties are much closer to in vivo tissue properties. However, tissue level experiments cannot be combined with incubation with the same time resolution and consistency as cell culture studies. Here we present a system in which contractile tissues can be incubated for days while intermittently being tested for their mechanical and contractile properties. A two-chamber system was developed with control of temperature in the outer chamber and CO2 and humidity control in the inner, sterile chamber. Incubation medium, to which biologically active components may be added, is reused after each mechanics test to preserve both added and released components. Mechanics and contractility are measured in a different medium to which, through a high accuracy syringe pump, up to 6 different agonists in a 100-fold dose range can be added. The whole system can be operated through fully automated protocols from a personal computer. Testing data shows accurate maintenance of temperature, CO2 and relative humidity at pre-set levels. Equine trachealis smooth muscle tissues tested in the system showed no signs of infection after 72 h with incubation medium replacement every 24 h. Methacholine dosing and electrical field stimulation every 4 h showed consistent responses. In conclusion, the developed system is a great improvement on the manual incubation techniques being used thus far, improving on time resolution, repeatability and robustness, while reducing contamination risk and tissue damage from repeated handling.

Molecular-level evidence of force maintenance by smooth muscle myosin during LC20 dephosphorylation.

Smooth muscle (SM) is found in most hollow organs of the body. Phasic SM, as found in the gut, contracts to propel content, whereas tonic SM, as found in most blood vessels, maintains tension. This force maintenance is referred to as the latch state and occurs at low levels of myosin activation (myosin light chain [LC20] phosphorylation). Molecular mechanisms have been proposed to explain the latch state but have been studied only at the whole-muscle level because of technological limitations. In the current study, an assay chamber was devised to allow injection of myosin light chain phosphatase (MLCP) during laser trap and in vitro motility assays, without creating bulk flow, to reproduce latch state conditions at the molecular level. Using the laser trap in a single-beam mode, an actin filament was brought in contact with several myosin molecules on a pedestal. Myosin pulled on the actin filament until a plateau force was reached, at which point, MLCP was injected. Force maintenance was observed during LC20 dephosphorylation, the level of which was assessed in a parallel in vitro motility assay performed in the same conditions. Force was maintained longer for myosin purified from tonic SM than from phasic SM. These data support the longstanding dogma of strong bonds caused by dephosphorylated, noncycling cross-bridges. Furthermore, MLCP injection in an in vitro motility mixture assay performed with SM and skeletal muscle myosin suggests that the maintenance of these strong bonds is possible only if no energy is provided by surrounding actively cycling myosin molecules.

System Identification and Two-Degree-of-Freedom Control of Nonlinear, Viscoelastic Tissues.

OBJECTIVE: This paper presents a force control scheme for brief isotonic holds in an isometrically contracted muscle tissue, with minimal overshoot and settling time to measure its shortening velocity, a key parameter of muscle function. METHODS: A two-degree-of-freedom control configuration, formed by a feedback controller and a feedforward controller, is explored. The feedback controller is a proportional-integral controller and the feedforward controller is designed using the inverse of a control-oriented model of muscle tissue. A generalized linear model and a nonlinear model of muscle tissue are explored using input-output data and system identification techniques. The force control scheme is tested on equine airway smooth muscle and its robustness confirmed with murine flexor digitorum brevis muscle. RESULTS: Performance and repeatability of the force control scheme as well as the number of inputs and level of supervision required from the user were assessed with a series of experiments. The force control scheme was able to fulfill the stated control objectives in most cases, including the requirements for settling time and overshoot. CONCLUSION: The proposed control scheme is shown to enable automation of force control for characterizing muscle mechanics with minimal user input required. SIGNIFICANCE: This paper leverages an inversion-based feedforward controller based on a nonlinear physiological model in a system identification context that is superior to classic linear system identification. The control scheme can be used as a steppingstone for generalized control of nonlinear, viscoelastic materials.

Intrapulmonary airway smooth muscle is hyperreactive with a distinct proteome in asthma.

Constriction of airways during asthmatic exacerbation is the result of airway smooth muscle (ASM) contraction. Although it is generally accepted that ASM is hypercontractile in asthma, this has not been unambiguously demonstrated. Whether airway hyperresponsiveness (AHR) is the result of increased ASM mass alone or also increased contractile force generation per unit of muscle directly determines the potential avenues for treatment.To assess whether ASM is hypercontractile we performed a series of mechanics measurements on isolated ASM from intrapulmonary airways and trachealis from human lungs. We analysed the ASM and whole airway proteomes to verify if proteomic shifts contribute to changes in ASM properties.We report an increase in isolated ASM contractile stress and stiffness specific to asthmatic human intrapulmonary bronchi, the site of increased airway resistance in asthma. Other contractile parameters were not altered. Principal component analysis (PCA) of unbiased mass spectrometry data showed clear clustering of asthmatic subjects with respect to ASM specific proteins. The whole airway proteome showed upregulation of structural proteins. We did not find any evidence for a difference in the regulation of myosin activity in the asthmatic ASM.In conclusion, we showed that ASM is indeed hyperreactive at the level of intrapulmonary airways in asthma. We identified several proteins that are upregulated in asthma that could contribute to hyperreactivity. Our data also suggest enhanced force transmission associated with enrichment of structural proteins in the whole airway. These findings may lead to novel directions for treatment development in asthma.

Contractile Properties of Intrapulmonary Airway Smooth Muscle in Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal-recessive disease caused by mutations in the CF transmembrane conductance regulator gene. Many patients with CF have asthma-like symptoms and airway hyperresponsiveness, which are potentially associated with altered airway smooth muscle (ASM) contractility. Our goal in this study was to assess the contractility of the CF intrapulmonary ASM. ASM strips were dissected from human control and CF intrapulmonary airways, and assessed for methacholine-induced shortening velocity, maximal force, and stress. We also assessed isoproterenol responses in maximally methacholine-contracted ASM. ASM strips were then incubated for 16 hours with IL-13 and measurements were repeated. Myosin light chain kinase (MLCK) expression was assessed by Western blotting. Airways were immunostained for morphometry. ASM mass was increased in CF airways, which likely contributes to airway hyperresponsiveness. Although ASM contractile properties were not intrinsically different between patients with CF and control subjects, CF ASM responded differently in the presence of the inflammatory mediator IL-13, showing impairment in ß-adrenergic-induced relaxation. Indeed, the percentage of relaxation measured at maximal isoproterenol concentrations in the CF ASM was significantly lower after incubation with IL-13 (46.0% ± 6.7% relaxation) than without IL-13 (74.0% ± 7.7% relaxation, P = 0.018). It was also significantly lower than that observed in control ASM incubated with IL-13 (68.8% ± 4.9% relaxation, P = 0.048) and without IL-13 (82.4% ± 9.9%, P = 0.0035). CF ASM incubated with IL-13 also expressed greater levels of MLCK. Thus, our data suggest that the combination of an increase in ASM mass, increased MLCK expression, and inflammation-induced ß-adrenergic hyporesponsiveness may contribute to airway dysfunction in CF.

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.

Rapid time-stamped analysis of filament motility.

The in vitro motility assay is a valuable tool to understand motor protein mechanics, but existing algorithms are not optimized for accurate time resolution. We propose an algorithm that combines trace detection with a time-stamped analysis. By tracking filament ends, we minimize data loss from overlapping and crossing filaments. A movement trace formed by each filament end is created by time-stamping when the filament either first (filament tip) or last (filament tail) occupies a pixel. 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 show, using generated mock motility videos, accurate detection of velocity and motile fraction changes for velocities < 0.05 pixels per frame, without manual trace dropping and regardless of filament crossings. Compared with established algorithms we show greatly improved accuracy in velocity and motile fraction estimation, with greatly reduced user effort. We tested two actual motility experiments: (1) adenosine triphosphate (ATP) added to skeletal myosin in rigor; (2) myosin light chain phosphatase (MLCP) added to phasic smooth muscle myosin. Our algorithm revealed previously undetectable features: (1) rapid increase in motile fraction paralleled by a slow increase in velocity as ATP concentration increases; (2) simultaneous reductions in velocity and motile fraction as MLCP diffuses into the motility chamber at very low velocities. Our algorithm surpasses existing algorithms in the resolution of time dependent changes in motile fraction and velocity at a wide range of filament lengths and velocities, with minimal user input and CPU time.

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

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, α-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.

Directional preference of airway smooth muscle mass increase in human asthmatic airways.

Airway smooth muscle (ASM) orientation and morphology determine the ability of the muscle to constrict the airway. In asthma, ASM mass is increased, but it is unknown whether ASM orientation and morphology are altered as well or whether the remodeling at the source of the mass increase is ongoing. We dissected human airway trees from asthmatic and control lungs. Stained, intact airway sections were imaged in axial projection to show ASM bundle orientation, whereas cross-sectional histological slides were used to assess ASM area, bundle thickness, and ASM bundle-to-basement membrane distance. We also used these slides to assess cell size, proliferation, and apoptosis. We showed that ASM mass increase in cartilaginous airways is primarily the result of an increase of ASM bundle thickness (as measured radially in an airway cross section) and coincides with an increased distance of the ASM bundles to the airway perimeter. ASM orientation was unchanged in all airways. Apoptosis markers and cell size did not show differences between asthmatics and controls. Our findings show that ASM mass increase likely contributes to the airway-constricting capacity of the muscle. Both the increased bundle thickness and increased thickness of the airway wall inwards of the ASM bundles could further enhance this capacity. Turnover of ASM appears to be the same in airways and biopsies, but the lack of correlation between different markers of proliferation casts doubt on the specificity of markers generally used to assess proliferation.

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.

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.

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.

Myosin filament polymerization and depolymerization in a model of partial length adaptation in airway smooth muscle.

Length adaptation in airway smooth muscle (ASM) is attributed to reorganization of the cytoskeleton, and in particular the contractile elements. However, a constantly changing lung volume with tidal breathing (hence changing ASM length) is likely to restrict full adaptation of ASM for force generation. There is likely to be continuous length adaptation of ASM between states of incomplete or partial length adaption. We propose a new model that assimilates findings on myosin filament polymerization/depolymerization, partial length adaptation, isometric force, and shortening velocity to describe this continuous length adaptation process. In this model, the ASM adapts to an optimal force-generating capacity in a repeating cycle of events. Initially the myosin filament, shortened by prior length changes, associates with two longer actin filaments. The actin filaments are located adjacent to the myosin filaments, such that all myosin heads overlap with actin to permit maximal cross-bridge cycling. Since in this model the actin filaments are usually longer than myosin filaments, the excess length of the actin filament is located randomly with respect to the myosin filament. Once activated, the myosin filament elongates by polymerization along the actin filaments, with the growth limited by the overlap of the actin filaments. During relaxation, the myosin filaments dissociate from the actin filaments, and then the cycle repeats. This process causes a gradual adaptation of force and instantaneous adaptation of shortening velocity. Good agreement is found between model simulations and the experimental data depicting the relationship between force development, myosin filament density, or shortening velocity and length.