Oleg S. Matusovsky

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.

Proteolytic substructure of myorod, a thick filament protein of molluscan smooth muscles

Myorod (MR), a new thick filament protein of molluscan smooth muscles, is an alternatively spliced product of the myosin (Mn) heavy chain gene. We studied digestion of MR and Mn from the posterior adductor of Crenomytilus grayanus and the outer portion of adductor of Mizuchopecten (Patinopecten) yessoensis by papain and constructed the proteolytic substructure of MR, that is an analogue to Mn substructure. There are a head domain (analogue of Mn S1) and a rod domain (analogue of Mn rod); the junction between them is split at low ionic strength. The rod, in turn, consists of a neck domain (analogue of Mn S2) and a tail domain (identical to light meromyosin); the junction between them is split at high ionic strength. The localization and possible function of MR are discussed.

Thin filaments of bivalve smooth muscle may contain a calponin-like protein

A novel 40-kDa calponin-like protein (CaP) was detected in thin filaments from catch muscles of the mussel Crenomytilus grayanus. The content of CaP in thin filaments depends on isolation conditions and varies from complete absence to the presence in amounts comparable with that of tropomyosin. The most significant factor that determines the CaP content in thin filaments is the temperature of solution in which thin filaments are sedimented by ultracentrifugation during isolation. At 22°C and optimal values of pH and ionic strength of the extraction solution, all CaP co-sediments with thin filaments. At 2°C it does not interact with thin filaments and remains in the supernatant. Like vertebrate smooth muscle calponin (33 kDa), the mussel CaP is thermostable, inhibits the Mg2+-ATPase activity of actomyosin, and can be phosphorylated, which is performed by endogenous (co-isolated) kinases in a Ca2+-independent manner. Thus, the C. grayanus CaP is a new member of the family of calponins, the function of which in muscle and nonmuscle cells is still obscure. We suggest that CaP is involved in Ca2+-independent regulation of smooth muscle contraction.

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.

Ileal smooth muscle dysfunction and remodeling in cystic fibrosis

Patients with cystic fibrosis (CF) often suffer from gastrointestinal cramps and intestinal obstruction. The CF transmembrane conductance regulator (CFTR) channel has been shown to be expressed in vascular and airway smooth muscle (SM). We hypothesized that the absence of CFTR expression alters the gastrointestinal SM function and that these alterations may show strain-related differences in the mouse. The aim of this study was to measure the contractile properties of the ileal SM in two CF mouse models. CFTR(-/-) and CFTR(+/+) mice were studied on BALB/cJ and C57BL/6J backgrounds. Responsiveness of ileal strips to electrical field stimulation (EFS), methacholine (MCh), and isoproterenol was measured. The mass and the cell density of SM layers were measured morphometrically. Finally, the maximal velocity of shortening (Vmax) and the expression of the fast (+)insert myosin isoform were measured in the C57BL/6J ileum. Ileal hyperreactivity was observed in response to EFS and MCh in CFTR(-/-) compared with CFTR(+/+) mice in C57BL/6J background. This latter observation was not reproduced by acute inhibition of CFTR with CFTR(inh)172. BALB/cJ CFTR(-/-) mice exhibited a significant increase of SM mass with a lower density of cells compared with CFTR(+/+), whereas no difference was observed in the C57BL/6J background. In addition, in this latter strain, ileal strips from CFTR(-/-) exhibited a significant increase in Vmax compared with control and expressed a greater proportion of the fast (+)insert SM myosin isoform with respect to total myosin. BALB/cJ CFTR(-/-) ilium had a greater relaxation to isoproterenol than the CFTR(+/+) mice when precontracted with EFS, but no difference was observed in response to exogeneous MCh. In vivo, the lack of CFTR expression induces a different SM ileal phenotype in different mouse strains, supporting the importance of modifier genes in determining intestinal SM properties.

CD4+ T cells enhance the unloaded shortening velocity of airway smooth muscle by altering the contractile protein expression

Activated CD4+ T cells enhance the contractility of airway smooth muscle. In order to enhance contractility, contact between CD4+ T cells and smooth muscle is required. The enhanced contractility is correlated with increased levels of fast myosin isoform. Our data suggest that inflammatory cells promote airway smooth muscle hypercontractility in airway hyper‐responsiveness and asthma.

Molluscan smooth catch muscle contains calponin but not caldesmon

We isolated Ca2+-regulated thin filaments from the smooth muscle of the mussel Crenomytilus grayanus and studied the protein composition of different preparations from this muscle: whole muscle, heat-stable extract, fractions from heat-stable extract, thin filaments and intermediate stages of thin filaments purification. Among the protein components of the above-listed preparations, we did not find caldesmon (CaD), although two isoforms of a calponin-like (CaP-like) protein, which along with CaD is characteristic of vertebrate smooth muscle, were present in thin filaments. Thus, CaD is not Ca2+-regulator of thin filaments of this muscle. On the other hand, the mussel CaP-like protein is also not such Ca2+-regulator since we have shown that this protein can be selectively removed from isolated mussel thin filaments without loss of their Ca2+-sensitivity. We suggest that thin filaments in the smooth catch muscle possess other type of Ca2+-regulation, different from that in vertebrate smooth muscles.

Twitchin can regulate the ATPase cycle of actomyosin in a phosphorylation-dependent manner in skinned mammalian skeletal muscle fibres

The effect of twitchin, a thick filament protein of molluscan muscles, on the actin-myosin interaction at several mimicked sequential steps of the ATPase cycle was investigated using the polarized fluorescence of 1.5-IAEDANS bound to myosin heads, FITC-phalloidin attached to actin and acrylodan bound to twitchin in the glycerol-skinned skeletal muscle fibres of mammalian. The phosphorylation-dependent multi-step changes in mobility and spatial arrangement of myosin SH1 helix, actin subunit and twitchin during the ATPase cycle have been revealed. It was shown that nonphosphorylated twitchin inhibited the movements of SH1 helix of the myosin heads and actin subunits and decreased the affinity of myosin to actin by freezing the position and mobility of twitchin in the muscle fibres. The phosphorylation of twitchin reverses this effect by changing the spatial arrangement and mobility of the actin-binding portions of twitchin. In this case, enhanced movements of SH1 helix of the myosin heads and actin subunits are observed. The data imply a novel property of twitchin incorporated into organized contractile system: its ability to regulate the ATPase cycle in a phosphorylation-dependent fashion by changing the affinity and spatial arrangement of the actin-binding portions of twitchin.

Catch muscle of bivalve molluscs contains myosin- and twitchin-associated protein kinase phosphorylating myorod.

We have shown previously that myorod, a molluscan thick filament protein of unknown function, is phosphorylated by vertebrate smooth myosin light chain kinase (MLCK) in N-terminal unique region. The aim of the present study was to clarify whether such phosphorylation may occur in molluscan muscles. We detected three kinases endogenous to molluscan catch muscle, namely, to the complex of surface thick filament proteins that consists of twitchin, myosin, and myorod. The first kinase was a protein kinase A because it was inhibited by a specific inhibitor; the second one was associated with twitchin and phosphorylated myorod at its N-terminal unique region independently of Ca(2+); and the third kinase was bound to myosin and phosphorylated myorod as well as myosin in the C-terminal part of both proteins. The myosin-associated kinase was inhibited by micromolar concentration of calcium ions. This enzyme could be separated from myosin by chromatography, whereas the kinase associated with twitchin could not be separated from twitchin. Since twitchin has a MLCK-like domain, it is possible that this domain was responsible for myorod phosphorylation. Phosphorylation of myorod within the twitchin-myosin-myorod complex increased the actin-activated Mg(2+)-ATPase activity of myosin. Taken together, these results indicate that phosphorylation of myorod by kinases associated with key proteins of catch contraction may contribute to the functional activity of myorod in molluscan smooth muscle.

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.