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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.

Background: There is evidence that the actin-activated ATP kinetics and the mechanical work produced by muscle myosin molecules are regulated by two surface loops, located near the ATP binding pocket (loop 1), and in a region that interfaces with actin (loop 2). These loops regulate force and velocity of contraction, and have been investigated mostly in single molecules. There is a lack of information of the work produced by myosin molecules ordered in filaments and working cooperatively, which is the actual muscle environment., Methods: We use micro-fabricated cantilevers to measure forces produced by myosin filaments isolated from mollusk muscles, skeletal muscles, and smooth muscles containing variations in the structure of loop 1 (tonic and phasic myosins). We complemented the experiments with in-vitro assays to measure the velocity of actin motility., Results: Smooth muscle myosin filaments produced more force than skeletal and mollusk myosin filaments when normalized per filament overlap. Skeletal muscle myosin propelled actin filaments in a higher sliding velocity than smooth muscle myosin. The values for force and velocity were consistent with previous studies using myosin molecules, and suggest a close correlation with the myosin isoform and structure of surface loop 1., General Significance: The technique using micro-fabricated cantilevers to measure force of filaments allows for the investigation of the relation between myosin structure and contractility, allowing experiments to be conducted with an array of different myosin isoforms. Using the technique we observed that the work produced by myosin molecules is regulated by amino-acid sequences aligned in specific loops.

Rationale: Airway smooth muscle (SM) of patients with asthma exhibits a greater velocity of shortening (Vmax) than that of normal subjects, and this is thought to contribute to airway hyperresponsiveness. A greater Vmax can result from increased myosin activation. This has been reported in sensitized human airway SM and in models of asthma. A faster Vmax can also result from the expression of specific contractile proteins that promote faster cross-bridge cycling. This possibility has never been addressed in asthma., Objectives: We tested the hypothesis that the expression of genes coding for SM contractile proteins is altered in asthmatic airways and contributes to their increased Vmax., Methods: We quantified the expression of several genes that code for SM contractile proteins in mild allergic asthmatic and control human airway endobronchial biopsies. The function of these contractile proteins was tested using the in vitro motility assay., Measurements and Main Results: We observed an increased expression of the fast myosin heavy chain isoform, transgelin, and myosin light chain kinase in patients with asthma. Immunohistochemistry demonstrated the expression of these genes at the protein level. To address the functional significance of this overexpression, we purified tracheal myosin from the hyperresponsive Fisher rats, which also overexpress the fast myosin heavy chain isoform as compared with the normoresponsive Lewis rats, and found a faster rate of actin filament propulsion. Conversely, transgelin did not alter the rate of actin filament propulsion., Conclusions: Selective overexpression of airway smooth muscle genes in asthmatic airways leads to increased Vmax, thus contributing to the airway hyperresponsiveness observed in asthma.