Naturally occurring groups of muscle myosin behave differently from individual myosins or small groups commonly assayed in vitro. Here, we investigate the emergence of myosin group behavior with increasing myosin group size. Assuming the number of myosin binding sites (N) is proportional to actin length (L) (N = L/35.5 nm), we resolve in vitro motility of actin propelled by skeletal muscle myosin for L = 0.2-3 μm. Three distinct regimes were found: L < 0.3 μm, sliding arrest; 0.3 μm ≤ L ≤ 1 μm, alternation between arrest and continuous sliding; L > 1 μm, continuous sliding. We theoretically investigated the myosin group kinetics with mechanical coupling via actin. We find rapid actin sliding steps driven by power-stroke cascades supported by postpower-stroke myosins, and phases without actin sliding caused by prepower-stroke myosin buildup. The three regimes are explained: N = 8, rare cascades; N = 15, cascade bursts; N = 35, continuous cascading. Two saddle-node bifurcations occur for increasing N (mono → bi → mono-stability), with steady states corresponding to arrest and continuous cascading. The experimentally measured dependence of actin sliding statistics on L and myosin concentration is correctly predicted.
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.
Animal studies show that the (ϩ)insert isoform is predominantly expressed in rapidly contracting phasic muscle and the (Ϫ)insert isoform is mostly found in slowly contracting tonic muscle. The expression of the (ϩ)insert isoform has never been demonstrated in human smooth muscle. We hypothesized that the (ϩ)insert isoform is present in humans and that its expression is commensurate with the organ's functional requirements. We report, for the first time, the sequence of the human (ϩ)insert isoform and quantification of its expression by real-time PCR and Western blot analysis in a panel of human organs. The (ϩ)insert isoform mRNA and protein expression levels are significantly greater in small intestine compared with all organs studied except for trachea and are significantly greater in trachea compared with uterus and aorta. To assess the functional significance of this differential myosin isoform expression between organs, we measured the rate of actin filament movement ( max) when propelled by myosin purified from rat organs, because the rat and human inserts are identical and their remaining sequences show 93% identity. max exhibits a rank correlation from the most tonic to the most phasic organ. The selective expression of the (ϩ)insert isoform observed among human organs suggests that it is an important determinant of tissue shortening velocity. A differential expression of the (ϩ)insert isoform could also account for altered contractile properties observed in human pathology. phasic and tonic smooth muscle; real-time polymerase chain reaction; in vitro motility assay SMOOTH MUSCLE IS FOUND in all hollow organs of the mammalian organism, and its function ranges from tone maintenance to content propulsion. Many studies point to the smooth muscle myosin heavy chain (SMMHC) as an important element contributing to these diverse contractile properties (see Ref. 16 for review). SMMHC is made up of a globular head, containing an ATPase site and an actin binding domain, and an ␣-helical tail to which regulatory and essential light chains are bound. SMMHC isoforms are generated by alternative splicing from a single gene (1,9,33,46). Four SMMHC isoforms have been described in various animal species. The first two isoforms identified differ in the carboxy terminus by distinct sequences of 43 (SM1) or 9 (SM2) amino acids (2, 33). The next two isoforms differ in the amino terminus by the presence [(ϩ)insert] or absence [(Ϫ)insert] of a seven-amino acid insert in a surface loop above the ATPase site (18, 46). The (ϩ) and (Ϫ)insert isoforms are also commonly referred to as SM-B and SM-A, respectively. All combinations of these isoforms are possible, i.e., (ϩ)insert SM1, (ϩ)insert SM2, (Ϫ)insert SM1, and (Ϫ)insert SM2. No difference in molecular mechanics has been observed between SM1 and SM2, but, as shown with myosin constructs, the sole presence of the amino-terminal insert doubles the actin-activated ATPase activity and the rate of actin filament movement ( max ) in the in vitro motility assay (17,22,36). Because of t...
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.
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