From amino-acid to disease: the effects of oxidation on actin-myosin interactions in muscle.

Actin-myosin interactions form the basis of the force-producing contraction cycle within the sarcomere, serving as the primary mechanism for muscle contraction. Post-translational modifications, such as oxidation, have a considerable impact on the mechanics of these interactions. Considering their widespread occurrence, the explicit contributions of these modifications to muscle function remain an active field of research. In this review, we aim to provide a comprehensive overview of the basic mechanics of the actin-myosin complex and elucidate the extent to which oxidation influences the contractile cycle and various mechanical characteristics of this complex at the single-molecule, myofibrillar and whole-muscle levels. We place particular focus on amino acids shown to be vulnerable to oxidation in actin, myosin, and some of their binding partners. Additionally, we highlight the differences between in vitro environments, where oxidation is controlled and limited to actin and myosin and myofibrillar or whole muscle environments, to foster a better understanding of oxidative modification in muscle. Thus, this review seeks to encompass a broad range of studies, aiming to lay out the multi layered effects of oxidation in in vitro and in vivo environments, with brief mention of clinical muscular disorders associated with oxidative stress.

New paradigms in actomyosin energy transduction: Critical evaluation of non-traditional models for orthophosphate release.

Release of the ATP hydrolysis product ortophosphate (Pi) from the active site of myosin is central in chemo-mechanical energy transduction and closely associated with the main force-generating structural change, the power-stroke. Despite intense investigations, the relative timing between Pi-release and the power-stroke remains poorly understood. This hampers in depth understanding of force production by myosin in health and disease and our understanding of myosin-active drugs. Since the 1990s and up to today, models that incorporate the Pi-release either distinctly before or after the power-stroke, in unbranched kinetic schemes, have dominated the literature. However, in recent years, alternative models have emerged to explain apparently contradictory findings. Here, we first compare and critically analyze three influential alternative models proposed previously. These are either characterized by a branched kinetic scheme or by partial uncoupling of Pi-release and the power-stroke. Finally, we suggest critical tests of the models aiming for a unified picture.

Cooperativity of myosin II motors in the non-regulated and regulated thin filaments investigated with high-speed AFM.

Skeletal myosins II are non-processive molecular motors that work in ensembles to produce muscle contraction while binding to the actin filament. Although the molecular properties of myosin II are well known, there is still debate about the collective work of the motors: is there cooperativity between myosin motors while binding to the actin filaments? In this study, we use high-speed AFM to evaluate this issue. We observed that the initial binding of small arrays of myosin heads to the non-regulated actin filaments did not affect the cooperative probability of subsequent bindings and did not lead to an increase in the fractional occupancy of the actin binding sites. These results suggest that myosin motors are independent force generators when connected in small arrays, and that the binding of one myosin does not alter the kinetics of other myosins. In contrast, the probability of binding of myosin heads to regulated thin filaments under activating conditions (at high Ca2+ concentration in the presence of 2 µM ATP) was increased with the initial binding of one myosin, leading to a larger occupancy of available binding sites at the next half-helical pitch of the filament. The result suggests that myosin cooperativity is observed over five pseudo-repeats and defined by the activation status of the thin filaments.

Oxidation alters myosin-actin interaction and force generation in skeletal muscle filaments.

The interaction between actin and myosin is the basis of contraction and force production in muscle fibers. Studies have shown that actin and myosin oxidation cause myofibrillar weakness in healthy and diseased muscles. The degree to which oxidation of each of these proteins contributes to an attenuated force in myofibrils is unclear. In this study, we show that exposure of actin and myosin to the chemical 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride (SIN-1), an NO and O2•- donor, affected actin-myosin interactions, as shown by a decreased myosin-propelled actin velocity in the in vitro motility assay. We also observed that oxidation of actin and myosin resulted in a decrease in force generated by myosin and actin filaments, as determined by a system of microfabricated cantilevers. Although myosin is more sensitive to oxidative modifications than actin, as indicated by a steeper decrease in velocity and force by the filaments, modifications on actin are sufficient to affect force and velocity and also contribute to a decrease in contractile activity in muscles.

Multistep orthophosphate release tunes actomyosin energy transduction.

Muscle contraction and a range of critical cellular functions rely on force-producing interactions between myosin motors and actin filaments, powered by turnover of adenosine triphosphate (ATP). The relationship between release of the ATP hydrolysis product ortophosphate (Pi) from the myosin active site and the force-generating structural change, the power-stroke, remains enigmatic despite its central role in energy transduction. Here, we present a model with multistep Pi-release that unifies current conflicting views while also revealing additional complexities of potential functional importance. The model is based on our evidence from kinetics, molecular modelling and single molecule fluorescence studies of Pi binding outside the active site. It is also consistent with high-speed atomic force microscopy movies of single myosin II molecules without Pi at the active site, showing consecutive snapshots of pre- and post-power stroke conformations. In addition to revealing critical features of energy transduction by actomyosin, the results suggest enzymatic mechanisms of potentially general relevance.

Millisecond Conformational Dynamics of Skeletal Myosin II Power Stroke Studied by High-Speed Atomic Force Microscopy.

Myosin-based molecular motors are responsible for a variety of functions in the cells. Myosin II is ultimately responsible for muscle contraction and can be affected by multiple mutations, that may lead to myopathies. Therefore, it is essential to understand the nanomechanical properties of myosin II. Due to the lack of technical capabilities to visualize rapid changes in nonprocessive molecular motors, there are several mechanistic details in the force-generating steps produced by myosin II that are poorly understood. In this study, high-speed atomic force microscopy was used to visualize the actin-myosin complex at high temporal and spatial resolutions, providing further details about the myosin mechanism of force generation. A two-step motion of the double-headed heavy meromyosin (HMM) lever arm, coupled to an 8.4 nm working stroke was observed in the presence of ATP. HMM heads attached to an actin filament worked independently, exhibiting different lever arm configurations in given time during experiments. A lever arm rotation was associated with several non-stereospecific long-lived and stereospecific short-lived (∼1 ms) HMM conformations. The presence of free Pi increased the short-lived stereospecific binding events in which the power stroke occurred, followed by release of Pi after the power stroke.

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.

High-speed AFM reveals subsecond dynamics of cardiac thin filaments upon Ca2+ activation and heavy meromyosin binding.

High-speed atomic force microscopy (HS-AFM) can be used to study dynamic processes with real-time imaging of molecules within 1- to 5-nm spatial resolution. In the current study, we evaluated the 3-state model of activation of cardiac thin filaments (cTFs) isolated as a complex and deposited on a mica-supported lipid bilayer. We studied this complex for dynamic conformational changes 1) at low and high [Ca2+] (pCa 9.0 and 4.5), and 2) upon myosin binding to the cTF in the nucleotide-free state or in the presence of ATP. HS-AFM was used to directly visualize the tropomyosin-troponin complex and Ca2+-induced tropomyosin movements accompanied by structural transitions of actin monomers within cTFs. Our data show that cTFs at relaxing or activating conditions are not ultimately in a blocked or activated state, respectively, but rather the combination of states with a prevalence that is dependent on the [Ca2+] and the presence of weakly or strongly bound myosin. The weakly and strongly bound myosin induce similar changes in the structure of cTFs as confirmed by the local dynamical displacement of individual tropomyosin strands in the center of a regulatory unit of cTF at the relaxed and activation conditions. The displacement of tropomyosin at the relaxed conditions had never been visualized directly and explains the ability of myosin binding to TF at the relaxed conditions. Based on the ratios of nonactivated and activated segments within cTFs, we proposed a mechanism of tropomyosin switching from different states that includes both weakly and strongly bound myosin.

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.

Crenomytilus grayanus 40kDa calponin-like protein: cDNA cloning, sequence analysis, tissue expression, and post-translational modifications.

Calponin-like protein (CaP-40), a third major protein after actin and tropomyosin, has recently been identified by us in the Ca2+-regulated thin filaments of mussel Crenomytilus grayanus. It contains calponin homology domain, five calponin family repeats and possesses similar biochemical properties as vertebrate smooth muscle calponin. In this paper, we report a full-length cDNA sequence of CaP-40, study its expression pattern on mRNA and protein levels, evaluate CaP-40 post-translational modifications and perform protein-protein interaction analysis. The full-length sequence of CaP-40 consists of 398 amino acids and has high similarity to calponins among molluscan species. CaP-40 gene is widely expressed in mussel tissues, with the highest expression in adductor and mantle. Comparison of these data with protein content established by mass-spectrometry analysis revealed that the high mRNA content is mirrored by high protein levels for adductor smooth muscles. To provide unbiased insight into the function of CaP-40 and effect of its over-expression in adductor smooth muscle, we built protein-protein interaction network of identified Crenomytilus grayanus proteome. In addition, we showed that CaP-40 is subjected to post-translational N- and C-terminal acetylation at N127, G229 and G349 sites which potentially regulates its function in vivo.

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.

Troponin-like regulation in muscle thin filaments of the mussel Crenomytilus grayanus (Bivalvia: Mytiloida).

Muscles of bivalve molluscs have double calcium regulation--myosin-linked and actin-linked. While the mechanism of myosin-linked regulation is sufficiently studied, there is still no consensus on the mechanism of actin-linked regulation. Earlier we showed a high degree of Ca2+-sensitivity of thin filaments from the adductor muscle of the mussel Crenomytilus grayanus (Mytiloida). In order to elucidate the nature of this regulation, we isolated the fraction of minor proteins from the mussel thin filaments, which confers Ca2+-sensitivity to reconstituted actomyosin-tropomyosin. Proteins of this fraction, ABP-19, ABP-20, and ABP-28, were chromatographically purified and identified. According to the results of mass spectrometry and Western blot analysis, as well as by their functional properties, these mussel actin-binding proteins appeared to correspond to the troponin components from the skeletal muscles of vertebrates (TnC, TnI and TnT). The reconstituted mussel troponin complex confers to actomyosin-tropomyosin more than 80% Ca2+-sensitivity. The in vivo molar ratio of actin/tropomyosin/troponin was calculated to be 7:1:0.5, i.e., the content of troponin in mussel thin filaments is two times lower than in thin filaments of skeletal muscles of vertebrates. These data demonstrate that troponin-like regulation found in the catch muscle of the mussel C. grayanus is present at least in two suborders of bivalves: Pectinoida and Mytiloida.

Catch muscle myorod modulates ATPase activity of Myosin in a phosphorylation-dependent way.

Myorod is expressed exclusively in molluscan catch muscle and localizes on the surface of thick filaments together with twitchin and myosin. Myorod is an alternatively spliced product of the myosin heavy-chain gene that contains the C-terminal rod part of myosin and a unique N-terminal domain. The unique domain is a target for phosphorylation by gizzard smooth myosin light chain kinase (smMLCK) and, perhaps, molluscan twitchin, which contains a MLCK-like domain. To elucidate the role of myorod and its phosphorylation in the catch muscle, the effect of chromatographically purified myorod on the actin-activated Mg2+-ATPase activity of myosin was studied. We found that phosphorylation at the N-terminus of myorod potentiated the actin-activated Mg2+-ATPase activity of mussel and rabbit myosins. This potentiation occurred only if myorod was phosphorylated and introduced into the ATPase assay as a co-filament with myosin. We suggest that myorod could be related to the catch state, a function specific to molluscan muscle.

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.

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

Abundant data indicate that pathogenesis in allergic airways disease is orchestrated by an aberrant T-helper 2 (Th2) inflammatory response. CD4(+) T cells have been localized to airway smooth muscle (ASM) in both human asthmatics and in rodent models of allergic airways disease, where they have been implicated in proliferative responses of ASM. Whether CD4(+) T cells also alter ASM contractility has not been addressed. We established an in vitro system to assess the ability of antigen-stimulated CD4(+) T cells to modify contractile responses of the Brown Norway rat trachealis muscle. Our data demonstrated that the unloaded velocity of shortening (Vmax) of ASM was significantly increased upon 24 h co-incubation with antigen-stimulated CD4(+) T cells, while stress did not change. Enhanced Vmax was dependent upon contact between the CD4(+) T cells and the ASM and correlated with increased levels of the fast (+)insert smooth muscle myosin heavy chain isoform. The levels of myosin light chain kinase and myosin light chain phosphorylation were also increased within the muscle. The alterations in mechanics and in the levels of contractile proteins were transient, both declining to control levels after 48 h of co-incubation. More permanent alterations in muscle phenotype might be attainable when several inflammatory cells and mediators interact together or after repeated antigenic challenges. Further studies will await new tissue culture methodologies that preserve the muscle properties over longer periods of time. In conclusion, our data suggest that inflammatory cells promote ASM hypercontractility in airway hyper-responsiveness and asthma.

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.

Molluscan smooth catch muscle contains calponin but not caldesmon.

We isolated Ca(2+)-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 Ca(2+)-regulator of thin filaments of this muscle. On the other hand, the mussel CaP-like protein is also not such Ca(2+)-regulator since we have shown that this protein can be selectively removed from isolated mussel thin filaments without loss of their Ca(2+)-sensitivity. We suggest that thin filaments in the smooth catch muscle possess other type of Ca(2+)-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.

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.