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.

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.

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.

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.

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.

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.

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.

High-speed AFM reveals subsecond dynamics of cardiac thin filaments upon Ca 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 [Ca] (pCa 9.

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.

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 Ca-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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

Molluscan catch muscle myorod and its N-terminal peptide bind to F-actin and myosin in a phosphorylation-dependent manner.

Myorod is expressed exclusively in molluscan catch muscle and localizes on the surface of thick filaments together with twitchin and myosin. This protein is an alternatively spliced product of the myosin heavy-chain gene containing the C-terminal rod part of myosin and a unique N-terminal domain. We have recently reported that this unique domain is a target for phosphorylation by gizzard smooth muscle myosin light chain kinase (MLCK) and molluscan twitchin, which contains a MLCK-like domain.

Myosin kinase of molluscan smooth muscle. Regulation by binding of calcium to the substrate and inhibition of myorod and twitchin phosphorylation by myosin.

Major contractile proteins were purified from relaxed actomyosin extracted from molluscan catch muscle myofibrils using ammonium sulfate fractionation and divalent cation precipitation. A fraction of this actomyosin was precipitated and purified as a supramolecular complex composed of twitchin (TW), myosin (MY), and myorod (MR). Another TW-MR complex was obtained via the removal of myosin.

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.

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan muscles: evidence that twitchin interacts with myosin, myorod, and paramyosin core and affects properties of actomyosin.

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan smooth muscles postulates in vivo existence of twitchin links between thin and thick filaments that arise in a phosphorylation-dependent manner [N.S. Shelud'ko, G.

Phosphorylation of myorod (catchin) by kinases tightly associated to molluscan and vertebrate smooth muscle myosins.

Myorod, also known as catchin, a newly discovered component of molluscan smooth muscle thick filaments, is an alternative product of the myosin heavy chain gene. It contains a C-terminal rod part that is identical to that part of myosin and a unique N-terminal domain that is very small relative to the myosin head domain. The role of myorod in contraction or relaxation of this muscle type is unknown.