The Myosin Inhibitor Blebbistatin Stabilizes the Super-Relaxed State in Skeletal Muscle

The super-relaxed state of myosin (SRX), in which the myosin ATPase activity is strongly inhibited, has been observed in a variety of muscle types. It has been proposed that myosin heads in this state are inhibited by binding to the core of the thick filament in a structure known as the interacting-heads motif. The myosin inhibitor blebbistatin has been shown in structural studies to stabilize the binding of myosin heads to the thick filament, and here we have utilized measurements of single ATP turnovers to show that blebbistatin also stabilizes the SRX in both fast and slow skeletal muscle, providing further support for the proposal that myosin heads in the SRX are also in the interacting-heads motif. We find that the SRX is stabilized using blebbistatin even in conditions that normally destabilize it, e.g., rigor ADP. Using blebbistatin we show that spin-labeled nucleotides bound to myosin have an oriented spectrum in the SRX in both slow and fast skeletal muscle. This is to our knowledge the first observation of oriented spin probes on the myosin motor domain in relaxed skeletal muscle fibers. The spectra for skeletal muscle with blebbistatin are similar to those observed in relaxed tarantula fibers in the absence of blebbistatin, demonstrating that the structure of the SRX is similar in different muscle types and in the presence and absence of blebbistatin. The mobility of spin probes attached to nucleotides bound to myosin shows that the conformation of the nucleotide site is closed in the SRX.     

Slow Myosin ATP Turnover in the Super-Relaxed State in Tarantula Muscle

We measured the nucleotide turnover rate of myosin in tarantula leg muscle fibers by observing single turnovers of the fluorescent nucleotide analog 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-triphosphate, as monitored by the decrease in fluorescence when 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-triphosphate (mantATP) is replaced by ATP in a chase experiment. We find a multiexponential process with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant (∼ 30 min). This slow-turnover state is termed the super-relaxed state (SRX). If fibers are incubated in 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-diphosphate and chased with ADP, the SRX is not seen, indicating that trinucleotide-relaxed myosins are responsible for the SRX. Phosphorylation of the myosin regulatory light chain eliminates the fraction of myosin with a very long lifetime. The data imply that the very long-lived SRX in tarantula fibers is a highly novel adaptation for energy conservation in an animal that spends extremely long periods of time in a quiescent state employing a lie-in-wait hunting strategy. The presence of the SRX measured here correlates well with the binding of myosin heads to the core of the thick filament in a structure known as the “interacting-heads motif,” observed previously by electron microscopy. Both the structural array and the long-lived SRX require relaxed filaments or relaxed fibers, both are lost upon myosin phosphorylation, and both appear to be more stable in tarantula than in vertebrate skeletal or vertebrate cardiac preparations.     

The Active State of Mammalian Skeletal Muscle

A new technique is proposed for computing the active state of striated muscle, based on the three component model of Fenn and Marsh (8) and of Hill (7). The method permits calculation of the time course of the active state from its peak to the time at which maximum isometric twitch tension is reached. The intormation required for the calculation can be obtained from a single muscle without moving it from its mount in the lever system. The time course of the active state proved to be a function of the length of the muscle. This length dependency led to the predictions that (a) the length at which maximum force is developed during tetanic stimulation is different from that at which it is developed during a twitch, and (b) the tetanus-twitch tension ratio is a function of length. Both predictions were verified in a series of experiments on the rat gracilis anticus muscle at 17.5°C.     

The Selective Myosin II Inhibitor Blebbistatin Reversibly Eliminates Gastrovascular Flow and Stolon Tip Pulsations in the Colonial Hydroid Podocoryna carnea

Blebbistatin reversibly disrupted both stolon tip pulsations and gastrovascular flow in the colonial hydroid Podocoryna carnea. Epithelial longitudinal muscles of polyps were unaffected by blebbistatin, as polyps contracted when challenged with a pulse of KCl. Latrunculin B, which sequesters G actin preventing F actin assembly, caused stolons to retract, exposing focal adhesions where the tip epithelial cells adhere to the substratum. These results are consistent with earlier suggestions that non-muscle myosin II provides the motive force for stolon tip pulsations and further suggest that tip oscillations are functionally coupled to hydrorhizal axial muscle contraction.     

The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II

The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25–30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis.     

In Vivo Orientation of Single Myosin Lever Arms in Zebrafish Skeletal Muscle

Cardiac and skeletal myosin assembled in the muscle lattice power contraction by transducing ATP free energy into the mechanical work of moving actin. Myosin catalytic/lever-arm domains comprise the transduction/mechanical coupling machinery that move actin by lever-arm rotation. In vivo, myosin is crowded and constrained by the fiber lattice as side chains are mutated and otherwise modified under normal, diseased, or aging conditions that collectively define the native myosin environment. Single-myosin detection uniquely defines bottom-up characterization of myosin functionality. The marriage of in vivo and single-myosin detection to study zebrafish embryo models of human muscle disease is a multiscaled technology that allows one-to-one registration of a selected myosin molecular alteration with muscle filament-sarcomere-cell-fiber-tissue-organ- and organism level phenotypes. In vivo single-myosin lever-arm orientation was observed at superresolution using a photoactivatable-green-fluorescent-protein (PAGFP)-tagged myosin light chain expressed in zebrafish skeletal muscle. By simultaneous observation of multiphoton excitation fluorescence emission and second harmonic generation from myosin, we demonstrated tag specificity for the lever arm. Single-molecule detection used highly inclined parallel beam illumination and was verified by quantized photoactivation and photobleaching. Single-molecule emission patterns from relaxed muscle in vivo provided extensive superresolved dipole orientation constraints that were modeled using docking scenarios generated for the myosin (S1) and GFP crystal structures. The dipole orientation data provided sufficient constraints to estimate S1/GFP coordination. The S1/GFP coordination in vivo is rigid and the lever-arm orientation distribution is well-ordered in relaxed muscle. For comparison, single myosins in relaxed permeabilized porcine papillary muscle fibers indicated slightly differently oriented lever arms and rigid S1/GFP coordination. Lever arms in both muscles indicated one preferred spherical polar orientation and widely distributed azimuthal orientations relative to the fiber symmetry axis. Cardiac myosin is more radially displaced from the fiber axis. Probe rigidity implies the PAGFP tag reliably indicates cross-bridge orientation in situ and in vivo.     

In?Vivo Orientation of Single Myosin Lever Arms in Zebrafish Skeletal Muscle

Cardiac and skeletal myosin assembled in the muscle lattice power contraction by transducing ATP free energy into the mechanical work of moving actin. Myosin catalytic/lever-arm domains comprise the transduction/mechanical coupling machinery that move actin by lever-arm rotation. In vivo, myosin is crowded and constrained by the fiber lattice as side chains are mutated and otherwise modified under normal, diseased, or aging conditions that collectively define the native myosin environment. Single-myosin detection uniquely defines bottom-up characterization of myosin functionality. The marriage of in vivo and single-myosin detection to study zebrafish embryo models of human muscle disease is a multiscaled technology that allows one-to-one registration of a selected myosin molecular alteration with muscle filament-sarcomere-cell-fiber-tissue-organ- and organism level phenotypes. In vivo single-myosin lever-arm orientation was observed at superresolution using a photoactivatable-green-fluorescent-protein (PAGFP)-tagged myosin light chain expressed in zebrafish skeletal muscle. By simultaneous observation of multiphoton excitation fluorescence emission and second harmonic generation from myosin, we demonstrated tag specificity for the lever arm. Single-molecule detection used highly inclined parallel beam illumination and was verified by quantized photoactivation and photobleaching. Single-molecule emission patterns from relaxed muscle in vivo provided extensive superresolved dipole orientation constraints that were modeled using docking scenarios generated for the myosin (S1) and GFP crystal structures. The dipole orientation data provided sufficient constraints to estimate S1/GFP coordination. The S1/GFP coordination in vivo is rigid and the lever-arm orientation distribution is well-ordered in relaxed muscle. For comparison, single myosins in relaxed permeabilized porcine papillary muscle fibers indicated slightly differently oriented lever arms and rigid S1/GFP coordination. Lever arms in both muscles indicated one preferred spherical polar orientation and widely distributed azimuthal orientations relative to the fiber symmetry axis. Cardiac myosin is more radially displaced from the fiber axis. Probe rigidity implies the PAGFP tag reliably indicates cross-bridge orientation in situ and in vivo.     

Reversal of the Myosin Power Stroke Induced by Fast Stretching of Intact Skeletal Muscle Fibers

Force generation and movement in skeletal muscle result from a cyclical interaction of overlapping myosin and actin filaments that permits the free energy of ATP hydrolysis to be converted into mechanical work. The rapid force recovery that occurs after a step release imposed on a muscle is thought to result from a synchronized tilting of myosin lever arms toward a position of lower free energy (the power stroke). We investigated the power stroke mechanism in intact muscle fibers of Rana esculenta using a fast stretch to detach forcibly cross-bridges. Stretches were applied either with or without a conditioning step release. Cross-bridge rupture tension was not significantly influenced by the release, whereas sarcomere elongation at the rupture point increased immediately after the release and returned to the prerelease condition within 15-20 ms, following a slower time course compared to the recovery of tension. These observations suggest that the rupture force of a bridge is unaltered by a conditioning release, but rupture must first be preceded by a power stroke reversal, which restores the prepower stroke state. The sarcomere extension at the rupture point indicates both the extent of this power stroke reversal and the time course of strained bridge replenishment.     

Expression of the Myosin Heavy Chain IIB Gene in Porcine Skeletal Muscle: The Role of the CArG-Box Promoter Response Element

Due to its similarity to humans, the pig is increasingly being considered as a good animal model for studying a range of human diseases. Despite their physiological similarities, differential expression of the myosin heavy chain (MyHC) IIB gene (MYH4) exists in the skeletal muscles of these species, which is associated with a different muscle phenotype. The expression of different MyHC isoforms is a critical determinant of the contractile and metabolic characteristics of the muscle fibre. We aimed to elucidate whether a genomic mechanism was responsible for the drastically different expression of MYH4 between pigs and humans, thus improving our understanding of the pig as a model for human skeletal muscle research. We utilized approximately 1 kb of the MYH4 promoter from a domestic pig and a human (which do and do not express MYH4, respectively) to elucidate the role of the promoter sequence in regulating the high expression of MYH4 in porcine skeletal muscle. We identified a 3 bp genomic difference within the proximal CArG and E-box region of the MYH4 promoter of pigs and humans that dictates the differential activity of these promoters during myogenesis. Subtle species-specific genomic differences within the CArG-box region caused differential protein-DNA interactions at this site and is likely accountable for the differential MYH4 promoter activity between pigs and humans. We propose that the genomic differences identified herein explain the differential activity of the MYH4 promoter of pigs and humans, which may contribute to the differential expression patterns displayed in these otherwise physiologically similar mammals. Further, we report that both the pig and human MYH4 promoters can be induced by MyoD over-expression, but the capacity to activate the MYH4 promoter is largely influenced by the 3 bp difference located within the CArG-box region of the proximal MYH4 promoter.     

Calsequestrin Is an Inhibitor of Skeletal Muscle Ryanodine Receptor Calcium Release Channels

We provide novel evidence that the sarcoplasmic reticulum calcium binding protein, calsequestrin, inhibits native ryanodine receptor calcium release channel activity. Calsequestrin dissociation from junctional face membrane was achieved by increasing luminal (trans) ionic strength from 250 to 500 mM with CsCl or by exposing the luminal side of ryanodine receptors to high [Ca²⁺] (13 mM) and dissociation was confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Calsequestrin dissociation caused a 10-fold increase in the duration of ryanodine receptor channel opening in lipid bilayers. Adding calsequestrin back to the luminal side of the channel after dissociation reversed this increased activity. In addition, an anticalsequestrin antibody added to the luminal solution reduced ryanodine receptor activity before, but not after, calsequestrin dissociation. A population of ryanodine receptors (∼35%) may have initially lacked calsequestrin, because their activity was high and was unaffected by increasing ionic strength or by anticalsequestrin antibody: their activity fell when purified calsequestrin was added and they then responded to antibody. In contrast to native ryanodine receptors, purified channels, depleted of triadin and calsequestrin, were not inhibited by calsequestrin. We suggest that calsequestrin reduces ryanodine receptor activity by binding to a coprotein, possibly to the luminal domain of triadin.     

Effect of Calcium Activated Factor on Properties of Actin and Myosin of Rabbit Skeletal Muscle

The effect of calcium activated factor (CAF) on enzymatic properties of actin and myosin was investigated. SDS polyacrylamide gel electrophoresis revealed that CAF did not degrade actin, but a slight degradation was found in myosin during CAF digestion, which might have been due to contaminated protease (s) in CAF preparation. No influence was found in EDTA ATPase of myosin and polymerization of G-actin during CAF digestion. However, heavy meromyosin (HMM) ATPase activating ability of actin was slightly decreased during CAF digestion. Although CAF digestion slightly decreased the biological activity of myofibrillar proteins, a single sarcomere prepared by CAF digestion is a useful model for studying muscle contraction because of its almost intact contractility.     

肌萎缩时的蛋白质降解通路

肌萎缩主要是因为蛋白质降解增强,主要涉及到泛素-蛋白酶体系统、依赖于钙离子的蛋白酶系统、溶酶体系统。在肌萎缩时这些蛋白质降解系统可能是平行作用,也可能按多步骤形式进行。弄清其机制无疑对开发抗肌萎缩药物是有帮助的。     

Membrane Ion Conductances of Mammalian Skeletal Muscle in the Post-Decompression State after High-Pressure Treatment

Exposure of excitable tissues to hyperbaric environments has been shown to alter membrane ion conductances, but only little is known about the state of the membranes of intact cells in the post-decompression phase following a prolonged high-pressure treatment. Furthermore, almost nothing is known about high-pressure effects on skeletal muscle membranes. Therefore, we investigated changes to the input resistances, membrane potentials and voltage-gated membrane currents for sodium (INa), potassium (IK) and calcium (ICa) ions under voltage-clamp conditions in enzymatically isolated intact mammalian single fibers following a 3-hr high-pressure treatment up to 25 MPa at +4 degrees C. After a 3-hr 20 MPa treatment, the input resistance was increased but declined again for treatments with higher pressures. The resting membrane potentials were depolarized in the post-decompression phase following a 20-MPa high-pressure treatment; this could be explained by an increase in the Na+- over K+-permeability ratio and in intracellular [Na+]i. Following a 10-MPa high-pressure treatment, INa, IK and ICa amplitudes were similar compared to controls but were significantly reduced by 25 to 35% after a 3-hr 20-MPa high-pressure treatment. Interestingly, the voltage-dependent inactivation of INa and ICa seemed to be more stable at high pressures compared to the activation parameters, as no significant changes were found up to a 20-MPa treatment. For higher pressure applications (e.g., 25 MPa), there seemed to be a marked loss of membrane integrity and INa, IK and ICa almost disappeared.     

Systems-based Discovery of Tomatidine as a Natural Small Molecule Inhibitor of Skeletal Muscle Atrophy

Skeletal muscle atrophy is a common and debilitating condition that lacks an effective therapy. To address this problem, we used a systems-based discovery strategy to search for a small molecule whose mRNA expression signature negatively correlates to mRNA expression signatures of human skeletal muscle atrophy. This strategy identified a natural small molecule from tomato plants, tomatidine. Using cultured skeletal myotubes from both humans and mice, we found that tomatidine stimulated mTORC1 signaling and anabolism, leading to accumulation of protein and mitochondria, and ultimately, cell growth. Furthermore, in mice, tomatidine increased skeletal muscle mTORC1 signaling, reduced skeletal muscle atrophy, enhanced recovery from skeletal muscle atrophy, stimulated skeletal muscle hypertrophy, and increased strength and exercise capacity. Collectively, these results identify tomatidine as a novel small molecule inhibitor of muscle atrophy. Tomatidine may have utility as a therapeutic agent or lead compound for skeletal muscle atrophy.     

Protease and Protease Inhibitor Activity in Rat Skeletal Muscle during Growth,Protein Deficiency and Fasting

Growing rats fed a protein deficient diet showed an increase in acid and alkaline protease activity and a decrease in protease inhibitor activity. Similar results were found after two and four day fasting periods. Animals on diets of 10% and 20% casein showed parallel protease and protease inhibitor activity when compared to each other at similar body weight periods.     

Myostatin in the Pathophysiology of Skeletal Muscle

Myostatin is an endogenous, negative regulator of muscle growth determining both muscle fiber number and size. The myostatin pathway is conserved across diverse species ranging from zebrafish to humans. Experimental models of muscle growth and regeneration have implicated myostatin as an important mediator of catabolic pathways in muscle cells. Inhibition of this pathway has emerged as a promising therapy for muscle wasting. Here we discuss the recent developments and the controversies in myostatin research, focusing on the molecular and cellular mechanisms underlying the actions of myostatin on skeletal muscle and the potential therapeutic role of myostatin on muscle-related disorders.     

Comparison of Orientation and Rotational Motion of Skeletal Muscle Cross-bridges Containing Phosphorylated and Dephosphorylated Myosin Regulatory Light Chain

Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca²⁺ binding, such as phosphorylation of myosin regulatory light chain (RLC) also controls contraction of vertebrate striated muscle (Cooke, R. (2011) Biophys. Rev. 3, 33–45). Electron paramagnetic resonance (EPR) studies using nucleotide analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC (Stewart, M. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 430–435). The slower ATPase activity in the dephosporylated moiety has been defined as a new super-relaxed state (SRX). It can be observed in both skeletal and cardiac muscle fibers (Hooijman, P., Stewart, M. A., and Cooke, R. (2011) Biophys. J. 100, 1969–1976). Given the importance of the finding that suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (i) relaxed cross-bridges, but not active ones, are statistically better ordered in muscle where the RLC is dephosporylated compared with phosphorylated RLC; (ii) relaxed phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (iii) active phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones during isometric contraction but the duty cycle remained the same, suggesting that both phosphorylated and dephosphorylated muscles develop the same isometric tension at full Ca²⁺ saturation. A simple theory was developed to account for this fact.     

The PDZ-Containing Unconventional Myosin XVIIIA Regulates Embryonic Muscle Integrity in Zebrafish

Myosin XVIIIA, or MYO18A, is a unique PDZ domain-containing unconventional myosin and is evolutionarily conserved from Drosophila to vertebrates. Although there is evidence indicating its expression in the somites, whether it regulates muscle function re- mains unclear. We show that the two zebrafish myo18a genes （myo18aa and myo18ab） are predominantly expressed at somite borders during early developmental stages. Knockdown of these genes or overexpression of the MYO18A PDZ domain disrupts myofiber integrity, induces myofiber lesions, and compromises the localization of dystrophin, ~-dystroglycan （~-DG） and laminin at the myotome boundaries. Cell transplantation experiments indicate that myo18a morphant myoblasts fail to form elongated myofibers in the myotomes of wild-type embryos, which can be rescued by the full-length MYO18A protein. These results suggest that MYO18A likely functions in the adhesion process that maintains the stable attachment of myofibers to ECM （extracellular matrix） and muscle integrity during early development.