Three-dimensional structure of the basketweave Z-band in midshipman fish sonic muscle.

Striated muscle enables movement in all animals by the contraction of myriads of sarcomeres joined end to end by the Z-bands. The contraction is due to tension generated in each sarcomere between overlapping arrays of actin and myosin filaments. At the Z-band, actin filaments from adjoining sarcomeres overlap and are cross-linked in a regular pattern mainly by the protein α-actinin.

Insights into Actin-Myosin Interactions within Muscle from 3D Electron Microscopy.

Much has been learned about the interaction between myosin and actin through biochemistry, in vitro motility assays and cryo-electron microscopy (cryoEM) of F-actin, decorated with myosin heads. Comparatively less is known about actin-myosin interactions within the filament lattice of muscle, where myosin heads function as independent force generators and thus most measurements report an average signal from multiple biochemical and mechanical states. All of the 3D imaging by electron microscopy (EM) that has revealed the interplay of the regular array of actin subunits and myosin heads within the filament lattice has been accomplished using the flight muscle of the large water bug sp.

Vancomycin Prescribing and Therapeutic Drug Monitoring in Children With and Without Acute Kidney Injury After Cardiac Arrest.

Background: Acute kidney injury (AKI) commonly occurs after cardiac arrest. Those subsequently treated with vancomycin are at additional risk for drug-induced kidney injury.

Objective: We aimed to determine whether opportunities exist for improved drug monitoring after cardiac arrest.

Stretch activation properties of Drosophila and Lethocerus indirect flight muscle suggest similar calcium-dependent mechanisms.

Muscle stretch activation (SA) is critical for optimal cardiac and insect indirect flight muscle (IFM) power generation. The SA mechanism has been investigated for decades with many theories proposed, but none proven. One reason for the slow progress could be that multiple SA mechanisms may have evolved in multiple species or muscle types.

A Population Pharmacokinetic Analysis to Study the Effect of Therapeutic Hypothermia on Vancomycin Disposition in Children Resuscitated From Cardiac Arrest.

Objectives: Limited data exist on the effects of therapeutic hypothermia on renal function and pharmacokinetics in pediatric patients after cardiac arrest. The objective was to describe the differences in vancomycin disposition in pediatric patients following cardiac arrest treated with either therapeutic hypothermia or normothermia using population pharmacokinetic modeling.

Design: Single-center, retrospective cohort study.

Structure of myosin filaments from relaxed flight muscle by cryo-EM at 6 Å resolution.

We describe a cryo-electron microscopy three-dimensional image reconstruction of relaxed myosin II-containing thick filaments from the flight muscle of the giant water bug . The relaxed thick filament structure is a key element of muscle physiology because it facilitates the reextension process following contraction. Conversely, the myosin heads must disrupt their relaxed arrangement to drive contraction.

Myosin S2 origins track evolution of strong binding on actin by azimuthal rolling of motor domain.

Myosin crystal structures have given rise to the swinging lever arm hypothesis, which predicts a large axial tilt of the lever arm domain during the actin-attached working stroke. Previous work imaging the working stroke in actively contracting, fast-frozen Lethocerus muscle confirmed the axial tilt; but strongly bound myosin heads also showed an unexpected azimuthal slew of the lever arm around the thin filament axis, which was not predicted from known crystal structures. We hypothesized that an azimuthal reorientation of the myosin motor domain on actin during the weak-binding to strong-binding transition could explain the lever arm slew provided that myosin's α-helical coiled-coil subfragment 2 (S2) domain emerged from the thick filament backbone at a particular location.

Structural changes in isometrically contracting insect flight muscle trapped following a mechanical perturbation.

The application of rapidly applied length steps to actively contracting muscle is a classic method for synchronizing the response of myosin cross-bridges so that the average response of the ensemble can be measured. Alternatively, electron tomography (ET) is a technique that can report the structure of the individual members of the ensemble. We probed the structure of active myosin motors (cross-bridges) by applying 0.

Electron microscopy and x-ray diffraction evidence for two Z-band structural states.

In vertebrate muscles, Z-bands connect adjacent sarcomeres, incorporate several cell signaling proteins, and may act as strain sensors. Previous electron microscopy (EM) showed Z-bands reversibly switch between a relaxed, "small-square" structure, and an active, "basketweave" structure, but the mechanism of this transition is unknown. Here, we found the ratio of small-square to basketweave in relaxed rabbit psoas muscle varied with temperature, osmotic pressure, or ionic strength, independent of activation.

X-ray diffraction evidence for myosin-troponin connections and tropomyosin movement during stretch activation of insect flight muscle.

Stretch activation is important in the mechanical properties of vertebrate cardiac muscle and essential to the flight muscles of most insects. Despite decades of investigation, the underlying molecular mechanism of stretch activation is unknown. We investigated the role of recently observed connections between myosin and troponin, called "troponin bridges," by analyzing real-time X-ray diffraction "movies" from sinusoidally stretch-activated Lethocerus muscles.

Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions.

Background: Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ.

Methodology: We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned.

Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle.

During active muscle contraction, tension is generated through many simultaneous, independent interactions between the molecular motor myosin and the actin filaments. The ensemble of myosin motors displays heterogeneous conformations reflecting different mechanochemical steps of the ATPase pathway. We used electron tomography of actively contracting insect flight muscle fast-frozen, freeze substituted, Araldite embedded, thin-sectioned and stained, to obtain 3D snapshots of the multiplicity of actin-attached myosin structures.

Similarities and differences between frozen-hydrated, rigor acto-S1 complexes of insect flight and chicken skeletal muscles.

The structure and function of myosin crossbridges in asynchronous insect flight muscle (IFM) have been elucidated in situ using multiple approaches. These include generating "atomic" models of myosin in multiple contractile states by rebuilding the crystal structure of chicken subfragment 1 (S1) to fit IFM crossbridges in lower-resolution electron microscopy tomograms and by "mapping" the functional effects of genetically substituted, isoform-specific domains, including the converter domain, in chimeric IFM myosin to sequences in the crystal structure of chicken S1. We prepared helical reconstructions (approximately 25 A resolution) to compare the structural characteristics of nucleotide-free myosin0 S1 bound to actin (acto-S1) isolated from chicken skeletal muscle (CSk) and the flight muscles of Lethocerus (Leth) wild-type Drosophila (wt Dros) and a Drosophila chimera (IFI-EC) wherein the converter domain of the indirect flight muscle myosin isoform has been replaced by the embryonic skeletal myosin converter domain.

Measuring the resolution of uncompressed plastic sections cut using an oscillating knife ultramicrotome.

Thin sections of biological tissue embedded in plastic and cut with an ultramicrotome do not generally display useful details smaller than approximately 50 A in the electron microscope. However, there is evidence that before sectioning the embedded tissue can be substantially better preserved, which suggests that cutting is when major damage and loss of resolution occurs. We show here a striking example of such damage in embedded insect flight muscle fibres.

Electron tomography of swollen rigor fibers of insect flight muscle reveals a short and variably angled S2 domain.

Subfragment 2 (S2), the segment that links the two myosin heads to the thick filament backbone, may serve as a swing-out adapter allowing crossbridge access to actin, as the elastic component of crossbridges and as part of a phosphorylation-regulated on-off switch for crossbridges in smooth muscle. Low-salt expansion increases interfilament spacing (from 52 nm to 67 nm) of rigor insect flight muscle fibers and exposes a tethering segment of S2 in many crossbridges. Docking an actoS1 atomic model into EM tomograms of swollen rigor fibers identifies in situ for the first time the location, length and angle assignable to a segment of S2.

The myosin filament superlattice in the flight muscles of flies: A-band lattice optimisation for stretch-activation?

Low-angle X-ray diffraction patterns from relaxed fruitfly (Drosophila) flight muscle recorded on the BioCat beamline at the Argonne Advanced Photon Source (APS) show many features similar to such patterns from the "classic" insect flight muscle in Lethocerus, the giant water bug, but there is a characteristically different pattern of sampling of the myosin filament layer-lines, which indicates the presence of a superlattice of myosin filaments in the Drosophila A-band. We show from analysis of the structure factor for this lattice that the sampling pattern is exactly as expected if adjacent four-stranded myosin filaments, of repeat 116 nm, are axially shifted in the hexagonal A-band lattice by one-third of the 14.5 nm axial spacing between crowns of myosin heads.

Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges.

As a first step toward freeze-trapping and 3-D modeling of the very rapid load-induced structural responses of active myosin heads, we explored the conformational range of longer lasting force-dependent changes in rigor crossbridges of insect flight muscle (IFM). Rigor IFM fibers were slam-frozen after ramp stretch (1000 ms) of 1-2% and freeze-substituted. Tomograms were calculated from tilt series of 30 nm longitudinal sections of Araldite-embedded fibers.

Ca-activation and stretch-activation in insect flight muscle.

Asynchronous insect flight muscle is specialized for myogenic oscillatory work, but can also produce isometric tetanic contraction. In skinned insect flight muscle fibers from Lethocerus, with sarcomere length monitored by a striation follower, we determined the relation between isometric force (F(0)) at serial increments of [Ca(2+)] and the additional active force recruited at each [Ca(2+)] by a stretch of approximately 12 nm per half-sarcomere (F(SA)). The isometric force-pCa relation shows that 1.

Cross-bridge number, position, and angle in target zones of cryofixed isometrically active insect flight muscle.

Electron micrographic tomograms of isometrically active insect flight muscle, freeze substituted after rapid freezing, show binding of single myosin heads at varying angles that is largely restricted to actin target zones every 38.7 nm. To quantify the parameters that govern this pattern, we measured the number and position of attached myosin heads by tracing cross-bridges through the three-dimensional tomogram from their origins on 14.

Concentric intermediate filament lattice links to specialized Z-band junctional complexes in sonic muscle fibers of the type I male midshipman fish.

Type I male midshipman fish produce high-frequency hums for prolonged durations using sonic muscle fibers, each of which contains a hollow tube of radially oriented thin and flat myofibrils that display extraordinarily wide ( approximately 1.2 microm) Z bands. We have revealed an elaborate cytoskeletal network of desmin filaments associated with the contractile cylinder that form interconnected concentric ring structures in the core and periphery at the level of the Z bands.

Myosin head configuration in relaxed insect flight muscle: x-ray modeled resting cross-bridges in a pre-powerstroke state are poised for actin binding.

Low-angle x-ray diffraction patterns from relaxed insect flight muscle recorded on the BioCAT beamline at the Argonne APS have been modeled to 6.5 nm resolution (R-factor 9.7%, 65 reflections) using the known myosin head atomic coordinates, a hinge between the motor (catalytic) domain and the light chain-binding (neck) region (lever arm), together with a simulated annealing procedure.