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X-ray patterns were recorded from bundles of 2-3 fibers of R. Esculenta at 4°C at rest and at the plateau of an isometric tetanus (T0) at sarcomere length (SL) 2.0 to 3.6 μm at the ID02 beamline at the European Synchrotron Radiation Facility, Grenoble. The patterns were normalized by the intensity of the 1,0 equatorial reflection at rest at each experimental SL to compensate for variation of diffracting mass with SL and between fibers. The axial diffraction pattern from resting fibers was independent of SL in the range 2.0-2.6 μm; in the range 2.6-3.0 μm the intensity and interference fine structure of the meridional M3 reflection from the axial repeat of the myosin heads along the filaments was constant, but its spacing (SM3) increased. The intensity of the first myosin layer line decreased in this SL range, indicating decreased helical ordering of the myosin heads. The intensity of the 44nm merdional reflection associated with myosin binding protein C was constant up to SL 2.7 μm, but much reduced for SL>2.7 μm. At T0, the M3 reflection intensity was smaller at longer SL, in proportion to the overlap between thick and thin filaments. The interference fine structure of the M3 was independent of SL up to 2.8 μm; at longer SL it varied between preparations and SM3 reduced with increasing SL. The SL-dependence of the M3 reflection at T0 indicates that the detached myosin heads in the non-overlap region of the thick filament are axially disordered compared with actin-attached heads in the overlap region, although the axial center of mass of the detached and attached heads is similar. Supported by Ente Cassa di Risparmio di Firenze, FIRB-Futuro in Ricerca, MRC and ESRF.

Force and shortening in muscle are generated by ATP-driven working strokes of myosin II motors, during their cyclic interactions with the actin filament. In vitro and in situ studies suggest that the working stroke is associated with the release of phosphate (Pi). We used nanometer-microsecond mechanics on skinned muscle fibers from rabbit psoas (2.4 µm sarcomere length, 12 °C) to record the velocity transient following a force step and found that the early rapid shortening, which represents the mechanical manifestation of the working stroke, is not affected by the increase in [Pi], while the subsequent transition to the steady shortening velocity is accelerated and the steady power at high loads is reduced. A new chemo-mechanical model has been proposed that reproduces the transient and steady state responses by assuming that biochemical and mechanical steps are not tightly coupled: (i) the release of the hydrolysis products (Pi and ADP) from the catalytic site of the myosin motor can occur at any stage of the working stroke and (ii) a myosin motor, in an intermediate state of the working stroke, can slip to a second actin monomer (the next monomer away from the center of the sarcomere) before terminating the biochemical cycle. This model explains the efficient action of muscle molecular motors working as an ensemble. Here we demonstrate the model ability to fit the force transients elicited by jumps in either [ATP] (Goldman et al., Nature 300, 701-705, 1982; Dantzig et al., J. Physiol. 432, 639-680, 1991) or [Pi] (Dantzig et al., J. Physiol. 451, 247-278, 1992; Homsher et al., Biophys. J. 72, 1780-1791, 1997). Supported by MIUR-PRIN and ECRF-2012 (Italy).

X-ray diffraction patterns were recorded from whole EDL muscle of 1-2 month old mice at rest and during isometric contraction (force T0) at temperatures between 10 and 35°C. Muscles were vertically mounted at the BioCAT beamline (APS, Argonne, USA, X-ray energy 12 keV, camera length 3.5m) in a trough containing physiological solution gassed with carbogen (95% O2 plus 5% CO2, pH=7.4) and at the in situ rest length. X-ray exposure time was 20 ms for both rest and T0, repeated twice at each temperature. The muscle was shifted axially between exposures to minimise radiation damage. T0 was 251±17 kPa(mean±S.E.M., n=3) at 30°C (T0,30) and not significantly different at 35°C. Below 30°C force decreased with decreasing temperature, reaching 0.25 T0,30 at 10°C. The M3 meridional reflection from the axial repeat of the myosin heads was sampled by X-ray interference between the two halves of the myosin filament. At rest the M3 reflection consisted of a main peak with two small satellite peaks; during activation it consisted of two main peaks with an intensity ratio (RM3; high angle/low angle) that varied from ∼0.8 at 30°C to ∼1.1 at 10°C. The total M3 intensity (IM3) was five times smaller at 10 than at 30°C, both at rest and during isometric contraction. The M3 spacing (SM3) increased by ∼1.3% from rest to isometric contraction at all temperatures; the spacing of its second order (SM6) had a markedly different temperature dependence at rest, but a roughly similar temperature dependence during isometric contraction, so that, from rest to T0, SM6 increased by ∼1.3% at 30°C but by only ∼0.5% at 10°C.Supported by Ente Cassa di Risparmio di Firenze, MIUR, CNISM, (Italy), MRC (U.K.), NIH.

Osmotic compression of skinned fibers from rabbit psoas muscle with 5% Dextran T-500, to recover the in vivo interfilament spacing, increases the Ca++ sensitivity of force without altering maximum force (Godt and Maughan, Pflugers Arch 391:334, 1981). We investigated the structural basis of this effect using X-ray diffraction at beam-line ID02 (ESRF, Grenoble, France). Bundles of 3-5 fibers were activated isometrically at different pCa by a temperature jump from 1°C to 12°C. In relaxed fibers at 12°C, addition of 5% Dextran induced (i) a three-fold increase in the intensity of the first myosin layer line, (ii) a four-fold increase in the intensity of the so-called forbidden reflections, and (iii) 0.5% reduction in the spacings of all the myosin-based meridional reflections. Thus osmotic compression to restore the in vivo interfilament spacing induces recovery of the thick filament structure observed in resting intact muscle, in particular the systematic axial perturbation of the three layers of myosin heads within the 43-nm repeat attributed to the presence of links between thick and thin filaments mediated by Myosin Binding Protein-C (MyBP-C). The [Ca++]-dependence of active force and the intensity of M3 reflection from the axial periodicity of the myosin heads had a pCa50 of 6.39 in the presence of 5% Dextran, 0.4 pCa units larger than in its absence. These results indicate that the increase in interfilament spacing in skinned fibers impairs the signalling pathway involved in normal Ca-activation of the contractile apparatus, probably by causing the loss of MyBP-C links to the thin filaments.Supported by FIRB-Futuro in Ricerca and MIUR-PRIN (Italy), MRC (UK).