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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.

The contracting muscle exhibits a quite high resistance to sudden increase in load up to twice the isometric force (Katz, J. Physiol. 96:45,1939). The increase in half-sarcomere (hs) stiffness and the changes in the x-ray interference of myosin-based reflections during muscle stretch indicate that the second motor domain of the myosin molecules with the first motor domain already attached to actin in the isometric contraction attaches within 2 ms following the stretch (Brunello et al., Proc. Natl. Acad. Sci. USA, 104:20114, 2007). The mechanism is further investigated here by using single frog fiber mechanics (Rana esculenta, 4 °C, 2.1 μm sarcomere length). Stretches between 2 and 8 nm hs−1, complete within 100 μs, were applied at the tetanus plateau (T0) and the fraction of new motors relative to the isometric number (f) was determined either at the peak of the force response to stretch (T1) or at the end of the quick phase of force recovery, 2 ms after the stretch (T2). We show that: 1) for stretches 5nm at T1 f reaches a maximum value of 0.3, while at T2 f reaches a maximum value of 1. These results support the idea that the distortion of the attached motor domain of one myosin molecule promotes the attachment of the partner motor domain and indicate an upper limit (∼104 s−1) for the rate of the recruitment process. Supported by NIH (Grant no. 5R01AR49033-4) and MiUR, 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).