Interference fine structure and sarcomere length dependence of the axial x-ray pattern from active single muscle fibers

Abstract

Axial x-ray diffraction patterns from single intact fibers of frog skeletal muscle were recorded by using a highly collimated x-ray beam at the European Synchrotron Radiation Facility. During isometric contraction at sarcomere lengths 2.2-3.2 m, the M3 x-ray reflection, associated with the repeat of myosin heads along the filaments, was resolved into two peaks. The total M3 intensity decreased linearly with increasing sarcomere length and was directly proportional to the degree of overlap between myosin and actin filaments, showing that it comes from myosin heads in the overlap region. The separation between the M3 peaks was smaller at longer sarcomere length and was quantitatively explained by x-ray interference between myosin heads in the two overlap regions of each sarcomere. The relative intensity of the M3 peaks was independent of sarcomere length, showing that the axial periodicities of the nonoverlap and overlap regions of the myosin filament have the same value, 14.57 nm, during active contraction. In resting fibers the periodicity is 14.34 nm, so muscle activation produces a change in myosin filament structure in the nonoverlap as well as the overlap part of the filament. The results establish x-ray interferometry as a new tool for studying the motions of myosin heads during muscle contraction with unprecedented spatial resolution. In resting skeletal muscles from vertebrates, the myosin head domains or cross-bridges have a roughly helical arrangement, with three layers of heads in each 43-nm helical repeat (1). During isometric contraction, this helical structure disappears, but the myosin heads retain a strong axial periodicity of 14.57 nm, 1.6% larger than the corresponding value at rest, 14.34 nm (1-7). Both structures produce an intense axial x-ray ref lection called the M3. The M3 ref lection during isometric contraction has been associated with force-generating myosin heads oriented roughly perpendicular to the filament axis (3, 4, 8 -11), and the intensity of this ref lection has been used to investigate the changes in myosin head conformation that drive force generation (8 -11). The transition between myosin head conformations with 14.34 and 14.57 nm axial periodicities has roughly the same time course as isometric force development after electrical stimulation (3, 4, 7), but its functional significance is not understood.We investigated this transition in isolated intact muscle fibers, by using the unprecedented spatial resolution of the small-angle x-ray scattering beamline ID2-SAXS at the European Synchrotron Radiation Facility (ESRF, Grenoble, France). At this resolution, the M3 reflection is split into closely spaced peaks by interference between the two arrays of myosin heads in each bipolar myosin filament (1, 2, 12). We recorded the interference fine structure of the M3 reflection during isometric contraction at different sarcomere lengths in the range 2.0 to 3.6 m, to vary the degree of overlap between the myosin-and actin-containing filaments. The sarcomere length controls both the nu...

Mechanism of force generation by myosin heads in skeletal muscle

“…1c). These equations would only apply exactly if each layer of myosin heads could be considered as a point mass, but a similar analysis using the axial mass projections of atomic models for the myosin head 5 -7 gave the same values of I HA /I LA and S M3 within the precision of the present measurements 18 . The changes in L can therefore be interpreted solely in terms of the average axial motion (Dc) of the centres of mass of the myosin heads with respect to their attachments to the myosin filament, independent of any other effects of changes in their conformation.…”

“…Although many studies of the mechanism of muscle contraction 9 -13 have been based on this hypothesis, the alternative view-that the fast force transients are caused by fast components of attachment and detachment 14 -17 has not been excluded definitively. Here we show that measurements of the axial motions of the myosin heads at ångström resolution by a new X-ray interference technique 18 rule out the rapid attachment/detachment hypothesis, and provide compelling support for the working stroke model of force generation.…”

“…The resulting fine structure of the X-ray reflection (Fig. 1d, green) provides an extremely sensitive measure of the axial motion of the myosin heads 18 .…”

“…3b, red circles, n = 9 fibres) and 0.34^0.05%/T 0 at T 2 (blue triangles, n = 18). The value of kS M l at T 1 represents the instantaneous compliance of the myosin filament, and the higher value at T 2 is likely to include a contribution from changes in the structure of the myosin filament 18,22 . The total instantaneous compliance of each set of overlapping myosin and actin filaments crosslinked by myosin heads during active isometric contraction is 5.1 nm/T 0 .…”

“…The calculated values of I HA /I LA , S LA and S HA are almost independent of the value of kS M l, because myosin filament compliance leads to proportional changes in d and L (ref. 18). The discrepancy is reduced by increasing the value of kS A l, but would not be eliminated even if kS A l were twice the measured value 20,21 , in which case almost the whole compliance of the muscle sarcomere would be in the filaments, with virtually no contribution left for the myosin heads.…”