Changes in conformation of myosin heads during the development of isometric contraction and rapid shortening in single frog muscle fibres

Abstract

1. Two-dimensional X-ray diffraction patterns were recorded at the European Synchrotron Radiation Facility from central segments of intact single muscle fibres of Rana temporaria with 5 ms time resolution during the development of isometric contraction. Shortening at ca 0.8 times the maximum velocity was also imposed at the isometric tetanus plateau. 2. The first myosin-based layer line (ML1) and the second myosin-based meridional reflection (M2), which are both strong in resting muscle, were completely abolished at the plateau of the isometric tetanus. The third myosin-based meridional reflection (M3), arising from the axial repeat of the myosin heads along the filaments, remained intense but its spacing changed from 14.34 to 14.56 nm. The intensity change of the M3 reflection, IM3, could be explained as the sum of two components, I14.34 and I14.56, arising from myosin head conformations characteristic of rest and isometric contraction, respectively. 3. The amplitudes (A) of the X-ray reflections, which are proportional to the fraction of myosin heads in each conformation, changed with half-times that were similar to that of isometric force development, which was 33.5 +/- 2. 0 ms (mean +/- s.d., 224 tetani from three fibres, 4 C), measured from the end of the latent period. We conclude that the myosin head conformation changes synchronously with force development, at least within the 5 ms time resolution of these measurements. 4. The changes in the X-ray reflections during rapid shortening have two temporal components. The rapid decrease in intensity of the 14.56 nm reflection at the start of shortening is likely to be due to tilting of myosin heads attached to actin. The slower changes in the other reflections were consistent with a return to the resting conformation of the myosin heads that was about 60 % complete after shortening of 70 nm per half-sarcomere.

Figure 1. X-ray diffraction patterns at rest and at the isometric tetanus plateau

A, two-dimensional diffraction patterns at rest (upper half) and at the plateau of the isometric tetanus (lower half). Total exposure time 11 s, obtained by adding 50 ms time frames collected during 224 tetani in three fibres. The equatorial reflections were attenuated with a vertical aluminium strip in front of the detector. Arrowheads: positions of the myosin-based reflections discussed in the text. White boxes: integration limits for reflection intensities. B, intensity distributions near the first myosin layer line (ML1), obtained by vertical integration of the patterns in A at rest (thick line) and at the isometric tetanus plateau (thin line). Dotted vertical lines: integration limits used for I_ML1, minimizing the contribution of the first actin layer line at 38 nm. C, intensity distribution along the meridian in the region of the M2 reflection; thick and thin lines as in B; dotted vertical lines: integration limits used for I_M2. D, intensity distribution along the meridian in the region of the M3 reflection; thick and thin traces as in B; dotted vertical lines: integration limits used for I_M3.

Figure 2. Changes in the X-ray pattern during isometric force development and rapid shortening

A, sarcomere length change in nanometres per half-sarcomere. B-D, force relative to isometric tetanus plateau value (continuous trace), and X-ray intensity (○) of ML1 (B), M2 (C) and M3 (D);I_ML1 and I_M2 are relative to their resting values, I_M3 is relative to the tetanus plateau value; dotted line: zero level for force and intensity. E, superimposed time courses of force and spacing of the M3 reflection; dotted line: zero force and 14.34 nm spacing. Diffraction patterns were collected in 50 ms frames just before the start of stimulation (1 frame), at the plateau of the isometric tetanus before imposed shortening (3 frames) and after force had completely redeveloped (2 frames), and in 5 ms time frames during force development (30 frames), and during the imposed shortening and force redevelopment (40 frames). Each point is the average of the data from three fibres. The total exposure time for the 5 ms time frames was 1.12 s (5 ms × 224 tetani). The dashed line in B, C and E was obtained by fitting the following equation to the X-ray points starting from the third one: where a and b are fixed parameters representing values at the isometric plateau and at rest, respectively. t_½ values in Table 1 were estimated from these fits. Mean force per cross-sectional area was 367 ± 16 kN m⁻² (means ±s.e.m.).

Figure 3. Separation of M3 reflection into two components and time course of the amplitudes of myosin-based reflections

A, time course of the intensity of the 14.34 nm reflection (○, normalized for the value at rest) and the 14.56 nm reflection (•, normalized for the value at the isometric plateau) calculated as described in the text. Continuous line, force; dashed lines superimposed on the X-ray data during force development were obtained as described in Fig. 2. B-E, time course of force development (continuous line) superimposed on that of the amplitude (A) of X-ray reflections (○). For the ML1 (B), M2 (C) and 14.34 (D) reflections the amplitude was inverted for comparison with force development. Dashed lines were obtained as described in Fig. 2.