Dual-filament regulation of relaxation in mammalian fast skeletal muscle

Fig. 1.. Mechanical protocol, experimental set-up and small-angle X-ray diffraction (SAXD) patterns. (A) Mechanical protocol. Force normalized to peak force (T/T0), the approximate time course of intracellular free calcium ion concentration [Ca2+]i from (19), and applied change in muscle length as a percentage of optimal length (L0). Plateau force in fixed-end tetani at L0 was 270.6 ± 21.8 kPa (mean ± SEM; n = 7). Colored segments of the force trace denote periods used for averaging the SAXD data: cyan, rest; red, peak force at longer muscle length; green, unloaded shortening; orange, peak force at shorter muscle length; blue, isometric relaxation; purple, mechanically relaxed. The blue-shaded region denotes isometric relaxation. Vertical black lines mark the start (at time zero) and end of electrical stimulation. The dashed horizontal black line denotes zero force. (B) Arrangement for mounting muscles vertically on the synchrotron beamline with minimized X-ray path in solution, perfusion with oxygenated Krebs solution, electrical stimulation, force measurement, and control of muscle length. (C) Left-hand side of SAXD pattern from resting muscle with a sample-to-detector distance 2.0 m;…

Fig. 2.. Structural signals associated with thick filament activation. (A) Intensity of the first myosin layer line (IML1). Inset: Helical arrangement of pairs of myosin motors (blue) folded back against their tails that gives rise to the strong ML1 reflection with axial periodicity 43 nm in resting muscle; disordered motors are shown in gray. (B) Intensity of the fourth myosin layer line (IML4). (C) Spacing of the sixth myosin-based meridional reflection (SM6) corresponding to an axial periodicity of 7.17 nm (Inset; red dashed lines) in resting muscle. (D) Spacing of the eleventh myosin-based meridional reflection (SM11) corresponding to the 3.9 nm axial periodicity of titin domains in the C-zone (Inset, magenta). Relative force (T/T0) is shown as the continuous black line. Color coding of the phases of the protocol is shown in Fig. 1A. Vertical lines denote the start and end of electrical stimulation; horizontal dashed lines denote the resting values of each parameter. Mean ± SEM for n = 4 muscles in (A) to (C), and n = 3 muscles in (D).

Fig. 3.. Structural signals associated with thin filament activation. (A) Intensity of the second actin layer line (IAL2). Inset: Schematic transverse section through the thin filament indicating azimuthal positions of tropomyosin (Tm) at rest (cyan; dark brown circles) and in active contraction (red; light brown circles). (B) Spacing of the thirteenth meridional actin-based reflection (SA13), calculated from the spacings of the sixth and seventh actin layer lines. Inset: Longitudinal thin filament schematic with ca 2.73 nm axial repeat of actin monomers associated with the A13 reflection, and genetic actin helices associated with the AL6 (5.9 nm) and AL7 (5.1 nm) reflections. Tropomyosin (dark brown) blocks myosin binding sites on actin (black) when troponin (dark purple) is not saturated by Ca2+. Relative force (T/T0) is shown as the continuous black line. Color coding of the phases of the protocol is shown in Fig. 1A. Vertical lines denote the start and end of electrical stimulation; horizontal dashed lines denote the resting values of each parameter. Mean ± SEM for n = 3 muscles in (A) and n = 4 muscles in (B).

Fig. 4.. Structural signals of myosin motor conformation and actin attachment. (A) Intensity of the third myosin-based meridional reflection (IM3). Inset: Schematic of overlapping thin and thick filaments with myosin motors in their resting (blue; axial periodicity 14.34 nm), actin-attached force-generating (light red), and disordered (dark gray) conformations. (B) Intensity of the first actin layer line (IAL1). Inset: Thin filament schematic showing the ca 37 nm helical periodicity associated with the AL1 reflection. (C) Intensity of the sixth actin layer line (IAL6) corresponding to the genetic helix with a periodicity of 5.9 nm (Inset). (D) Intensity of the seventh actin layer (IAL7) corresponding to the genetic helix with a periodicity of 5.1 nm (Inset). Relative force (T/T0) is shown as the continuous black line. Color coding of the phases of the protocol is shown in Fig. 1A. Vertical lines denote the start and end of electrical stimulation; horizontal dashed lines denote the resting values of each parameter. Mean ± SEM for n = 4 muscles (A), (C), and (D) and n = 7 muscles for (B).

Fig. 5.. Equatorial X-ray reflections. (A) Equatorial intensity ratio (I1,1/I1,0). (B) Lattice spacing (d1,0) indicated in the inset as the separation of the 1,0 planes of the filament lattice. (C) Volume of the filament lattice (ΔV) expressed as a percentage change relative to resting volume. Closed and open squares correspond to ΔV calculated from the short and long sarcomere length (SL) populations, respectively. “Average” denotes the average volume of the short and long sarcomere populations. Relative force (T/T0) is shown as the continuous black line. Color coding of the phases of the protocol is shown in Fig. 1A. Vertical lines denote the start and end of electrical stimulation; horizontal dashed lines denote the resting values of each parameter. Mean ± SEM for n = 7 muscles in (A) and (B), and n = 4 muscles in (C).

Fig. 6.. Sarcomere reflections. (A) Ultra-low-angle meridional intensity distribution after subtraction of the diffuse background, showing the X-ray reflections corresponding to the first three orders of the sarcomere repeat; colors denote protocol periods shown in Fig. 1A. (B) First-order sarcomere reflection profiles during isometric relaxation (thicker black), and at the indicated later times. (C) Diffracted intensity distribution without removing the diffuse background plotted on a log scale; colors as in (A). (D) SL is determined from the centroid of the first-order sarcomere reflections. (E) Fractional intensities of the first-order reflections from the long and short sarcomere populations. Vertical lines denote the start and end of electrical stimulation. In (D) and (E), open/closed squares denote long/short sarcomere populations, respectively. Color coding for (A) and (C) to (E) is the same as in Fig. 1A. Vertical black lines denote the start and end of electrical stimulation; the horizontal dashed line denotes the resting SL. Gray and yellow shaded regions as in Fig. 1A. (A) to (C) show the average of n = 4 muscles; (D) and (E) are mean ± SEM for n = 4 muscles.

Fig. 7.. Thick and thin filament activation states and myosin motor conformations. Upper panel: Force normalized to maximal force (T/T0). Vertical black lines denote the start and end of electrical stimulation. Lower panels: Schematic of thin and thick filaments and myosin motor conformations at the five periods marked on the force trace. The filament components are identified in the key box. The activation state of the thick filament is coded as gray (OFF) to green (ON); that of the thin filament as gray (OFF) to orange (ON).