The Closed State of the Thin Filament Is Not Occupied in Fully Activated Skeletal Muscle

Abstract

Muscle contraction is powered by actin-myosin interaction controlled by Ca²⁺ via the regulatory proteins troponin (Tn) and tropomyosin (Tpm), which are associated with actin filaments. Tpm forms coiled-coil dimers, which assemble into a helical strand that runs along the whole ∼1 μm length of a thin filament. In the absence of Ca²⁺, Tn that is tightly bound to Tpm binds actin and holds the Tpm strand in the blocked, or B, state, where Tpm shields actin from the binding of myosin heads. Ca²⁺ binding to Tn releases the Tpm from actin so that it moves azimuthally around the filament axis to a closed, or C, state, where actin is partially available for weak binding of myosin heads. Upon transition of the weak actin-myosin bond into a strong, stereo-specific complex, the myosin heads push Tpm strand to the open, or O, state allowing myosin binding sites on several neighboring actin monomers to become open for myosin binding. We used low-angle x-ray diffraction at the European Synchrotron Radiation Facility to check whether the O- to C-state transition in fully activated fibers of fast skeletal muscle of the rabbit occurs during transition from isometric contraction to shortening under low load. No decrease in the intensity of the second actin layer line at reciprocal radii in the range of 0.15-0.275 nm^-1 was observed during shortening suggesting that an azimuthal Tpm movement from the O- to C-state does not occur, although during shortening muscle stiffness is reduced compared to the isometric state, and the intensities of other actin layer lines demonstrate a ∼2-fold decrease in the fraction of myosin heads strongly bound to actin. The data show that a small fraction of actin-bound myosin heads is sufficient for supporting the O-state and, therefore the C-state is not occupied in fully activated skeletal muscle that produces mechanical work at low load.

Figure 1

(A, top to bottom) A typical time course of temperature, length change, tension and x-ray shutter opening signal during a run of the experimental protocol in a bundle of three muscle fibers (from top to bottom, (A)). Step stretches were applied to the bundle immediately after each x-ray frame to measure instantaneous stiffness. (B and C) Expanded views of the parts of the record indicated by ellipses (B) and (C), respectively, in (A).

Figure 2

Off-meridional x-ray intensity plotted against meridional reciprocal spacing was collected from 43 runs of the experimental protocol in six bundles of muscle fibers during isometric contraction and steady shortening (solid lines, see color code). The intensity was integrated along equatorial direction at reciprocal radii from 0.005 to 0.15 nm⁻¹. Some myosin (M) and actin (A) layer lines are labeled. Inset: intensity profiles in the region of the M1 and A1 layer lines, and their breakdown into their Gaussian components, namely, the M1 (dash-dot lines) and A1 (dashed lines) layer lines during isometric contraction and shortening (see color code). To see this figure in color, go online.

Figure 3

The x-ray intensity profiles during isometric contraction and at the end of shortening (color code is shown at the top) were plotted against reciprocal meridional spacing. The intensities were obtained by integration along the equator at reciprocal radial ranges shown above each panel (see also Materials and Methods section). The positions of the actin (A1, A2, A6, and A7), myosin (M1, M2, and M3) and actin-myosin (AM₊₁) layer lines are labeled. To see this figure in color, go online.

Figure 4

The intensities of A1, A2, A6, and A7 actin layer lines calculated at different positions of the tropomyosin strand in the overlap zone of thin filament and with different fractions of myosin heads bound to actin. Solid, dashed, and dotted lines correspond to 40%, 20%, and 0% of myosin heads strongly bound to actin in the “open” (O-state) and “closed” (C-state) positions of Tpm, the color code is shown on the left. The O-state is the original structural model (17) while the C-state was obtained by azimuthal movement of the Tpm strand around the thin filament axis by 10°. To see this figure in color, go online.