Intensity Changes of Myosin Meridional Reflections in Frog Skeletal Muscle

[Specimen]

A sartorius muscle (a muscle in the thigh) of a Japanese frog (Rana porosa) in a Ringer solution cooled to about 4 degrees. The muscle was mounted vertically.

[X-ray setup]

The X-ray energy was 12.4 keV (undulator gap 14.3mm). The front-end slit was closed to 0.50 mm (horizontal) x 0.15 mm (vertical). The beam at the sample was approximately 0.20 mm x 0.04 mm. The ring current was about 90 mA. A galvanometer-based x-ray shutter was used to limit the exposure to about 30 msec.

[Detector]

An x-ray image intensifier with a CCD digital camera was used to record the diffraction pattern. The x-ray image intensifier has a 15-cm diameter Be window (V5445P, Hamamatsu Photonics). The phosphor in the entrance window is CsI:Na, while that in the exit window is P46 (YAG). Since both CsI and P46 have a short decay, no persistence is detectable at this time resolution.
The CCD camera (C7770, Hamamatsu Photonics) is made of a prism and three fast CCD chips. All three CCDs view the same image but each reads an image every three frames. With 640 x 480 pixels (full frame), the rate of readout is 290 frames per second (0.34 msec per frame). Images can be recorded continuously up to 20 sec. The frame rate can be increased by reducing the number of lines to be readout: with 12 lines, 0.12 msec per frame is achieved.

[Experimental protocol]

The time resolution was 0.52 msec per frame. To achieve this, only the central (meridional) 72 lines were recorded.
The muscle was stimulated with a train of electrical pulses to induce tetanus. At time 0 in the figure, about 1% release (complete within 1 msec) was applied to the muscle using a servo motor.
The figure below shows the intensity changes of two reflections arising from the array of myosin heads in the muscle. One is the third myosin meridional reflection at 1/14.3 nm^-1 (at 1/14.45 nm^-1 during contraction), the other is the second myosin meridional reflection at about 1/21.4 nm^-1. It is observed that the third meridian drops in intensity after the release of muscle while the second increases in intensity. There is a short (about 0.5 msec) delay after the beginning of the release before the intensity changes take place. The intensity of the third meridian increases during this period.

Note that this was recorded in a single experiment (one contraction of one muscle). No summation or averaging was applied to the data.

Movie of the above data : the intensity of the third-order meridional reflection drops extensively upon a quick stretch.


Other studies on skeletal and cardiac muscles:

N. Yagi. An X-ray Diffraction Study on Early Structural Changes in Skeletal Muscle Contraction. Biophys. J. 84, 1-10 (2003).

N. Yagi, J. Shimizu, S. Mohri, J. Araki, K. Nakamura, H. Okuyama, H. Toyota, T. Morimoto, Y. Morizane, M. Kurusu, T. Miura, K. Hashimoto, K. Tsujioka, H. Suga, F. Kajiya. X-ray Diffraction from a Left Ventricular Wall of Rat Heart. Biophys. J. 86, 2286-94 (2004).

J. T. Pearson, M. Shirai, H. Ito, N. Tokunaga, H. Tsuchimochi, N. Nishiura, D. O. Schwenke, H. Ishibashi-Ueda, R. Akiyama, H. Mori, K. Kangawa, H. Suga and N. Yagi In Situe Measurements of Crossbridge Dynamics and Lattice Spacing in Rat Hearts by X-ray Diffraction: Sensitivity to regional ischemia. Circulation 109, 2983-2986 (2004).

J. Wakayama, T. Tamura, N. Yagi and H. Iwamoto. Structural transients of contractile proteins upon sudden ATP liberation in skeletal muscle fibers. Biophys. J. (in press)