Rapid cryofixation of rabbit muscle fibres after a temperature jump

We describe a procedure whereby structural changes that occur in muscle fibres after a rapid temperature jump can be captured by cryofixation. In the thick filament from rabbit and other mammalian skeletal muscles there is a rapid transition from a non-helical to a helical structure as the temperature is raised from 273 K towards physiological levels. This transition is accompanied by characteristic intensity changes in the X-ray diffraction pattern of the muscle. In our experiments to capture these changes, single fibres of glycerinated psoas muscle were subjected to a Joule temperature jump of 15-30 K from approximately 278 K in air. We have developed a freezing method using a modified Gatan cryosnapper in which a pair of liquid nitrogen-cooled copper jaws were projected under pressure and closed on the fibre between 50 and 100 ms after the temperature jump. The frozen fibres were freeze-substituted and embedded for electron microscopy. Transverse and longitudinal sections of relaxed 'cold' (approximately 278 K) and temperature-jumped fibres as well as rigor fibres were obtained. Fourier transforms of the images from the three preparations showed differences in the relative intensities of the reflections from the hexagonal filament lattice and in those of the helix-based layer lines, similar to the differences seen by X-ray diffraction. We conclude that we have preserved the 'hot' structure and that cryofixation is sufficiently fast to prevent the transition back to the 'cold' state.