Ultrastructure of skeletal muscle fibers studied by a plunge quick freezing method: myofilament lengths

Abstract

We have set up a system to rapidly freeze muscle fibers during contraction to investigate by electron microscopy the ultrastructure of active muscles. Glycerinated fiber bundles of rabbit psoas muscles were frozen in conditions of rigor, relaxation, isometric contraction, and active shortening. Freezing was carried out by plunging the bundles into liquid ethane. The frozen bundles were then freeze-substituted, plastic-embedded, and sectioned for electron microscopic observation. X-ray diffraction patterns of the embedded bundles and optical diffraction patterns of the micrographs resemble the x-ray diffraction patterns of unfixed muscles, showing the ability of the method to preserve the muscle ultrastructure. In the optical diffraction patterns layer lines up to 1/5.9 nm-1 were observed. Using this method we have investigated the myofilament lengths and concluded that there are no major changes in length in either the actin or the myosin filaments under any of the conditions explored.

Deleting titin’s I-band/A-band junction reveals critical roles for titin in biomechanical sensing and cardiac function

“…The A-band width that we obtained (∼1.52 μm) is slightly less than the 1.6 μm that others have reported in cardiac and skeletal muscle (11,22). Tissue shrinkage that occurs during sample preparation for TEM is a likely explanation (11). We can gain insights in shrinkage during the preparatory steps specific for TEM (dehydration, embedding, sectioning) by comparing the I103 epitope to M-band distances measured with transmission electron microscopy (TEM) to that measured with SIM (Fig.…”

“…Discussion Thick Filament Length. The A-band width that we obtained (∼1.52 μm) is slightly less than the 1.6 μm that others have reported in cardiac and skeletal muscle (11,22). Tissue shrinkage that occurs during sample preparation for TEM is a likely explanation (11).…”

Contractile properties of mouse single muscle fibers, a comparison with amphibian muscle fibers

“…On the same assumptions, the intersection of the regression line with the abscissa at 3.88·µm sarcomere length is compatible with an average thick filament length of 1.63·µm. The values of the thick and thin filament lengths so derived agree exceedingly well with recent electron microscopical measurements of the two filaments in rabbit psoas muscle (Sosa et al, 1994).…”

Contractile Performance of Striated Muscle

“…2, the length-tension relationship has a shape that, similar to the situation in amphibian muscle fibres, can be fitted well with the sliding-filament hypothesis. The slope of the descending limb predicts that the average length of the thin filaments is 1.10 µm and the length of the thick filaments is 3.88 µm, both values being in excellent agreement with recent electron microscopical measurements of the two filaments in rabbit psoas muscle (Sosa et al, 1994). …”