Structural features of unfixed mammalian smooth and striated muscle prepared by glycol dehydration

Abstract

Unfixed rat skeletal muscle, as well as smooth muscle from the jejunum, aorta, and splenic artery have been prepared for electron microscopy by “inert dehydration” (controlled glycol substitution at room temperature). Also, arteriolar smooth muscle has been studied after “freeze-substitution” at −50°C with eutectic ethylene glycol (70%). These procedures provide an identical preservation of many macromolecular systems. Most importantly, in the present experiments a system of relatively coarse filaments unlike anything previously described in vertebrate smooth muscle was seen in all the varieties of mammalian smooth muscle studied. These coarse filaments resembled the myosin filaments of striated muscle in their staining reactions, but most closely resembled the “paramyosin” systems of invertebrate smooth muscle in other aspects, including their variable thickness, reaching diameters of over 300 . The myosin system of invertebrate smooth muscle is thought to be stabilized by the relatively insoluble tropomyosin A. It is postulated that the coarse filaments seen in the mammalian smooth muscle represent a homologous system, but utilizing soluble tropomyosin B. Evidence is presented in an analysis of striated muscle that much tropomyosin B easily can be lost by solubilization. Presumably, the filamentous structures it might stabilize then would disaggregate, and thus not be visualized. This is thought probably to occur when vertebrate smooth muscle is preserved conventionally. On the other hand, the present techniques of preservation retained the tropomyosin B in place, and then the population of coarse filaments was preserved. The inference is that the contractile mechanism of vertebrate smooth muscle may, indeed, be homologous with the much better known invertebrate smooth muscle system in which it is thought that thin actin filaments slide over thick filaments, which have exposed myosin upon their surface, and tropomyosin A cores. In relation to vertebrate striated muscle, the present technique demonstrated relatively electron-lucid cores within myosin filaments. What was thought to be tropomyosin B ordinarily was retained in such quantities in the I-discs that these could barely be differentiated into zones. Thus, the Z- and N-lines could hardly be distinguished unless there was some differential extraction. Actin filaments were not clearly seen except sometimes as fragments. It is thought that they generally were depolymerized, perhaps by the declining ionic strength of the substituting medium inherent in the technique. (Thin actin filaments were not to be seen either in the smooth muscles studied.) The contents of the transverse system of invaginated sarcolemmal membranes in striated muscle was quite evidently different from that of the associated cisterns of sarcoplasmic reticulum, substantiating discontinuity between the systems. Material in the clefts between the two systems resembled that found within the sarcoplasmic reticulum. The dense cisternal material was concentrated in the zone of the triads, and did not appear to extend uniformly throughout the sarcoplasmic reticulum.