THE ULTRASTRUCTURE OF STRIATED MUSCLE AT VARIOUS SARCOMERE LENGTHS

1. Rest and equilibrium length muscle sarcomeres are composed of thin filaments (actin) which traverse the sarcomeres from the Z membranes up to the H band; at this level the filaments are considerably thicker and less numerous. 2. Shortening of muscle is associated with a transformation of thin into thick filaments in the A band. 3. These observations are discussed in terms of interaction of actin and myosin to form a supercoiled structure as the basis of contraction.     

MECHANISM OF SUPERCONTRACTION IN A STRIATED MUSCLE

The phenomenon of contraction of a striated muscle down to below 50 per cent rest length has been examined for the scutal depressor of the barnacle Balanus nubilus by a combination of phase contrast and electron microscopy. It was found that neurally evoked contraction down to 60 per cent rest length results from the shortening of the I band. At the same time the Z disc changes in structure by an active process which results in spaces opening up within it. Thick filaments can now pass through these spaces from adjacent sarcomeres, interdigitating across the discs. Interdigitation permits repetitive contraction in the living muscle to below 30 per cent rest length. In non-neurally evoked contractions most thick filaments do not find spaces in the Z disc and bend back, giving rise to contraction band artifacts. Expansion of the Z disc can be produced in glycerinated material by the addition of solutions containing a high concentration of ATP.     

IMMUNOHISTOCHEMICAL LOCALIZATION OF CONTRACTILE PROTEINS IN LIMULUS STRIATED MUSCLE

Limulus paramyosin and myosin were localized in the A bands of glycerinated Limulus striated muscle by the indirect horseradish peroxidase-labeled antibody and direct and indirect fluorescent antibody techniques. Localization of each protein in the A band varied with sarcomere length. Antiparamyosin was bound at the lateral margins of the A bands in long (~ 10.0 µ) and intermediate (~ 7.0 µ) length sarcomeres, and also in a thin line in the central A bands of sarcomeres, 7.0–~6.0 µ. Antiparamyosin stained the entire A bands of short sarcomeres (<6.0). Conversely, antimyosin stained the entire A bands of long sarcomeres, showed decreased intensity of central A band staining except for a thin medial line in intermediate length sarcomeres, and was bound only in the lateral A bands of short sarcomeres. These results are consistent with a model in which paramyosin comprises the core of the thick filament and myosin forms a cortex. Differential staining observed using antiparamyosin and antimyosin at various sarcomere lengths and changes in A band lengths reflect the extent of thick-thin filament interaction and conformational change in the thick filament during sarcomeric shortening.     

ANOMALOUS CONTRACTION OF INVERTEBRATE STRIATED MUSCLE

The phenomenon of A band shortening or contraction has been investigated in glycerinated myofibrils of Pecten irradians, Homarus americanus, Cambarus virilis, and Limulus polyphemus through the techniques of ultraviolet microbeam inactivation and polarization microscopy. With the former method, it has been shown that these muscles, even though exhibiting the shortening effect, contract in a manner consistent with only the sliding filament model. Intrinsic birefringence studies have indicated no significant changes in mass distribution or orientation within the shortened A bands. Except in the case of Limulus muscle, the shortening effect was seen only in contraction under tension. The magnitude of this anomalous phenomenon was dependent upon glycerination time and has been duplicated in rabbit psoas muscle through brief trypsin treatment. A band shortening could not be observed in glutaraldehyde-fixed muscle or in myofibrils glycerinated for only short periods. It has been concluded that the phenomenon of A band contraction is an artifact induced by the glycerination procedure, possibly through weakening of the sarcomere structure. However, the fact that the A band shortens under tension rather than lengthens poses an interesting paradox.     

ULTRASTRUCTURAL ORGANIZATION OF OBLIQUELY STRIATED MUSCLE FIBERS IN ASCARIS LUMBRICOIDES

The somatic musculature of the nematode, Ascaris, is currently thought to consist of smooth muscle fibers, which contain intracellular supporting fibrils arranged in a regular pattern. Electron microscopic examination shows that the muscle fibers are, in fact, comparable to the striated muscles of vertebrates in that they contain interdigitating arrays of thick and thin myofilaments which form H, A, and I bands. In the A bands each thick filament is surrounded by about 10 to 12 thin filaments. The earlier confusion about the classification of this muscle probably arose from the fact that in one longitudinal plane the myofilaments are markedly staggered and, as a result, the striations in that plane of section are not transverse but oblique, forming an angle of only about 6° with the filament axis. The apparent direction of the striations changes with the plane of the section and may vary all the way from radial to longitudinal. A three-dimensional model is proposed which accounts for the appearance of this muscle in various planes. Z lines as such are absent but are replaced by smaller, less orderly, counterpart "Z bundles" to which thin filaments attach. These bundles are closely associated with fibrillar dense bodies and with deep infoldings of the plasma membrane. The invaginations of the plasma membrane together with intracellular, flattened, membranous cisternae form dyads and triads. It is suggested that these complexes, which also occur at the cell surface, may constitute strategically located, low-impedance patches through which local currents are channeled selectively.     

THE LOCATION OF ADENINE NUCLEOTIDE IN THE STRIATED MUSCLE OF THE TOAD

The location of tritiated adenine nucleotide was studied by autoradiography in toad muscle which had been fixed in an acetic acid/ethanol mixture, with lead acetate present to act as the nucleotide precipitant. The muscles were embedded in Araldite. The autoradiographs were examined in both the light microscope and the electron microscope. In confirmation of earlier work, it was found that at least 50 per cent of the adenine nucleotide is concentrated in the I band, near the A-I boundary, and evidence is now available to suggest that this fraction is located in the interfibrillar spaces rather than in the substance of the fibrils. Electron micrographs of unstained sections failed to show any structural features at the site in question, though the triads were made visible by the lead. However, when sections were stained with an alkaline solution containing lead, or with uranyl acetate, "vesicles" were revealed at the appropriate site and it is thought that these may be elements of a transverse network (reticulum) of tubules containing the adenine nucleotide. The location of phosphocreatine could not be investigated because this substance was lost from the muscle during the preparative procedure.     

THE RELATIONSHIP BETWEEN MYOFILAMENT PACKING DENSITY AND SARCOMERE LENGTH IN FROG STRIATED MUSCLE

In cross-sections of single fibers from the frog semitendinosus muscle the number of thick myofilaments per unit area (packing density) is a direct function of the sarcomere length. Our data, derived from electron microscopic studies, fit well with other data derived from in vivo, low-angle X-ray diffraction studies of whole semitendinosus muscles. The data are consistent with the assumption that the sarcomere of a fibril maintains a constant volume during changes in sarcomere length. The myofilament lattice, therefore, expands as the sarcomere shortens. Since the distance between adjacent myofilaments is an inverse square root function of sarcomere length, the interaction of the thick and the thin myofilaments during sarcomere shortening may occur over distances which increase 70 A or more. The "expanding-sarcomere, sliding-filament" model of sarcomere shortening is discussed in terms of the current concepts of muscle architecture and contraction.     

THE FILAMENT LATTICE OF COCKROACH THORACIC MUSCLE

The fine structure of the tergo-coxal muscle of the cockroach, Leucophaea maderae, has been studied with the electron microscope. This muscle differs from some other types of insect flight muscles inasmuch as the ratio of thin to thick filaments is 4 instead of the characteristic 3. The cockroach flight muscle also differs from the cockroach femoral muscle in thin to thick filament ratios and diameters and in lengths of thick filaments. A comparison of these latter three parameters in a number of vertebrate and invertebrate muscles suggests in general that the diameters and lengths of the thick filaments and thin to thick filament ratios are related.     

THE SARCOPLASMIC RETICULUM OF STRIATED MUSCLE OF A CYCLOPOID COPEPOD

The fine structure of the abdominal musculature of the copepod Macrocyclops albidus was investigated by electron microscopy. Tubules penetrate into the muscle fibers from the sarcolemma, continuity between the wall of the tubules and the sarcolemma being clear. A dense network of tubules envelops the myofibrils, its interstices being occupied by cisternal elements. At the Z lines the tubules traverse the interior of myofibrils, giving off branches which course longitudinally within the substance of the myofibrils. These branches are also accompanied by elongate, non-intercommunicating cisternae. Comparison of this fast acting copepod muscle with other vertebrate and invertebrate muscles indicates that the complexity of the tubular system is a function of the myofibrillar geometry, whereas the degree of development of the cisternal system is related to the contraction speed of the muscle.     

THE FINE STRUCTURE OF STRIATED MUSCLE : A COMPARISON OF INSECT FLIGHT MUSCLE WITH VERTEBRATE AND INVERTEBRATE SKELETAL MUSCLE

The available evidence from phase contrast, polarization optical, and electron microscopic studies on vertebrate skeletal muscle, insect skeletal muscle, and dipteran flight muscle is interpreted as favoring the following general structure of striated muscle. A continuous array of filaments (actin) runs through all bands of the sarcomere. These are linked by an axially periodic system of transverse filamentous bridges. Myosin (and probably other substances) are localized in the A bands. The system of transverse bridges compensates the birefringence of actin and is thus responsible for the isotropy of the I band. Myosin is responsible for the birefringence of the A bands. On strong contraction, A band material migrates to the Z bands to form contraction bands. It is not yet certain whether this migration involves myosin or another A band component.     

STUDIES ON THE ENDOPLASMIC RETICULUM : III. ITS FORM AND DISTRIBUTION IN STRIATED MUSCLE CELLS

Several types of striated muscle have been examined by the technics of electron microscopy and the findings in myotome fibers of Amblystoma larvae, the sartorius, and cardiac muscle of the rat are reported on in some detail. Particular attention has been given to structural components of the interfibrillar sarcoplasm and most especially to a finely divided, vacuolar system known as the sarcoplasmic reticulum. This consists of membrane-limited vesicles, tubules, and cisternae associated in a continuous reticular structure which forms lace-like sleeves around the myofibrils. It shows a definable organization which repeats with each sarcomere of the fiber so that the entire system is segmented in phase with the striations of the associated myofibrils. Details of these repetitive patterns are presented diagrammatically in Text-figs. 1, 2, and 3 on pages 279, 283, and 288 respectively. The system is continuous across the fiber at the H band level and largely discontinuous longitudinally because of interruptions in the structure at the I and Z band levels. The structure of the system relates it to the endoplasmic reticulum of other cell types. The precise morphological relation of the reticulum to the myofibrils, with specializations opposite the different bands, prompts the supposition that the system is functionally important in muscle contraction. In this regard it is proposed that the membrane limiting the system is polarized like the sarcolemma and that the corresponding potential difference is utilized in the intracellular distribution of the excitatory impulse.     

OBLIQUELY STRIATED MUSCLE : III. Contraction Mechanism of Ascaris Body Muscle

Segments of the obliquely striated body muscle of Ascaris were fixed at minimum body length after treatment with acetylcholine and at maximum body length after treatment with piperazine citrate and then studied by light and electron microscopy. Evidence was found for two mechanisms of length change: sliding of thin filaments with respect to thick filaments such as occurs in cross-striated muscle, and shearing of thick filaments with respect to each other such that the degree of their stagger increases with extension and decreases with shortening. The shearing mechanism could account for great extensibility in this muscle and in nonstriated muscles in general and could underlie other manifestations of "plasticity" as well. In addition, it is suggested that the contractile apparatus is attached to the endomysium in such a way that the sarcomeres can act either in series, as in cross-striated muscle, or individually. Since the sarcomeres are virtually longitudinal in orientation and are almost coextensive with the muscle fiber, it would, therefore, be possible for a single sarcomere contracting independently to develop tension effectively between widely separated points on the fiber surface, thus permitting very efficient maintenance of isometric tension.