Fine Structure of Cardiac Sarcomeres in the Black Widow Spider Latrodectus mactans

Fine structural characteristics of the cardiac muscle and its sarcomere organization in the black widow spider, Latrodectus mactans were examined using transmission electron microscopy. The arrangement of cardiac muscle fibers was quite similar to that of skeletal muscle fibers, but they branched off at the ends and formed multiple connections with adjacent cells. Each cell contained multiple myofibrils and an extensive dyadic sarcotubular system consisting of sarcoplasmic reticulum and T‐tubules. Thin and thick myofilaments were highly organized in regular repetitive arrays and formed contractile sarcomeres. Each repeating band unit of the sarcomere had three apparent striations, but the H‐zone and M‐lines were not prominent. Myofilaments were arranged into distinct sarcomeres defined by adjacent Z‐lines with relatively short lengths of 2.0 μm to 3.3 μm. Cross sections of the A‐band showed hexagon‐like arrangement of thick filaments, but the orbit of thin filaments around each thick filament was different from that seen in other vertebrates. Although each thick filament was surrounded by 12 thin filaments, the filament ratio of thin and thick myofilaments varied from 3:1 to 5:1 because thin filaments were shared by adjacent thick filaments.     

CORRELATED MORPHOLOGICAL AND PHYSIOLOGICAL STUDIES ON ISOLATED SINGLE MUSCLE FIBERS : I. Fine Structure of the Crayfish Muscle Fiber

Single fibers isolated from walking leg muscles of crayfish have 8- to 10-µ sarcomeres which are divided into A, I, and Z bands. The H zone is poorly defined and no M band is distinguishable. Changes in the width of the I band, accompanied by change in the overlap between thick and thin myofilaments, occur when the length of the sarcomere is changed by stretching or by shortening the fiber. The thick myofilaments (ca. 200 A in diameter) are confined to the A band. The thin myofilaments (ca. 50 A in diameter) are difficult to resolve except in swollen fibers, when they clearly lie between the thick filaments and run to the Z disc. The sarcolemma invaginates at 50 to 200 sites in each sarcomere. The sarcolemmal invaginations (SI) form tubes about 0.2 µ in diameter which run radially into the fiber and have longitudinal side branches. Tubules about 150 A in diameter arise from the SI and from the sarcolemma. The invaginations and tubules are all derived from and are continuous with the plasma membrane, forming the transverse tubular system (TTS), which is analogous with the T system of vertebrate muscle. In the A band region each myofibril is enveloped by a fenestrated membranous covering of sarcoplasmic reticulum (SR). Sacculations of the SR extend over the A-I junctions of the myofibrils, where they make specialized contacts (diads) with the TTS. At the diads the opposing membranes of the TTS and SR are spaced 150 A apart, with a 35-A plate centrally located in the gap. It appears likely that the anion-permselective membrane of the TTS which was described previously is located at the diads, and that this property of the diadic structures therefore may function in excitation-contraction coupling.     

Ultrastructurally different muscle cell types in Eisenia foetida (Annelida,Oligochaeta)

Muscles in the body wall, intestinal wall, and contractile hemolymphatic vessels (pseudohearts) of an oligochaete anelid (Eisenia foetida) were studied by electron microscopy. The muscle cells in all locations, except for the outer layer of the pseudohearts, are variants of obliquely striated muscle cells. Cells comprising the circular layer of the body wall possess single, peripherally located myofibrils that occupy most of the cytoplasm and surround other cytoplasmic organelles. The nuclei of the cells lie peripherally to the myofibrils. The sarcomeres consist of thin and thick myofilaments that are arranged in parallel arrays. In one plane of view, the filaments appear to be oriented obliquely to Z bands. Thin myofilaments measure 5–6 nm in diameter. Thick myofilaments are fusiform in shape and their width decreases from their centers (40–45 nm) to their tips (23–25 nm). The thin/thick filament ratio in the A bands is 10. The Z bands consist of Z bars alternating with tubules of the sarcoplasmic reticulum. Subsarcolemmal electron-dense plaques are found frequently. The cells forming the longitudinal layer of the body wall musculature are smaller than the cells in the circular layer and their thick filaments are smaller (31–33 nm centrally and 21–23 nm at the tips). Subsarcolemmal plaques are less numerous. The cells forming the heart wall inner layer, the large hemolymphatic vessels, and the intestinal wall are characterized by their large thick myofilaments (50–52 nm centrally and 27–28 nm at the tips) and abundance of mitochondria. The cells forming the outer muscular layer of the pseudohearts are smooth muscle cells. These cells are richer in thick filaments than vertebrate smooth muscle cells. They differ from obliquely striated muscle cells by possessing irregularly distributed electron-dense bodies for filament anchorage rather than sarcomeres and Z bands and by displaying tubules of smooth endoplasmic reticulum among the bundles of myofilaments. © 1995 Wiley-Liss, Inc.     

Cytological differentiation of human fetal skeletal muscle.

The ultrastructural differentiation of several different muscles was investigated in human fetuses ranging in age from 13 weeks to neonatal. At approximately 16 weeks of gestation cell cluster containing both myotubes and satellite cells lie enclosed by a newly formed basal lamina and show evidence of fusion. The development of organelles is evident in myoblasts, proceeds as the cells transform into myofibers, and continues in the neonate. Filament synthesis occurs primarily in the cell periphery where thin filaments appear to align themselves in relations to parallel arrays of ribosome-studded thick filaments: Z line formation follows the appearance of thin filaments. Intermediate filaments, approximately 10-12 nm thick, were also consistently observed in perinuclear regions and distal to filament assembly. Although sarcoplasmic reticulum (SR) development is closely related to fibril formation, connections between Z lines and SR are not consistent, thus supporting the conclusion that SR does not evoke the formation of the Z line. Bristlecoated vesicles appear to be the precursors of elements of the SR, possibly the lateral sacs. Development of the transverse tubules, as invaginations of the sarcolemma, is closely associated with the formation of lateral sacs since the latter occur along the sarcolemma as soon as transverse tubules appear. Cytological differentiation is similar, though not identical, in several different muscles. During the last trimester muscle fibers show some evidence of diversity mainly of variation in Z line width. In gerneral the results suggest that the sequence and stages of human myogenesis are similar to those of other species.     

AN ELECTRON MICROSCOPE STUDY OF MYOFIBRIL FORMATION IN EMBRYONIC CHICK SKELETAL MUSCLE

The formation of myofibrils in the developing leg muscle of the 12-day chick embryo was studied by electron microscopy. Myofilaments of two varieties, thick (160–170 A in diameter) and thin (60–70 A in diameter), which have been designated myosin and actin filaments, respectively, on the basis of their similarity to natural and synthetic myosin and actin filaments, appear in the cytoplasm of developing muscle cells. There is a greater than 7:1 ratio of thin to thick filaments in these young myofibers. The free myofilaments become aligned in the long axis of the cells, predominantly in subsarcolemmal locations, and aggregate into hexagonally packed arrays of filaments. The presence of Z band material or M band cross-bridges do not appear to be essential for the formation or spacing of these aggregates of filaments. Formation of the Z band lattices occurs coincidentally with the back-to-back apposition of thin filaments. An hypothesis concerning myofibril growth, based on the self-assembly characteristics of the filaments, is presented.     

CARDIAC MUSCLE OF THE HORSESHOE CRAB, LIMULUS POLYPHEMUS : I. Ultrastructure

The fine structure of the cardiac muscle of the horseshoe crab, Limulus polyphemus, has been studied with respect to the organization of its contractile material, and the structure of its organelles and the cell junctions. Longitudinal sections show long sarcomeres (5.37 µ at L_max), wide A bands (2.7 µ), irregular Z lines, no M line, and no apparent H zone. Transverse sections through the S zone of the A band show that each thick filament is ca. 180 A in diameter, is circular in profile with a center of low density, and is surrounded by an orbit of 9–12 thin filaments, each 60 A in diameter. Thick filaments are confined to the A band: thin filaments originate at the Z band, extend through the I band, and pass into the A band between the thick filaments. The sarcolemmal surface area is increased significantly by intercellular clefts. Extending into the fiber from these clefts and from the sarcolemma, T tubules pass into the fiber at the A-I level. Each fibril is enveloped by a profuse membranous covering of sarcoplasmic reticulum (SR). Sacculations of the SR occur at the A-I boundary where they make diadic contact with longitudinal branches of the T system. These branches also extend toward the Z, enlarge at the Z line, and pass into the next sarcomere. Infrequently noted were intercalated discs possessing terminal insertion and desmosome modifications, but lacking close junctions (fasciae occludentes). These structural details are compared with those of mammalian cardiac and invertebrate muscles.     

ELECTRON MICROSCOPIC STUDIES ON THE INDIRECT FLIGHT MUSCLES OF DROSOPHILA MELANOGASTER : I. Structure of the Myofibrils

The myofibrils in Drosophila have thick and thin types of myofilaments arranged in the hexagonal pattern described for Calliphora by Huxley and Hanson (15). The thick filaments, along most of their length in the A band, seem to be binary in structure, consisting of a dense cortex and a lighter medulla. In the H zone, however, they show more uniform density; lateral projections (bridges) also appear to be absent in this region. The M band has a varying number of granules (probably of glycogen) distributed between the myofilaments. The myofilaments on reaching the Z region appear to change their hexagonal arrangement and become connected to one another by Z filaments. The regular arrangement of the filaments found in most regions of the fibrils is not seen in the terminal sarcomeres of some flight muscles; the two types of filaments appear to be intermingled in an irregular pattern in these parts of the fibrils. The attachment of myofibrils to the cuticle through the epidermal cells is described.     

The fine structure of differentiating muscle in the salamander tail

Summary Thin methacrylate sections of developing tails of Amblystoma opacum larvae were examined in the electron microscope and a series of stages in the differentiation of the myotome musculature was reconstructed from electron micrographs and earlier light microscopic studies of living muscle. The earliest muscle cell precursor that can be clearly identified is a round or oval cell with abundant cytoplasm containing scattered myofilaments and free ribonucleoprotein granules, but little endoplasmic reticulum. These cells sometimes form a syncytium and they may also be fused with adjacent formed muscle fibers by lateral processes. Nuclei are large and nucleoli are prominent. This cell, called a myoblast here, is distinctly different in its appearance from the adjacent mesenchymal cells which have abundant granular endoplasmic reticulum. The earliest myofilaments are of both the thick and thin varieties and are distributed in a disorganized fashion in the cytoplasm. These filaments are similar to the actin and myosin filaments described by Huxley and they are present in the cytoplasm at an earlier stage of differentiation than heretofore suspected from light microscopy studies. The first myofibrils are a heterogeneous combination of thick and thin filaments and dense Z bands and are not homogeneous as so many light microscopists have contended. As development progresses, cross striations become more orderly and definitive sarcomeres are formed. Thereafter, new myofilaments and Z bands seem to be added to the lateral surfaces and distal ends of existing myofibrils.Free ribonucleoprotein granules are a prominent part of the myoblast cytoplasm and are found in close association with the differentiating myofilaments in all stages of development. In early muscle fibers and some of the formed fibers, similar granules are often concentrated in the I bands. A theory of myofilament differentiation based on current concepts of the role of ribonucleoprotein in protein synthesis is presented in the discussion. Stages in myofibril formation and possible relationships of the filaments in developing muscle cells to other types of cytoplasmic filaments are also discussed.Supported by grant C-5196 from the United States Public Health Service.     

A study of the fine structure of the accessory muscle of the horseshoe crab,Tachypleus gigas

The accessory muscle of the walking leg of the horseshoe crab, Tachypleus gigas, was examined electron microscopically. The muscle fibers vary in size but are small in diameter, when compared with other arthropod skeletal muscles. They are striated with A, I, Z and poorly defined H bands. The sarcomere length ranges from 3-10 μm with most sarcomeres in the range of about 6 μm. The myofilaments are arranged in lamellae in larger fibers and less well organized in the smaller ones. Each thick filament is surrounded by 9-12 thin filaments which overlap. The SR is sparse but well organized to form a fenestrated collar around the fibrils. Individual SR tubules are also seen among the myofibrils. Long transverse tubules extend inward from the sarcolemma to form dyads or triads with the SR at the A-I junction. Both dyads and triads coexist in a single muscle fiber, a feature believed to have evolutionary significance. The neuromuscular relationship is unique. In the region of synaptic contact, the sarcolemma is usually elevated to form a large club-shaped structure containing no myofilaments and few other organelles. The axons or axon terminals and glial elements penetrate deep into the club-shaped sarcoplasm and form synapses with the fiber. As many as 13 terminals have been observed within a single section. Synaptic vesicles of two types are found in the axon terminals.     

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 FINE STRUCTURE OF THE VENTRAL INTERSEGMENTAL ABDOMINAL MUSCLES OF THE INSECT RHODNIUS PROLIXUS DURING THE MOLTING CYCLE : II. Muscle Changes in Preparation for Molting

The development of the ventral intersegmental abdominal muscles of Rhodnius prolixus is triggered by feeding. The early muscle (1 day after feeding) contains essentially nonstriated fibrils. However, in cross-sections, areas indicating early I bands, Z lines, and A bands can be recognized. Interdigitating thick and thin myofilaments do not assemble into a precise lattice until sometime between 4 and 5 days after feeding. As development continues, the number of fibrils increases, the region corresponding to the Z line increases in density, and the fibrils contain more recognizable striations. The newly formed fibrils broaden as myofilaments are added peripherally. At all stages throughout development, the ratio of thin to thick myofilaments is always 6:1. The formation of fibrils in the abdominal muscles of Rhodnius is different from that in chick embryo skeletal muscle. The major differences are that at all stages in Rhodnius there are (1) a constant ratio of thin to thick myofilaments, and (2) detectable Z-line material. Other findings in Rhodnius suggest (1) that fusion of mononucleated cells with the multinucleated muscle cell occurs, (2) that microtubules develop in the tendon cell concomitantly with development of myofibrils in the associated muscle cell, and (3) that filaments 55A in diameter aggregate into microtubules.     

THE ULTRASTRUCTURE OF FROG VENTRICULAR CARDIAC MUSCLE AND ITS RELATIONSHIP TO MECHANISMS OF EXCITATION-CONTRACTION COUPLING

Frog ventricular cardiac muscle has structural features which set it apart from frog and mammalian skeletal muscle and mammalian cardiac muscle. In describing these differences, our attention focused chiefly on the distribution of cellular membranes. Abundant inter cellular clefts, the absence of tranverse tubules, and the paucity of sarcotubules, together with exceedingly small cell diameters (less than 5 µ), support the suggestion that the mechanism of excitation-contraction coupling differs in these muscle cells from that now thought to be characteristic of striated muscle such as skeletal muscle and mammalian cardiac muscle. These structural dissimilarities also imply that the mechanism of relaxation in frog ventricular muscle differs from that considered typical of other striated muscles. Additional ultrastructural features of frog ventricular heart muscle include spherical electron-opaque bodies on thin filaments, inconstantly present, forming a rank across the I band about 150 mµ from the Z line, and membrane-bounded dense granules resembling neurosecretory granules. The functional significance of these features is not yet clear.     

Fine structure of smooth muscle and neuromuscular junctions in the foot of Helix aspersa

Summary The smooth muscle cells in the foot of Helix aspersa are arranged in bundles which interweave to form a complex mesh. In the peripheral cytoplasm of the muscle cells there is a system of interconnected obliquely and longitudinally orientated tubules. The full extent of this system has not been determined; its possible function in relation to Ca⁺⁺ storage and excitation-contraction coupling is discussed. Longitudinal tubules are present among the myofilaments and in association with mitochondria. Distributed throughout the myofilaments are elliptically shaped dense bodies, the fine structure of which resembles an accumulation of thin filaments. Located on the plasma membrane of the muscle cells are dense areas; the fine structure and relationships of these cellular elements resemble desmosomes. They may serve as attachment points for thin, cytoplasmic filaments (not necessarily myofilaments). The muscle cells are innervated by axons which diverge from a coarse, neural plexus (the sole plexus). The axons initially come into close contact with the muscle cells and then pass over their surfaces for up to 35 before being gradually enveloped by flange-like protrusions of the muscle cells. These axons contain either, (i) agranular vesicles (600 Å in diameter), (ii) agranular and very dense granular vesicles (1000 Å in diameter) or (iii) agranular and less dense, granular vesicles (1000 Å in diameter). The possible role of these inclusions as sites of excitatory and inhibitory transmitters is discussed.I wish to thank Professor G. Burnstock for making laboratory facilities available. This work has been supported by the Australian Research Grants Committee.     

CYTOLOGICAL AND ULTRASTRUCTURAL STUDIES ON MUSCLE DIFFERENTIATION IN THE ASCIDIAN,PEROPHORA ORIENTALIS*,**

Muscle cell differentiation in the tail of the ascidian, Perophora orientalis, from early tail-bud embryos to swimming larvae, were studied cytologically and ultrastructurally. Myogenic cells did not form multinucleated myotubes, but remained as mononucleated cells. Nucleolar component increased prior to a marked increase in cytoplasmic RNA. Cytoplasmic RNA appeared first around nucleus and later concentrated in the peripheral cytoplasm. The fine filaments measuring 20–30 Å in their thin parts and 30–45 Å in their thick parts in diameter appeared initially, forming loose networks, in the peripheral cytoplasm where ribosome clusters had been concentrated. These filaments were tightly attached by particles of various size and density. These filaments tended to be arranged in parallel as they increased in their size. They seemed to be precursors of both actin and myosin filaments of formed myofibrils. Z band precursors were found as dense patches in association with loosely arranged myofilaments and consisted of particulate and filamentous materials. The myofibrils seemed to grow further by organizing free filaments into bundles and further by aligning bundles of myofilaments at both ends.     

Ultrastructure of the neurogenic heart of Limulus polyphemus

Summary The ultrastructure of Limulus cardiac muscle was examined. The hearts were fixed in situ by perfusion with isotonic glutaraldehyde solution while in relaxed, contracted, or stretched states. The sarcomeres are relatively long, varying in length from about 2.5 to 6.6 . The average A-band length is 2.46 . M lines are absent, and H zones are poorly distinguished. Thick and thin filament diameters average about 200 Å and 50 Å, respectively; each thick filament is surrounded by 8–12 thin ones. Superficial invaginations of the sarcolemma occur, making contact with the Z lines of the outermost myofibrils. There is an extensive sarcoplasmic reticulum and transverse (T) tubules. Some T tubules run longitudinally and some open into deep sarcolemmal invaginations which extend into the fiber interior. The T tubules swell markedly in hypertonic solution. Single neurons and small bundles of neurons are observed in close apposition with myocardial cells. Intercalated disks are found in Limulus heart at regions of contact between contiguous myocardial cells lying end to end; semitight or gap junctions are essentially absent. Prominent differences in sarcomere lengths sometimes occur across the disk, thus indicating that the disks demarcate cells functionally. Hence, in addition to direct motoneuron activation, there may be some transfer of excitation across the intercalated disks in accord with our previous finding that propagating, overshooting action potentials can be induced in this heart.Supported by grants from the American Heart Association and from the Public Health Service (HE-11155 and HE-05815). I thank Mrs. Jan Redick for expert technical assistance.     

Immunocytochemical electron microscopic study and Western blot analysis of paramyosin in different invertebrate muscle cell types of the fruit flyDrosophila melanogaster, the earthwormEisenia foetida, and the snailHelix aspersa

Summary The presence and distribution pattern of paramyosin have been examined in different invertebrate muscle cell types by means of Western blot analysis and electron microscopy immunogold labelling. the muscles studied were: transversely striated muscle with continuous Z lines (flight muscle fromDrosophila melanogaster), transversely striated muscle with discontinuous Z lines (heart muscle from the snailHelix aspersa), obliquely striated body wall muscle from the earthwormEisenia foetida, and smooth muscles (retractor muscle from the snail and pseudoheart outer muscular layer from the earthworm). Paramyosin-like immunoreactivity was localized in thick filaments of all muscles studied. Immunogold particle density was similar along the whole thick filament length in insect flight muscle but it predominated in filament tips of fusiform thick filaments in both snail heart and earthworm body wall musculature when these filaments were observed in longitudinal sections. In obliquely sectioned thick filaments, immunolabelling was more abundant at the sites where filaments disappeared from the section. These results agree with the notion that paramyosin extended along the whole filament length, but that it can only be immunolabelled when it is not covered by myosin. In all muscles examined, immunolabelling density was lower in cross-sectioned myofilaments than in longitudinally sectioned myofilaments. This suggests that paramyosin does not form a continuous filament. The results of a semiquantitative analysis of paramyosin-like immunoreactivity indicated that it was more abundant in striated than in smooth muscles, and that, within striated muscles, transversely striated muscles contain more paramyosin than obliquely striated muscles.     

Fine structure of the nephridial muscle layer cells of Phascolosoma granulatum (Sipuncula)

The nephridial muscle layer of Phascolosoma granulatum consists of a network of longitudinal and circular cells separated by connective tissue matrix. The muscle fibers are densely packed with thick and thin myofilaments, among which are scattered cytoplasmic dense bodies. The nucleus and noncontractile cytoplasmic organelles occupy a lateral projection from the contractile portion of the fiber. Cytoplasmic dense bodies are the result of a clustering of an indeterminate number of the thin actin filaments that fill the cytoplasm between thick filaments. Attached to the cytoplasmic face of the cell membrane are membrane-associated electron-dense plaques. These sites are linked to the contractile myofilaments by narrow filamentous bridges. Extracellular narrow filaments extend from these plaques to collagen fibers of the connective tissue matrix. Differences in length of the dense plaques may be related to differences in thick myofilament diameter in three types of muscle fiber, types A, B and C, statistically distinguished by mean fiber size differences. The plaques may serve as connecting links for the transmission of tension from contractile units to the connective tissue of the muscle layer. © 1993 Wiley-Liss, Inc.     

Fine structure of smooth muscle and neuromuscular junctions in the optic tentacles of Helix aspersa and Limax flavus

Summary The smooth muscle cells studied contain a central core of thick and thin myofilaments surrounded by a peripheral layer of myofilament-free cytoplasm. Numerous vesicles, tubules, microfilaments, mitochondria and fine granules are present in the peripheral cytoplasm. Glycogen particles are distributed in large or small groups in both the peripheral cytoplasm and among the myofilaments. In contracted muscle cells the peripheral cytoplasm bulges out at regular intervals into the intercellular connective tissue. Numerous close contacts between single, usually naked, axons and these cytoplasmic protrusions occur. The axons at these contacts contain numerous small (500 Å in diameter) and large vesicles (800–1000 Å in diameter). Sometimes a number of axons simultaneously form close contacts with a muscle cell. These close contacts are considered to be the sites at which transmitter is released and acts on the muscle cell membrane.I wish to thank Professor G. Burnstock for making laboratory facilities available. This work has been supported by the Australian Research Grants Committee.     

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.     

Compositional studies of myofibrils from rabbit striated muscle

The localization of high-molecular-weight (80,000-200,000-daltons) proteins in the sarcomere of striated muscle has been studied by coordinated electron-microscopic and sodium dodecyl sulfate (SDS) gel electrophoretic analysis of native myofilaments and extracted and digested myofibrils. Methods were developed for the isolation of thick and thin filaments and of uncontracted myofibrils which are devoid of endoproteases and membrane fragments. Treatment of crude myofibrils with 0.5% Triton X-100 results in the release of a 110,000-dalton component without affecting the myofibrillar structure. Extraction of uncontracted myofibrils with a relaxing solution of high ionic strength results in the complete disappearance of the A band and M line. In this extract, five other protein bands in addition to myosin are resolved on SDS gels: bands M 1 (190,000 daltons) and M 2 (170,000 daltons), which are suggested to be components of the M line; M 3 (150,000 daltons), a degradation product; and a doublet M 4, M 5 (140,000 daltons), thick-filament protein having the same mobility as C protein. Extraction of myofibrils with 0.15% deoxycholate, previously shown to remove Z-line density, releases a doublet Z 1, Z 2 (90,000 daltons) with the same mobility as alpha-actinin, as well as proteins of 60,000 daltons and less, and small amounts of M 1, M 2, M 4, and M 5; these proteins were not extracted with 0.5% Triton X-100. The C, M-line, and Z-line proteins and/or their binding to myofibrils are very sensitive to tryptic digestion, whereas the M 3 (150,000 daltons) component and an additional band at 110,000 daltons are products of proteolysis. Gentle treatment of myofibrils with an ATP relaxing solution results in the release of thick and thin myofilaments which can be pelleted by 100,000-g centrifugation. These myofilaments lack M-and Z-line structure when examined with the electron microscope, and their electrophoretograms are devoid of the M 1, M 2, Z 1, and Z 2 bands. The M 4, M 5 (C-protein doublet), and M 3 bands, however, remain associated with the filaments.