The relationship between the transverse tubular system and other tubules at the Z disc levels of myocardial cells in the ferret

Apart from cytoplasmic microtubules, which exist in cardiac muscle as in most other tissues, there are two tubular systems in mammalian myocardial cells — the transverse (T) tubules which are invaginations of the sarcolemma (i.e., plasma membrane and basement membrane) and the finer tubules which appear to be separate from the transverse system and which have hitherto been known as longitudinal (L) tubules. Because so many of these finer tubules are transversely orientated it seems better to call them simply “sarcotubules.” The sarcotubular system is best envisaged as a three-dimensional network extending throughout the cell, with specialized areas of flattened sarcotubular sacs at points of contact with the plasma membrane or its extensions in the transverse tubular system. A particularly constant element of the sarcotubular system is found encircling the myofibrils at the Z regions.     

Biomechanics of the sarcolemma and costameres in single skeletal muscle fibers from normal and dystrophin-null mice

We studied the biomechanical properties of the sarcolemma and its links through costameres to the contractile apparatus in single mammalian myofibers of Extensor digitorum longus muscles isolated from wild (WT) and dystrophin-null (mdx) mice. Suction pressures (P) applied through a pipette to the sarcolemma generated a bleb, the height of which increased with increasing P. Larger increases in P broke the connections between the sarcolemma and myofibrils and eventually caused the sarcolemma to burst. We used the values of P at which these changes occurred to estimate the tensions and stiffness of the system and its individual elements. Tensions of the whole system and the sarcolemma, as well as the maximal tension sustained by the costameres, were all significantly lower (1.8-3.3 fold) in muscles of mdx mice compared to WT. Values of P at which separation and bursting occurred, as well as the stiffness of the whole system and of the isolated sarcolemma, were ~2-fold lower in mdx than in WT. Our results indicate that the absence of dystrophin reduces muscle stiffness, increases sarcolemmal deformability, and compromises the mechanical stability of costameres and their connections to nearby myofibrils.     

Transverse stiffness of myofibrils of skeletal and cardiac muscles studied by atomic force microscopy

The transverse stiffness of single myofibrils of skeletal and cardiac muscles was examined by atomic force microscopy. The microscopic images of both skeletal and cardiac myofibrils in a rigor state showed periodical striation patterns separated by Z-bands, which is characteristic of striated muscle fibers. However, sarcomere patterns were hardly distinguishable in the stiffness distributions of the relaxed myofibrils of skeletal and cardiac muscles. Myofibrils in a rigor state were significantly stiff compared with those in a relaxed state, and in each state, cardiac myofibrils were significantly stiffer compared with skeletal myofibrils. By proteolytic digestions of sarcomere components of myofibrils, it was suggested that cardiac myofibrils are laterally stiffer than skeletal myofibrils because Z-bands, connectin (titin) filament networks, and other components of sarcomere structures for the former myofibrils are stronger than those for the latter.     

Immunofluorescent localization of desmin and vimentin in developing cardiac muscle of Syrian hamster

The distributions of desmin and vimentin were examined in frozen sections of cardiac muscle from embryonic, newborn, and adult Syrian hamster by using immunofluorescent methods. Frozen sections of newborn and adult skeletal muscle were used for comparison. Cardiac myocytes from day 9 in utero embryos already show a clear association of desmin with the sarcomeric myofibrils. In newborn hearts, desmin is localized in the myofibrillar Z-line areas as well as in the peripheral cytoplasm of the cell. Three days after birth, desmin is associated with the intercalated discs. Thus, in adult cardiac muscle, desmin is present in both Z-bands and intercalated discs. Skeletal muscle of newborn and adult hamster also contains desmin associated with the Z-lines of myofibrils. Vimentin is associated with the myofibrils of day 9 in utero cardiac muscle cells. The protein remains associated with the myofibrillar Z-lines in the newborns and adults. No detectable staining for vimentin was observed in newborn or adult hamster skeletal muscle. The existence of vimentin as well as desmin in differentiated cardiac muscle may be a consequence of the somewhat more epithelial-like nature of cardiac cells as compared to skeletal muscle syncitia.     

The fine structure of the Necturus (Amphibia) heart

The fiber diameter in the heart of Necturus maculosus is not significantly different from that of other amphibian myocardial cells, averaging approximately 5 μ over most of its length and about 10 μ in the nuclear region. No transverse (T) tubular system was found. Instead, there were shallow invaginations of the sarcolemma and many pinocytotic vesicles at the periphery of the myocytes. Other intracellular membrane systems such as the Golgi apparatus and sarcoplasmic reticulum were also sparse and poorly organized. However, subsarcolemmal cisterns, specialized segments of the sarcoplasmic reticulum in close apposition with the sarcolemma, were clearly demonstrated. Nexal junctions were recognized at points of contact between myocytes, although they occupied smaller areas than those of mammalian myocardial cells. Mitochondria were small and few. Light cells with few organelles or myofibrils, which resembled Purkinje fibers of mammalian and avian hearts, were often observed. A nerve ending in close contact with muscle cells was described.     

An electron microscope study of differentiation of the molting muscles of Rhodnius prolixus

The abdominal intersegmental molting muscles of Rhodnius prolixus undergo a repeated differentiation and dedifferentiation correlated with the molting cycle of the insect. In the dedifferentiated phase, the muscle fibers lack myofibrils but contain a few bundles of 50–60Å filaments associated with amorphous dense plaques on the sarcolemma. Differentiation of the muscle fibers begins when a starved Rhodnius nymph takes a blood meal. The first morphological signs of differentiation at ten hours after feeding are a dispersal of ribosomes from the nuclear envelopes and a formation of polysomes in the sarcoplasm. The initial myofilaments appear within 10 to 15 hours after the blood meal and are preferentially deposited in the cortices of the muscle fibers in association with the 50–60Å filament bundles and the dense plaques on the sarcolemma. Striated myofibrils are present after five days of differentiation. Developmental continuity between Z-band segments and sarcolemmal dense plaques is suggested. The formation of cross-bridges between thick and thin myofilaments appears to be the primary mechanism of myofibril organization. Disruption of microtubules with colchicine does not significantly alter myofilament deposition and organization during early stages of differentiation. Dyads, consisting of smooth sarcoplasmic reticulum and invaginations of the sarcolemma, are present in the dedifferentiated muscle fiber, and increase in number as differentiation proceeds.     

Fine structure of the myotendinous junction and “terminal coupling” in the skeletal muscle of the lamprey,Lampetra japonica

The myotendinous junction in the skeletal muscle of adult lamprey Lampetra japonica was studied with an electron microscope. Numerous finger-like sarcolemmal invaginations were present at the ends of muscle fibers to form the myotendinous junction. Parietal fibers of each muscle unit showed more closely distributed sarcolemmal invaginations than central fibers. Features of the myotendinous junction. Parietal fibers of each muscle unit showed more closely distributed sarcolemmal invaginations than central fibers. Features of the myotendinous junction generally conform to the accounts in the literature. The sarcolemmal invagination was covered on its sarcoplasmic aspect by the connecting filament layer and the dense amorphous attachment layer, and on the extracellular aspect by the intermediary layer and the external lamina with collagen fibrils arising from the myosepta. Sarcolemmal invaginations were sometimes seen to consist of a pair of sarcolemmas of adjacent muscle fibers within a muscle unit, which is characteristic to the myotendinous junction of lamprey. It is noteworthy that the connecting filament layer is much thinner than that, e.g., in the tadpole tail muscles (Nakao, '74). Furthermore, it is much thicker in the parietal fibers than in the central fibers. The sarcolemma of the terminal segment of the invagination frequently showed specific coupling with cisterns of the sarcoplasmic reticulum (terminal coupling). The external lamina is partially or completely deficient in the terminal segment of sarcolemmal invaginations which form terminal couplings so that collagen fibrils contained in the invagination appear to be in direct contact with the sarcolemma; however, definite relationships of collagen fibrils with the sarcolemma and the external lamina in the terminal segment of invagination still remain obscure. This type of coupling is considered to play a role in the coupling of excitation to contraction of muscle fibers as triads and diads.     

Fine structure of the myotendinous junction and "terminal coupling" in the skeletal muscle of the lamprey, Lampetra japonica.

The myotendinous junction in the skeletal muscle of adult lamprey Lampetra japonica was studied with an electron microscope. Numerous finger-like sarcolemmal invaginations were present at the ends of muscle fibers to form the myotendinous junction. Parietal fibers of each muscle unit showed more closely distributed sarcolemmal invaginations than central fibers. Features of the myotendinous junction generally conform to the accounts in the literature. The sarcolemmal invagination was covered on its sarcoplasmic aspect by the connecting filament layer and the dense amorphous attachment layer, and on the extracellular aspect by the intermediary layer and the external lamina with collagen fibrils arising from the myosepta. Sarcolemmal invaginations were sometimes seen to consist of a pair of sarcolemmas of adjacent muscle fibers within a muscle unit, which is characteristic to the myotendinous junction of lamprey. It is noteworthy that the connecting filament layer is much thinner than that, e. g., in the tadpole tail muscles (Nakao, '74). Furthermore, it is much thicker in the parietal fibers than in the central fibers. The sarcolemma of the terminal segment of the invagination frequently showed specific coupling with cisterns of the sarcoplasmic reticulum (terminal coupling). The external lamina is partially or completely deficient in the terminal segment of sarcolemmal invaginations which form terminal couplings so that collagen fibrils contained in the invagination appear to be in direct contact with the sarcolemma; however, definite relationships of collagen fibrils with the sarcolemma and the external lamina in the terminal segment of invagination still remain obscure. This type of coupling is considered to play a role in the coupling of excitation to contraction of muscle fibers as triads and diads.     

Three dimensional arrangement of mitochondria and endoplasmic reticulum in the heart muscle fiber of the rat

The three-dimensional arrangement of mitochondria and endoplasmic reticulum was studied in thick sections of the heart left ventricle fixed in glutaraldehyde and impregnated with the Ur-Pb-Cu technique and in thin sections of glutaraldehyde-fixed tissue post-fixed in potassium ferrocyanide-reduced osmium. Squarish flattened mitochondria, approximately the size of a sarcomere, were arranged in longitudinal columns in the clefts between the myofibrils. At the periphery of the fiber, the endoplasmic reticulum took the appearance of a subsarcolemmal network of plate-like and tubular cisternae running parallel to the cell surface. Between the myofibrils, the ER network formed longitudinally oriented repetitive units whose structure varied according to their position in relation to the A- or I- bands of the myofibrils. In front of the A-band, the endoplasmic reticulum appeared as a single layered network of anastomotic tubules compressed between the adjacent myofibrils. In front of the I-band, it formed a multilayered network the three-dimensional arrangement of which was dependent upon the presence or absence of the T-tubule. In the absence of the T-tubule, the ER cisternae were loosely anastomosed and occasionally displayed bulbous terminal swellings. In the presence of T-tubules, tubular ER cisternae were seen running parallel on both sides of the T-tubules and were continuous with sheetlike cisternae sandwiched between the distended T-tubule and adjacent extremities of longitudinally arranged mitochondria. These tubular or flattened cisternae were connected to each other by numerous bridging cisternae around the T-tubules.     

Incorporation of microinjected biotin-labelled actin into nascent myofibrils of cardiac myocytes: an immunoelectron microscopic study

Summary Incorporation of microinjected biotin-labelled actin into nascent myofibrils of cultured cardiac muscle cells was investigated by immunogold electron microscopy. At the proximal parts of myofibrils, gold labelling was first found (at about 4 min after injection) around the A-band level. This observation suggests that polymerization of actin or the addition of newly-formed actin filaments occurs preferentially in association with myosin filaments to increase the myofibrillar girth. The distal terminals of developing myofibrils were also labelled at about 4 min after injection. This rapid incorporation of actin subunits at the myofibrillar ends suggests the continued reorganization and/or de novo formation of myofibrils at these positions. Along the extending direction of the myofibrillar terminals, gold particles were arranged in rows on the inner surface of the sarcolemma. These rows of particles continued to become longer with incubation. It appears that actin subunits are added at the membrane-associated ends of pre-existing actin filaments to increase the length of myofibrils.     

The ultrastructure of myofibers in a reptilian heart: The boa constrictor

Electron microscopic observation of cardiac myofibers from the boa constrictor depict several features that differ from other vertebrate cardiac and striated muscle cells. The indentations in the sarcolemma do not penetrate into the deep sarcoplasmic matrix to form a transverse tubular system, instead the regular indentations extend the circumference of the myofiber at the regions of the Z line and are continuous with the latter via a dense homogeneous material that occurs at regular intervals along the subsarcolemmal surface. This dense material provides a direct contact between the sarcolemma and the contractile system. A complex sarcoplasmic reticulum was not observed around the myofibrils or in the sarcoplasmic matrix, instead there were small vesicles dispersed about the sarcoplasm. These myofibers are compared with the slow or tonic muscle fiber (Felderstruktur) which also lacks a transverse tubular system and contains an irregular sarcoplasmic reticulum.     

Sarcotubules and subsarcolemmal caveolae and their continuity with the sarcolemma in frog striated muscle

After using tannic acid mordanting, T-tubules and subsarcolemmal caveolae show a dense staining with lead citrate, with some increase in contrast also of the sarcolemma, in frog muscle. While a few T-tubules show direct continuity with the extracellular space, the majority open indirectly via caveolae. Caveolae lie immediately beneath the sarcolemma, mainly in a single row, and are more numerous in relation to I-bands.     

Heart development in the mexican salamander,Ambystoma mexicanum. II. Ultrastructure

Heart development in the Mexican axolotl, Ambystoma mexicanum, from early embryonic stages to the adult was studied by electron microscopy. During early development, myocardial cells exhibit scanty cytoplasmic matrixes which contain numerous large yolk platelets and lipid droplets. As development progresses the yolk and lipid materials become reduced; the cytoplasmic matrixes increase in glycogen content and membranous organelles. Degradation of yolk platelets appears to take place by an “unraveling” process. The Golgi complex becomes well-developed just prior to the first heart beats and its many associated vesicles are suggestive of secretory activity. Degeneration and death of certain myocardial cells are evident at the onset of trabeculation. Several mechanisms for myofibrillogenesis appear to operate both simultaneously and at different developmental stages. Pre-heart-beat myocardial cells display ribosome-containing amorphous masses and unorganized 60 Å and 140 Å filaments in their cytoplasm. They also have electron-opaque plaques on their plasma membranes. Sarcomeric myofibrils first appear parallel to and immediately beneath the sarcolemma. This occurs at stage 34, the heart-beat initiation stage (6 days). During later development “isolated” Z band-myofilament complexes, numerous loosely-organized 60 Å filaments and a few 140 Å filaments are observable in the cell matrixes. This suggests that Z bands may form centers for myofibril organization at these stages. Also at later stages, polysomes are parallel to the already-formed myofibrils. These polysomes are possibly synthesizing contractile proteins “in situ” and may represent a mechanism for myofibril diameter growth. In late embryonic and juvenile stages 100 Å—110 Å filaments appear to be continuous between Z bands of adjacent myofibrils. It is possible that such filaments of intermediate size are involved in aligning myofibrils into register during advanced developmental stages.     

The application of various electron microscopic techniques for ultrastructural characterization of the human papillary heart muscle cell in biopsy material

Summary Various electron microscopical techniques have been applied to biopsy material obtained from patients suffering from mitral stenosis in order to characterize the subcellular organization of the hypertrophied papillary muscle. Small pieces of the same sample were processed for correlative transmission - (TEM) and scanning -(SEM) electron microscopical studies. TEM was carried out on conventionally fixed tissue with or withouten bloc staining with a Cu-Pb citrate solution, and on freeze fracture replicas, while cryofractured material was studied by SEM. Stereo electron micrographs of the Cu-Pb impregnated tissue and of the cryofractured material were especially useful for studying the spatial distribution and relationship between various cell organelles.The myofilaments of the hypertrophied cells were arranged in a normal hexagonal pattern. Regions with irregular orientation of the myofibrils were occasionally seen. Accumulations of interfilamentous glycogen particles adjacent to the Z-bands were characteristic patterns of the contracted muscle cells. The extensive nexuses frequently observed in the subsarcolemmal regions may reflect functional alterations of the intercommunication between hypertrophied cells. The T-tubules were relatively few and irregularly distributed, and the complexity of the sarcotubular system (SR) revealed regional variations. Excellent visualization of the interior couplings between the SR and the T-tubules was achieved by studying thick sections of Cu-Pb impregnated tissue in the TEM.The dense staining of the various intracellular membranes when compared with the almost unstained external membranes including the free cell surface, intercalated disc and T-system, strongly indicates differences in chemical and functional properties of the two membrane systems.En bloc staining resulted also in contrasted glycogen as well as components of the nucleolus and the heterochromatin. The biochemical basis for the selective staining remains obscure; it may be a result of binding of heavy metal ions to carboxyl groups of specific proteins, and/or it may represent deposits of lead phosphate.     

Distribution of connectin (titin), nebulin and α-actinin at myotendinous junctions of chicken pectoralis muscles: an immunofluorescence and immunoelectron microscopic study

Summary The distribution of connectin (titin), nebulin and -actinin in the areas of myotendinous junctions of chicken pectoralis muscles was examined by immunocytochemical methods. Staining with antibodies against connectin (4C9, SM1 and P1200) and nebulin formed doublets flanking nonterminal Z-bands; near the end of muscle fibres singlets were seen within the terminal sarcomere on the side adjacent to the terminal Z-bands. The apical regions of muscle processes, where no myosin filaments are present although actin filaments exist, were reactive with anti-nebulin but not with anti-connection. Antibodies against pectoralis (skeletal muscle type) -actinin stained non terminal Z-bands and that against gizzard (smooth muscle type) the sarcolemma. Terminal Z-bands were unreactive with both of these antibodies. These findings indicate that, although terminal and nonterminal Z-bands differ in their molecular composition, connectin and nebulin filaments appear to link myosin and actin filaments, respectively, to both Z-band types.     

Fine structural alterations of muscle fibers in diseases accompanied by myotonia.

The authors have reported the results of examination by electron microscopy of two muscle biopsy specimens from cases of myotonia congenita and three cases of myotonia dystrophica. They have stated that "peripheral annular formation" was a frequently observed alteration in the myotonia congenita cases. In the myotonia dystrophica cases there were additionally disorganized myofibrils in the subsarcolemmal region and inclusion body vacuoles morphologically connected with the sarcolemma. The term "peripheral annular formation" refers to the situation in which the peripheral myofibrils of the muscle fiber fracture and the fragments retract and form a helical sheath around the central myofibrils of the same muscle fiber.     

Reduction of density and anisotropic distribution of intermediate filaments occur during avian skeletal myogenesis

Chicken skeletal muscle taken from embryos in ovo was examined by thin-section electron microscopy. Measurements of filament diameters reveal three nonoverlapping groups of filaments: thin (actin myofibrillar) filaments with mean diameters of 5.3 ± 0.6 nm (S.D.), thick (myosin myofibrillar) filaments with mean diameters of 15 ± 1.4 nm, and intermediate filaments with mean diameters of 9.3 ± 0.9 nm. During muscle development these diameters do not change. By counting the number of filaments observed in the sarcoplasm at different stages, we find that the spatial density of intermediate filaments decreases during avian myogenesis in ovo, from 91 intermediate filaments/μm² at 6 days to 43 intermediate filaments/μm² at 17 days in ovo. Initially randomly arranged, some intermediate filaments become associated with Z discs, sarcoplasmic reticulum, nuclear membrane, and the sarcolemma between 6 and 10 days in ovo. These associated intermediate filaments course both parallel and transverse to myofibrils, forming lateral connections between myofibrillar Z discs and longitudinal connections from Z disc to Z disc within myofibrils. Intermediate filaments also appear to connect Z discs with the nuclear membrane. The intermediate filament associations persist through day 17 of development, after which the presence of cytoskeletal filaments is obscured by the densely packed myofibrils and membranes. Intermediate filament distribution becomes anisotropic during development. A greater proportion of intermediate filaments in the immediate perimyofibrillar area are oriented parallel to myofibrils than in other areas, so that the majority of the intermediate filaments nearest the myofibrils course parallel to them. The longitudinal intramyofibrillar intermediate filaments persist throughout development, as shown by their existence in KI-extracted adult myofibrils.     

Morphometric and autoradiographic study of developing ventricular and atrial myocardium in fetal rats.

The ventricular and atrial myocardia of 19- to 21-day-old rat fetuses have been studied by electron microscopic morphometry and quantitative autoradiography following the injection of 3H-leucine. The myocytes in both these regions contain numerous myofibrils located predominantly near the sarcolemma and oriented parallel to each other and to the long axis of the cell. The width of myofibrils at this stage of development is 0.428 +/- 0.240 mum. in the ventricle and 0.365 +/- 0.185 mum. in the atrium. The volume fractions of myofibrils, mitochondria, smooth endoplasmic reticulum, sarcoplasmic matrix, and sarcolemma in ventricular myocytes are nearly equal to the corresponding fractions in atrial cells. A similarity between ventricular and atrial myocytes was also found in the distribution of newly synthesized proteins as indicated autoradiographically by the concentration of grains over various sarcoplasmic structures. In both tissues the concentration of grains was consistently above average over the sarcolemma, smooth endoplasmic reticulum, and myofibrils; average over the mitochondria; and below average in association with the sarcoplasmic matrix. The specificity of this autoradiographic procedure for labeled amino acids actually incorporated into protein was tested biochemically, and it was found that glutaraldehyde-paraformaldehyde fixation leads to a negligible contamination with non-specifically bound leucine. It is suggested that the high concentration of protein labeling near the sarcolemma is related to the imminent development of the T-system and its associated channels of sarcoplasmic reticulum.     

Adult-derived liver stem cells acquire a cardiomyocyte structural and functional phenotype ex vivo

We examined the differentiation potential of an adult liver stem cell line (WB F344) in a cardiac microenvironment, ex vivo. WB F344 cells were established from a single cloned nonparenchymal epithelial cell isolated from a normal male adult rat liver. Genetically modified, WB F344 cells that express beta-galactosidase and green fluorescent protein or only beta-galactosidase were co-cultured with dissociated rat or mouse neonatal cardiac cells. After 4 to 14 days, WB F344-derived cardiomyocytes expressed cardiac-specific proteins and exhibited myofibrils, sarcomeres, and a nascent sarcoplasmic reticulum. Further, rhythmically beating WB F344-derived cardiomyocytes displayed calcium transients. Fluorescent recovery after photobleaching demonstrated that WB F344-derived cardiomyocytes were coupled with adjacent neonatal cardiomyocytes and other WB F344-derived cardiomyocytes. Fluorescence in situ hybridization experiments suggested that fusion between WB F344 cells and neonatal mouse cardiomyocytes did not take place. Collectively, these results support the conclusion that these adult-derived liver stem cells respond to signals generated in a cardiac microenvironment ex vivo acquiring a cardiomyocyte phenotype and function. The identification ex vivo of microenvironmental signals that appear to cross germ layer and species specificities should prove valuable in understanding the molecular basis of adult stem cell differentiation and phenotypic plasticity.     

High-resolution scanning electron microscopic studies on the three-dimensional structure of the transverse-axial tubular system, sarcoplasmic reticulum and intercalated disc of the rat myocardium

The three-dimensional structure of the transverse-axial tubular system, sarcoplasmic reticulum (SR), and intercalated disc of the rat left ventricle was examined by high-resolution scanning electron microscopy after removal of the cytoplasmic matrices by the osmium-DMSO-osmium procedure. In the intermyofibrillar space, the transverse tubules (T-tubules) are accompanied by longitudinally oriented axial tubules and together form a transverse-axial system. The junctional SR is usually small but occasionally medium or large in size and couples with the T- or with the axial tubules. On the surface of the junctional SR facing the T- or the axial tubule, tiny junctional processes are seen. One or two sarcotubules, the so-called Z-tubules, frequently run parallel to the T-tubule. The sarcotubules derived from the junctional SR or from the Z-tubule run longitudinally or obliquely and form polygonal meshes around the myofibrils. On the surface of the SR at the H-band level, small fenestrations of 12-40 nm in diameter, and tiny hollows 8-20 nm in diameter are seen. Bulbous swellings of the SR, the corbular SR, are preferentially seen near the Z-band. The large and flat SR, known as the cisternal SR, intercalates among the SR meshes. In the subsarcolemmal space, the sarcotubules form a multilayered network (peripheral SR). The cisternal SR is frequently intercalated in these meshes and closely associated with the inner surface of the sarcolemma. The intercalated disc appears as a prominently undulated membrane demarcating the border between two adjacent heart muscle cells, and occasionally small projections 60-90 nm in diameter and 200-600 nm in length display on its surface.