Adult mammalian cardiac muscle cells in culture

Adult rat cardiac muscle cells were isolated from the ventricle by a retrograde perfusion technique through the aorta (Nag and Zak, 1979). These single, isolated cardiac muscle cells were cultured for 4 weeks. Throughout the culture period, a small number of muscle cells retained their cylindrical shape, while the rest exhibited alterations in shape and size assuming a flattened body of irregular shape with pseudopodia-like processes and thereby resembling embryonic/neonatal cardiac muscle cells in culture. Transmission electron microscopy revealed that the cylindrical muscle cells contained compactly arranged myofibrils and cellular organelles, similar to those of freshly isolated and in vivo cells. A few irregularly shaped cardiac muscle cells were similar to the cylindrical cells in their internal structural organization. Most of the irregular cells exhibited less myofibrillar content than that of the freshly dissociated and in vivo cells. Myofibrils in the irregular cells were widely spaced and myofilament of some of the myofibrils were loosely bunched. In addition, scattered patches of myofibrils and free myofilaments were observed in many of these cells. The internal structural organization of these irregularly shaped cardiac muscle cells closely resembled the embryonic and neonatal cardiac muscle cells in vitro and in vivo. Most of the muscle cells in culture continued to contract spontaneously, and electron microscope studies clearly indicated that they underwent dedifferentiation. Autoradiography studies demonstrated that the cylindrical and irregularly shaped cardiac muscle cells underwent DNA synthesis and cell division in culture.     

Isolation,long-term culture,and ultrastructural characterization of adult cardiomyopathic cardiac muscle cells

Summary A long-term cell culture system for adult cardiomyopathic hamster cardiac muscle cells has been established. The diseased and control hearts were dissociated into single cell suspension with the modifications of our previous technique using collagenase and hyaluronidase as applied to the dissociation of the adult rat heart. The postperfusion of the diseased heart with Krebs-Ringer phosphate buffer and bovine serum albumin was very helpful in obtaining greater yield of viable diseased muscle cells; the cells were cultured for 4 wk. Approximately 60% of the myocytes from the diseased heart and 85% of the myocytes from the normal heart attached to the substrates and survived throughout the culture period. Approximately 60 to 70% of the cardiac myocytes from the diseased and control hearts were bi- or multinucleated; 30% of the diseased and 80% of the normal myocytes showed rhythmic contractility. Electron microscopy revealed the presence of two kinds of cardiac muscle cells in the diseased cell culture on the basis of their myofibril content: one with scanty myofibrils and another with abundant myofibrils. Myocytes with sparse myofibrils showed certain characteristic features that included autophagic vacuoles, amorphous matrix of fine filamentous texture, scattered strips of myofibrils, and abnormal organization of the Z-line. Cardiac muscle cells with abundant myofibrillar content contained unorganized myofibrils in certain sarcomeres. These studies demonstrate the feasibility of maintaining diseased cardiac muscle cells from adult cardiomyopathic hamsters for at least 4 wk in monolayer culture. This study was supported by a grant from the American Heart Association of Michigan, National Institutes of Health grant HL-25482, and by an Oakland University Biomedical Research Support Grant.     

Myofibrillogenesis in the first cardiomyocytes formed from isolated quail precardiac mesoderm

De novo assembly of myofibrils was investigated in explants of precardiac mesoderm from quail embryos to address a controversy about different models of myofibrillogenesis. The sequential expression of sarcomeric components was visualized in double- and triple-stained explants before, during, and just after the first cardiomyocytes began to beat. In explants from stage 6 embryos, cultured for 10 h, ectoderm, endoderm, and the precardiac mesoderm displayed arrays of stress fibers with alternating bands of the nonmuscle isoforms of alpha-actinin and myosin IIB. With increasing time in culture, mesoderm cells contained fibrils composed of actin, nonmuscle myosin IIB, and sarcomeric alpha-actinin. Several hours later, before beating occurred, both nonmuscle and muscle myosin II localized in some of the fibrils in the cells. Concentrations of muscle myosin began as thin bundles, dispersed in the cytoplasm, often overlapping one another, and progressed to small, aligned A-band-sized aggregates. The amount of nonmuscle myosin decreased dramatically when Z-bands formed, the muscle myosin became organized into A-bands, and the cells began beating. The sequential changes in protein composition of the fibrils in the developing muscle cells supports the model of myofibrillogenesis in which assembly begins with premyofibrils and progresses through nascent myofibrils to mature myofibrils.     

Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin

Fluorescently labeled alpha-actinin, isolated from chicken gizzards, breast muscle, or calf brains, was microinjected into cultured embryonic myotubes and cardiac myocytes where it was incorporated into the Z-bands of myofibrils. The localization in injected, living cells was confirmed by reacting permeabilized myotubes and cardiac myocytes with fluorescent alpha-actinin. Both living and permeabilized cells incorporated the alpha-actinin regardless of whether the alpha-actinin was isolated from nonmuscle, skeletal, or smooth muscle, or whether it was labeled with different fluorescent dyes. The living muscle cells could beat up to 5 d after injection. Rest-length sarcomeres in beating myotubes and cardiac myocytes were approximately 1.9-2.4 microns long, as measured by the separation of fluorescent bands of alpha-actinin. There were areas in nearly all beating cells, however, where narrow bands of alpha-actinin, spaced 0.3-1.5 micron apart, were arranged in linear arrays giving the appearance of minisarcomeres. In myotubes, alpha-actinin was found exclusively in these closely spaced arrays for the first 2-3 d in culture. When the myotubes became contraction-competent, at approximately day 4 to day 5 in culture, alpha-actinin was localized in Z-bands of fully formed sarcomeres, as well as in minisarcomeres. Video recordings of injected, spontaneously beating myotubes showed contracting myofibrils with 2.3 microns sarcomeres adjacent to noncontracting fibers with finely spaced periodicities of alpha-actinin. Time sequences of the same living myotube over a 24-h period revealed that the spacings between the minisarcomeres increased from 0.9-1.3 to 1.6-2.3 microns. Embryonic cardiac myocytes usually contained contractile networks of fully formed sarcomeres together with noncontractile minisarcomeres in peripheral areas of the cytoplasm. In some cells, individual myofibrils with 1.9-2.3 microns sarcomeres were connected in series with minisarcomeres. Double labeling of cardiac myocytes and myotubes with alpha-actinin and a monoclonal antibody directed against adult chicken skeletal myosin showed that all fibers that contained alpha-actinin also contained skeletal muscle myosin. This was true whether alpha-actinin was present in Z-bands of fully formed sarcomeres or present in the closely spaced beads of minisarcomeres. We propose that the closely spaced beads containing alpha-actinin are nascent Z-bands that grow apart and associate laterally with neighboring arrays containing alpha-actinin to form sarcomeres during myofibrillogenesis.     

Studies of adult amphibian heart cells in Vitro: DNA synthesis and mitosis

The ventricle of the adult newt heart was excised and cut into several pieces of approximately 0.5 – 1.0 mm. These heart pieces were then cultured for 60 days at 25 °C in a modified Leibovitz medium (L-15). Approximately 37% of the explants were attached to the substrate and more than 33% of the attached explants and approximately 15% of the unattached explants established pulsation rates which ranged 3–67 beats/min. The explants were labeled with 1 μCi/ml of ³H-thymidine for 24 hr at 7, 15, 21, 30, 45 and 60 days of culture initiation, and processed for electron microscopic autoradiography. The examination of the autoradiograms revealed that as the culture continued, the cardiac muscle cells altered their morphology, resembling embryonic cardiac muscle cells. These altered muscle cells were termed dedifferentiated cardiac muscle cells. The number of these dedifferentiated cells increased over the period of culture, showing 10.3–94% dedifferentiated cells after 7–60 days of culture respectively. DNA synthesis and mitosis were observed in the dedifferentiated cardiac muscle cells, apart from the non-muscle cells. The quantitation of the autoradiograms revealed that the number of labeled nuclei in the cardiac muscle cells gradually increased over the period of culture, and a maximum number of labeled cardiac muscle cells (30%) was observed in the third week. The peak was followed by a decline in the eighth week which exhibited 1.5 % labeled cardiac muscle cells. The trend of mitosis was similar to that of DNA synthesis. The maximum number of mitotic figures (9%) was observed in the third week of culture, which was followed by a decline and finally absent in the eighth week. The cardiac non-muscle cells, mostly fibroblasts and endothelial cells, also showed incorporation of ³H-thymidine in their nuclei. The number of labeled non-muscle cells nuclei and the mitotic index were highest (61 and 15% respectively) in the first week of culture, but then they decreased gradually over the eight-week period in culture. This study provides evidence for the first time that the adult amphibian cardiac myocytes can undergo DNA synthesis and mitosis when explanted and cultured. The significance of this cell replication is discussed.     

DNA synthesis, mitosis, and differentiation in cardiac myogenesis

Cardiac muscle cells obtained by trypsinizing 5-day chick embryonic heart were cultured as single cells in separate culture dishes. Using this technique, problems of heterotypic cell interactions, “overgrowth” of one cell type, etc., are eliminated. Experiments performed on these single cell cultures show that the muscle cells in the embryonic chick hearts differ in morphology, including content of cross-striated myofibrils; in ability to synthesize DNA and undergo mitosis; and in frequency of contraction. Contracting cells containing cross-striated myofibrils undergo mitosis in vitro, giving rise to spontaneously beating daughter cells. These daughter cells contain cytoplasmic fibrils, which bind fluorescein-labeled antimyosin immediately after cytokinesis. Some cardiac muscle cells from 5-day heart do not divide in culture; the rest undergo 1–5 doublings. This preliminary investigation suggests that the new muscle cells formed during cardiac growth are derived from mitotically active “overtly” differentiated cardiac muscle cells.     

LONG-TERM ORGAN CULTURE OF THE SALAMANDER HEART

Beating salamander hearts were maintained in tissue culture for periods ranging from 1 to 6 months. After 1, 3, or 6 months of culture, six hearts, along with six control hearts, were fixed for electron microscopy. In control tissue, the sarcoplasmic reticulum usually demonstrated the normal pattern of paired, linearly arranged membranes, although in some cases, the reticulum showed a variation from these membranes to a series of small vesicles. There was no evidence of a T-system of tubules in any of the material examined. Desmosome-Z band complexes were observed in almost all sections of both control and experimental material. A possible role of these complexes in the excitation-contraction mechanism is discussed. In 3 month cultured material, alterations in normal myofibrillar pattern occurred. Small segments of myofibrils branched from one Z band to join the Z band of an adjacent myofibril, or appeared to be fraying out into the sarcoplasm. In 6 month cultured material, myofibrils were fragmented into short segments from which myofilaments frayed out into the sarcoplasm. This filamentous material may be remnants of myofilaments. Despite the morphological changes in myofibrils, the heart pulsation rate, established at the beginning, was maintained throughout the culture period. It is suggested that the alterations, observed in the experimental material, occurred in elements not essential for heart beat maintenance, or that these alterations have not yet progressed to a critical point of affecting the heart beat.     

Mitosis and intermediate-sized filaments in developing skeletal muscle

A new class of filaments intermediate in diameter between actin and myosin filaments has been demonstrated in skeletal muscle cells cultured from chick embryos. These filaments, which account for the majority of free filaments, average 100 A in diameter. They may run for more than 2 µ in a single section and can be distinguished in size and appearance from the thick and thin filaments assembled into myofibrils. The 100-A filaments are seen scattered throughout the sarcoplasm at all stages of development and show no obvious association with the myofibrils. The 100-A filaments are particularly conspicuous in myotubes fragmented by the mitotic inhibitors, colchicine and Colcemid. In addition, filaments similar in size and appearance to those found in myotubes are present in fibroblasts, chondrocytes, and proliferating mononucleated myoblasts. The 100-A filaments are present in cells arrested in metaphase by mitotic inhibitors. Definitive thick (about 150 A) or thin (about 60 A) myofilaments are not found in skeletal myogenic cells arrested in metaphase. Myogenic cells arrested in metaphase do not bind fluorescein-labeled antibody directed against myosin or actin. For these reasons, it is concluded that not all "thin" filaments in myogenic cells are uniquely associated with myogenesis.     

Intracellular assembly of newly synthesized canine cardiac myosins

To investigate how newly synthesized cardiac myosins are assembled into myofilaments, we analysed the distribution of newly produced alpha-myosin heavy chain isozyme in sarcomeres by immunoelectron microscopy using a monoclonal antibody (CMA19), which is specific for alpha-myosin heavy chain. Isozymic changes in myosin heavy chains from beta to alpha type were induced in canine ventricular muscles and cultured ventricular myocytes by administration of 1-thyroxine. We incubated the glycerinated ventricular muscles or cultured ventricular myocytes with the enzyme (horseradish peroxidase) labelled Fab fragment of CMA19. After the reaction with 3, 3'-diaminobenzidine and osmification, we prepared ultrathin sections of the ventricular muscles or cultured ventricular myocytes and analysed their staining patterns by electron microscopy. There was apparent heterogeneity in the staining intensity of the myofilaments among different cells, among different myofibrils and even intramyofibrillarly. Higher magnification revealed that there were scattered foci of strong reaction which appeared to be foci of assembly of the newly synthesized alpha-myosin heavy chain. Immunocytochemical study also showed heterogeneous reactions within myofilaments and that there were scattered foci of myofilament assembly, which were closely associated with polyribosomes producing newly induced alpha-myosin heavy chain. These data suggest that newly synthesized cardiac myosins are assembled into myofilaments from the sites of synthesis, that is polyribosomes. This may explain the heterogeneity of the assembly pattern of newly synthesized cardiac myosins at the subcellular level.     

Suppression of cardiac troponin T induces reduction of contractility and structural disorganization in chicken cardiomyocytes

We herein examine the effect of cardiac troponin T (CTnT) suppression in cultured chicken cardiomyocytes derived from embryonic cardiac ventricular muscle. TnT is an important protein participating in regulation of striated muscle contraction, but it is not clear whether TnT contributes to the formation of sarcomere structure in myofibrils. Double-stranded RNA homologous to the nucleotide sequence of CTnT (CTnT-siRNA) was introduced into cultured muscle cells two days after plating. Transfection efficiency was above 80%. Immunoblot analyses suggested that the expression of CTnT progressively falls for the three consecutive days after transfection, but partly reappears on the fourth day. Maximum suppression occurs three days after transfection, with almost invisible CTnT protein on immunoblots in all the examined conditions: 0.5-2 nmol CTnT-siRNA towards 1-3 x 10(6) cells. The suppression was specific to CTnT, and the other myofibrillar proteins such as myosin, connectin/titin, tropomyosin, alpha-actinin, and troponin I were all present in transfected cells. The following functional and morphological changes were detected in CTnT-suppressed cells. The population of beating cells decreased significantly after transfection, when compared to control cells. A part of CTnT-suppressed cells showed two non-overlapping types of morphological changes: 1) myofibrils presenting unusually long Z-Z intervals; 2) myofibrils with irregular small striations in cells not connected at their adhesion interfaces of a jagged-appearance. Thus, our results reveal that CTnT is important for stable beating in cultured ventricular muscle cells, and also to some extent, for maintaining myofibrillar structure and cell-to-cell adhesion.     

Development of Vertebrate Skeletal Muscle

An investigation of developing skeletal muscle necessitatesthe study of three categories; the derivation of muscle cellsor fibers, myofilament synthesis and interactions, assemblyof myofilaments into functional sarcomeres of striated myofibrils.With few exceptions, skeletal muscle cells are of mesodermalorigin, and consist of rounded mononucleated cells which elongateand fuse with one another to become myotubes. Within the sarcoplasm,myofibrillar proteins are synthesized and grouped into interactingthick and thin filaments. Crude, non-striated myofibrils resultfrom linear arrangements of thick and thin filaments which arehorizontally aligned by the invaginating sarcotubular system.After Z-lines form, providing attachment sites for thin filaments,a typical banding pattern follows. The newly formed Z-linespull apart, followed by the attached thin filaments, and repeating"relaxed" sarcomeres are the resulting striated myofibrillarpattern.     

Rhabdomyoma of the soft palate. Fine structural details of a highly differentiated muscle tumor

An adult rhabdomyoma was light and electron microscopically studied. The lesional cells presented well-known structural details, such as abundance of mitochondria and glycogen, myofilaments and myofibrils, hypertrophied Z-bands and masses of Z-band material. Triads were randomly scattered in the cytoplasm and also related to the individual sarcomeres. In sarcomeres the triads were regularly placed near to the A-I-junctions. This peculiarity of mammalian skeletal muscle fibers may yield a criterion to distinguish between cardiac and extracardiac rhabdomyomas. Circumscribed surface areas of the tumor cells were provided with elaborate infoldings of plasma membrane and basal lamina. These areas were interpreted as imitating myotendinous junctions. Satellite cells were regularly found.     

Ribosome distribution in normal and infarcted rat hearts

Summary Distribution of ribosomes throughout the myocardium of normal and infarcted rat hearts was studied by immunofluorescence and laser confocal scanning microscopy. In addition, sections were labelled with peroxidase or immunogold particles for electron microscopic examination. Ligation of the proximal free left coronary artery produced severe myocardial ischaemia, and after 6 days of ligation most of the left ventricular wall was necrotic and partially replaced by granulation tissue. Immunofluorescence microscopy revealed the presence of ribosomes throughout the non-necrotic myocardium. Some cardiac muscle cells located in subendocardial areas and in the border areas surrounding the infarct were particularly intensely stained. Cells constituting the granulation tissue frequently exhibited strong ribosomal immunostaining. Within longitudinally sectioned cardiac muscle cells, ribosomes were organized in strands oriented along the long axis of the cell as well as in a cross-striated pattern. By double labelling of muscle cells with antibodies against ribosomes and Z-line-associated proteins (desmin or α-actinin), it was shown that the cross-striated bands of anti-ribosomal staining coincided with the I-bands along the myofibrils. Immunoelectron microscopy confirmed a wide distribution of ribosomes throughout the intermyofibrillar and subsarcolemmal sarcoplasm, and some labelling was also observed within the I-band. The present results indicate that ribosomes are distributed in a characteristic pattern throughout the sarcoplasm of cardiac muscle cells in association with the myofibrils. Furthermore, it is suggested that within viable cardiac muscle cells located adjacent to the infarct, protein synthesis is increased; this might be an important factor in regional development of compensatory hypertrophy of the surviving cardiac muscle cells.     

Myofibril organisation and mitosis in cultured cardiac muscle cells.

Isolated cardiac muscle cells grown in vitro have been studied with respect to their ability to contract spontaneously and maintain myofibrillar organisation during division. These cells do not round up to undergo mitosis; division is achieved by the cell pinching itself in two in a selected area. This adaptation minimises disturbance to cell attachment sites and to myofibrils running between them. We correlated this with the persistence of beating during division and the maintenance of myofibrils with intact Z bands, even in close proximity to the nucleus, through division in many cells. Cessation of beating and disorganisation of myofibrils are therefore not prerequisites for division of cardiac muscle cells, as reported previously.     

血管内皮细胞和心脏组织块的立体培养

本文旨在对比研究二维平面与三维立体培养模式下,内皮细胞和心脏组织形态学的差异。采用胶内、胶上、三明治模式、玻片培养小室模型等多种I型胶原立体培养模型,通过免疫荧光技术及显微形态学观察组织和细胞的生长情况。在二维平面培养中,原代心脏血管内皮细胞呈铺路石样排列;而在三维胶原培养模式中,内皮细胞呈长梭状形态,并迁入胶原培养介质中,和体内血管新生及血管生成过程中的内皮细胞活化表型相似。加入血管内皮生长因子(vascular endo- thelial growth factor VEGF)能增强内皮细胞管状结构的形成。在三维胶原中,心脏组织块生长良好,迁出的细胞将相邻组织块连接起来,组织块有自发的搏动。本工作表明,改进的薄层胶原培养、玻片培养小室模型和动脉条模型是较好的研究血管生成和血管新生的工具。在三维培养的情况下,内皮细胞通过空间增殖、迁移和锚定,可形成管状结构,比二维平面培养更适合用于血管新生的研究。不同的立体培养模型可用于不同目的的研究。     

Fine structure and differentiation of Ascidian muscle. I. Differentiated caudal musculature of Distaplia occidentalis tadpoles

The structure of the caudal muscle in the tadpole larva of the compound ascidian Distaplia occidentalis has been investigated with light and electron microscopy. The two muscle bands are composed of about 1500 flattened cells arranged in longitudinal rows between the epidermis and the notochord. The muscle cells are mononucleate and contain numerous mitochondria, a small Golgi apparatus, lysosomes, proteid-yolk inclusions, and large amounts of glycogen. The myofibrils and sarcoplasmic reticulum are confined to the peripheral sarcoplasm. Myofibrils are discrete along most of their length but branch near the tapered ends of the muscle cell, producing a Felderstruktur. The myofibrils originate and terminate at specialized intercellular junctional complexes. These myomuscular junctions are normal to the primary axes of the myofibrils and resemble the intercalated disks of vertebrate cardiac muscle. The myofibrils insert at the myomuscular junction near the level of a Z-line. Thin filaments (presumably actin) extend from the terminal Z-line and make contact with the sarcolemma. These thin filaments frequently appear to be continuous with filaments in the extracellular junctional space, but other evidence suggests that the extracellular filaments are not myofilaments. A T-system is absent, but numerous peripheral couplings between the sarcolemma and cisternae of the sarcoplasmic reticulum (SR) are present on all cell surfaces. Cisternae coupled to the sarcolemma are continuous with transverse components of SR which encircle the myofibrils at each I-band and H-band. The transverse component over the I-band consists of anastomosing tubules applied as a single layer to the surface of the myofibril. The transverse component over the H-band is also composed of anastomosing tubules, but the myofibrils are invested by a double or triple layer. Two or three tubules of sarcoplasmic reticulum interconnect consecutive transverse components. Each muscle band is surrounded by a thin external lamina. The external lamina does not parallel the irregular cell contours nor does it penetrate the extracellular space between cells. In contracted muscle, the sarcolemmata at the epidermal and notochordal boundaries indent to the level of each Z-line, and peripheral couplings are located at the base of the indentations. The external lamina and basal lamina of the epidermis are displaced toward the indentations. The location, function, and neuromuscular junctions of larval ascidian caudal muscle are similar to vertebrate somatic striated muscle. Other attributes, including the mononucleate condition, transverse myomuscular junctions, prolific gap junctions, active Golgi apparatus, and incomplete nervous innervation are characteristic of vertebrate cardiac muscle cells.     

On the origin of Z-band material and myofilaments in myoblasts from the human atrial wall

Summary The origin of cardiac myofibrils in cells from the atrial wall in human embryos was studied. Z-band substance appears throughout the cytoplasm as irregular electron dense patches in a network of thin filaments. The thin and thick filaments are synthesized as separate units in the sarcoplasm and are later aggregated into myofibrils. Complexes of Z substance and thin filaments occur numerously at different stages of myofibrillar organisation. Thick filaments are formed in close proximity to free ribosomes and are later incorporated in an hexagonal pattern into the Z-band/thin filament complex.This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanities     

Mitosis in Beating Cardiac Muscle Cells from Newt Embryos In Vitro

Cardiac muscle cells from newt embryos were cultured at relatively low cell density. Within 10 days in culture, 2 cell types (spindle and flat type) were distinguished both among beating and non-beating cells. Mitosis in single beating cells was frequently observed both in spindle and flat cells. Some cells maintained almost constant contractile activities throughout the mitotic stages, while the others transiently stopped beating during mitosis, which accords well to the case in chick embryos (1). Ultra-thin section shows the presence of myofibril's structure in a dividing cell, as shown in newborn rats (2, 3, 4), chick embryos (1, 5, 6, 7) and adult newts (8, 9). As a consequence of mitosis, 3 types (spindle, flat and mixed type) of beating colonies developed after 34 weeks in culture. Cell proliferation was accompanied with pulsation and could be directly pursued till the 4th division, suggesting that differentiated myocardiac cells with myofibrils proliferate by their mitoses in vivo , maintaining rhythmic contraction.     

The effect of 5-bromodeoxyuridine (BrdU) on cardiac muscle differentiation

Cultured cardiac muscle cells undergo cell division and form beating progeny. Incorporation of BrdU into the nuclei of daughter cells does not suppress their ability to beat and form cross-striated myofibrils. Fluorescence microscopy of clones derived from single beating cells fed with BrdU-treated medium for over 2 weeks reveal cytoplasmic fibrils stainable with fluorescein-labeled antimyosin. The effect of BrdU on the emergence of cardiac muscle phenotype was also investigated by utilizing cardiac myogenic precursor cells from precardiac mesoderm in early embryos (stage 4–stage 9). These studies show that the cardiac myogenic cells fall into the following categories with respect to their ability to express the differentiated phenotype in the presence of BrdU: (1) precardiac mesodermal cells that are inhibited; (2) precardiac mesodermal cells that are not inhibited; and (3) beating cardiac muscle cells that are not inhibited. The entry of precardiac cells from the first category to the second and to the third appears to be unsynchronized.     

Subcellular localization of dystrophin and vinculin in cardiac muscle fibers and fibers of the conduction system of the chicken ventricle

The subcellular localization of dystrophin and vinculin was investigated in cardiac muscle fibers and fibers of the conduction system of the chicken ventricle by immunofluorescence confocal microscopy. In ventricular cardiac muscle fibers, strong staining with antibody against dystrophin appeared as regularly arranged transverse striations at the sarcolemmal surface, and faint but uniform staining was seen in narrow strips between these striations. In fibers of the ventricular conduction system, the sarcolemma was stained uniformly with this antibody, but strong staining was found as regular striations in many areas and as scattered patches in other areas of the sarcolemma. These intensely stained striations and scattered patches of dystrophin were colocalized with those of vinculin. Because dystrophin striations were located at the level of Z bands of the underlying myofibrils, they were regarded as the concentration of this protein at costameres together with vinculin. In fibers of the conduction system, myofibrils were close to the sarcolemma where dystrophin and vinculin assumed a striated pattern, at some distance from the cell membrane where these proteins exhibited a patchy distribution, and distant from the sarcolemma where dystrophin was uniformly distributed. These data suggest that the distribution patterns of dystrophin reflect the degree of association between the sarcolemma and underlying myofibrils.