Changes in alpha-actinin localization and myofibrillogenesis in rat cardiomyocytes under cultivation

By indirect immunofluorescence with monoclonal anti-alpha-actinin antibodies the localization of this contractile protein was studied in ventricular cardiac myocytes from newborn 2-4-day old rats in the course of their cultivation. In freshly isolated heart muscle cells a predominant longitudinal orientation of myofibrils was observed; in some cells on the periphery of cytoplasm the contours of Z-lines are indistinct. During cell spreading, in the areas of intercalated discs, growing processes were observed mostly containing no contractile structures at earlier stages of cultivation. On days 3 to 14, the cytoplasmic processes and ruffles are filled with developing myofibrils. The cultures are heterogeneous in the morphology of contractile apparatus of individual cells. In most cardiomyocytes mature myofibrils are well-developed in the central part of the cytoplasm, whereas in its peripheral areas non-myofibrillar stress-fiber-like structures and bundles with continuous distribution of alpha-actinin frequently connected to myofibrils are more typical. In the areas of active myofibrillogenesis, located mainly on the cell periphery, numerous alpha-actinin dots are observed; most of them are arranged linearly and periodically at a distance of 0.3-1.5 microns and seem to be structural precursors of Z-lines. The data obtained show that the cultures of mammalian cardiac cells may be a convenient object for studying myofibrillogenesis in the course of cardiomyogenic differentiation.     

ULTRASTRUCTURAL ANALYSES OF STONE HEART SYNDROME AT ONSET AND SIX DAYS LATER FOLLOWING TOTAL SUPPORT OF THE CIRCULATION WITH A PARTIAL ARTIFICIAL HEART OR LEFT VENTRICULAR ASSIST DEVICE (ALVAD)

Ischemic myocardial contracture developed in a 21-year-old man following aortic and mitral valve replacement. The patient's circulation was supported totally for 6 days with an abdominal left ventricular assist device (ALVAD). Cardiac allografting was then undertaken. Samples of myocardium taken at the original operation and 6 days later at transplantation were analyzed ultrastructurally. At the onset of ischemic cortracture, left ventricular abnormalities included hypercontraction of myofibrils, loss of normal A-band and Z-band patterns, mitochondrial swelling with fusion of cristae, interfibrillar edema and glycogen depletion. Capillaries demonstrated swelling of endothelial cells and basement membrane disruption. Six days later, ultrastructural morphology showed further degeneration. The myofibrils remained hypercontracted, but were more fragmented. Degenerative changes in mitochondria were more advanced and calcium deposition in cristae was present. No glycogen was seen. The right ventricular myocardium exhibited significantly fewer ultrastructural abnormalities. The principal right ventricular changes were endothelial swelling and basement membrane disruption. Glycogen granules were present. Ischemic contracture affects the left ventricle more than the right, and the morphology becomes more abnormal with time. To our knowledge, this is the first instance wherein morphologic progressions of the ultrastructural alterations of ischemic contracture have been documented.     

On the ultrastructure of specific heart granules in teleosts

The ultrastructure and chemical reactivity of teleostean specific ('atrial') heart granules (SHG) are described. In atrial cells of Glyptocephalus cynoglossus and Gadus morhua , large aggregations of SHG occur in areas of the sacroplasm rich in glycogen and poor in myofibrils. The SHG often appear to be in a process of lysis. A number of heart granules were also noted in atrial cells of Cichlasoma meeki, Pistella riddlei and Sebastes viviparus . although large aggregations were not seen. The mean diameters of SHG in G. cynoglossus and S. viviparus are 110 and 210 nm, respectively. SHG are regularly found in ventricular cells, although less frequently than in the atrium. Ultrahistochemical tests suggest that the teleostean SHG mainly contain proteins.     

Occurrence of unusual heterogeneous lipid-containing granule-storing cells in the main excretory duct epithelium of the male mouse submandibular gland

Summary The fine structure of the main excretory duct epithelium of the male mouse submandibular glands was investigated by scanning and transmission electron microscopy. Three principal cell-types were observed: type I and II, and basal cells. This epithelium was characterized by the presence of intercellular canaliculi. Type-I cells were the most numerous. They had an abundance of mitochondria, well-developed Golgi apparatus, a few electron-lucent lipid-containing granules and poorly developed basal infoldings. These cells were also characterized by many glycogen granules throughout the cytoplasm and abundant smooth endoplasmic reticulum in the apical cytoplasm. Type-II cells were the second most numerous. Their most characteristic feature was the presence of abundant heterogeneous lipid-containing granules having acid phosphatase activity at the periphery. They were concentrated in the infra- and supranuclear cytoplasm. The granules may be derived from mitochondrial transformation and seem to be a special kind of secondary autolysosome. Type-II cells also contained abundant mitochondria throughout the cytoplasm, much smooth endoplasmic reticulum in the apical cytoplasm, a well developed Golgi apparatus adjacent to the heterogeneous lipid-containing granules and no basal infoldings. Basal cells were situated adjacent to the basal lamina. They had a large nucleus and the cytoplasm was filled with glycogen granules.     

2D and 3D-organized cardiac cells shows differences in cellular morphology, adhesion junctions, presence of myofibrils and protein expression

Cardiac cells are organized in vivo in a complex tridimensional structural organization that is crucial for heart function. While in vitro studies can reveal details about cardiac cell biology, usually cells are grown on simplified two-dimensional (2D) environments. To address these differences, we established a cardiac cell culture composed of both 2D and three-dimensional (3D)-organized cells. Our results shows significant differences between the two culture contexts in relation to the overall morphology of the cells, contraction ability, proliferation rate, presence of intercellular adhesion structures, organization of myofibrils, mitochondria morphology, endoplasmic reticulum contents, cytoskeletal filaments and extracellular matrix distribution, and expression of markers of cardiac differentiation. Cardiac cells grown in 2D-context displayed a flattened and well spread shape, were mostly isolated and their cytoplasm was filled with a large network of microfilaments and microtubules. In contrast, 3D-cells were smaller in size, were always in close contact with each other with several cellular junctions, and displayed a less conspicuous cytoskeletal network. 3D-cells had more mitochondria and myofibrils and these cells contract spontaneously more often than 2D-cells. On the other hand, endoplasmic reticulum membranes were present in higher amounts in 2D-cells when compared to 3D-cells. The expression of desmin, cadherin and alpha-actinin was higher in 3D-aggregates compared to 2D-spread cells. These findings indicate that the tridimensional environment in which the cardiac cells are grown influence several aspects of cardiac differentiation, including cell adhesion, cell shape, myofibril assembly, mitochondria contents and protein expression. We suggest that the use of this cardiac culture model, with 2D and 3D-context cells, could be useful for studies on the effects of different drugs, or growth factors, giving valuable information on the biological response of cells grown in different spatial organizations.     

Ultrastructure of the ovary of Dermatobia hominis (Diptera: Cuterebridae). I. Development during the 3rd larval instar

The ultrastructure and distribution of gonial and somatic cells in the ovary of Dermatobia hominis was studied during the 3rd larval instar. In larvae weighing between 400 and 500 mg, the ovary is partially divided into basal and apical regions by oblong somatic cells that penetrate from the periphery; these cells show ovoid nucleus and cytoplasm full of microtubules. In both regions, gonial cells with regular outlines, large nucleus and low electron-density cytoplasm are scattered among the interstitial somatic cells. These later cells have small nucleus and electrondense cytoplasm. Clear somatic cells with small nucleus and cytoplasm of very low electron-density are restrict to the apical region of the gonad. Degenerating interstitial somatic cells are seen in the basal portion close to the ovary peduncle. During all this larval period the morphological features of the ovary remain almost the same. At the end of the period there is a gradual deposition of glycogen in the cytoplasm of the somatic cells, increase in the number and density of their mitochondria plus nuclear modification as membrane wrinkling and chromatin condensation in masses.     

Myocardial lysosomes in pressure-overload hypertrophy

Summary Combined electron microscopic and cytochemical studies were used to investigate the effects of chronic-pressure overload hypertrophy on myocardial lysosomes, mitochondria, and myofibrils in the left ventricle of the cat. Myocardial hypertrophy was induced by an 84% banding constriction of the ascending aorta. After one month of aortic constriction the experimental animals demonstrated a 51% increase in left ventricular mass. No qualitative ultrastructural differences were noted between the myocardial tissues of the hypertrophy and normal group. However, the cytochemical reaction product to acid phosphatase appeared more frequently in the myocardium of the hypertrophy group compared to that of the normal group. By use of quantitative morphometry the percentage of mitochondria, myofibrils and lysosomes per myocardial cell was determined in both hypertrophy and normal groups of animals. Despite significant increases in the left ventricular mass of hypertrophy animals, a normal balance of mitochondria and myofibrils was maintained within the myocardium. Further analysis indicated an enhanced lysosomal population in the hypertrophy group compared to the normal group.This research was supported by a grant from the California Affiliate of the American Heart Association     

东方杯叶吸虫卵黄腺和卵巢的超微结构研究

本文应用透射电镜观察了东方杯叶吸虫卵黄腺和卵巢的超微结构,并与体外培养成虫进行比较。根据形态特征和内含物的存在情况,将卵黄细胞和卵母细胞的发育均分为不同时期,详细描述了各期的形态特征,探讨了卵黄球和皮质颗粒等内含物的生理功能。体外培养成虫成熟卵黄细胞中有散在的卵黄物质,成熟卵母细胞中线粒体囊泡化,这些可作为体外培养的评价指标。     

Ultrastructure of the corpus luteum of antarctic seals during pregnancy

Summary The fine structure of granulosa lutein cells from three crabeater seals, Lobodon carcinophagus, and two leopard seals, Hydrurga leptonyx, has been studied from early through mid-pregnancy. Analysis of the arrangement and modifications of the cytoplasmic organelles and inclusions has revealed three types of lutein cells throughout the corpus. Type I cell typically possesses a central nucleus and cytoplasm containing very large amounts of smooth and/or fenestrated endoplasmic cisternae which frequently extend from the juxta-nuclear to the periphery of the cell. Type II cell contains a central or eccentric nucleus, moderate amounts of peripheral, smooth and fenestrated cisternae which often form large and concentric membranous whorls, numerous mitochondria and small lipid droplets. Frequently these cells show polarity in the arrangement of the cytoplasmic organelles and inclusions. Type III cell contains predominant large lipid droplets, many mitochondria, and small amounts of smooth and fenestrated cisternae. In light microscopy the type I cell is evenly granular, while the type III cell is highly vacuolated. Type II cells have both granular and vacuolated conditions. Ultrastructural features of type I and II cells suggest that they probably secrete most of the steroids, whereas the primary role of the type III cells appear to be lipid storage.This research was supported by National Science Foundation, Grant No. 1325 from the Office of Antarctic Biology.     

A light and electron microscopic study of he heart of a crayfish, Procambarus clarkii (Giraud). II. Fine structure

The adventitia of the crayfish heart is composed of cells that are separated from each other by an intercellular space about 280 Å wide. Desmosomes are present on apposing surfaces of adjacent cells. A basal lamina underlies the adventitia and consists of a dense, amorphous substance that contains numerous fine filaments. The myocardial cells are striated and an external lamina 0.1 μ thick is present on the surface of the plasma membrane. The nuclei and most of the cytoplasm, glycogen and mitochondria are located at the cell periphery. The myofibrils are composed of thick and thin filaments and confined to the core of the cell. A T system and a well-developed SR are present. Elements of these organelles form dyads at levels that correspond to the H bands, and triads at levels that correspond to the Z bands of the peripheral myofibrils. The relationship of the T tubules to the myofibrils is discussed. Locus cells exhibit a unique pattern of intracellular myofibrillar branching. They branch from a region which has a structure similar to the Z band material. The myofibrils radiate outwardly in various directions and form numerous cellular branches which form intercalated discs with adjacent myocardial cells. These discs are more complex than those observed in poikilothermic vertebrates but are simpler than those in mammals. An endocardium is lacking in the crayfish heart but interstitial cells are present in close association with the myocardial cells and neural elements. Terminal nerve processes deeply embedded in the myocardial cells are described.     

Organization of calsequestrin-positive sarcoplasmic reticulum in rat cardiomyocytes in culture

The sarcoplasmic reticulum (SR) regulates the levels of cytoplasmic free Ca²⁺ ions in muscle cells. Calsequestrin is a major Ca²⁺ -storing protein and is localized at special sites in the SR. To investigate the development of calsequestrin-positive SR and its interaction with the cytoskeleton, we examined the distribution of calsequestrin in cultured cardiomyocytes from newborn rats by immunofluorescence with anticalsequestrin and antitubulin antibodies and rhodamine-phalloidin. In frozen sections of neonatal rat heart, anticalsequestrin immunostaining was apparent as cross-striations at Z-lines. When newborn cardiomyocytes were isolated, calsequestrin-positive SR was disorganized and was apparent as small vesicles beneath the sarcolemma, whereas myofibrils accumulated in the center of the cells. As the cells spread in culture, calsequestrin-positive vesicles spread to the periphery of the cytoplasm, becoming associated with the developing myofibrils. In mature cells, calsequestrin was closely associated with myofibrils, showing cross-striations at the Z-lines. Double-labeling using anticalsequestrin and antitubulin antibodies demonstrated that the distribution of calsequestrin-positive structures was similar to that of the microtubular arrays. When the microtubules were depolymerized by nocodazole at an early stage, the extension of the SR to the cell periphery was inhibited. In mature cardiomyocytes, nocodazole appeared not to affect the distribution of the SR. These results indicate that the calsequestrin-positive SR in cardiomyocytes is organized at the proper sites of myofibrils during myofibrillogenesis and that the microtubules might serve as tracts for the transport of components of the SR. © 1994 Wiley-Liss, Inc.     

THE ULTRASTRUCTURE OF THE CAT MYOCARDIUM : I. Ventricular Papillary Muscle

The ultrastructure of cat papillary muscle was studied with respect to the organization of the contractile material, the structure of the organelles, and the cell junctions. The morphological changes during prolonged work in vitro and some effects of fixation were assessed. The myofilaments are associated in a single coherent bundle extending throughout the fiber cross-section. The absence of discrete "myofibrils" in well preserved cardiac muscle is emphasized. The abundant mitochondria confined in clefts among the myofilaments often have slender prolongations, possibly related to changes in their number or their distribution as energy sources within the contractile mass. The large T tubules that penetrate ventricular cardiac muscle fibers at successive I bands are arranged in rows and are lined with a layer of protein-polysaccharide. Longitudinal connections between T tubules are common. The simple plexiform sarcoplasmic reticulum is continuous across the Z lines, and no circumferential "Z tubules" were identified. Specialized contacts between the reticulum and the sarcolemma are established on the T tubules and the cell periphery via subsarcolemmal saccules or cisterns. At cell junctions, a 20 A gap can be demonstrated between the apposed membranes in those areas commonly interpreted as sites of membrane fusion. In papillary muscles worked in vitro without added substrate, there is a marked depletion of both glycogen and lipid. No morphological evidence for preferential use of glycogen was found.     

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.     

Quantitative studies on the ultrastructural differentiation and growth of mammalian cardiac muscle cells. The atria and ventricles of the cat

Ultrastructural differentiation of cardiac muscle cells in the bilateral atria and ventricles of the cat at 1, 16, 25 and 40 days and 6 months after birth was studied by morphometry on electron micrographs. At the newborn stage, no T-tubule was found in the ventricular muscle cells, but specific granules were already noted in the atrial myocytes. The cell diameter of the ventricular myocardium was greater than that of the atrium at this stage. The T-tubule was first recognized in the ventricular muscle cells at day 16, at which stage the area occupied by the mitochondria and glycogen in the atrial muscle cells was definitely found to differ from that in the ventricular muscle cells. Thereafter, the differences in the ultrastructure between the atria and ventricles became more remarkable, particularly in the cell diameter and in the mitochondrial area. The cat cardiac muscle cells are characterized by numerous lipid droplets within the cytoplasm in contrast to those of the rat and the guinea pig.     

The pedal muscle of the land snail Megalobulimus oblongus (Gastropoda, Pulmonata): an ultrastructure approach

M. Cristina Faccioni-Heuser, Denise M. Zancan, Christiane Q. Lopes and Matilde Achaval. 1999. The pedal muscle of the land snail Megalobulimus oblongus (Gastropoda, Pulmonata): an ultrastructure approach. — Acta Zoologica (Stockholm) 80: 325–337
The ultrastructure of the pedal muscle of the Megalobulimus oblongus is described. This muscle consists of transverse, longitudinal and oblique bundles ensheathed in collagenous tissue. Each muscle cell is also ensheathed by collagen. The smooth muscle cells contain thin and thick filaments; the thin filaments are attached to dense bodies. These cells contain a simple system of sarcoplasmic reticulum, subsarcolemmal caveolae and mitochondria with dense granules in the matrix, and glycogen. Three types of muscle cells were identified. Type A cells exhibited densely packed myofilaments, abundant glycogen rosettes, numerous mitochondria and sarcoplasmic reticulum profiles. Type B cells exhibited scanty glycogen and mitochondria, few cisternae of sarcoplasmic reticulum and large intermyofibrillar spaces. Type C cells exhibited intermediate characteristics between type A and type B cells. Neither nexus nor desmosomes were observed between the muscle cell membranes. The muscle contains well developed connective tissue and blood vessels. These structures and the distribution of muscle cells are probably involved in the muscular-hydrostat system. The muscle is richly innervated, having neuromuscular junctions with clear and electron-dense synaptic vesicles. The clear vesicles probably contain acetylcholine because the axons to which they are connected arise from acetylcholinesterase positive neurones of the pedal ganglion. The other vesicles may secrete monoamines such as serotonin and/or neuropeptides such as substance P.     

The fine structure of pigeon breast muscle

The pectoralis major muscle of the pigeon is composed of two types of muscle fibre. In the Type I fibres, the myofibrils are closely packed and there are few mitochondria. The myofibrils in the Type II fibres are separated by numerous columns of large mitochondria and lipid droplets. The membrane systems of the two types of fibre are similar. The triads occur at the Z-line; the sarcoplasmic reticulum is in the form of large terminal cisternae which are joined by narrow longitudinal tubules to a broad central cisterna. The value of morphological criteria in the classification of muscle fibres is discussed.     

Glycogen in the central neurons of insects: Massive aggregations induced by anoxia or axotomy

Large nerve cell bodies in the metathoracic ganglion of the cockroach, Periplaneta americana were examined for glycogen content. Periodic Acid Schiff (PAS) staining of frozen sections, together with electron microscopic evidence, confirmed the low glycogen content in normal neuron somata. Subjecting animals to a variety of stressful factors including axotomy and CO₂ or N₂ anaesthesia resulted in a rapid and substantial increase in neuronal glycogen. The glycogen appeared as large aggregates 1–3 μm in diameter closely associated with numerous mitochondria. These structures were principally restricted to the trophospongial regions of the nerve cell cytoplasm. Glycogen was rarely encountered in the trophospongial glia either in normal or experimental animals. The glycogen response, which persisted in large nerve cell bodies for at least 6.25 days, is discussed in relation to fluctuations in circulating trehalose resulting from the rapid mobilization of fat body glycogen.     

Timing and sequence of the events in the development of extraocular muscles in staged human embryos: ultrastructural and histochemical study.

The ultrastructure and the appearance of glycogen were studied in the extraocular muscles of 14 externally normal human embryos (Carnegie stages 13-21). At stage 16, myofibrils with an immature Z line and glycogen granules appeared in the cytoplasm of the myoblast. The myoblasts came into cluster at stage 18, and fusion between the myotubes was observed at stage 20. At this stage, an M line appeared in the myofibrils. At stage 21, an A band with a Z line and an H band with an M line were observed, the sarcoplasmic reticulum appeared in the cytoplasm of the muscle fibers and glycogen increased in volume in the cytoplasm. In the previous study, we showed that the muscle-specific isoenzymes, such as creatine kinase, beta-enolase and glycogen phosphorylase, appeared from stage 18 to 20 in the extraocular muscles. The previous findings and the present results suggest that the fusion of the muscle cells occurs in the period when some molecular markers of muscle differentiation are expressed in vivo.     

Internal defenses of the snail Biomphalaria glabrata.

We describe three distinct types of cells among Biomphalaria glabrata hemocytes: large cells with a tubulo-vesicular compartment, a component of the endocytic system, and with numerous mitochondria and large aggregates of glycogen particles; medium-size cells poor in organelles and glycogen; and small cells with organelles and few secretory granules. Other small hemocytes can be interpreted as juvenile cells. B. glabrata hemocytes contain few enzymes and do not show specific secretory granules, except for a subpopulation of large cells richer in acid phosphatase vesicles. Hemocytes have different aspects corresponding to different physiological states and their transitions: in quiescent hemocytes, the cell cortex is narrow and organelles are scattered in the cytoplasm, both in circulating cells characterized by thin-folded filopods and large macropinocytic vacuoles and in sedentary cells in which extended filopods connect to the extracellular matrix. In stress-activated hemocytes, the cortical region is thickened by polymerization of actin, and organelles are gathered around the nucleus. Fixed phagocytes are components of the connective tissue; the presence of numerous lysosomes and residual bodies and of acid phosphatase and peroxidase activities suggests a high phagocytic activity.     

Ultrastructural heterogeneity of the mitochondrial population in rat embryonic and induced pluripotent stem cells

Even though rats are popular model animals, the ultrastructure of their pluripotent cells, that is, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), remains unexplored, although fine structure of pluripotent stem cells of mice and humans and its changes during differentiation have been investigated well. In the present study, we carried out ultrastructural and morphometric analyses of three lines of rat ESCs and two lines of rat iPSCs. The rat pluripotent stem cells were found to have the main typical morphological features of pluripotent cells: large nuclei of irregular or nearly round shape, scanty cytoplasm with few membrane organelles, and a poorly developed Golgi apparatus and endoplasmic reticulum. The cytoplasm of the rat pluripotent cells contains clusters of glycogen, previously described in human ESCs. To identify possible differences between rat ESCs and iPSCs, we performed a morphometric analysis of cell parameters. The mean area of cells and nuclei, the nuclear/cytoplasmic ratio, distributions of glycogen and diversity of mitochondria showed marked variations among the lines of rat pluripotent stem cells and were more pronounced than variations between rat ESCs and iPSCs as separate types of pluripotent stem cells. We noted morphological heterogeneity of the mitochondrial population in the rat pluripotent stem cells. The cells contained three types of mitochondria differing in the structure of cristae and in matrix density, and our morphometric analysis revealed differences in cristae structure.