Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. I. Presence of immunofluorescent titin spots in premyofibril stages

Our initial attempts to immunolabel intact myocardial walls of 4-12 somite stage chick embryos were hindered by the presence of the cardiac jelly that covers the inner myocardial wall surface and prevents the access of antibodies to that surface. We overcame this difficulty by treating the specimens with hyaluronidase, which made the cardiac jelly permeable to the antibodies. An additional nonionic detergent treatment made the two or more cell layers of the myocardial wall accessible to the antibodies from both surfaces of the wall. Specimens treated in this manner were fluorescently labeled with antibodies to titin, myosin, or actin or with NBD-phallacidin for F-actin and examined as whole mount preparations or cut into semithin sections after resin embedding. These preparations and sections revealed that titin, a putative scaffolding protein of sarcomeres, is present in a punctate state and also in a diffuse form throughout the cytoplasm of cardiac myocytes in the premyofibril stages (4-7 somite stages) as well as in the early stages of myofibril formation. We interpreted the punctate and diffuse states to represent an aggregated state of several titin molecules and a dispersed state of individual titin molecules, respectively. In the 4-7 somite cardiac primodia, myosin and actin show only a uniform labeling throughout the cytoplasm of the myocytes. These observations are in contrast to a previous report that titin and myosin are tightly linked during in vitro skeletal myofibrillogenesis (Hill, C. S., S. Duran, Z. Ling, K. Weber, and H. Holtzer, 1986, J. Cell Biol., 103:2185-2196). In the 8-11 somite stage hearts, the number of individual titin spots rapidly reduces, while the number of myofibrils with periodically aligned titin spots increases, which strongly suggests that the titin spots are incorporated into the newly arising myofibrils. Titin spots were seen as doublets only after titin spots were incorporated into the first myofibrils. However, the fact that the distance between the components of the narrowest doublet was close to the resolution limit of the light microscope left open the possibility that undiscernible doublets of submicroscopic separations might exist in the premyofibril stages. The myosin labeling revealed the sarcomeric periodicity in an earlier stage of myofibril development than the F- actin labeling. In addition, we made two morphogenic observations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Organization of Actin Filaments and Zonula Adherens during Somitogenesis in the Chick Embryo

The organization of F-actin during somitogenesis in the chick embryo was studied by use of rhodamine-conjugated phalloidin and transmission electron microscopy (TEM). Separation of a somite from the segmental plate proceeded simultaneously with the organization of segmental plate cells into a hemispherical epithelial sheet whose open side was directed antero-laterally. At the same time, intense staining of F-actin appeared in the apical surface of the epithelial sheet. Observations by TEM showed that zonulae adherentes associated with many actin filaments increased in the apical region of cells being organized into an epithelial sheet while this junctional apparatus was only sparsely distributed in the segmental plate cells. The hemispherical sheet subsequently closed to form an epithelial vesicle, with increase in curvature of its apical surface, and narrowing of cellular apices. At the same time, the zonulae adherentes and actin filaments in the cellular apices further increased, and many cellular processes formed on the apical surface of the epithelial somites. These findings suggest that segmentation involves organization of zonulae adherentes and a contractile process caused by acin filaments anchored to the zonulae adherentes.     

The association of desmin with the developing myofibrils of cultured embryonic rat heart myocytes

The association of desmin, a 55,000-dalton intermediate-filament protein, with the developing cardiac myofibril was studied by immunocytochemical methods in primary cultured myocytes isolated from embyronic rat hearts at different ages. In the earliest contractile myocytes obtained from 10-day-old embryonic hearts, desmin exists as an extensive cytoskeletal network with little or no association with the myofibrils. As the heart develops the cytoskeletal desmin undergoes the myofibrils. Initially, the cytoskeletal desmin appears to outline the developing myofibril as short, discontinuous filaments. At intermediate stages of heart development, desmin filaments in 12- to 16-day-old embryonic myocytes continue to outline the forming myofibrils. Associated with these filaments are crossbridges and foci of desmin spaced at a frequency equal to that of the Z-line spacing. Desmin becomes progressively associated with the myofibril from the central region of the cell toward the cell margin. Desmin filaments at this stage begin to coalesce in the region of the intercalated disk. In the early neonatal heart, desmin of the Z lines becomes continuous across the sarcomere and appears to integrate the myofibrils into a unit. These observations suggest that desmin is not required in the early stages of mammalian heart development for the initial assembly of cardiac sarcomeres or the initiation of cardiac myofibrillar contractions. In later stages of mammalian heart development, desmin is found associated with the cardiac myofibrils in such a manner as to stably integrate these elements into the cytoplasm. Additionally, desmin, in the Z lines of the more mature myocytes appears to maintain the myofibrils in close registry to each other and to the intercalated disk.     

Ultrastructural and biochemical localization of N-RAP at the interface between myofibrils and intercalated disks in the mouse heart.

N-RAP is a recently discovered muscle-specific protein found at cardiac intercalated disks. Double immunogold labeling of mouse cardiac muscle reveals that vinculin is located immediately adjacent to the fascia adherens region of the intercalated disk membrane, while N-RAP extends approximately 100 nm further toward the interior of the cell. We partially purified cardiac intercalated disks using low- and high-salt extractions followed by density gradient centrifugation. Immunoblots show that this preparation is highly enriched in desmin and junctional proteins, including N-RAP, talin, vinculin, beta1-integrin, N-cadherin, and connexin 43. Electron microscopy and immunolabeling demonstrate that N-RAP and vinculin are associated with the large fragments of intercalated disks that are present in this preparation, which also contains numerous membrane vesicles. Detergent treatment of the partially purified intercalated disks removed the membrane vesicles and extracted vinculin and beta1-integrin. Further separation on a sucrose gradient removed residual actin and myosin and yielded a fraction morphologically similar to fasciae adherentes that was highly enriched in N-RAP, N-cadherin, connexin 43, talin, desmin, and alpha-actinin. The finding that N-RAP copurifies with detergent-extracted intercalated disk fragments even though beta-integrin and vinculin have been completely removed suggests that N-RAP association with the adherens junction region is mediated by the cadherin system. Consistent with this hypothesis, we found that recombinant N-RAP fragments bind alpha-actinin in a gel overlay assay. In addition, immunofluorescence shows that N-RAP remains bound at the ends of isolated, detergent-treated cardiac myofibrils. These results demonstrate that N-RAP remains tightly bound to myofibrils and fasciae adherentes during biochemical purification and may be a key constituent in the mechanical link between these two structures.     

Structure and protein composition of sites of papillary muscle attachment to chordae tendineae in avian hearts

Summary Most cardiac myocytes transmit force across fasciae adherentes, specialized sites of cell-cell adhesion. However, some cardiac myocytes in papillary muscle terminate on collagenous connective tissue in the chordae tendineae. These papillary myotendinous junctions (MTJs) are specialized for force transmission from myocytes to extracellular matrix. In the present study, we compared structural molecules at papillary MTJs to those at fasciae adherentes and skeletal MTJs. By using indirect immunofluorescence, we found that papillary MTJs more closely resemble skeletal MTJs in their molecular composition in that they are enriched in talin, vinculin, integrin, and fibronectin. Zeugmatin and -actinin, both components of fasciae adherentes, are absent from papillary MTJs. Although papillary MTJs and skeletal MTJs display strong similarities in structural protein composition, ultrastructural organization of the two junctions is different. Papillary MTJs display little folding of the junctional membrane and, according to morphological criteria, more closely resemble sites of thin filament-membrane association in smooth muscle than skeletal MTJs. Thus, papillary MTJs display a combination of structural characteristics described previously in skeletal and smooth muscles but exhibit few structural features observed previously in cardiac fasciae adherentes.     

Developmental changes in the subcellular localization of R-cadherin in chick retinal pigment epithelium

 It has been reported that in the chick embryonic retina, N-cadherin first appears at the very early stages and is subsequently substituted by R-cadherin at the middle to late stages of development. To examine the role of R-cadherin in the morphogenesis of chick retinal pigment epithelium (RPE), the distribution of this adhesion molecule was studied by immunofluorescence cytochemistry and immunoelectron microscopy from embryonic day (E) 6 to hatching. R-cadherin immunoreactivity was detected at E6, and was strongest at E12–13. During these stages, R-cadherin was expressed uniformly on the lateral plasma membranes of RPE cells in contact with each other. Thereafter, R-cadherin immunoreactivity was markedly decreased, with intense immunoreactivity restricted to zonulae adherentes in latero-apical regions at E16. R-cadherin immunoreactivity was no longer detectable in the newly hatched chick RPE, even though morphologically well developed zonulae adherentes were present in latero-apical regions. No immunoreactivity was detected on the apical side facing the neural retina or on the basal side facing the basal lamina at any stage of development. These findings indicate that R-cadherin plays an important role as a major cadherin subtype in the morphogenesis of chick embryo RPE, and is involved initially in non-specific cell-cell adhesions, and subsequently in the formation and maintenance of developing zonulae adherentes. Accepted: 11 April 1997     

Contractile activity and cell-cell contact regulate myofibrillar organization in cultured cardiac myocytes

Adult feline ventricular myocytes cultured on a laminin-coated substratum reestablish intercellular junctions, yet disassemble their myofibrils. Immunofluorescence microscopy reveals that these non- beating heart cells lack vinculin-positive focal adhesions; moreover, intercellular junctions are also devoid of vinculin. When these quiescent myocytes are stimulated to contract with the beta-adrenergic agonist, isoproterenol, extensive vinculin-positive focal adhesions and intercellular junctions emerge. If solitary myocytes are stimulated to beat, an elaborate series of vinculin-positive focal adhesions develop which appear to parallel the reassembly of myofibrils. In cultures where neighboring myocytes reestablish cell-cell contact, myofibrils appear to reassemble from the fascia adherens rather than focal contacts. Activation of beating is accompanied by a significant reduction in the rate of total and cytoskeletal protein synthesis; in fact, myofibrillar reassembly, redevelopment of focal adhesions and fascia adherens junctions require no protein synthesis for at least 24 h, implying the existence of an assembly competent pool of cytoskeletal proteins. Maturation of the fasciae adherens and the appearance of vinculin within Z-line/costameres, does require de novo synthesis of new cytoskeletal proteins. Changes in cytoskeletal protein turnover appear dependent on beta agonist-induced cAMP production, but myofibrillar reassembly is a cAMP-independent event. Such observations suggest that mechanical forces, in the guise of contractile activity, regulate vinculin distribution and myofibrillar order in cultured adult feline heart cells.     

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.     

Myogenesis and contraction in the early embryonic heart of the rainbow trout

Summary Myogenesis in the embryonic heart of the rainbow trout, Salmo galrdneri (Rich.), was investigated electron microscopically from the 29th to the 41st somite stage. Thick and thin myofilaments are formed simultaneously as well as precursors of Z-lines, to which the thin filaments are attached. The genesis of filaments takes place in the region around the intracellular yolk droplets. The first myofibrils appear by the 33rd somite stage, probably formed by a mechanism of self-assembly in which the binding sites of actin and myosin participate. A- and I-bands do not develop before the 38th somite stage. The contraction already begins during the 33rd somite stage in the middle of the tubular heart. Gradually, the peristaltic waves spread increasingly to other parts of the heart. In the 41st somite stage the entire heart is contractile and all myocytes contain myofibrils.     

Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. II. Generation of alpha-actinin dots within titin spots at the time of the first myofibril formation

In whole mount preparations of the 9 somite stage chick embryonic hearts that were immunofluorescently double labeled for titin and alpha- actinin, presumptive myofibrils were recognized as rows of several periodically aligned titin spots. Within these titin spots, smaller alpha-actinin dots were observed. These periodical arrangements of titin spots and alpha-actinin dots were not found in the 7 somite stage hearts. In wide myofibrils in the 10 somite stage hearts, the alpha- actinin dots and titin spots simultaneously became 'lines.' To study the ultrastructural features of the titin-positive regions in the 6-9 somite stage hearts, the thoracic portions of the embryos were immunofluorescently labeled for titin and embedded in resin. Ultrathin sections were mounted on electron microscopic grids and examined in immunofluorescence optics. The titin-positive regions thus identified were then examined in the electron microscope. No readily discernable specific ultrastructural features were found in titin-positive regions of the 6 somite stage cardiac primodia. Examination of the sections of the 9 somite stage hearts, on the other hand, revealed the occasional presence of small dense bodies, Z bodies, in the titin-positive regions. These observations strongly suggest that these Z bodies are the ultrastructural counterparts of the alpha-actinin dots seen by immunofluorescence optics and that they are formed nearly at the time of the formation of the first myofibrils. In some of the nascent myofibrils the Z bodies were found to be considerably narrower than the myofibrils, implying that the Z bodies are required not for the assembly of myofibrils per se but for their stabilization. Immunofluorescent labeling for titin and alpha-actinin revealed that the length of the shortest sarcomeres in the first myofibrils is approximately 1.5 micron, approximately the width of the A bands of mature myofibrils. The possibility that the A bands might define the initial length of nascent sarcomeres was indicated.     

Functional studies of the domains of talin

The protein talin has two domains of approximately 200 and 47 kD, which can be cleaved apart by a variety of proteases. To examine the function of these two structural domains of talin, we have digested purified talin with a calcium-dependent protease and separated the resulting fragments chromatographically. Both fragments were radioiodinated and used to probe Western blots of whole fibroblasts and chicken gizzard extracts. The large talin fragment bound to vinculin and metavinculin. The small fragment did not demonstrate any binding in this assay. The fragments were labeled fluorescently and microinjected into fibroblasts in tissue culture. The large talin fragment incorporated quickly into focal adhesions where it remained stable for at least 14 h. The small fragment associated with focal adhesions of fibroblasts but was also distributed diffusely in the cytoplasm and the nucleus. These experiments suggest that talin has at least two sites that contribute to its localization in focal adhesions. Intact talin microinjected into Madin-Darby bovine kidney epithelial cells localized to the focal adhesions but was excluded from the zonulae adherentes, despite the localization of vinculin to both of these sites. In contrast, the large talin fragment, when microinjected into these epithelial cells, incorporated into both focal adhesions and zonulae adherentes. The difference in localization between the large talin fragment and intact talin seems to be due to the removal of the small domain. This difference in localization suggests that talin binding sites in zonulae adherentes have limited accessibility.     

SYNTHETIC STRANDS OF CARDIAC MUSCLE : Formation and Ultrastructure

Spontaneously active bundles of cardiac muscle (synthetic strands) were prepared from isolated cells of 11–13-day old embryonic chick hearts which were disaggregated with trypsin. Linear orientation of the cells was obtained by plating them on agar-coated culture dishes in which either grooves were cut in the agar film or a thin line of palladium was deposited over the agar. The influence of cell-to-cell and cell-to-substrate interactions was observed with time lapse cinematography and the formation of the synthetic strand was shown to involve both random and guided cell movements, enlargement of aggregates by accretion and coalescence, and the compact linear arrangement of cells along paths of preferential adhesion. Electron microscope investigations of these strands showed that a dispersed population of heart cells organized into an inner core of muscle cells and an outer sheath of fibroblast-like cells. The muscle cells contained well-developed, but widely spaced myofibrils, a developing sarcoplasmic reticulum associated in part with the myofibrils and in part with the sarcolemma, an abundance of nonmembrane bound ribosomes and glycogen, and a prominent Golgi complex. Numerous specialized contacts were observed between the muscle cells in the strand, e.g., fasciae adherentes, desmosomes, and nexuses. A distinct type of muscle cell characterized by its pale appearance was regularly observed in the strand and was noted to be similar to Purkinje cells described in the adult avian conduction system and in developing chick myocardium. The present findings were compared with other observations of the developing myocardium, in situ, and it was concluded that, by a number or criteria, the muscle cells of the strand were differentiating normally and suitably organized for electrophysiological studies.     

Mechanism of oleic acid-induced myofibril disassembly in rat cardiomyocytes

This study investigated the mechanism of oleic acid (OA)-induced disassembly of myofibrils in cardiomyocytes. OA treatment disrupted myofibrils, as revealed by the disorganization of several sarcomeric proteins. Since focal adhesions (FAs) are implicated in myofibril assembly, we examined structural changes in FAs after OA treatment. Immunofluorescence studies with antibodies against FA proteins (vinculin, integrin beta1D, and paxillin) showed that FAs and costameres disintegrated or disappeared after OA treatment and that the changes in FA proteins occurred prior to myofibril disassembly. The effects of OA on FAs and myofibrils were reversed after removal of OA. OA decreased expression of integrin beta1D, paxillin, vinculin, and actin, and induced tyrosine dephosphorylation of FA kinase (FAK) and paxillin. These effects were blocked by pretreatment with sodium orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor. This inhibitor also prevented OA-induced myofibril disassembly, indicating the involvement of PTP in myofibril disassembly. Furthermore, OA increased protein levels of PTP-PEST. The upregulation of this phosphatase correlated with the tyrosine dephosphorylation of paxillin and FAK, which are targets for PTP-PEST. In addition, OA decreased RhoA activity and the phosphorylation of cofilin, a downstream target of RhoA. Cofilin dephosphorylation increased its actin-severing activity and led to the depolymerization of F-actin, which might provide another potential mechanism for OA-induced myofibril disassembly.     

The vinculin/sarcomeric-alpha-actinin/alpha-actin nexus in cultured cardiac myocytes

Experiments are described supporting the proposition that the assembly of stress fibers in non-muscle cells and the assembly of myofibrils in cardiac cells share conserved mechanisms. Double staining with a battery of labeled antibodies against membrane-associated proteins, myofibrillar proteins, and stress fiber proteins reveals the following: (a) dissociated, cultured cardiac myocytes reconstitute intercalated discs consisting of adherens junctions (AJs) and desmosomes at sites of cell-cell contact and sub-sarcolemmal adhesion plaques (SAPs) at sites of cell-substrate contact; (b) each AJ or SAP associates proximally with a striated myofibril, and conversely every striated myofibril is capped at either end by an AJ or a SAP; (C) the invariant association between a given myofibril and its SAP is especially prominent at the earliest stages of myofibrillogenesis; nascent myofibrils are capped by oppositely oriented SAPs; (d) the insertion of nascent myofibrils into AJs or into SAPs invariably involves vinculin, alpha-actin, and sarcomeric alpha-actinin (s-alpha-actinin); (e) AJs are positive for A-CAM but negative for talin and integrin; SAPs lack A-CAM but are positive for talin and integrin; (f) in cardiac cells all alpha-actinin-containing structures invariably are positive for the sarcomeric isoform, alpha-actin and related sarcomeric proteins; they lack non-s-alpha-actinin, gamma-actin, and caldesmon; (g) in fibroblasts all alpha-actinin-containing structures are positive for the non-sarcomeric isoform, gamma-actin, and related non-sarcomeric proteins, including caldesmon; and (h) myocytes differ from all other types of adherent cultured cells in that they do not assemble authentic stress fibers; instead they assemble stress fiber-like structures of linearly aligned I-Z-I-like complexes consisting exclusively of sarcomeric proteins.     

Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes

Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating sites for the assembly of new sarcomeres. How 1.7-2.0 microns nascent sarcomeres can be added distally during elongation while the tips of the myofibrils remain inserted into submembranous adhesion plaques is unknown.     

Polysome structure in sea urchin eggs and embryos: an electron microscopic analysis

Primary cultures of cardiac myocytes from newborn normal and genetically cardiomyopathic (strain UM-X7.1) hamsters were analyzed by electron microscopy and immunofluorescent staining for myosin, actin, tropomyosin, and alpha-actinin. Antibody staining of these contractile proteins demonstrates that both normal and cardiomyopathic (CM) myocytes contain prominent myofibrils after 3 days in culture, although the CM myofibrils are disarrayed and not aligned as those in normal cells. The disarray becomes even more pronounced in CM cells after 5 days in culture. The immunofluorescent staining patterns of individual myofibrils in normal and CM cells were similar for myosin, actin, and tropomyosin. However, alpha-actinin staining reveals that the CM myofibrils have abnormally wide and irregularly shaped Z bands. Electron microscopy confirms the irregular Z-band appearance as well as the myofibril disarray. Thus, CM cardiomyocytes clearly show an aberrant pattern of myofibril structure and organization in culture.     

The relationship between stress fiber-like structures and nascent myofibrils in cultured cardiac myocytes

The topographical relationship between stress fiber-like structures (SFLS) and nascent myofibrils was examined in cultured chick cardiac myocytes by immunofluorescence microscopy. Antibodies against muscle-specific light meromyosin (anti-LMM) and desmin were used to distinguish cardiac myocytes from fibroblastic cells. By various combinations of staining with rhodamine-labeled phalloidin, anti-LMM, and antibodies against chick brain myosin and smooth muscle alpha-actinin, we observed the following relationships between transitory SFLS and nascent and mature myofibrils: (a) more SFLS were present in immature than mature myocytes; (b) in immature myocytes a single fluorescent fiber would stain as a SFLS distally and as a striated myofibril proximally, towards the center of the cell; (c) in regions of a myocyte not yet penetrated by the elongating myofibrils, SFLS were abundant; and (d) in regions of a myocyte with numerous mature myofibrils, SFLS had totally disappeared. Spontaneously contracting striated myofibrils with definitive Z-band regions were present long before anti-desmin localized in the I-Z-band region and long before morphologically recognizable structures periodically link Z-bands to the sarcolemma. These results suggest a transient one-on-one relationship between individual SFLS and newly emerging individual nascent myofibrils. Based on these and other relevant data, a complex, multistage molecular model is presented for myofibrillar assembly and maturation. Lastly, it is of considerable theoretical interest to note that mature cardiac myocytes, like mature skeletal myotubes, lack readily detectable stress fibers.     

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.     

Photoreceptor fine structure in the southern fiddler ray (Trygonorhina fasciata).

The fine structure of the retinal photoreceptors has been studied by light and electron microscopy in the southern fiddler ray or guitarfish (Trygonorhina fasciata). The duplex retina of this species contains only rods and single cones in a ratio of about 40:1. No multiple receptors (double cones), no repeating pattern or mosaic of photoreceptors and no retinomotor movements of these photoreceptors were noted. The rods are cylindrical cells with inner and outer segments of the same diameter. Cones are shorter, stouter cells with a conical outer segment and a wider inner segment. Rod outer segment discs display several irregular incisures to give a scalloped outline to the discs while cone outer segment discs have only a single incisure. In all photoreceptors a non-motile cilium joins the inner and outer segments. The inner segment is the synthetic centre of photoreceptors and in this compartment is located an accumulation of mitochondria (the ellipsoid), profiles of both rough and smooth endoplasmic reticulum, prominent Golgi zones and frequent autophagic vacuoles. The nuclei of rods and cones have much the same chromatin pattern but cone nuclei are invariably located against or particularly through the external limiting membrane (ELM). Numerous Landolt's clubs which are ciliated dendrites of bipolar cells as well as Müller cell processes project through the ELM, which is composed of a series of zonulae adherentes between these cells and the photoreceptors. The synaptic region of both rods (spherules) and cones (pedicles) display both invaginated (ribbon) synapses and superficial (conventional) synapses with cones showing more sites than the rods.     

Phospholipid asymmetry during erythropoiesis. A study on Friend erythroleukemic cells and mouse reticulocytes

The distribution of phospholipids over the outer and inner layers of the plasma membranes of differentiated Friend erythroleukemic cells (Friend cells) and mouse reticulocytes has been determined. Phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol were found to be distributed symmetrically over both layers, sphingomyelin was found to be enriched in the outer layer (80-85%) and phosphatidylserine appeared to be present mainly in the inner layer (80-90%) of the plasma membranes of differentiated Friend cells. The outer layer of reticulocyte membranes contains 50-60% of the phosphatidylcholine, 20% of the phosphatidylethanolamine, 82-85% of the sphingomyelin and 40-42% of the phosphatidylinositol. All of the phosphatidylserine is present in the inner layer. The results show, that the asymmetric distribution of phospholipids, typical for erythrocyte membranes, is partially apparent already at an early stage of erythropoiesis, the proerythroblast, while the final organization of phospholipid distribution takes place at some stage during enucleation of the enormoblast and release of the reticulocyte into the blood stream.