ASSEMBLY AND REMODELLING OF MYOFIBRILS AND … · Formation of intercalated discs in culture 235...

J. Cell Sci. 86, 233-248 (1986) 233Printed in Great Britain © The Company of Biologists Limited 1986

ASSEMBLY AND REMODELLING OF MYOFIBRILS

AND INTERCALATED DISCS IN CULTURED

NEONATAL RAT HEART CELLS

B. T. ATHERTON, D. M. MEYER ANDD. G. SIMPSONDepartment of Cell Biology and Anatomy, Northwestern University Medical School,303 E. Chicago Avenue, Chicago, Illinois 60611, USA

SUMMARY

The reorganization of myofibrils and the re-formation of intercalated discs was examined inneonatal rat cardiac muscle cells during the first 72 h of culture. Rhodamine phalloidin was used tomonitor the organizational state of the myofibrils and antibodies to desmoplakin and vinculin wereused as markers for the presence of desmosomes and fasciae adherentes, respectively. Tinypunctate desmosomes were observed between muscle cells after 24 h and apparently increased innumber and/or size between 24 and 48 h in culture. Fasciae adherentes were not detectable withantibodies to vinculin until after 48 h in culture. Well-defined sarcomeres were restored after 48 h inculture. Once formed the sarcomeric organization of the myofibrils was found to be stable providedthey were attached to the sarcolemma via intercalated discs. However, if the myofibrils attachedto regions of the membrane that lacked intercalated discs the sarcomeres appeared to break downgradually centripetally. When myofibrils attached to the membrane at the free edges of cells thatwere not in contact with other muscle cells, the striations stopped abruptly at a considerabledistance before the myofibril attached to the membrane. These non-striated terminals elongatedbetween 48 and 72 h and were associated with focal contacts that contained vinculin. Overall theresults suggest that cell-cell contact may be critical for the stabilization of normal myofibrillarstructure in the heart.

INTRODUCTION

The intercalated disc is a specialized region of the cardiac muscle cell membranecharacterized by a grouping of three specialized intercellular junctions, whichinclude the macula adherens, the fascia adherens and the gap junction (McNutt,1970). The intercalated disc is vital for the integrity and function of the heart. Forexample, gap junctions mediate the electrical coupling between heart muscle cells(Sheridan & Atkinson, 1985). The fascia adherens and the macula adherens arebelieved to mediate the adhesions between adjacent muscle cells and also provideattachment sites for cytoskeletal elements to the cell membrane (Severs, 1985).Little is known concerning the molecular constituents of the two adherens junctionsor what molecules mediate membrane-cytoskeletal interactions. However, currentevidence indicates that the macula adherens and fascia adherens are biochemicallyand functionally distinct junctions. The macula adherens contains desmoplakin(Mueller & Franke, 1983) in an electron-dense plaque on the cytoplasmic side of themembrane where intermediate filaments appear to attach to the membrane (Arnn &

Key words: intercalated disc, vinculin, desmoplakin.

234 B. T. Atherton, D. M. Meyer and D. G. Simpson

Staehelin, 1981). In contrast, the dense plaque of the fascia adherens containsvinculin and alpha-actinin, which may be involved in the insertion of the thinfilaments of the contractile apparatus into the membrane (Tokuyasu et al. 1981).

Enzymic dissociation of neonatal heart tissue not only separates the cells atintercalated discs but also disrupts the sarcomeric structure of the myofibrils, leavingthem in a state of disarray (Wollenberger, 1967; Fischmann & Moscona, 1971).Presumably the connections of the myofibrils to the membrane via the fasciaadherens are also cleaved. With time in culture intercalated discs re-form and themyofibrils reorganize (Legato, 1972; Perissel et al. 1980). However, the role ofvarious cytoskeletal elements in this reorganization has not been delineated.

In the studies reported here fluorescence microscopy was used to examine thedegree of organization of the myofibrils in large numbers of whole cells. Thereappearance of the fascia adherens and desmosome components of the intercalateddisc was examined using antibodies specific for vinculin and desmoplakin, whichserved as distinctive molecular markers for the two types of adherens junctions incardiac muscle (Geiger et al. 1985; Cowin et al. 1985). We followed the re-formationof these structures during the first 3 days of culture of neonatal rat heart cells in orderto evaluate their potential as an in vitro system in which to investigate the assemblyof intercalated discs. The results indicate that re-formation of desmosomes andfasciae adherentes follow different time courses and that the stability of myofibrils incultured cardiac muscle cells may depend in part on the establishment of cell-cellcontacts.

MATERIALS AND METHODS

MaterialsPancreatin and Dulbecco's modified Eagle's medium (DMEM) were obtained from GIBCO

Laboratories (Grand Island, NY). Rhodamine phalloidin was from Molecular Probes, Inc.(Junction City, OR), normal goat serum, goat anti-mouse and goat anti-rabbit secondary anti-bodies conjugated with fluorescein were obtained from Cooper Biomedical, Inc. (Malvern, PA).Mouse monoclonal antibody to chicken gizzard vinculin (Geiger, 1981) was obtained from MilesScientific (Naperville, IL). Rabbit antiserum to bovine desmoplakin I was a generous gift fromDr James Arnn, San Diego State University, San Diego, California (Arnn, 1983; Jones &Goldman, 1985).

Cell cultureHearts were removed from 3-day-old neonatal rats and prepared for tissue culture according to

the method of Decker et al. (1985) with modifications. Heart ventricles were minced and subjectedto 30-min cycles of exposure to 0-6mgml~' pancreatin. Cells released in the first two cycles werediscarded. In order to remove some of the non-muscle cells and enrich cell suspensions formyocytes, the freshly dissociated cells were incubated in tissue culture dishes for 2h at 37°C inDMEM containing 15 % horse serum and 5 % foetal bovine serum in an atmosphere of 95 % air,5% CO2. The myocyte-enriched cell suspension was seeded into 35 mm tissue culture dishescontaining glass coverslips at densities ranging from 2x 10s to 7x 105 cells per dish.

The degree of enrichment for muscle cells was assessed after 24 h in culture for three separatetissue dissociations by periodic acid-Schiff (PAS) staining (Pollinger, 1973). In this proceduremuscle cells stain with PAS by virtue of their glycogen content, while non-muscle cells are un-stained. Cell suspensions seeded into culture without modification were composed of 54-5 ± 3-9%myocytes. After enrichment for muscle cells by differential adhesion, cultures contained 72-8 ±

Formation of intercalated discs in culture 235

1-4% myocytes, whereas the other 27-2% were mostly fibroblasts and endothelial cells. However,the distinctive differences between myocytes and non-muscle cells in actin content and organization(see below) suggested that rhodamine phalloidin staining may be a useful alternative to the PASmethod for assessing the proportion of muscle cells in primary cultures when the cells are wellspread throughout the culture.

Indirect immunofluorescent stainingAfter 24, 48 and 72 h in culture cells attached to glass coverslips were stained for indirect

immunofluorescence. The cells were fixed with a 3-7% formaldehyde in Dulbecco's phosphate-buffered saline for lOmin at room temperature. The cells were then permeabilized by incubationfor 2min in each of the following: ice-cold 50% acetone-water, acetone at —20°C, 50% acetone-water and finally returned to phosphate-buffered saline. Rhodamine phalloidin and all antibodieswere used at a final dilution of 1:20 in phosphate-buffered saline containing lOmgmP1 bovineserum albumin. For double-label staining, rhodamine phalloidin and antibodies were each dilutedto 1:20 as a single mixture and incubated on coverslips for 30 min at 37°C in a moist chamber. Afterwashing in PBS, cells were incubated for 30 min at 37°C with goat anti-mouse or goat anti-rabbitimmunoglobulin G (IgG) secondary antibodies conjugated with fluorescein isothiocyanate diluted1:20. Finally, the coverslips were washed and mounted on glass slides in Gelvatol containinglOOmgml"1 of l,4-diazabicyclo-[2.2.2]-octane to retard fading of fluorescein. Replicate coverslipsstained with normal rabbit serum, or normal mouse serum in lieu of immune sera gave negativestaining reactions except for weak nuclear staining seen with all polyclonal sera used. The non-specific nuclear fluorescence was exaggerated in photographs where long exposure times wererequired (e.g. see Fig. 3). Monoclonal antibodies to desmoplakin I and II (Boehringer-Mannhein;Cowin et al. 1985) gave the same results as rabbit polyclonal antibodies to desmoplakin.

The slides were viewed with a Leitz Dialux 20 microscope equipped for epifluorescence. Forfluorescein viewing the L2.1 cube was used, which is a very narrow-band blue excitation filter withselective barrier at 525 nm. For rhodamine an N2 narrow-band green excitation filter was used,which excluded FITC excitation. The objective was a 63X/1.40 piano apochromatic phase-contrast lens. Photographs were taken with the Leitz Vario-orthomat camera system using KodakTri-X film developed with Diafine (Acufine Inc., Chicago, IL).

Focal contacts were observed with a Zeiss Photo III microscope equipped for interference reflec-tion using a 63X/1.25 Neofluar Antiflex lens. The cells were prepared for immunofluorescence asdescribed above except fixation in formaldehyde was for 30 min. Permeabilization of the cells withacetone or O'l % Triton X-100 for 4 min gave similar results. Glass coverslips with adherent cellswere mounted on slides by placing them on pieces of no. 1 coverglass and the gap filled with PBS.The edges were sealed with wax to prevent evaporation.

RESULTS

Primary cultures of heart cells from 3-day-old neonatal rats contained at least threedifferent types of cells that exhibited marked differences in their pattern of stainingwith rhodamine phalloidin (Fig. 1). Muscle cells exhibited a regular cross-bandingpattern after staining with phalloidin and in well-developed cells the sarcomeres weresilhouetted to reveal Z lines, A bands and I bands (Fig. 1A). In the non-musclecells the actin was disposed in various arrangements of non-striated actin cables(Fig. 1A,B). In fibroblasts stress fibres were often seen throughout the cytoplasm ofthe cell (Fig. 1A), while in endothelial cells actin cables were restricted to the cellperiphery and were arranged circumferentially (Fig. IB).

Cellular organization after 24 h in culture

Rhodamine phalloidin was used to monitor the organizational state of the myo-fibrils, and antibodies to vinculin and desmoplakin were used as markers to detect


Fig. 1. Contrast between the organization of actin in the three predominant cell typesfound in cultures of dissociated neonatal rat heart. Staining of 72 h cultures withrhodamine phalloidin. A. A single cardiac muscle cell sitting on a bed of heart fibroblasts.Note that Z lines A and I bands are highlighted in the myofibrils. B. Four presumptiveendothelial cells in the centre of the field. The actin cables in non-muscle cells are notstriated. Bar, lOjun.

the presence of fasciae adherentes and desmosomes, respectively. Twenty-four hoursafter seeding there was considerable heterogeneity in the degree of organization ofthe myofibrils in muscle cells (Fig. 2). The most common morphology observedat 24 h was regular arrangements of fluorescent bands or stripes of actin, whichprobably corresponded to developing I bands (Fig. 2A). The gaps between thebands appeared to be devoid of actin. Other myocytes apparently revealed the firstsigns of actin bridging the gaps (Fig. 2B). A smaller subset of cells contained variablenumbers of striated myofibrils subjacent to the sarcolemma (Fig. 2C). These mayrepresent cells in which the myofibrils were not completely disrupted during dis-sociation or which had reassembled their myofibrils more rapidly than the majorityof the cells. Still other cells contained actin arranged like beads on a string, whichlooked like myofibrils of very small diameter (Fig. 2D).

The first elements of the intercalated disc that developed within the first 24 h inculture were desmosomes and gap junctions. Although no probes specific for gapjunction proteins were used in this study, nexal junctions between muscle cells had

Fig. 2. Heterogeneity in the organization of the myofibrils in muscle cells 24 h afterseeding into culture. A-D. Stained with rhodamine phalloidin. A. The staining patterntypical of the majority of the cells. Note that gaps between parallel bands of fluorescenceappear to be devoid of actin. B. Sarcomeres appear to be assembling in central regionsof the cell. C. Representative of a small subpopulation of cells with well-developedmyofibrils adjacent to the sarcolemma, but absent from the deeper cytoplasm. D. Pre-sumptive myocyte with myofibrils of very small diameter following tortuous andbranching paths through the cytoplasm. Bar, 10 fun.


apparently formed since groups of muscle cells beat synchronously (De Mello,1982). Antibodies to desmoplakin stained very thin continuous or dotted lines offluorescence at sites of cell-cell contact (Fig. 3B). Vinculin, a component of thefascia adherens, was not detected at sites of cell-cell contact in 24 h cultures.

Changes in cellular organization between 24 h and 48h

There were marked changes in the staining of desmosomes and in the degreeof organization of the myofibrils between 24 and 48 h in culture. Well-definedsarcomeres were not present in the majority of cells at 24h (Figs 2A, 3A). Duringthe next day in culture, other elements of the sarcomere were inserted in the gapsbetween the bands of fluorescence as evidenced by the presence of A and I bandsvisualized with phalloidin staining in 48 h cultures (Fig. 3C). The banding patternof the sarcomeres in the cells shown in Fig. 3C (bracket) were comparable to thoseshown in Fig. 6A (bracket) when viewed at higher magnification. Clearly a furtherreorganization of the contractile proteins had taken place between 24 and 48 h inculture.

Changes in the staining of desmosomes with antibodies to desmoplakin were alsodramatic between 24 and 48h in culture. As shown in Fig. 3D, the staining ofdesmosomes had become markedly more intense by 48 h in culture. This mightsuggest an increase in the number and/or surface area of the junctions. In addition,spots of fluorescence were seen in the cytoplasm of the cells (Fig. 3D). The spots,also present in 24 h cultures, could represent desmosomes that had been internalizedafter tissue dissociation (Overton, 1968) or precursors for new desmosomes en routeto the cell surface (Jones & Goldman, 1985) or both. No further change in thestaining of desmosomes was detected between 48 and 72 h in culture. Desmoplakinwas detected only in muscle cells; non-muscle cells were not stained by this antibody.

While desmosomes were first detected after 24 h in culture, fasciae adherentes werenot detected by immunofluorescent staining with antibodies to vinculin until after48 h in culture (Fig. 4A,B). Sites of cell—cell contact were labelled with very finelines of fluorescence that were difficult to capture on film. Furthermore, there was nochange in the intensity of staining up to 72 h in culture.

When myofibrils terminated in the free edges of cells that were not in contact withother muscle cells the striations of the myofibrils stopped abruptly a considerabledistance before the myofibril attached to the sarcolemma (Fig. 4C). The non-striatedterminals appeared continuous with striated portions of the myofibril deeper in thecytoplasm. Furthermore, elongated membrane plaques that stained with antibodiesto vinculin were associated with the non-striated terminals of myofibrils but not withstriated regions of myofibrils (Fig. 4D). The vinculin plaques corresponded exactlyto the location and extent of non-striated terminations of myofibrils where theyattached to the membrane at the free edges of muscle cells (Fig. 4C,D). Interferencereflection combined with double-label immunofluorescent staining showed a clearassociation between non-striated myofibril terminals, vinculin plaques and areas offocal contact with the substratum (Fig. 5). The focal contacts in the myocytes werevery similar to those seen in fibroblasts (Izzard & Lochner, 1976).


Fig. 3. Changes in desmosomes and the degree of organization of the myofibrils between24 and 48 h in culture. A,C. Myocytes stained with rhodamine phalloidin 24 h and 48 hafter seeding into culture, respectively. Well-defined sarcomeres can be seen in themyofibrils after 48 h that are apparently lacking after 24 h in culture. Details in thebanding pattern of sarcomeres in the myofibrils marked with a bracket (C) are compar-able to those in a similar area in brackets in Fig. 6A, shown at higher magnification.B,D. Immunofluorescent staining of the same two fields of cells shown in A and C withantibodies to desmoplakin to visualize desmosomes. B. After 24 h in culture desmosomesare seen as very fine dotted lines of fluorescence at sites of cell-cell contact (arrowheads).D. The staining of desmosomes is markedly more intense in 48 h cultures (arrowheads).Tiny spots of fluorescence are also present in the cytoplasm of muscle cells in 24 h and48 h cultures. Bar,


Fig. 4. The distribution of vinculin in 48 h cultures of cardiac myocytes. A. Phase-contrast view of a group of three muscle cells. B. The same cells after immunofluorescentstaining with monoclonal antibodies to vinculin. Lines of fluorescence at sites of cell-cellcontact mark the presence of fasciae adherentes junctions (arrows). C,D. Double-labelstaining of a muscle cell with rhodamine phalloidin (C) and antibodies to vinculin (D).Note that the terminals of myofibrils are not striated and that they are associated withplaque-like deposits of vinculin where they attach to the sarcolemma (arrowheads).Bar, 10/im.

Fig. 5. Correspondence between non-striated myofibril terminals, vinculin plaques andfocal contacts. A,C. 48 h and 72 h cardiac myocytes, respectively, stained with rhodaminephalloidin. B,D. Same cells stained for immunofluorescence with antibodies to vinculin.Insets show the interference reflection images of selected regions of the two cells. Darkareas in interference reflection mark the location of focal contacts of the cells with thesubstratum. Arrowheads indicate some of the sites of correspondence between the threecell structures. All panels are at the same magnification. Bar, 10 fim.


In non-muscle cells vinculin was localized in tiny focal adhesion plaques at sites ofcell-to-substratum contacts (Geiger, 1979). In endothelial cells vinculin plaqueswere located along the cell border while in fibroblastic cells the plaques were oftenscattered all over the ventral cell surface in association with focal contacts seen byinterference reflection (unpublished observations).

Cellular organization after 72 h in culture

After 72 h, greater than 90% of the cells contained what appeared to be matureparallel myofibrils in register with one another (Fig. 6A). Thick transverse bands ofactin marked the location of sites of cell-cell contact. The bands corresponded withthe presence of intercalated discs as manifested by immunofluorescent staining withantibodies to desmoplakin (Fig. 7) and vinculin (not shown). The actin bands atsites of cell—cell contact were occasionally seen after 48 h in culture, but they werepresent in large numbers after 72 h in culture.

When the bands at sites of cell-cell contact were present, the myofibrils of eachcell were striated continuously right up to the intercalated disc on both sides of thejunctions (Figs 6A, 7A). At the opposite end of the cells where the same myofibrilsterminated at the free edge of the cell, they were not striated (Fig. 7A). In additionthe length of the non-striated terminal region of myofibrils was of the order of two tothree times longer at 72 h than those seen at 48 h (cf. Figs 4C, 6B). In 48 h cultures,some of the myofibrils were striated all the way to the end (Fig. 3C), while others hadnon-striated terminals up to 10-12/zm in length. After 72h in culture, non-striatedterminals approximately 20 fim in length were common and they ranged up to30 (Um long (Fig. 6B). These values were based on measurements of terminals whoseboundaries were clearly defined. Accurate measurements of the remaining terminalscould not be made because they overlapped and crossed over one another, makingit difficult to determine precisely where they ended. Nevertheless, examination ofhundreds of cells from several different preparations indicated that the increase inthe length of the non-striated terminals between 48 and 72 h was quite obvious evenwithout extensive quantitative measurements. They continued to be associated withmembrane deposits of vinculin along much of their length (Fig. 6C).

DISCUSSION

When neonatal rat heart was dissociated into single cells with proteolytic enzymes,intercellular junctions were cleaved and the sarcomeric organization of the myofibrilswas disrupted. During the first two days in culture, the cells gradually reassembledtheir myofibrils, aligned them into parallel arrays and regenerated intercalated discs.

In 24 h cultures, the predominant pattern observed after staining actin in musclecells with rhodamine phalloidin was parallel bands of fluorescence spaced approxi-mately 2/im apart. This is similar to the distance between Z lines in maturemyofibrils. The gaps between the bands appeared to be devoid of actin. This patternsuggested that the bands represented Z discs in the process of developing into Ibands in the early stages of the reassembly of sarcomeres. This interpretation is

Fig. 6. Myocytes 72 h after seeding into culture. A. Rhodamine phalloidin staining ofmuscle cells showing intercalated discs marked by thick transverse bands of actin(arrows). Note that the myofibrils are striated continuously where they attach to themembrane at intercalated discs (bracket). B. Myocytes stained with rhodamine phalloidinshowing very long non-striated terminals of myofibrils at free edges of the cells. Centrally,the myofibrils have retained their sarcomeric organization. C. The same field double-label-stained with antibodies to vinculin showing continued association of plaques withmyofibril terminals. Bar, 10/im.


Fig. 7. Double-label immunofluorescent staining of cardiac muscle cells after 72 h inculture. A. Rhodamine phalloidin. B. The same field stained with antibodies to desmo-plakin. The thick transverse bands of actin seen in A (arrows) correspond to the locationof desmosomes between cells seen in B. Bar,

supported by electron-microscopic observations of others. For example, Cedergren& Harary (1964) saw Z material with clusters of thin filaments emanating from themin opposite directions in cultured neonatal rat heart cells. Similar structures wereseen in vivo by Chacko (1973) in cardiac muscle cells in 10-day rat embryos. Suchstructures could give rise to the staining pattern observed with rhodamine phalloidinin the light microscope. Furthermore, double-label immunofluorescent staining ofcultured embryonic chick heart muscle with rhodamine phalloidin and antibodiesto alpha-actinin indicate that both label Z lines (unpublished observations). Inaddition, the former stains actin in the I bands of myofibrils.

Intercalated discs were assembled during the first 48 h in culture. Gap junctionspresumably had formed during the first 24 h in culture, since groups of muscle cellswere seen to beat synchronously. Desmosomes were present at 24 h, but fasciaeadherentes could not be detected by immunofluorescent staining with antibodies tovinculin until 48 h in culture. Electron microscopic observations of cultures similarto ours by Perissel et al. (1980) indicate that cell junctions morphologically con-sistent with fasciae adherentes had formed between muscle cells after 24 h in culture.Furthermore, they found that developing desmosomes were often quite smallcompared to fasciae adherentes in 24 h cultures. Our own electron-microscopic


observations corroborate their findings; all three types of junction were present in24h cultures (unpublished observations). These results combined with the fact thatvinculin was not detectable by immunofluorescent staining until after 48 h in culturesuggests that the fasciae adherentes present in 24 h cells were not biochemicallycomplete. The disparity between our immunofluorescence results and electron-microscopic data raises two important considerations in studies of the genesis ofspecialized cell junctions. First, although cell junctions may appear to be maturemorphologically, in fact they may not have completed their development at themolecular level. Second, more molecular markers which distinguish between junc-tions with similar morphologies are needed in order to identify unambiguouslyparticular junctions (Geiger et al. 1983), especially in developing systems.

The wide transverse bands of actin stained with rhodamine phalloidin cor-responded to intercalated discs at sites of contact between muscle cells. Thus,rhodamine phalloidin appears to be a useful and convenient probe with which tofollow the organizational state of the myofibrils of cultured rat cardiac cells and itmay also provide a marker for the presence of intercalated discs.

Reassembly of sarcomeres took place during the first 48 h in culture as manifestedby the gradual delineation of sarcomeres in the myofibrils. Beginning at 48 h, theterminals of some of the myofibrils were not striated at the free edges of cells thatwere not in contact with other muscle cells. The non-striated terminals elongatedbetween 48 and 72 h strongly suggesting that a gradual remodelling or disassembly ofsarcomeres was occurring centripedally (cf. Figs 4C, 6B). At the same time, thesarcomeric organization of the myofibrils remained stable at sites of cell-cell contactwhere intercalated discs had formed.

Non-striated terminal regions of myofibrils were observed in two previous studiesof cultured cells. One was an electron-microscopic study of amphibian myotome(Peng et al. 1981) and the other was an immunofluorescence study of embryonicchick heart cells (Dlugosz et al. 1984). In both studies the non-striated terminalswere interpreted to be regions of myofibril assembly. However, the possibility thatthe terminals represented instead sites of myofibril breakdown was not ruled out.The distribution of vinculin was not examined in these experiments and no changesin the length of the non-striated terminals were noted. It is possible that similarterminals in rat cells may provide a scaffold for the assembly of myofibrils underappropriate conditions. However, in our cultures of neonatal cells the thick, non-striated cables of actin appeared to elongate with time in culture, suggesting thatthey were being left in the wake of sarcomeric disassembly along myofibrils. Thus,sarcomeric assembly may be a reversible process, which somehow involves actincables continuous with the terminals of myofibrils.

In vivo the muscle cells in the heart make contact with other muscle cells viaintercalated discs and the myofibrils are striated all the way up to their terminals,where they attach to the membrane at fasciae adherentes junctions (McNutt, 1970).In the intact heart the distribution of vinculin is restricted to the fascia adherens(Tokuyasu et al. 1981) and rib-like deposits on the cytoplasmic side of the sarco-lemma in association with Z discs (Pardo et al. 1983). Precisely what role vinculin


plays in these membrane-cytoskeletal interactions is not known since recent dataindicate that vinculin does not bind actin (Wilkins & Lin, 1986). Therefore, it maybe only indirectly involved in the attachment of thin filaments to the cell membrane.

In culture, vinculin was found not only in intercalated discs but also in extra-junctional sites in association with actin in non-striated terminals of myofibrils wherethey attach to the cell membrane at the free edges of muscle cells. When myocyteswere unable to form junctional contacts with other muscle cells, they adapted byattaching the distal ends of myofibrils to membrane plaques that contained vinculin.The fact that the non-striated terminals of myofibrils were associated with focalcontacts that contained vinculin suggests a structural and functional homology withfocal adhesion contacts described in non-muscle cells such as fibroblasts. Actin cablesin fibroblasts attach to the cell membrane at focal contact sites that contain vinculin(Geiger, 1979), alpha-actinin (Lazarides & Burridge, 1975), talin (Burridge &Connell, 1983), and a number of other proteins (Mangeat & Burridge, 1984). Theplaques present in cultured rat heart myocytes have not been analysed for thepresence of talin, and the immunofluorescent localization of alpha-actinin was in-conclusive due to the poor cross-reactivity with rat tissue of antibodies to chickenalpha-actinin. It is noteworthy that vinculin and alpha-actinin have been shown tobe present in the fascia adherens in intercalated discs in vivo, but talin is not aconstituent of this structure (Geiger et al. 1985). Thus, the homology between thefascia adherens and focal adhesion contacts appears to be incomplete.

Although the membrane plaques and fascia adherens junctions appear to be struc-turally and functionally related membrane specializations, myofibrils in culturedcells that attached to the membrane via the fascia adherens appeared to be stable,while those that attached to the membrane at extrajunctional sites were not, asevidenced by their lack of sarcomeric organization. This suggests that fundamentaldifferences exist between the two types of myofibrillar attachment sites, which mayrelate to changes in the membrane associated with the establishment of cell contacts.Apparently, the plaques lack structures or constituents present at cell—cell contactsites that are necessary for the maintenance or stabilization of the sarcomericorganization of myofibrils in rat cardiac muscle. Cell contacts do not appear to benecessary for the initial reassembly of myofibrils, since isolated single cells containthem. However, our results do suggest that cell-cell contact may have a critical rolein stabilizing normal myofibrillar structure in cultured cardiac muscle cells. There-fore, it will be important to explore further structural and functional relationshipsbetween the two membrane specializations by extending this work to include ultra-structural and molecular analyses.

If our proposal that cell contact is important for the stability of myofibrils provestrue, then it has major implications concerning the mechanisms that may underliediseases affecting cardiac muscle, such as muscular dystrophy (Jasmin & Eu, 1979)and hypertrophic obstructive cardiomyopathy (Ferrans et al. 1972), where a break-down or disorganization of the myofibrils is associated with detachment of theintercalated discs or altered cell-cell interactions.


This project was supported in part by BRSG S07RR 05370-24 awarded by the BiomedicalResearch Support Grant program, Division of Research Resources, the National Institutes ofHealth and by the Chicago Heart Association. The authors thank Dr Robert S. Decker for readingthe manuscript and for the enthusiasm he expressed during the course of this work. We also thankDr James Arnn for generously providing us with antibodies to desmoplakin.

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{Received 16 June 1986 - Accepted 27 August 1986)

ASSEMBLY AND REMODELLING OF MYOFIBRILS AND … · Formation of intercalated discs in culture 235...

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