neurofilament proteins are co-expressed with desmin in ... · panel of five anti-nf antibodies,...

Neurofilament proteins are co-expressed with desmin in heart conduction

system myocytes

MAUEIZIO VTTADELLO, MICHELA MATTEOLI

C.N.R.-Istituto di Fisiologia dei Centri Nervosi, Milano, Italy

and LUISA GORZA*

C.N.R.-Unit for Muscle Biology and Physiopathology, Institute of General Pathology, University ofPadova, Padova, Italy

* Author for correspondence at: Institute of General Pathology, Department of Biology, via Trieste 75, 36132 Padova, Italy

Summary

We have recently shown that specialized myocytes ofthe rabbit heart express a cytoskeletal protein simi-lar to the M subunit of neurofilaments (NF). Since thisresult was obtained using a single anti-NF-M mono-clonal antibody, we tested on conduction myocytes apanel of five anti-NF antibodies, specific for each ofthe three NF subunits and for phosphorylated andnon-phosphorylated epitopes. Two antibodies, onespecific for the L subunit and one for phosphorylatedM subunit of NF, reacted with specialized myocytesin immunohistochemistry. In immunoblots on con-duction tissue homogenates the two antibodiesrecognized two polypeptides with electrophoreticmobility and solubility properties identical to thoseof NF-L and NF-M in the sciatic nerve.

The subcellular distribution of NF immunoreac-tivity in specialized myocytes was very similar todesmin localization; namely, it was distributed onlarge filamentous bundles and on fine filaments local-ized transversely at the level of the Z line. At theultrastructural level, immunoreactive filaments were

localized in the intermyofibrillar space and connec-ted myofibrils with mitochondria. Co-expression ofNF proteins and desmin was also observed in vitro ina minor population of cardiac myocytes culturedfrom embryonic rabbit heart. In most cases NFimmunoreactivity co-localized with desmin, es-pecially where filaments were well organized, but insome cells anti-NF and anti-desmin antibodieslabelled different filamentous structures.

These results indicate that NF proteins are struc-tural components of the cytoskeleton of specializedmyocytes and show a subcellular distribution verysimilar to desmin. Such a composition of intermedi-ate filaments indicates that in these cardiac cellsmuscle differentiation is compatible with the ex-pression of neuronal proteins.

Key words: heart conduction system, neurofilaments, desmin,cardiac myocyte culture.

Introduction

In the heart, a specialized system initiates and conductselectrical impulses for the precise sequential contractionof atria and ventricles. Electrical impulses are generatedby pacemaker cells at the sino-atrial (SA) node. Conduct-ing myocytes of the atrio-ventricular (AV) node, of the AVbundle and the peripheral Purkinje system, regulate andcoordinate the activation of the ordinary ventricular myo-cardium, by slowing or accelerating the diffusion of thestimuli (Carmeliet and Verecke, 1979). In most mammals,heart conduction system myocytes do not appear morpho-logically different from surrounding ordinary myocytes(De Haan, 1961). Nevertheless, specialized myocytes canbe distinguished from ordinary myocytes, since they ex-press unique myosin types, distinct from those found inadult and embryonic ordinary myocytes (Sartore et al.1978; Gonzalez-Saanchez and Bader, 1985; Gorza et al.1986,1988a). In addition, electron microscopy showed thatspecialized myocytes do not present transverse tubulesand contain short myofibrils, variably oriented and inter-Journal of Cell Science 97, 11-21 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

mingled with a huge amount of intermediate filaments(IFs) (see Sommer and Jennings, 1986, for a review).

Desmin, the muscle IF protein, is the major componentof EFs in specialized myocytes (Thornell and Eriksson,1981). Five differently phosphorylated forms of desminwere resolved on two-dimensional gels of bovine AVbundle myocytes, whereas only two isoforms were found inbovine ordinary myocardium (Kjorell et al. 1987). Thevariable degree of desmin phosphorylation observed inthese myocytes (Kjorell et al. 1987) might play an import-ant role on the organization and the turnover of desminfilaments (Geisler and Weber, 1988). In addition to that ofdesmin, vimentin immunoreactivity has been described inavian Purkinje system myocytes, but not in mammals(Thornell et al. 1986).

Using an immunological approach, we have recentlydemonstrated that conduction system myocytes of theadult and developing rabbit heart express a cytoskeletalprotein similar to NF-M (Gorza et al. 19886; Gorza andVitadello, 1989). Mammalian neurofilaments are com-posed of three different protein subunits with molecular

11

weights of about 68, 150 and 200 (xlO3) (NF-L, M and H)(Hoffman and Lasek, 1975; Liem et al. 1978; Schlaepferand Freeman, 1978). These proteins are the product ofdistinct genes (Czosnek et al. 1980), whose expression isdifferently regulated during neuronal development(Julien et al. 1986). All three proteins can be phosphoryl-ated at multiple sites (Julien and Mushinski, 1982) andthe extent of phosphorylation might play a role in theinteractions of NFs with other neuronal components(Carden et al. 1985, 1987; Lee et al. 1987; Sternberger andSternberger, 1983).

To rule out the possibility that in specialized myocytesthe reaction with a single anti-NF-M monoclonal antibodycould be due to non-specific cross-reaction and to comparethe NF subunit composition of cardiac conduction tissuewith that of nervous tissue, five antibodies directedagainst the three NF protein subunits were tested in thepresent study on rabbit heart conduction tissue. Wedemonstrate that two monoclonal antibodies, directedagainst NF-M and L, recognize two cytoskeletal proteinsin rabbit specialized myocytes, that are similar in molecu-lar weight and solubility to neuronal NF-M and NF-L,respectively. The subcellular distribution of NF proteinswas studied in vivo, at the light and at the electronmicroscope levels. In addition, we compared the expressionand the localization of NF proteins and desmin in primarycultures of rabbit embryonic hearts and under differentconditions.

Materials and methods

Tissue preparationsSciatic nerve and selected heart regions such as the SA noderegion, the AV node and bundle regions, and the free wall of theright ventricle were removed from adult New Zealand Whiterabbits and frozen in liquid nitrogen or fixed for immunoelectronmicroscopy. Tissue samples from AV bundle regions were fixedwith Methacarnoy and embedded in paraffin as previously de-scribed (Gorza and Vitadello, 1989).

Whole hearts were excised from embryos obtained from 11pregnant rabbits at different gestational ages (2 at embryonic day(E) 10; 8 at 11 days and one at E13) and were processed for tissuecultures.

AntibodiesSix monoclonal anti-NF antibodies were used in this study: iC8,which reacts with a non-phosphorylated epitope on NF-M (Vita-dello et al. 1986); 1G2, 2F8 and 4E5, which react with phosphoryl-ated epitopes on NF-M (1G2) and NF-H (2F8 and 4E5), respect-ively (Vitadello and Denis-Donini, 1990); N52, which recognizesboth phosphorylated and non-phosphorylated forms of NF-H(Shaw et al. 1986); and NR4, which reacts with a non-phosphoryl-ated epitope on NF-L (Debus et al. 1983); they were purchasedfrom Sigma (St Louis, MO).

Polyclonal anti-vimentin antibody V4630 was purchased fromSigma (St Louis, MO). Polyclonal (A 611) and monoclonal (cloneDE-B-5) anti-desmin antibodies were obtained from Dakopatts(Glostrup, Denmark) and from Boheringer-Manheim (FRG), re-spectively. The monoclonal antibody BF-49, specific for the myo-sin heavy-chain subunit, was used in double-labelling exper-iments, as previously described (Gorza et al. 19886).

Secondary rabbit anti-mouse IgG conjugated with peroxidaseand goat anti-rabbit IgG conjugated with FITC were from Dako-patts (Glostrup, Denmark); goat anti-mouse IgG conjugated withTRITC were from Cappel (West Chester, PA); FITC-conjugatedrabbit anti-goat IgG were from Sigma (St Louis, MO).

Light-microscopic immunohistochemistryParaffin sections (4 /an) or thick cryosectdons (10 /on) were used.

Sections were incubated with various dilutions of monoclonalantibodies (mAbs) iC8 (1:500 to 1:1000), 1G2 (1:400 to 1:800),NR4 (1:40 to 1:100), 2F8 and 4E5 (undiluted supernatants), anti-desmin (1:4) and polyclonal anti-vimentin (1:40) in phosphate-buffered saline (PBS) with 0.5% bovine serum albumin. Incu-bation was carried out in a humidified chamber at 37 °C for30 min. After rinsing in PBS for 30 min, sections were incubatedwith appropriated dilutions of secondary antibodies coupled withFITC, TRITC or peroxidase. Peroxidase was revealed by incu-bation with the substrate solution (Hanker-Yates Reagent, Poly-science Inc., in 50nm Tris-HCl, pH 7.6, containing 0.01 % hydro-gen peroxide), as previously described (Gorza et al. 1988a). Somesections of the SA node were incubated with 100 and 400/igml"1

alkaline phosphatase (type IS, Sigma), following the proceduresdescribed by Sternberger and Sternberger (1983), and thenprocessed for indirect immunofluorescence.

Controls were performed using pre-immune mouse and rabbitimmunoglobulins or hybridoma supernatant in the first step. AZeiss Photomicroscope HI and a Leitz Dialux microscope,equipped with epifluorescence optics, were used. Pictures weretaken with nford HP5 and Kodak Technical Pan Film and TMaxfilms.

Tissue culturesPrimary heart cultures were prepared as described by Dlugosz etal. (1984) with minor modifications. Briefly, hearts were removedand rinsed in Ca2+-free PBS. Cells were dissociated with 0.05 %trypsin (Gibco Labs, UK) in Ca2+- and Mg2+-free balanced saltsolution. Dissociated cells were placed in 25 ml of chilled nutrientmedium (10 % fetal calf serum, 2 mM glutamine in 199 Medium)(Flow Labs, UK) and centrifaged for 10 min at 200 g. Pelleted cellswere resuspended in nutrient medium and were grown on cover-slips precoated with gelatin in 24-well Linbro plates (Flow Labs,UK). Cells were plated at densities varying from 3.3X104 to27xl04cellml~1. Cultures were maintained in a humidifiedatmosphere containing 5% CO2 at 37 °C. The day after plating,nutrient medium was replaced with glutamine-free medium orwith serum-free medium (20 nM progesterone, 30nM sodiumselenite, lOO/jgml"1 transferrin, 100 /IM putrescine (Sigma),5/igml"1 insulin (Calbiochem) in 199 medium) (Bottenstein andSato, 1979) with or without lOOngmP1 7S NGF (Sigma). After 7days in vitro, some cultures were grown for 24 or 48 h in mediumcontaining 10~ 6 M colchicine (Colcemid, Sigma).

After 2, 4, 7, 9 and 11 days cultures were rinsed with chilledPBS and fixed with 95% acetone at -20°C for 10min. Doubleimmunofluorescence staining was performed using polyclonalrabbit anti-desmin antibody (1:100) and monoclonal mouse anti-NF antibodies. Briefly, cultures were incubated with a mixture ofanti-desmin and anti-NF antibodies for 30 min at 37 °C in ahumidified incubator. After several rinses in PBS, cultures wereincubated with secondary antibodies (goat anti-rabbit IgG conju-gated with FITC and goat anti-mouse IgG conjugated withTRITC) in two separate steps. Before use, anti-rabbit IgG wereabsorbed with non-immune mouse IgG and anti-mouse IgG withnon-immune rabbit IgG to eliminate any cross-reactivity.

Electron-microscopic immunocytochemistrySamples were fixed by treatment with 0.25 % glutaraldehyde/2 %paraformaldehyde in 0.1 M phosphate buffer, pH 7.2, for 60 min atroom temperature. Small tissue blocks were washed in 0 . 1 Mphosphate buffer overnight and then infiltrated in 2.3 M sucroseand frozen in Freon cooled with liquid nitrogen. Ultrathin frozensections were prepared with an Ultracut Microtome equippedwith an FC4 attachment (Reichert, Vienna) and were immunola-belled with a mixture of iC8 and 1G2 mAbs (1:1) at finalconcentrations of 2/igml"1 and 20mgml~1 followed by rabbitanti-mouse-colloidal gold (10 run in diameter, Sera-Labo) conju-gates as previously described (Keller et al. 1984; Matteoli et al.1988). Observations were performed with a Philips EM 300electron microscope.

ImmunoblottingTissue samples were homogenized (50mg wet weightml"1) in

12 M. Vitadello et al.

Laemmli (1970) sample buffer (3% SDS, 65 I M Tris-HCl,pH 6.8, 2 mM EDTA, 5 % 2-mercaptoethanol) heated for 5 min inboiling water and centrifuged for 15 min at 15000#. TritonX-100-insoluble cytoskeletal proteins from rabbit heart ordinarymyocardium and conduction tissue were prepared following theprocedure described by Eriksson and Thornell (1979), with minormodifications. Briefly, SA node, His bundle region and right atrialordinary myocardium were homogenized and stirred at 4 °C in alow ionic strength solution (60 min KC1, 20 mM imidazole, lmMcysteine, 2 mM EDTA and 10 mM ATP) with 0.2 % Triton X-100.After 2h the homogenate was centrifuged for 15 min at 15 000 gand the supernatant was discarded. Subsequently, the pellet wasresuspended in high ionic strength solution (0.6 M KC1, 20 mMimidazole, lmM cysteine, 2mM EDTA and 10mM ATP) andcentrifuged. This extraction procedure was repeated four times.The final pellet was solubilized in Laemmli buffer, heated andcentrifuged. Samples were run in 7.5% to 15% gradient poly-acrylamide gels and polypeptides were electrotransferred onnitrocellulose paper as described by Towbin et al. (1979). Blotswere incubated with 1G2 (1:500) and NR4 (1:100) ascites fluid.Bound antibodies were revealed by incubation with the substratesolution (3,3'-diaminobenzidine in 100 mM Tris-imidazole,pH7.6, containing 0.05% hydrogen peroxide).

Results

NF-M and NF-L subunits are expressed in heartconduction system myocytesImmunohistochemistry on serial cryosections of adultrabbit heart showed that 1G2 (anti-NF-M) and NR4 (anti-NF-L) reacted with conduction tissue myocytes of the SAnode, the AVnode, the AV bundle and Purkinje fibers with

a pattern identical to that previously described with iC8(anti-NF-M) (Gorza et al. 19886) (Fig. 1A-C). No reac-tivity was observed with ordinary atrial or ventricularmyocytes. Pretreatment of sections with alkaline phospha-tase abolished immunoreactivity with 1G2 (results notshown). Specialized cardiac myocytes as well as ordinaryones were labelled by anti-desmin antibodies (Fig. ID). Incontrast, specialized myocytes showed no immunoreac-tivity with antibodies directed against phosphorylated andnon-phosphorylated NF-H and vimentin (results notshown).

The antigens recognized by 1G2 and NE4 were ident-ified by Western blot analysis on homogenates and TritonX-100-insoluble cytoskeletal extracts from specializedmyocardial regions (Fig. 2). On homogenates from special-ized cardiac tissue 1G2 recognized a protein having thesame electrophoretic mobility as NF-M in the sciatic nerve(Fig. 2A). NK4 reacted with a polypeptide in conductingtissue homogenates with the same apparent electrophor-etic mobility as NF-L in the sciatic nerve (Fig. 2B). 1G2and NR4 immunoreactive proteins in specialized tissuemyocytes were insoluble in Triton X-100, as indicated bythe strong reaction observed on cytoskeletal extracts(Fig. 2, lanes 2, 7 and 8). No reactivity was observed onhomogenate (Fig. 2, lanes 4 and 9) and Triton-insolublefraction (not shown) of ordinary myocardium.

Subcellular localization of NF subunits in specializedmyocytesIn transversely cut sections of specialized myocytes, such

Wi W^^

Fig. 1. Serial cryosections of the AV bundle region of adult rabbit heart stained with anti-NF monoclonal antibodies (A-C) andanti-desmin antibodies (D) with indirect immunoperoxidase. The section includes portions of the AV bundle (left side of each section)and ordinary ventricular myocardium (u). AV bundle conduction tissue myocytes are labelled by anti-phosphorylated NF-M (1G2)(A), anti-NF-M (iC8) (B) and anti-NF-L (NR4) (C) antibodies, as well as by anti-desmin antibodies (D). Ordinary ventricular (v)myocytes, which are desmin-immunoreactive, are negative for neurofilaments. Bar, 54 fun.

Co-expression of NF proteins and desmin in myocytes 13

1 2 3 4 5 6 7 8 9

B

Fig. 2. A. Immunoblotting of anti-phosphorylated NF-M (1G2)on homogenate from rabbit sciatic nerve (lane 1); TritonX-100-insoluble fractions from SA node region (lane 2);homogenates from SA node region (lane 3) and right atrialordinary myocardium (lane 4). B. Immunoblotting of anti-NF-L(NR4) on homogenates from rabbit sciatic nerve (lane 5) andfrom SA node region Qane 6), Triton X-100-insoluble fractionsfrom SA node region (lane 7) and AV bundle region (lane 8);homogenate from right atrial ordinary myocardium (lane 9).Arrowheads indicate NF-M and NF-L in rabbit sciatic nerverecognized by anti-NF-M and anti-NF-L, respectively (lanes1—5; note that in this species anti-NF-L reacts weakly also withNF-M). In heart conduction tissue homogenates the twoantibodies reacted with polypeptides having identicalelectrophoretic mobility as neuronal NF subunits. A verystrong reaction with the same polypeptides was observed onTriton X-100 cytoskeletal extracts (lanes 2, 7, 8). No reactionwas observed on homogenates from ordinary myocardiumregions (lanes 4, 9) with both antibodies.

as Purkinje fibers (Fig. 3A), NF-L and NF-M immunoreac-tivity was localized in the center of the cytoplasm andaround myofibrils, with a distribution identical to that ofdesmin (Fig. 3B).

In longitudinal sections (Fig. 3C) NF-immunoreactivefilaments were observed in longitudinally orientedbundles localized beneath sarcolemma and in fine stripesdisplaying sarcomeric periodicity on myofibrils. Doubleimmunostaining experiments with anti-myosin heavychain and anti-NF antibodies showed that myofibrillaranti-NF immunoreactivity was localized at the level of theI band (not shown). Phase-contrast observations indicatedthat reactivity with anti-NF antibodies was located at theZ line (Fig. 3D and E).

Immunoelectron microscopy on ultrathin frozen sectionof the AV node with anti-NF antibodies showed that thelabelling was not as abundant as described for desmin(Tokuyasu et al. 1983). This finding should be not surpris-ing, since NF proteins in conduction myocytes are presentin a very small amount as indicated by immunoblotanalysis. Most gold particles were observed on filamentous

bundles localized in the intermyofibrillar spaces and con-necting myofibrils with mitochondria (Fig. 4). Sparselabelling was seen also on sarcomeres, but in this case itwas often due to the overlap of labelled filaments on thesectioned myofibril (arrows, Fig. 4A). In fact, on ultrathinfrozen sections morphology is not perfectly preserved andintermediate filament network especially may be des-troyed by surface tension, as described by Tokuyasu(1983).

In vitro expression of NF proteinsPrimary cultures of heart myocytes from rabbit embryoswere prepared for studying the influence of several con-ditions, such as the age of the donor, the density of cells attime of plating, the time in culture, and the effects of fetalserum and/or nerve growth factor on the expression ofneurofilament proteins M and L in cardiac myocytes.

In every condition studied, most cells stained for desminbut not for NF (Fig. 5). A small proportion of desmin-positive myocytes (about 1-5 %) was also labelled by anti-NF-L (not shown) and anti-NF-M (Fig. 5 A-D) antibodies.NF-immunoreactive myocytes were often clustered andgenerally smaller than myocytes decorated only by anti-desmin. The number of myocytes showing NF immuno-reactivity was not influenced by exposure to serum-freenutrient medium, or by the presence of NGF. Very rarecells displayed only NF-immunoreactivity and were nega-tive for desmin. These cells were usually small, polygonaland did not extend neurite-like processes, even whencultured for 11 days in the presence of NGF (Fig. 5 E-F).

Myocytes positive for anti-NF and anti-desmin anti-bodies showed that, in most cases, both antibodies labelledfilamentous bundles with the same intracellular distri-bution (Fig. 6). NF antibodies decorated a perinuclearwhorl of filaments from which a network of finer filamentsemanated; desmin-immunolabelling was observed on fila-mentous bundles localized around the nucleus, and also onfine filamentous structures localized at the very peripheralborder of the cell.

In myocytes with abundant myofibrillar network, anti-NF, as well as anti-desmin antibodies, also decorated finefilaments localized transversely with sarcomeric period-icity (Fig. 7 A and B). In rare myocytes different filamen-tous bundles were decorated only by anti-desmin or onlyby anti-NF antibodies (Fig. 7 C and D).

We tested whether NF-immunoreactive filaments incultured myocytes collapsed after addition of Colcemid tothe culture medium. We found that, after a 24- or 48-hexposure to the drug, the filaments reacting with anti-desmin and anti-NF disappeared and perinuclear masses,brightly stained by both antibodies, were observed: nodifference in the distribution of the labelling was detectedbetween anti-desmin and anti-NF antibodies in theseconditions (results not shown).

Discussion

The present results show that rabbit specialized heartmyocytes express two cytoskeletal proteins that displayimmunoreactivity, electrophoretic mobility in SDS-con-taining gels and solubility properties identical to NF-Mand NF-L in the sciatic nerve. Analogous results, indi-cating similarity between neuronal NF-M and an NF-M-like cytoskeletal protein expressed in conduction myo-cytes, have been described in a previous study, using themonoclonal anti-NF-M antibody iC8 (Gorza et al. 1988b).


Fig. 3. A and B. Serial transverse cryosections of AV bundle region from adult rabbit heart labelled in indirect immunofluorescencewith anti-NF-L (NR4) (A) and anti-desmin (B). Both antibodies stain AV bundle myocytes and labelling is mainly localized in thecenter of the cytoplasm. Myofibrils are unstained and appear as dark spots localized at the periphery of the myocyte and surroundedby labelled filaments. C. Longitudinal cryosection of a peripheral Purkinje fiber labelled in indirect immunofluorescence with anti-NF-L; transverse labelled stripes, with sarcomeric periodicity, can be observed. D. Longitudinal paraffin section of an AV bundlerabbit myocyte stained in indirect immunofluorescence with anti-NF-M (iC8). E. Phase-contrast view of the same field. Arrowheadsindicate transverse labelling of anti-NF corresponding to the Z line. Bars: A and B, 17/an; C, 12/an; D and E, 4.5/on.

In the same report, we also excluded the possibility thatthe reaction, which we observed on immunoblots, couldresult from contamination by cardiac nerves. In thepresent study, we used a new and different anti-NF-Mantibody (1G2) that recognizes a phosphorylated epitopeon neuronal NF-M (Vitadello and Donini, 1990). Theepitope recognized by this antibody on the cytoskeletalNF-M-like protein expressed in rabbit specialized myo-cytes is also phosphorylated, since immunoreactivity wasabolished by incubation with alkaline phosphatase. Thus,1G2 reacts with an epitope different from the one recog-nized by iC8, whose reactivity with NF-M is not affectedby alkaline phosphatase (Vitadello et al. 1986). Theseresults suggest that in rabbit specialized myocytes themiddle and the low molecular weight subunits of neuro-filaments are expressed.

To our knowledge, this is the first example of non-neuralexpression of NF-M and NF-L proteins in adult animals.Previous studies in chicken embryos showed a transientexpression of NF-M in the majority of cardiac myocytes(Bennett and Di Lullo, 1985). In contrast, in the rabbitembryonic heart, at any stage examined, the expression ofNF-M (Gorza and Vitadello, 1989) and NF-L (Gorza andVitadello, unpublished results) was restricted to a minor

population of myocytes showing the typical distribution ofthe conduction tissue. The same distribution was main-tained in myocytes of the adult heart. In vitro experimentsgave results consistent with in vivo observations: NFprotein expression was observed only in a minor pro-portion of embryonic cardiac myocytes and was notinfluenced by the presence of NGF and/or fetal calf serumin the medium.

Specialized heart myocytes did not react with mono-clonal antibodies that recognize phosphorylated and non-phosphorylated NF-H. Such a negative result does notnecessarily rule out the presence of NF-H in the special-ized myocytes, since the protein could be present in such alow amount that it was not revealed by anti-NF-H anti-bodies used in this study. Alternatively, NF subunitexpression in conducting myocytes might resemble the so-called 'immature' expression of NF protein subunits M andL, which has been described in developing neurons (Car-den et al. 1987; Pachter and Liem, 1984; Shaw and Weber,1982) and in adult cerebellar granule cells (Vitadello andDenis-Donini, 1990). However, in developing neurons theexpression of NF-L and NF-M subunits was not ac-companied by formation of filaments (Pachter and Liem,1984; Bignami et al. 1985). This does not seem to be the


Fig. 4. Electron micrographs of ultrathin cryosections of rabbit AV node myocytes immunostained with a mixture of anti-NF-Mantibodies, iC8 and 1G2, followed by 10 run colloidal gold-conjugated antibodies. A. Gold particles are observed on filamentousbundles in the intermyofibrillar space and are especially abundant around mitochondria. Arrows indicate a decorated filamentoverlapping a sarcomere. B. Higher magnification of decorated filamentous structures that appear to connect myofibrils tomitochondria. Bars: A, 231 nm; B, 267 run.

16 M. Vitadello et at.

Fig. 5. Double immunofluorescence labelling with anti-NF-M (iC8) (A, C and E) and polyclonal anti-desmin (B, D and F) of rabbitheart primary cultures. A and B were obtained from ElO embryos after 7 days of culture in presence of serum. C-F were obtainedfrom E l l embryos and cultured for 9 days in presence of NGF. NF- and desmin-immunoreactive myocytes are usually smaller thanNF-negative desmin-positive myocytes (A—D). Rare cardiac cells, that do not form neurites, are labelled by anti-NF, but not by anti-desmin (E and F). Bars: A-D, 28 /an; E-F, 18 /an.


Fig. 6. E l l (A and B) and E13 (C and D) rabbit heart primary cultures maintained in vitro for 9 days were double stained with anti-NF-M (iC8) (A and C) and polyclonal anti-desmin (B and D). Anti-NF and anti-desmin antibodies stain a perinuclear whorl offilaments from which finer filaments emanated; A and B show a cell in which filaments are equally well stained by both antibodies;in C and D only a portion of the cellular filamentous bundles labelled by anti-desmin are also labelled by anti-NF. Bar, 18 /an.


Fig. 7. E l l rabbit heart primary cultures maintained in vitro for 9 days. Double immunofluorescence staining with anti-NF-M (iC8)(A and C) and polyclonal antd-desmin (B and D). A. In addition to filamentous bundles, anti-NF antibody stains transverse stripes,which are also labelled by anti-desmin (B). C. Anti-NF labels strongly perinuclear bundles of filaments. D. Anti-desmin does notstain all filaments decorated by anti-NF in C; arrows indicate bundles of filaments reactive with anti-NF and apparently negativefor desmin. Bars: A and B, 9 /an; C and D, 18 fan.

case for specialized myocytes, where filaments decoratedby anti-NF antibodies were observed also at the ultra-structural level. This fact might imply that componentsnecessary for the assembly of NF proteins in filaments arepresent in conduction myocytes. Specialized, as well asordinary, cardiac myocytes express desmin, the muscle-specific IF protein. The results presented here show thatthe NF-M and NF-L immunoreactivity on filamentousbundles co-localized with desmin. In addition, the stainingwith anti-NF antibodies could be localized at the Z line,both in vivo and in vitro. By immunoelectron microscopy,anti-NF antibodies were also found to decorate filamen-tous structures localized in the intermyofibrillar space andsurrounding mitochondria. In cardiac myocytes, desmin isthe major constituent of a wide filamentous network thatfills the space between myofibrils and mitochondria andconnects adjacent Z disks (Tokuyasu etal. 1983; Tokuyasu,1983). These results suggest the possibility that NFproteins and desmin might form heteropolymers. It isusually accepted that no interactions occur among thedifferent types of IF proteins except for acidic and basic

cytokeratins (type I and type II IF proteins), which areobligate heteropolymers (for a review, see Steinert andRoop, 1988). However, the possibility that type HI IFs(namely, vimentin, desmin, glial fibrillary acidic proteinand 57 000 Mr IF) and type IV IFs (neurofilament tripletproteins) may co-assemble was recently suggested by thedemonstration that in fibroblasts transfected with cDNAsfor NF proteins, NF-L and -M subunits were localized inthe same filamentous structures in which endogenousvimentin was detected (Chin and Liem, 1989). Intermedi-ate filaments thus produced had solubility propertiesintermediate between those of vimentin and of NFs (Mon-teiro and Cleveland, 1989). On the other hand, the possi-bility of an interaction between desmin and NF proteins inspecialized myocytes does not exclude the possibility thatNF protein subunits can also be assembled in filamentsdistinct from those constituted by desmin, as we observedin some cells ire vitro. Since no other type HI IF proteins,either vimentin or the 57 000Mr IF protein (L.Gorza,unpublished observations), was detected in specializedmyocytes of the adult rabbit heart, other factors, such as


the amount of NT protein subunits, might play an import-ant role in conditioning filament formation (Chiu andGoldman, 1984).

Major problems arising concern the significance and thefunctions of NF protein expression in specialized myocar-dial cells. The expression of tissue-specific IF proteins hasbeen often used as a tool to determine the embryonic originof cells during development and for the diagnosis of poorlydifferentiated neoplasms (Osborn and Weber, 1983; Virta-nen et al. 1985). Recently, Kuruc and Franke (1988) havedescribed the expression of cytokeratins 8 and 18 incardiac myocytes during the early phases of development:the transient expression of these epithelial EF proteins hasbeen related to the origin of the myocardium from theprecardiac splanchnic mesoderm, namely from an epi-thelial cell layer. In this line, NF protein expression inheart conduction tissue cells might reveal a differentembryonic origin for these myocardial cells. Like mostneurons, specialized myocytes also display acetylcholin-esterase activity (Bojsen-Moller and Tranum-Jensen,1972) and the hyperpolarizing-activated current if (for areview, see Di Francesco, 1985). In a previous study, on thebasis of the concomitant expression of HNK-1 surfaceantigen, a marker for neural crest-derived cells, in thedeveloping specialized myocytes, we suggested that thesecells originate from the neuroectoderm (Gorza et al.19886). The finding that in vitro NF expression is restric-ted to a minor subpopulation of cardiac myocytes isconsistent with this interpretation. The rare NF-positiveand desmin-negative cells could correspond to precursorsof specialized myocytes in which desmin synthesis had notattained a sufficient level to be revealed by anti-desminantibodies. In fact, we can exclude the possibility thatthese cells are neurons, since they never snowed neurites,even when cultured for long times in the presence of NGF.

At variance with cytokeratins, which are only tran-siently expressed in cardiac myocytes and apparently donot form filaments (Kuruc and Franke, 1988), NF proteinsare expressed in specialized myocardial cells, persist intoadulthood and might not represent a simple vestigialexpression. In fact, NF proteins were not observed in someother neural crest derivatives, like Schwann cells andmelanocytes, while they are present in some other deriva-tives, like adrenal chromaffin cells (Trojanowski and Lee,1985). This fact suggests that NF protein expression inspecialized myocytes might be functionally relevant. Thestructure and the relative insolubility of NFs, like otherIFs, have been generally interpreted in the context of acytoskeletal function. Thus, NFs have been suggested tobe major determinants of axonal diameter (Hoffman et al.1987). Similarly, the abundant network of desmin ex-pressed in the specialized myocytes has been explained asa determinant of the structural integrity of these cells,favoring the distribution of mechanical stress duringheart beat. Such a mechanism might be relevant inreducing the effects of stretching on myocyte membranes(Thornell and Eriksson, 1981). It seems unlikely that inspecialized myocytes NF proteins perform the same func-tions as desmin. The unique IF composition of conductingmyocytes suggests that these proteins might have otherroles in addition to their structural ones. Thus, these cellsrepresent a very interesting model for investigating theexpression and interaction of neuronal and muscularcomponents.

We thank Professor S. Schiaffino and Dr S. Denis-Donini forhelpful discussion; Dr L. Parysek for the generous gift of the

57 000 Mr EF antiserum; Dr I. Mussini for useful advice; Mr A.Buso, L. Chiumiento, M. Fabbri and Mrs L. Aquino for theirskilful technical assistance.

This work was supported by grants from Consiglio Nazionaledelle Ricerche, C.N.R.-Gruppo Cardiorespiratorio (grant no.89.02608.04) and Ministero dell'Universita e della Ricerca Scien-tifica.

References

BENNETT, G. S. AND DI LULLO, C. (1985). Transient expression of aneurofilament protein by replicating neuroepithelial cells of theembryonic chick brain. Devi Biol. 107, 107-127.

BIGNAMI, A., GROSSI, M. AND DAHL, D. (1985). Transient expression ofneurofilament protein without filament formation in Purkinje cellsdevelopment. Immunohistological and electron microscopic study ofchicken cerebellum. Int. J. devl Neurosci. 3, 365-377.

BOJSEN-MOLLER, F. AND TRANUM-JENSKN, J. (1972). Rabbit heart nodaltissue, sinuatrial ring bundle and atrioventricular connexionsidentified as a neuromuscular system. J. Anat. 112, 367-382.

BOTTENSTEIN, J. E. AND SATO, G. H. (1979). Growth of a ratneuroblastoma cell line in serum-free supplemented medium. Proc.natn Acad. Sci. U.SA. 76, 514-517.

CARDEN, M. J., SCHLAEPFER, W. W. AND LEE, V. M-Y. (1985). Thestructure, biochemical properties and immunogenicity ofneurofilament peripheral regions are determined by phoaphorylationstate. J. biol. Chem. 260, 9805-9817.

CARDEN, M. J., TROJANOWSKI, J. Q., SCHLAEPFER, W. W. AND LEE, V. M-Y. (1987). Two-stage expression of neurofilament polypeptides duringrat neurogenesis with early establishment of adult phosphorylationpattern. J. Neurosci. 7, 3489-3504.

CARMELIET, E. AND VEERECKE, J. (1979). Electrogenesis of the actionpotential and automaticity. In Handbook of Physiology, Section 2: TheCardiovascular System (ed. R. M. Berne, N. SperelakiB and B. S.Geiger), pp. 269-334. Bethesda: American Physiological Society.

CHIN, S S . M AND LIEM, R. K. H. (1989). Expression of ratneurofilament proteins NF-L and NF-M in transfected non-neuronalcells. Eur. J. Cell Biol. SO, 475-490.

CHIU, F. C. AND GOLDMAN, J. E. (1984). Synthesis and turnover ofcytoskeletal proteins in cultured astrocytes. J. Neurochem. 42,166-174.

CZOSNEK, H., SOIFER, D. AND WISNIEWSKI, H. (1980). Studies on thebiosynthesis of neurofilament proteins. J. Cell Biol. 86, 726-734.

DEBUS, E., WEBER, K. AND OSBORN, M. (1983). Monoclonal antibodiesspecific for glial fibrillar acidic (GFA) protein and for each of theneurofllament triplet polypeptides. Differentiation 25, 193-203.

DE HAAN, R. L. (1961). Differentiation of the atrio-ventricular conductionsystem of the heart. Circulation 24, 458-470.

Di FRANCESCO, D. (1985). The cardiac hyperpolariring-activated currentif, origins and developments. Prog. Biophys. molec. Biol. 46, 163-183.

DLUGOSZ, A. A., PARKER, B. A., NACHMIAS, V. T. AND HOLTZER, H. (1984).The relationship between stress fiber-like structures and nascentmyofibrils in cultured cardiac myocytes. J. Cell Biol. 99, 2268-2278.

ERIKSSON, A. AND THORNELL, L. E. (1979). Intermediate (skeletin)filaments in heart Purkinje fibers. A correlative morphological andbiochemical identification with evidence of a cytoskeletal function. J.Cell Biol. 80, 231-247.

GEISLBR, N. AND WEBER, K. (1988). Phosphorylation of desmin in vitroinhibits formation of intermediate filaments; identification of threekinase A Bites in the aminoterminal head domain. EMBO J. 7, 15—20.

GONZALEZ-SAANCHEZ, A. AND BADER, D. (1985). Characterization of amyosin heavy chain in the conductive system of the adult anddeveloping chicken heart. J. Cell Biol. 100, 270-275.

GORZA, L , SAGGIN, L., SARTORE, S. AND AUSONI, S. (1988a). Anembryonic-like myosin heavy chain is transitorily expressed in the ratnodal conduction tissue. J. molec cell Cardiol 20, 931-941.

GORZA, L., SCHIAFFINO, S., THORNELL, L.-E. AND SARTORE, S. (1986).Myosin types and fiber types in cardiac muscle. LU. Nodal conductiontissue. J. Cell Biol. 102, 1758-1766.

GORZA, L , SCHIAFFINO, S. AND VITADELLO, M (19886). Heart conductionsystem: a neural crest derivative? Brain Res. 457, 360-366.

GORZA, L. AND VITADELLO, M. (1989). Distribution of conduction systemfibers in the developing and adult rabbit heart revealed by anantineuronlament antibody. Circ. Res. 65, 360-369.

HorFMANN, P. N., CLEVELAND, D. W., GRIFFIN, J. W., LANDES, P. W.,COWAN, N. J. AND PRICE, D. L. (1987). Neurofilament gene expression:a major determinant of axonal caliber. Proc. natn. Acad. Sci. U.S-A.84, 3472-3476.

HOFFMANN, P. N. AND LASEK, R. J. (1975). The slow component of axonaltransport Identification of major structural polypeptides of the axonsand their generality among mammalian neurons. J. Cell Biol. 66,351-366.


JULJBN, J. P., MEYBR, D., HURST, J. AND GROSVBLD, F. (1986). Cloningand developmental expression of the murine neurofilament genefamily. Molec. Brain Res. 1, 243-260.

JULIEN, J. P. AND MUSHYNSKI, W. E. (1982). Multiple phosphorylationsites in mammalian neurofilament polypeptides. J. Neurochem. 37,1579-1585.

KELLER, G. A., TOKUYASU, K. T., DUTTON, A. H. AND SINGER, S. J. (1984).An improved procedure for immuno-electronmicroscopy: ultrathinplastic embedding of immunolabelled ultrathin frozen sections. Proc.natn. Acad. Sci. U.SA. 81, 5744-5747.

KJORBLL, V., THORNELL, L -E., LEHTO, V.-P , VIRTANEN, I AND WHALKN,R. G. (1987). A comparative analysis of intermediate filament proteinsin bovine heart Purkinje fibers and gastric smooth muscle. Eur. J. CellBiol. 44, 68-78.

KURUC, N. AND FEANKE, W. W. (1988). Transient co-expression of desminand cytokeratins 8 and 18 in developing myocardial cells of somevertebrate species. Differentiation 38, 177-193.

LAEMMLI, U. K. (1970). Cleavage of structural protein during theassembly of the head of the bacteriophage T4. Nature 227, 680-685.

LKB, V. M.-Y., CARDEN, M. J., SCHLAEPFER, W. W. AND TROJANOWSKI, J.Q. (1987). Monoclonal antibodies distinguish several differentiallyphosphorylated states of the two largest rat neurofilament subunits(NF-H and NF-M) and demonstrate their existence in the normalnervous system of adult rats. J. Neurosci. 7, 3474-3488.

LIEM, R. K. H., YEN, S. H., SALOMON, G. D. AND SHBLANSKI, M. L. (1978).Intermediate filaments in nervous tissues. J. Cell Biol. 79, 637—645.

MATTEOLJ, M., HAIMANN, C, TORRI-TARBLU, F., POLAK, J. M.,CECCARELLI, B. AND DE CAMILLI, P. (1988). Differential effect of o>latrotoxin on exocytosis from small Bynaptic vesicles containingcalcitonin gene-related peptide at the frog neuromuscular junction.Proc. natn. Acad. Sci. U.SA. 86, 7366-7370.

MONTBIRO, M. J. AND CLEVELAND, D. W. (1989). Expression of NF-L andNF-M in fibroblasts reveals co-assembly of neurofilaments andvimentin subunits. J. Cell Biol. 108, 579-593.

OSBORN, M. AND WEBER, K. (1983). Tumor diagnosis by intermediatefilaments typing: a novel tool for surgical pathology. Lab. Invest 48,372-394.

PACHTER, J. S. AND LIEM, R. H. K. (1984). The differential appearance ofneurofilament triplet polypeptides in the developing rat optic nerve.Devi Biol. 103, 200-210

SARTORE, S., PIBROBON-BORMIOLI, S. AND SCHIAFFINO, S. (1978). Immuno-histochemical evidence for myosin polymorphism in the chicken heart.Nature 274, 82-83.

SCHLABPFKR, W. W AND FHEEMAN, L. A. (1978). Neurofllament proteinsof rat peripheral nerve and spinal cord. J. Cell Biol. 78, 663-662.

SHAW, G., OSBORN, M. AND WIBIR, K (1986) Reactivity of a panel of

neurofilament antibodies on phosphorylated and dephosphorylatedneurofilaments. Eur. J. Cell Biol. 42, 1-9.

SHAW, G. AND WEBER, K. (1982). Differential expression of neurofilamenttriplet proteins in brain development. Nature 328, 277-279.

SOMMER, J. R. AND JENNINGS, R. B. (1986). Ultrastructure of cardiacmuscle. In The Heart and Cardiovascular System (ed. H. A. Fozzard etal. ), pp. 61-100. New York: Raven Press.

STIINERT, P. M. AND ROOP, D. R. (1988). Molecular and cellular biology ofintermediate filaments. A. Rev. Biochem. 57, 593-625.

STERNBERGER, L. A. AND STERNBERGER, N. H. (1983). Monoclonalantibodies distinguish phosphorylated and non-phosphorylated formsof neurofilaments in situ. Proc. natn. Acad. Sci. U.SA. 80, 6126-6130.

THORNELL, L.-E. AND ERIKSSON, A. (1981). Filament systems in thePurkinje fibers of the heart. Am. J. Physiol. 10, H291-H305.

THORNELL, L.-E., ERIKSSON, A., JOHANSSON, B., KJORELL, U., FRANKS, W.W., VIRTANEN, I. AND LEHTO, V.-P. (1986). Intermediate filament andassociated proteins in heart Purkinje fibers: a membrane-myonbrilanchored cytoskeletal system. Ann. N. Y. Acad. Sci. 468, 213-240.

TOKUYASU, K. T. (1983). Visualization of longitudinally-orientedintermediate filaments in frozen sections of chicken cardiac muscle bya new staining method. J. Cell Biol. 97, 572-565.

TOKUYASU, K. T., DUTTON, A. H. AND SINGER, S. J. (1983).Immunoelectron microscopic study of desmin (skeletin) localizationand intermediate filament organization in chicken cardiac muscle. J.Cell Biol. 96, 1736-1742.

TOWBIN, H., STAEHILJN, TH. AND GORDON, J. (1979). Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulose sheets.Procedure and some applications. Proc. natn. Acad. Sci. U.SA. 76,4350-4364.

TROJANOWSKI, J. Q AND LEU, V. M-Y. (1985). Expression ofneurofilament antigens by normal and neoplastic human adrenalchromaffin cells. New Engl. J. Med. 313, 101-104.

VIRTANEN, I., MIETTINEN, M., LEHTO, V-P., KARINIEMI, A. L. ANDPAASIVUO, R. (1985). Diagnostic application of monoclonal antibodiesto intermediate fllamenta. Ann. N.Y. Acad. Sci. 465, 635-648.

VITADELLO, M. AND DENIS DONINI, S. (1990). Expression ofneurofilaments proteins in granule cells of the cerebellum. Brain Res.609, 47-54.

VITADELLO, M., TRIBAN, C, FABRIS, M., GORIO, A. AND SCHIAFTINO, S.(1986). Heterogeneity of rat neurofilament polypeptides revealed by amonoclonal antibody. J. Neurochem. 46, 665-670.

(.Received 20 March 1990 - Accepted 29 May 1990)


neurofilament proteins are co-expressed with desmin in ... · panel of five anti-nf antibodies,...

Documents