Gen Pharmac Vol 23, No 2, pp 249-255, 1992 0306-3623/92 $5 00+000 Pnnted m Great Britain All rights reserved Copyright © 1992 Pergamon Press plc

ISOLATION OF PROTEOGLYCANS SYNTHESIZED BY RAT HEART: EVIDENCE FOR THE PRESENCE OF

SEVERAL DISTINCT FORMS

ROLANDO GONZALEZ, 2 RODRIGO URREA, 1 MAURICIO GONZALEZ, 1 NIBALDO C INESTROSA L2 and ENRIQUE BRANDAN l*

tMolecular Neuroblology Umt, Department of Cell and Molecular Biology, Faculty of Biological Soences and 2Department of Cardiology, Faculty of Medicine, Cathohc Umverslty of Chile, P.O Box 114-D,

Santiago, Chile [Fax 56-2-222-5515]

(Recezved 9 July 1991)

Abstract--1 The proteoglycans (Ps) synthesized by auricle and ventricle from adult rat heart were studied 2 Auricle tissue incorporated over two t~mes radioactive sulfate compared to ventricle t~ssue and the

Ps were mainly found m the detergent insoluble fraction 3 The Ps from both t~ssues were ~solated by ~on-exchange chromatography on DEAE-Sephacel,

followed by gel filtration on Sepharose CL-6B and SDS-PAGE electrophoresis 4 Enzymatic and chemical degradatmn of these Ps suggest that at least three and probably four different

speoes of Ps can be observed m heart t~ssue 4 A h~gh molecular weight chondromn sulfate-P, a h~gh molecular weight heparan sulfate-P, a

chondromn/dermatan sulfate-P of 240-200 kDa and a dermatan sulfate of 115 kDa 5 Th~s latter P was specifically ~mmunopreopttated using rat deconn anUserum

INTRODUCTION

Proteoglycans (Ps) are major components of the extracellular matrix (ECM) and comprise a large family m which they differ indwidually m their core protein or m the chemical nature of glycosammogly- can (GAG) side chains (Poole, 1986; Gallagher, 1986, Ruoslaht~, 1988). Special interest has been focused to the study of Ps present at the basal lamina which surround skeletal muscle cells, however those studies has been oriented to particular type(s) of Ps and there ~s not a clear wew of the total P population in muscle tissue. Thus, three major h~gh-buoyant-dens~ty chon- dromn sulfate Ps has been ~solated from embryomc chick skeletal muscle (Carnno and Caplan, 1982, 1984); a low molecular weight chondromn sulfate P has been Isolated from rabbit skeletal muscle (Parthasarathy and Tanzer, 1987) and two heparan sulfate P (Brandan and Inestrosa, 1987a) and a dermatan sulfate P, d~splaced by heparin have been isolated from rat skeletal muscle (Brandan and Inestrosa, 1987b)

Several observations suggest that Ps present at the basal lamina might play important functional roles Of specml interest is a P of heparan sulfate that is concentrated at the neuromuscular junction (Bayne et aL, 1984). The asymmetric form of acetylcholin- esterase, one of the major components of the neuro- muscular juncuon, is anchored to the basal lamina through an interaction mediated by an heparan sulfate P (Brandan and Inestrosa, 1984; Brandan et aL, 1985). Immunocytolocahzat~on experiments

*To whom all correspondence and reprint requests should be addressed

have shown that aggregates of acetylchohne receptors are associates with plaques of basement membranes heparan sulfate P on the surface of skeletal muscle fibers (Anderson and Fambrough, 1983).

There is httle reformation regarding the control of the expression of muscle Ps. It has been demonstrated that durang in wtro myogenesls of primary chick skeletal muscle, the synthesis of Ps is under develop- mental regulation (Miller et a l , 1987). In vitro culture experiments indicates that transforming growth factor B increases the expression of Ps re- leased to the medmm by a skeletal muscle cell line (Bassols and Massagu~, 1988) and recently we have shown that denervatlon of skeletal muscles influences their expression (Fadlc et a l , 1990) specifically a chondroltin/dermatan sulfate P known as deconn (Brandan et al., 1990). These observations are of specml interest because muscle tissue are composed of more than one cell type. Flbroblasts and other con- nectwe t~ssue cells are present between muscle fibers, and those cells might synthesize and/or influence the expression of Ps m multinucleated muscle fibers This heterologous influence ~s reinforced by the obser- vauon of Gross (1982), which indicated that dunng card~omyogenes~s zn vttro there is physical interacUon between fibroblast and myocyte.

Besides skeletal muscles, Ps have been ~solated and characterized from smooth-muscle cells and corre- spond to a large and small chondroltm sulfate type (Wight and Ross, 1985; Rauch et a l , 1986) Very httle is known regarding heart muscle P synthesis It ~s known that embryomc chick heart synthesize Ps wRh some structural s~milantles and d~fferences, to those made m skeletal muscle (Camno and Caplan, 1984) but no information is available w~th respect to the

249

250 ROLANDO GONZALEZ et al

Table 1 Incorporation of radlolabeled sulfate into ventricle and auricle isolated from rat heart

Auricle Ventricle CPM/G CPM/G A/V

TCA CPC TCA CPC TCA CPC

Experiment 1 714,220 420,000 285,640 171,280 2 5 2 5 Experiment 2 537,640 405,056 270,785 160,617 2 0 2 5 Experiment 3 354,433 233,514 131,142 113,927 2 7 2 1

Auricle and ventr, cle obtained from two rats prewously injected wRh [aSS]sulfate, were homogemzed as described m Matermls and Methods Ahquots of the different tissues were either precipitated with cold TCA or spotted on CPC-filters After processing of the samples the radmact~wty was deter- mined The results of three different experiments are presented

s y n t h e s i s o f P s by a d u l t h e a r t t i s sue T h e p u r p o s e o f th~s s t u d y w a s to e x a m i n e t he synthes~s o f P s p r e s e n t m the aur ic le a n d t he vent r ic le o f r a t h e a r t O u r r e su l t s ind ica te s t h a t the synthes~s o f P s is e n h a n c e d m aur ic le c o m p a r e d to v e n t n c u l a r t~ssue T h e p res - ence o f c h o n d r o m n , h e p a r a n a n d a d e r m a t a n su l fa t e P s~mllar to d e c o n n is d e m o n s t r a t e d a n d t h a t the level o f c h o n d r o l t m su l fa t e P d~ffer m e a c h t i s sue type

MATERIALS AND METHODS

Matertals

Benzamidme hydrochlonde, 6-ammohexanolc acid, N- ethylmaleimlde, guamdme hydrochlorlde, DEAE-Sephacel , Sepharose CL~SB, Sephadex G-75, Chondromnase ABC and AC and Protein A-Sepharose, were obtained from Sigma Chemicals C o , St Louis, M o , U S A [35S]-Na2SO4, carrier free was obtained from New England Nuclear, Boston, Mass, U S A Polyclonal rabbit antiserum against deconn punfied from rat skin fibroblast was a generous gift of Professor Hans Kresse (University of Munster , Fed Rep Germany) This ant iserum was produced by mjectlng deconn core protein purified from the secretmn of rat skin fibroblasts as described by Glossl et al (1984) Other reagents were obtained from commercml sources

Methods

Male rats (Sprague-Dawley) weighing 200-225 g were rejected lntrapentoneally with a total of 5 mCl of [35S]- sulfate in saline The hearts were removed 1, 6 or 18 hr after the injection

lsolatton of heart proteoglycans After labeling, the rats were killed and the heart removed The auricle and ventricle were separated and homogenized in 10raM Tns-HC1 buffer, pH 7 4, containing 5 m M benzamidme-HC1, 0 1 M 6-ammohexanolc acid, 0 1 m M N-ethylmaleimide and 0 5% (v/v) Triton X-100 and centrifuged in a Sorvall SS-34 rotor at 12,000g for 15 m m at 4°C The pellet was resuspended in the same homogenization medium and centrifuged again This last step was repeated once The supernatants were pooled and the labeled Ps present in the final pellet were extracted as described by Brandan and Inestrosa (1987a)

The detergent msoluble pellet was solubfltzed with 4 M guamdlne-HCl and 50 m M sodtum acetate, pH 5 8 contazn- mg proteases mhtbttors, for 12 hr at 4°C Unincorporated radtoacttve precursors, guamdme-HC1 and other salts were removed by dtalysts against 100 vol o f 4 M urea, 0 2 M NaC! and 5 0 m M sodmm acetate, pH 5 8, for 6 h r and then agamst 100 vol of the same solutton but containing 8 M urea, for another 6 hr After d m l y m the radioactive matenal was apphed to a DEAE-Sephacel column, 3 0 ml o f bed volume pre-eqmhbrated in the same 8 M urea buffer with the addition of 0 5% (v/v) Triton X-100 (Yanaglshlta and Hascall, 1984) After the sample application, the column was washed with 15 ml of the same buffer and then eluted with a continuous NaCI gradient, 0 2 to 1 1 M NaCI, dissolved m the same buffer (60 ml total vol) The column was eluted at a flow rate o f 3 5 ml/hr and fractions of 1 0 were collected The NaC1 gradient was momtored by measuring conductivity of the fractmns The labeled bound material was pooled and first dtalyzed for 12 hr against 10 m M Tns-HC1, 0 5% Triton X-100, 0 1 M NaCI contmn- mg proteases mhlbitors and prec~pitated by the addmon of 2 5 vol of cold ethanol The labeled-Ps were collected by cenmfugat ion and resuspended m 0 5 m l of 10mM Tns-HC1, 0 1 M NaCI

Fdtratwn chromatography Pooled fraction from the DEAE-Sephacel were fractionated on analytical Sephadex G-75 and Sepharose CL-6B prepared m 1% sodium dode- cyl sulfate (SDS), 0 1 M NaCI and 50 m M T n s - H C I buffer, pH 7 5 Samples (0 5 ml) were apphed to the columns to- gether with previously fracttonated blue Dextran (2000) and phenol red, to mark void and total volumes respectively Columns were runned at a flow rate o f 5 0 ml/hr and effluent fractions of 0 7-0 9 ml were collected and analyzed

Enzymatic treatments and chemwal analyses ChondroRt- nase ABC and AC treatments o f labeled-Ps was done exactly as described previously (Brandan and Inestrosa, 1987a) Degradation of heparan sulfate-P was done by nitrous acid degradation using reacuon "A" of Lmdahl et al (1973), except that the reaction was stopped by the addition of two vol of 2 M a m m o m u m sulfamate Acid precipitation was done by the addition of cold trichloroacetic acid (12% final), followed by washing and collection of the precipitates which were dissolved in I 0 N

Table 2 Distribution of sulfate labeled macromolecules in soluble and insoluble fractions isolated from rat auricle and ventricle

Auricle Ventricle (%) (%)

Muscle fraction TCA CPC TCA CPC

Soluble m detergent 20 3 + 3 6 16 3 + 2 7 36 0 + 7 6 35 8 + 8 9 Insoluble material 79 5 + 3 7 83 8 + 2 8 64 0 + 7 6 64 2 _+ 9 0

Auricle and ventricle from rat previously rejected with [3SS]sulfate were extracted three times m Tns-HCI buffer containing 0 5% Tmon X-100 The extracts were centnfuged m a Sorvall SS-34 rotor at 12,000g for 15 mm at 4°C Ahquots from the different samples were processed as described m Matertals and Methods Each determmaUon was carried out m tnphcate The number of expenments was 5 The values are expressed as percentage of the total radloactlwty recovered m the respecttve fractmn

Proteoglycans m heart tassue 251

NaOH pnor scmtdlatmn counting The presence of labeled- Ps was also quantified by binding to filter paper disc impregnated in cetylpyndmmm chlonde (CPC), as de- 300 scnbed prewously (Rapraeger and Bemfield, 1985, Brandan and Inestrosa, 1987a). 200

SDS-PAGE analysis of proteoglycans Samples eluted 100 from DEAE-Sephacel column were analyzed by electro- phorem on 3-10% polyacrylanude-dodecyl sulfate gradi- ents gels (SDS-PAGE) as prewously described (Carlson and 30o Wnght, 1987) and ttuorograplued

Immune prec~pUatwn ofdecorm Deconn was specifically ~ 200 tmmunopreclpltated with Protein A-Sepharose coated E with IgG anhbodles against rat deconn, from the mix- o Ioo ture of Ps present in the auricle- and ventrlcle-ECM, ,$ exactly as described by Glbssl et al (1984) The 300 lmmunoprecipltates were subjected to electrophoresis m cn SDS-PAGE as described above Control expenments were 2o0 done using the serum obtained from a non-immune New Zealand rabbit

RESULTS

Incorporation of [zaS] sulfate into heart tissue

Auricle and ventncle tissue from rats previously injected with [3SS]sulfate for 18 hr were homogemzed m T n s - T n t o n buffer and the presence of sulfate- labeled macromolecules evaluated by precapitatmn with cold T C A to detect sulfate transferred to macro- molecules or binding to CPC-filters to detect the

8!

o* 4

'o w "

K

E 2 =o

ttt~ "'it"

to) /...~//. ,'

(b)

/ f f

10 20

/ "

st S /

t t

~1 ...... I 3 0 4 0

Fraction number

0 8

0 6

O 4 ~

i.a

i

o o I I I

O6

0 4

02

Fig 1. Gradient chromatography on DEAE-Sephacel of rat heart Ps The Ps sample from rat heart auncle (top) and ventricle (bottom) obtained after guantdme solubihzauon were dmlyzed against 8 M urea, 0 2 M NaCI and 50 mM sodium acetate, pH 5 8, before being placed on a column. After a wash with the eqmhbratlng solution, a linear NaCI gradient (0.2-1.1 M) was apphed Fractmns of 1 0 ml were collected at a flow rate of 4 0 ml/hr Ahquots of each fraction were counted and the conductivity determined. The

dashed hne indicates the NaC1 gradient profle.

J

AuricLe VentricLe

Control VO ~o v~

Chondroltlnose ABC

V t

Chondroltlnor~ AC

Nitrous acid 3OO

A 0 5 10 15 0 5 10 15

Fraction number

Ftg 2 Sensitivity of rat heart Ps to mucopolysaccharidases and mtrous acid deammatmn Ahquots contmmng Ps from the DEAE-Sephacel were incubated voth the indicated enzymes or treated with nitrous acid The sensitivity to these treatments was deternuned by separation of digested prod- ucts from undigested material on Sephadex G-75 Left side,

auricle, nght side, ventricle

presence of sulfate transferred to Ps Table 1 indicates that auricle tissue incorporated more than twice of sulfate to macromolecules and Ps than ventricle. This h~gh incorporation was also seen if the t~ssues were processed 1 or 6 hr after the sulfate rejection (data not shown). To evaluate the distnbuUon of sulfate labeled macromolecules and Ps, the tissues were extracted three times with T n s - T n t o n buffer and the radioactivity evaluated in this fraction and in the detergent-insoluble one. Table 2 indicates that in auricle most of the labeled-macromoleculcs arc found in the insoluble fraction whereas m ventricle about one third of the labeled macromolecules were soluble m detergent

lsolatwn of proteoglycans from heart t~ssue by DEAE-Sephacel chromatography

To charactenze the Ps present m heart extra- cellular matrix, the insoluble fraction from auncle and ventncle were extracted separately with 4 M guanldme-HCl protease lnhlbLtors. Unincorporated radioactive sulfate was removed by dialysm against 8 M urea buffer as explained under Materials and Methods, and chromatograplued on DEAE-Sephace l m the presence of 0.5% Tn ton X-100. The eluuon patterns are shown m F~g 1. In both cases, more than 85% of the sulfated-molecules bound to the columns and a mean peak of radloactwe material eluted at 0 42 M NaC1

252 ROLANDO GONZALEZ et al

Glycosammoglyean composmon present tn heart pro- teoglyeans

The nature of the GAG composltmn of the Ps present m each tissue was determmed by treatment of the Ps with chondromnase ABC and AC as well as by mtrous acid degradation The sensmwty of the Ps from the mdlwdual tissues to the various treatments was assessed by separation of &gested products from undigested material on Sephadex G-75 F~gure 2 shows the chromatographic profiles for each case A quanuficatlon of these profiles indicated that the Ps from the auricle were 77% sensitive to chondromnase ABC but only 49% to chondromnase AC, suggesting the presence of chondromn sulfate (49%) and der- matan sulfate (28%) chains assocmted to the auricle Ps Only 10% was sensmve to nitrous actd degra- dation suggesting the presence of low amounts of heparan sulfate On the other hand, the chondromn sulfate present m ventricle was lower (32%) and a s~mllar amount of dermatan sulfate was also found (24%), m this tissue also a small proporUon of the sulfated Ps were sensmve to mtrous aod degradauon (24%) Thus, both heart tissues contain a slgmficant amount of chondromn sulfate Ps together with lower amount of dermatan and heparan sulfate Ps

Analysts of the proteoglycans from DEAE-Sephacel

In order to determine the relatwe sizes of the Ps present m each tissue, the materml bound to the DEAE-Sephacel was pooled, concentrated and chro- matographled on a Sepharose CL~SB column Figure 3 indicates that the eluUon profile was slmdar

Auric le Vent, r )c le

ZOO

200,

100

3O0

20O

I 0 0

Control

Chondrolt lnose AC

o o'2o'~o'6o~i 'o o o'2o'4o'6o'81"o KAv KAy

Fig 3 Sepharose CL-6B profile of Ps extracted from rat heart and their sensitivity towards chondroltmase AC Ahquots of rat heart Ps from DEAE-Sephacel column were incubated with and without chondromnase AC and applied to a Sepharose CL-6B column (0 7 x 100cm), which was equlhbrated and eluted with 1% SDS, 0 1 M NaCl and 50 mM Tris-HC1 buffer, pH 7 5, at a flow rate of 5 0 ml/hr Ahquots from each fraction was analyzed for ra&oactlvlty

Left side, auricle, right side, ventricle

'1 2 3

'180 - -

~ .,,,m---CSID$

,1~16

8 4 - -

DS

5 8 - -

Fig 4 Fluorogram of 3-10% SDS-PAGE of[35S]Ps isolated from rat heart Ps isolated from ventricle were purified by DEAE-Sephacel chromatography and fracUonated by SDS-PAGE Lane 1, un&gested sample Lane 2, &gested with chondromnase ABC Lane 3, &gested with chondroltl- nase AC The molecular weight standard corresponds to ct2-macroglobuhn (180kDa), fl-galactosldase (ll6kDa), fructose-6-phosphate kmase (84 kDa) and pyruvate kmase (58 kDa) CS, chondromn sulfate P, HS, heparan sulfate P,

DS, dermatan sulfate-P

for both tissues Three peaks were found in each case which eluted with a Kav of 007, 02 and 035 respectwely When the material was incubated with chondromnase AC, about 50% and 25% of the radloactwe material from the auricle and ventricle were &splaced to the Vt of the column, consistent w~th the prewous results (Fig 2) The &splacement was accompamed with a complete &sappearance of the peak which eluted w~th a Kav of 0.07, suggesting that the heart chondroltln sulfate-P correspond to the high molecular weight species The figure also shows that the Ps w~th Kav of 0 2 and 0 35 were resistant to the treatment

To wsuahze the &fferent Ps present m heart tissue an ahquot of ventricle material obtained after DEAE-Sephacel chromatography was fractlonated on SDS-PAGE as shown m F~g 4 At least three species of &fferent sizes can be &stmgmshed one with a broad high molecular weight, a 240-200 kDa and a l l 5kDa Chondromnase ABC treatment removed the label associated with the 240-200 and 115 kDa species and only partially to the high molecular weight band On the other hand chon- droltmase AC treatment partmlly ehmmates the

Proteoglycans

radioactwe sulfate assocmted to the bagh molecular weight P These results confirm the presence of at least three classes of different Ps assocmted with the heart ussue. A high molecular weight chondrottm sulfate P, a high molecular wetght heparan sulfate-P, a chondroltin/dermatan sulfate-P of 24(k-200 kDa and a dermatan sulfate-P of 115 kDa

Aurtc le and ventricle syn thes t ze a P s very s imilar to decorln

To further charactenze the Ps synthesized by rat heart, samples containing all the Ps isolated from auricle and ventricle were incubated with an anti- serum agamst rat decorm, a chondromn/dermatan sulfate P Figure 5 shows that a P of 115 kDa was specifically immunopreclpltated from both Ussues. Ttus result indicates that the chondroltln/dermatan sulfate synthesized by rat heart corresponds to deconn

DISCUSSION

This is the first report on the different ECM-Ps synthesized by adult rat heart Prewous studies have examined specific populations of Ps present e~ther at skeletal muscles (Brandan and Inestrosa, 1987a, b, Parthasarathy and Tanzer, 1987) or dunng develop- ment or d~fferentiatlon or myogenes~s (Carrino and Caplan, 1982, Carrlno and Caplan, 1984, Noonan et al , 1986; Mdler et al , 1987, Brandan et al , 1991). Our study extends these observations and describes the dlstnbution of newly synthesized Ps m rat heart tissue. The main d]fference between the population of Ps present in auricle and ventricle resides m the

in heart tissue 253

presence of a higher amount of chondromn sulfate-P m the former, however the hydrodynamics behavior on Sepharose columns suggests that the same species are present in both tissues.

An unexpected result was the fact that auntie ussue incorporated more than twice the amount of radioactwe sulfate to Ps compared to ventricular tissue (Table 1) This increase was observed in am- mals injected with radioactive sulfate for 1, 6 or 18 hr, suggesting that the auricle has a higher rate of Ps synthes~s than the ventricle. Very httle mformatmn ~s avadable about catabohsm of Ps (Morales and Roberts, 1988, Fadlc et a l , 1990) and it would be interesting to evaluate the rate of degradatmn of Ps m these tissues

Analys~s of rat heart Ps indicates the presence of several different species. They consist of a large P with chondroitm sulfate chains and a large heparan sulfate P, a chondromn/dermatan sulfate-P of 240-200 kDa and a dermatan sulfate-P of 115 kDa. Prewously we have described the presence of two heparan sulfate-Ps in the ECM from skeletal muscle which present a molecular weight slmdar to the one described m this paper (Brandan and Inestrosa, 1987a) The relatwe proportmn for the beparan sulfate-P m skeletal muscle Ussue is around 20-30%, a comparable value was found for the Ps isolated from ventricle and the auricle. Functional roles have been postulated for the heparan sulfate-P at the mature neuromuscular june- tmn, where they co-locahze with the acetylchohne receptors (Anderson and Fambrough, 1984) or anchor the asyrnmetnc acetylchohnesterase form (Brandan et al., 1985) Recently it has been shown that the presence of asymmetric acetylchohnesterase

1 2 3 4

1 8 0 - -

1 1 6 - -

8 4 - -

5 8 ~

4 8 ~

3 6 ~

2 7 ~

Fig 5 A P of 115 kDa Is specifically ]mmunopreopltated from a mixture of total Ps with an anUserum against rat deconn Samples containing total Ps population from auricle (1 and 2) and ventnde (3 and 4) were incubated w]th antiserum against rat deconn The lmmunoprec]p~tates were fractlonated on a

SDS-PAGE followed by fluorography

254 ROLANDO GONZALEZ el al

in human auricles (Gonzfilez et al., 1990) and the heparan sulfate-P found here might be also involved in the immobilization of this enzyme to the ECM.

A high molecular weight chondroltln sulfate-P present in heart have been described dunng m ovo embryogenesls (Camno and Caplan, 1984) and dunng In vi tro myogenesis of skeletal muscle (Miller et a l , 1987) The characteristics of the chondroitIn sulfate-P present m adult heart, descnbed in this report, are similar in size and relative proportion to the one's described during development (Miller et a l , 1987) opening the possibility that the same chon- droltm sulfate-P is expressed during development and in the adult life

The presence of small dermatan sulfate-P with a Kav of 0 35 in a Sepharose CL-6B with an apparent molecular weight of 115 kDA is described A similar P has been described in rabbit skeletal muscle (Parthasarathy and Tanzer, 1987) and fibroblast from several sources including skeletal muscle and skin (Miller et a l , 1987, Glbssl et a l , 1984) Using anti- serum against rat decorm we demonstrated in this work that the P synthesized by rat heart tissue indeed correspond to decorln We have previously descnbed that thls P can be solublhzed from skeletal muscle ECM by heparln (Brandan and Inestrosa, 1987b) and that the level of expression m skeletal muscle, is dependent on the lnnervation of the muscle (Fadlc et a l , 1990) and that differentmted skeletal muscles myotubes synthesize this P (Brandan et a l , 1991) The exact function of th~s P ~s unknown, however an important point to elucidate ~s the precise type of cell that synthesizes th~s P in the muscle tissue It has been suggested, although not proven, that lnterstltml Ps present at the myocardlum are produced by fibroblast (Borg et a l , 1985) Furthermore, It has been shown that the number of fibroblasts present in skeletal muscle depend on the presence of the nerve (Connor and McMahan, 1987, Gatchahan et a l , 1989) and an Interaction between fibroblast and myocyte has been described during car&omyogenesis m vitro (Gross, 1982), giving support to the idea that the synthesis and expression of functional macromolecules present at the muscle-ECM might be coordmated by more than one cell type

Taking these results together, it ~s tempting to postulate that the Ps constitutents of ECM from skeletal muscles and heart tissues are similar This opens important questions regarding the functional roles of these kind of macromolecules at the level of the organization of the ECM and/or as Integrative elements among different cell types

Acknowledgements--We wish to thank Dr Rlcardo Fa&c for the part~cipatmn m prehmmary experiments This study was supported by a Umted States of America-Chile Science m Developing Countries grant from the National Science Foundation and by grants from FONDECYT (726/87, 569/89), Internatmnal Foundation for Sciences (IFS 1407/1) and Fundacl6n Glldemelster

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