Cardiac myocyte cell surface: Identification and expression of differentiation antigens during chick heart development

DEVELOPMENTAL

BIOLOGY

81,

315-323 (1981)

Cardiac Myocyte Cell Surface: Identification and Expression of Differentiation Antigens during Chick Heart Development MARK Z. HOLODY,’ R. ZAK, and M. RABINOWITZ Departments

of Biology

and Medicine, Received

University

of Chicago and Pritzker

School of Medicine,

October 4, 1979; accepted in revised form July

Chicago, Illinois

60657

7, 1980

Cardiac myocytes from various stages of chick development were examined for specific cell-surface antigens not found on skeletal muscle to determine if specification of this striated muscle’s cell-surface antigenicity accompanies differentiation of cardiac tissue. Rabbit antisera raised against lo-day chick embryonic cardiac cells were assayed for specific anti-cell-surface antibodies with the complement-dependent 51Cr release and indirect immunofluorescence assays. Identification of specific surface antigens was possible following removal of cross-reactive antibodies by absorption of a l/25 dilution of anti-cardiac serum with skeletal muscle cells; a significant titer remained for cardiac cultures, while the absorbed antiserum no longer lysed skeletal muscle cells. A second class of shared surface antigens common to both heart and skeletal muscle cells was detectable at higher dilutions (l/250). These shared antigens were expressed in quantitatively similar amounts of both cell surfaces, although one lot of antisera appeared to detect a particular shared antigen that was slightly enriched on cardiac cells. Indirect immunofluorescence further corroborated the specificity found with the 51Cr release assay; fluorescent staining specific for cardiac cell surfaces remained after removal of all detectable fluorescence on skeletal muscle cells. Preferential lysis of cardiac myocytes but not cardiac fibroblasts by the absorbed antiserum demonstrated that the cardiac myocyte expresses a unique antigenic composition specific for its differentiated state. Titration of the absorbed antiserum against various developmental ages indicates that these specific antigens are expressed, and at a constant level, from Day 4 to Day 15 of chick embryogenesis. Last, cytolytic tests against a panel of embryonic cell types indicates that the absorbed antiserum is not absolutely tissue specific, but cross-reacts to a limited extent with kidney and some endodermal organs. Nonetheless, the strongest reactivity remains for cardiac cells, the tissue against which the antiserum was raised.

entiated products exist on the cell surface of cardiac myocytes. In other developmental systems, such as The cardiac myocyte, as it differentiates from the slime mold aggregation (Gerisch, 1977) or differentiasplanchnic mesoderm, acquires a complex array of sur- tion of subpopulations of T lymphocytes (Cantor and face properties, including sensitivity to neurotransmitBoyse, 1977), the acquisition of unique surface moleters (Barry, 1950; Coraboeuf et al., 1970; Roberts et al., cules detected in the form of “differentiation antigens” 1965), sugar and amino acid transport systems (Gui- (Boyse and Old, 1969) coincides with cellular differendotti et al., 1966, 1968), mucopolysaccharide cell coats tiation and marks the onset of particular recognitive (Manasek, 1976), specialized nexus junctions (McNutt, events and physiological states of the cell. Likewise, 1970), neuromuscular junctions (Manasek, 1976), cell putative specific antigens on cardiac cell surfaces may recognitive properties (Lesseps, 1973), etc. Many of serve to identify particular differentiated states of the these surface properties exhibit considerable fluctua- cardiac myocyte and prove critical in regulation of diftion as cardiac differentiation proceeds; glucose trans- ferentiative patterns of striated muscle myogenesis port rates (Clark, 1976; Guidotti et al., 1966), ion per- specific for cardiac tissue. meability (Carmeliet et al., 1975; Sperelakis et al., 1975), In this study, the antigenicity of cardiac myocyte cell sensitivity to tetrodotoxin (Shigenobu and Sperelakis, surfaces was examined to determine if specific “differ1971) or to neurotransmitters (Barry, 1950; Coraboeuf entiation antigens” are expressed on cardiac cell suret al., 1970), beating rates (Sachs and DeHaan, 1973), faces which are not found on skeletal muscle surfaces. and cholinergic binding sites (Guidotti et al., 1968) all Prior reports of cardiac-specific antigens (Espinosa and change considerably during cardiac development. The Kaplan, 1971; Kushner and Kaplan, 1967; Masaki, 1974) complexity of these properties and the developmental have not localized these moieties to the myocyte surpattern of surface differentiation suggests that differface. Previous investigations (Dewey et al., 1975; GoldSchneider and Moscona, 1972) of muscle surface antigenicity have noticed an antigenic makeup of cardiac i Present address: Bio-Rad, Inc., Diagnostic Systems Division, 2400 myocytes exceedingly similar to skeletal muscle cell Wright Ave., Richmond, Calif. 94803. INTRODUCTION

315 0012-1606/81/020315-09.$02.00/O Copyright All riahts

0 1981 by Academic Press. Inc. of reproduction in any form reserved.

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DEVELOPMENTALBIOLOGY

surfaces, finding no indication of any surface antigens preferentially expressed on the cardiac surface. Utilizing the 51Cr release assay (Sanderson, 1964; Wigzell, 1965) for detection of anti-cell-surface antibodies, our data reveal an antibody activity specific for cardiac cell surfaces once cross-reactive antibodies are removed by prior absorption of the anti-cardiac antiserum with skeletal muscle cells. Further specificity tests indicate that these determinants are expressed on the cardiac myocyte cell surface and not found on the fibroblast surface. Although not absolutely tissue specific, this absorbed antiserum still shows the greatest reaction with cardiac surfaces. Unaltered expression of these specific antigens from Day 4 to Day 15 of chick embryogenesis implies that these specific antigens are expressed at even earlier stages of cardiac differentiation which are critical for cellular differentiation of splanchnic mesoderm to cardiac myocytes. MATERIALS

AND METHODS

Tissue Dissociation Cardiac cells were dissociated from the hearts of loday White Leghorn chick embryos (Sharp Sales, Ill.) by incubating tissue fragments in 0.25% trypsin (Sigma Chemical Co., St. Louis, MO.) plus 0.05% collagenase (Worthington Biochemical Co.) for 30 min at 37°C (Clark, 1976). The resulting cell suspension was incubated in F12K medium (Gibco) containing 5% fetal bovine serum (Microbiological Associates) and 1% penicillin-streptomycin, 100 U/ml (Gibco). Skeletal muscle from 12-day chick embryos and stomach, lung, liver kidney, cerebrum, optic tectum, and neural retina from lo-day embryos were dissociated by sequential incubations in calcium- and magnesium-free Tyrode’s solution for 15 min at 37°C and then in 0.5% trypsin (Tryptar, Armour Pharmaceuticals) for 15 min at 37°C (Doering and Fischman, 1974). Skeletal muscle cells were suspended in Eagle’s basal medium (Gibco) containing 10% horse serum (Gibco), 5% embryo extract, 1% penicillin-streptomycin, 100 U/ml (Gibco), and 1% glutamine, 200 mM (Gibco). All other cells were suspended in the same media except 5% fetal calf serum, no horse serum nor embryo extract. Freshly trypsinized cells were cultured for 3 hr in Erlenmeyer flasks that were gassed with 5% CO/i2-95% air and rotated at 100 rpm for 4 hr at 37” C to prevent formation of aggregates. Cells were collected by centrifugation, washed three times with phosphate-buffered saline (PBS)2 (pH 7.5), and used for absorptions and immunizations. ’ Abbreviations used: PBS, phosphate-buffered saline; BSA, bovine serum albumin.

VOLUME81, 1981

Anti-Cardiac

Antiserum

Adult New Zealand white rabbits were injected intramuscularly and subcutaneously with 20 X lo6 cultured cardiac cells in 1 ml PBS (pH 7.5) emulsified with 1.0 ml of Freund’s complete adjuvant. Booster injections of 20 X lo6 cells were given intravenously at 2-week intervals. After three or four booster injections, the animals were bled lo-14 days after the last injection. The antiserum was heated for 30 min at 56°C to inactivate complement, filtered through 0.25~pm Millipore filters, and stored at -20°C. Absorption of Anti-Cardiac Suspensions

Serum with Cell

Aliquots of anti-cardiac antiserum were absorbed with increasing numbers of cultured cells for 30 min at room temperature in 10 X 75-mm centrifuge tubes. The cells were centrifuged for 2 min at 1OOOgin an Adams serofuge; the aliquots of absorbed anti-cardiac antiserum were removed and tested for remaining antibody activity. IgG Fraction from Tissue-Specific Anti-Cardiac

Sera

A pool of four anti-cardiac antisera (one-fifth dilution) was absorbed with twice the amount of skeletal muscle homogenate needed to absorb all lytic activity for skeletal muscle cell cultures and precipitated with 40% ammonium sulfate. The precipitate was suspended in 0.01 M phosphate buffer (pH 8.0). The sample was chromatographed on a DEAE-cellulose column equilibrated with the same buffer. The first eluted peak was collected, reprecipitated, and reconstituted in 2.0 ml PBS (pH 7.5). Cross-reactivity of the eluate with antirabbit serum or anti-rabbit IgG demonstrated one arc on immunoelectrophoresis. Complement-Dependent 51Cr Release Assay A procedure described previously (Sanderson, 1964; Wigzell, 1965) was modified for use on chick embryonic muscle cell cultures (Friedlander and Fischman, 1977, 1979). Freshly trypsinized cells (3 X 105) were cultured in 100 ~1 medium in flat-bottom microtiter plates (Falcon Plastics Co.) in a 5% Coz-95% air atmosphere at 37°C. At 24 hr, each well received 1 &i 51Cr (New England Nuclear Co.). After 48 hr incubation, the microtiter plates were washed with medium without the isotope; 50 ~1 anti-cardiac sera, normal sera, or media alone were added to each well, and the plates were incubated for 20 min at 37°C. Next, 50 ~1 guinea pig complement at l/10 dilution (Beckman Diagnostics Co.) were added to each well. After a 20-min incubation pe-

Cardiac

HOLODY, ZAK, AND RABINOWITZ

riod, 100 ~1 ice-cold Tyrode’s solution were added to each well; the plates were centrifuged at 50009 for 10 min. Aliquots of 100 ~1 of culture supernatants were transferred into 10 X ‘75~mm centrifuge tubes, and counted in a gamma counter. The percentage of 51Cr released was calculated as follows: % 51Cr Released = cpm(experimenta1 - background) cpm(maximum - background)

x 1oo ’

Background counts represent the amount of 51Cr released in the presence of complement alone; experimental counts measure the amount released in the presence of normal sera or antisera; and maximum counts measure the amount released in the presence of 20% Triton X-100. Indirect Immunojluorescence Assay Prior coating of glass coverslips with 1% poly-L-lysine and 1% gelatin was required for attachment of cardiac and skeletal muscle cells, respectively. Freshly trypsinized cells (1.5 X 105) in 1.0 ml medium were cultured on the coated coverslips in Falcon multiwells (16 mm diameter) in a 5% COz-95% air atmosphere at 37°C. After a 48-hr incubation period, the coverslips were washed three times for 5 min each with ice-cold PBS (pH 7.5) containing 1% bovine serum albumin (BSA-PBS) and then incubated for 30 min on ice with 50 yl antiserum or normal serum. The cells were again washed three times for 5 min each with ice-cold BSA-PBS (pH ‘7.5), and each coverslip was incubated with 50 ~1 fluorescein-conjugated goat anti-rabbit IgG at l/80 dilution (Cappel Labs) for 30 min on ice. The coverslips were washed three times with ice-cold PBS (pH 7.5), and the cultures were fixed in formyl:alcohol (1.10) for 15 min and the coverslips were then mounted with glycerol:PBS (1.10) onto glass microscope slides. Fluorescent micrographs were taken with a Zeiss microscope equipped with an ultraviolet light source, Ploem illumination, barrier filter No. 53, and excitation filters UG5 and BG3.

Cell-Surface

317

Antigens

TABLE 1 DETECTION OF CELL-SURFACE ANTIGENS BY THE COMPLEMENTDEPENDENT %r RELEASE ASSAY Assay conditions

5’Cr released (cpm)

Medium alone Complement Normal serum Normal serum + complement Antiserum Antiserum + complement 20% Triton X-100

174 190 201 253 205 2013 2217

Note. treated normal pled for released

Cultures of ‘ICr-labeled lo-day embryonic heart cells were with various combinations of 10-l dilution of antiserum or serum, and complement; aliquots of culture media were samthe amount of the isotope released. The above value of counts represents an average of three replicate cultures.

in Table 1, the addition of antiserum and complement to cardiac cell cultures resulted in the release of 90% of the maximal amount of 51Cr into the culture medium, while control cultures lacking both antiserum and complement released only 10% of that amount. All lots of antisera maintained maximal lytic activity at l/100 dilutions; the titer of lytic activity started to decline only after higher dilutions, generally l/250 to l/500 dilution (Fig. 1). To determine whether specific antibodies were present in a number of lots of anti-cardiac antisera, l/25 dilutions of anti-cardiac antisera were absorbed with sufficient numbers of skeletal muscle cells to remove all cross-reacting antibody for skeletal muscle cell cultures. Afterward, the absorbed antisera were tested on ‘lCr-labeled heart and skeletal muscle cell cultures to assess the level of lytic antibody which remained after absorption. As shown by one representative lot in Fig. 2a, unabsorbed anti-cardiac antiserum effectively lysed

RESULTS

Detection of Specific and Shared Cell Surface Antigens Our tests designed to detect cell surface antibodies utilized the complement-dependent 51Cr release assay to measure lytic antibody activity by quantitating the amount of isotope released into the culture medium after incubation of 51Cr-labeled muscle cell cultures with anti-cardiac antiserum and complement. As shown

10-s

IODilution

IO-'

of Antisera

FIG. 1. Titration of antiserum against cardiac cell cultures. Serial dilutions of anti-cardiac antiserum were tested on 48-hr cultures of lo-day chick embryonic heart cells with the complement-dependent ‘rCr release assay. Each point is an average of three replicate cultures.

318

DEVELOPMENTAL BIOLOGY

a

VOLUME 81, 1981

100

b

;fy--r !;;fyj-glz,, SO

‘0%

IO' No. of Absorbmg

Cells

(I I61

No. of

IO' Absorbing

IO' Cells

FIG. 2.(a) Detection of specific antibodies at low dilution. Aliquots (500 ~1) of anti-cardiac antiserum (l/25 dilution) were absorbed with increasing numbers of skeletal muscle cells; 50-pl aliquots of the absorbed antiserum were tested on 4%hr cultures of heart (0) and skeletal muscle cells (0) with the complement-dependent 51Cr release assay. Each point is an average of three replicate cultures. (b) Preferential absorption of specific antibodies by heart but not skeletal muscle cells. Aliquots (200 ~1) of absorbed anti-cardiac antiserum (l/10 dilution) (pooled from three lots) were absorbed quantitatively with increasing numbers of heart (0) or skeletal muscle (0) cells; 50-~1 aliquots of the absorbed antisera were tested on 48-hr cultures of heart cells with the complement-dependent 51Cr release assay. Each point is an average of three replicate cultures.

skeletal muscle cell cultures, demonstrating cross-reactivity between cardiac and skeletal muscle cell surfaces. Absorption with skeletal muscle cells removed all crossreactivity for skeletal muscle cell cultures. Cardiac cell cultures, however, still exhibit significant release of 51Cr when treated with the absorbed antiserum. Continued absorption with skeletal muscle cells removed no further reactivity for heart cell cultures, demonstrating that a set of specific antibodies exists that resists absorption with heterologous skeletal muscle. As a further demonstration of the specificity of the absorbed antiserum, a quantitative absorption test was used to verify that only heart cells were able to absorb the remaining antibody activity. Calculated on a percell basis, absorption by heart cells removed all reactivity for heart cultures whereas an equivalent number of skeletal muscle cells removed almost no antibody activity (Fig. 2b). Thus, only heart cells are unable to finally remove the remaining specific antibody. Repetition of the absorption tests for specific antibodies at l/250 dilution revealed a class of antibody-antigen reactions which did not exhibit the same degree of specificity as that found at low dilution (l/ 25). As anti-cardiac antiserum (l/250 dilution) was absorbed with skeletal muscle cells (Fig. 3a), a concomitant drop in reactivity for heart cultures appeared which coincided with the loss seen for skeletal muscle cell cultures. Thus, the antibody-antigen reactions detected at the high dilutions (l/250) were not specific but apparently represented antigens shared by both heart and skeletal muscle cell surfaces. The quantitative absorption test was used to further evaluate whether the shared antigens detected at high dilution (l/250) were expressed in quantitatively different amounts on heart and skeletal muscle cell sur-

faces. As seen in Fig. 3b, the quantitative absorption curves indicated that heart and skeletal muscle cells showed an equal capacity on a per-cell basis for absorbing the anti-cardiac antibodies, indicating that this class of shared antigens is present in quantitatively similar levels on both cell surfaces. One lot of antisera, however, identified a shared antigen which was slightly enriched on cardiac cells; fewer numbers of cardiac cells than skeletal muscle cells were required to absorb a similar amount of lytic antibody activity (Fig. 4). ImmunoJluorescent Staining Indirect immunofluorescence was used to corroborate the tissue specificity found with the “Cr release assay. Since our preliminary experiments indicated that indirect immunofluorescence was not as sensitive as the lysis assay? the IgG was purified from the absorbed anti-cardiac antiserum and concentrated lo-fold in order to raise titer. Unabsorbed IgG antibodies (data not shown) exhibited a uniform outline of fluorescence over the entire surface of both cardiac and skeletal ‘muscle cells. After absorption of IgG anti-cardiac antibody (10-l dilution) with 10’ skeletal muscle cells, the absorbed antiserum stained skeletal muscle cells no more brightly than normal serum control. In contrast, the fluorescence of cardiac cell surfaces was significantly above the control, and randomly distributed in fluorescent patches, further supporting the specific nature of the absorbed anti-cardiac antiserum (Fig. 5). Myocyte Specificity Although the myocyte is the predominant cell type (about 65%) in cardiac cell cultures (Polinger, 1973), 3Mark Z. Holody, Doctoral Dissertation, University of Chicago, Chicago, 111.

Department

of Biology,

HOLODY,ZAK, AND RABINOWITZ

Cardiac CeU-Surface Antigens

319

100 a

5

10

No. of Absorbing

1.5 Cells

2.0 25 (I IO-*1

No. of Absorbmg

Cells

FIG. 3.(a) Detection of shared antigens at high dilution. Aliquots (500 ~1) of anti-cardiac antiserum (l/250 dilution) were absorbed with increasing numbers of skeletal muscle cells; 50-pl aliquots of the absorbed antiserum were tested on 48-hr cultures of heart (0) and skeletal muscle (0) cells with the complement-dependent ‘rCr release assay. (b) A shared antigen expressed equally on heart and skeletal muscle cells. Aliquots (400 ~1) of high dilution anti-cardiac antiserum (l/250 dilution) were absorbed quantitatively with increasing numbers of heart (0) and skeletal muscle (0) cells; 509~1aliquots of the absorbed antiserum were tested on 48-hr cultures of heart cells with the complementdependent ‘ICr release assay. Each point is an average of three replicate cultures.

the presence of nonmuscle cells, mostly fibroblasts, pre- contrasts sharply with the significant killing of precludes an immediate identification of which cell type dominantly myocyte cultures, strongly suggesting that reacts with the absorbed antiserum. As pure myocyte this absorbed anti-cardiac antiserum is specific for the cell cultures cannot be acquired by existing techniques myocytes and does not cross-react with fibroblasts. The (Polinger, 19’73), pure cardiac fibroblast cell cultures cardiac myocyte cell surface, therefore, appears to poswere cultured to evaluate possible cross-reactivity with sess a distinct and specific antigenic composition not the nonmuscle cell population. Pure fibroblast cultures found on the cardiac fibroblast. can be readily obtained by taking advantage of their rapid proliferation rate in tissue culture. Therefore, to Antigen Expression during Striated Muscle Development determine if the absorbed antiserum reacted preferentially with the cardiac myocyte, it was titrated To determine whether the expression of these specific against both (1) predominantly myocyte, and (2) pure antigens is altered during cardiac development, the abfibroblast cell cultures. As shown in Fig. 6, the absence sorbed antiserum was titrated against cardiac cell culof any lytic activity against pure fibroblast cultures tures prepared from hearts of 4, 7, 10, and 15 days of embryonic chick development. As shown in Figs. ‘la and b, the absorbed antiserum reacted with all stages, indicating that the specific antigens are expressed across a wide range of embryonic development; second, the similar lytic titers for each stage suggest that no quantitative fluctuation occurs in the level of these specific antigens during the 4th through the 15th day of cardiac differentiation. In a second set of experiments we examined whether skeletal muscle cells express these putative cardiac-specific antigens at later stages of skeletal muscle differentiation when myotube formation is complete, even though the antigens are not detectable on the cell surface of prefusion myoblasts. Therefore, the tissue speNo. of Absorbing Cells cific anti-cardiac antiserum was tested on l-, 2-, and 5FIG. 4. A shared antigen enriched on cardiac cells. Aliquots (400 day-old cultures of skeletal muscle from 12-day em~1) of a second lot of high-dilution anti-cardiac antiserum (l/250 dibryos. By the fifth day of culture, the myoblasts are lution) were absorbed quantitatively with increasing numbers of completely fused and the culture consists primarily of heart (0) and skeletal muscle (0) cells; 507~1aliquots of the absorbed well-differentiated myotubes. Table 2 shows that the antiserum were tested on 48-hr cultures of heart cells with the complement-dependent %r release assay. Each point is an average of absorbed antiserum did not lyse skeletal muscle cells three replicate cultures. at any stage of differentiation; on the other hand, un-

320

DEVELOPMENTAL BIOLOGY

FIG. 5. Immunofluorescent staining of chick embryonic heart and 12-day skeletal muscle cells. Bar, 50 pm. (a) Anti-cardiac muscle. (d) Normal serum IgG tested on

VOLUME 81, 1981

cardiac and skeletal muscle cell cultures. Indirect immunofluorescence of 4%hr cultures of lo-day muscle after treatment with l/10 dilution of IgG anti-cardiac antibodies absorbed with 10’ skeletal IgG tested on heart. (b) Normal serum IgG tested on heart. (c) Anti-cardiac IgG tested on skeletal skeletal muscle.

absorbed anti-cardiac antiserum lysed cells at all stages. Thus, the cardiac-specific antigens are not expressed on skeletal muscle cells during the course of

skeletal muscle fusion; also, the antigens shared by cardiac and skeletal muscle cells do not undergo any apparent staging with differentiation of skeletal muscle.

Tissue Spec$icitg

o-..-..

,, T-p.--..

IO-'

I

10-Z

Dilution of Antisera

FIG. 6. Myocyte specificity of the absorbed anti-cardiac antiserum. Aliquots (50 ~1) of serial dilutions of tissue-specific anti-cardiac antiserum were tested on 4%hr cultures of predominantly cardiac myocytes (0) pure cardiac fibroblasts (Cl), and skeletal muscle (0) cells with the complement-dependent ‘iCr release assay. Each point is an average of three replicate cultures.

The absorbed anti-cardiac antiserum was tested against a panel of tissues of mesodermal, endodermal, and ectodermal origin to determine the degree of tissue specificity of the absorbed antiserum which shows no crossreactivity with skeletal muscle. As noted in Table 3, unabsorbed anti-cardiac antiserum effectively lysed all tissue types. The absorbed antisera did not crossreact with any of the three neural tissues tested, i.e., cerebrum, neural retina, and optic tectum. Very low levels of lytic activity were present for three endoderma1 organs (lung, liver, and stomach). Considerable cross-reactivity was present for kidney cells, a tissue of mesodermal origins. Nonetheless, the strongest crossreactivity was exhibited for heart cells. Thus, this specific cardiac antigen not present on skeletal muscle cell surfaces is not completely tissue-specific but is, in fact, limited to a discrete subset of tissue types which are

HOLODY, ZAK, AND RABINOWITZ

Km I

a

321

Cardiac CeU-Surface Antigens TABLE

2

EFFECTOFABSORBEDANTISERUMONSKELETALMUSCLECULTURES

80

!, s z

Percentage N

Days in vitro ‘b

i

‘iCr released

Assay conditions Unabsorbed anti-cardiac antiserum Normal serum Absorbed anti-cardiac antiserum l/3 Dilution l/12 Dilution l/24 Dilution

1

2

5

80 0

84 2

83 0

2 0 0

1 0 0

3 1 0

Note. Cultures of %r-labeled 12-day chick embryonic skeletal muscle cells were assayed after 1,2, and 5 days of culture with absorbed anti-cardiac antisera, unabsorbed anti-cardiac antiserum, and normal serum (l/3 dilution).

FIG. 7. (a) Developmental expression of specific antibodies: Days 4, ‘7, and 10 of cardiac development. Aliquots (50 ~1) of serial dilutions of the absorbed antiserum were tested on 4%hr cultures of 4 (0), 7 (o), and 10 (@)-day chick embryonic heart cells with the complementdependent ‘iC!r release assay. Each point is an average of three replicate cultures. (b) Developmental expression of specific antibodies: Days 10 and 15 of cardiac development. Aliquots (50 ~1) of serial dilutions of the cardiac-absorbed antiserum were tested on 48-hr cultures of 10 (0)- and 15 (@)-day chick embryonic heart cells with the complement-dependent 51Cr release assay. Each point is an average of three replicate cultures.

either of mesodermal origins or nonmesodermal tissues populated with mesodermally derived cells.

ward cardiac cultures. Second, a quantitative absorption test certified that only cardiac cells absorbed the specific antibody, whereas an equivalent number of skeletal muscle cells failed to do so. After absorption of concentrated IgG anti-cardiac antibodies with sufficient skeletal muscle cells, cardiac cell surfaces showed clear immunofluorescent staining whereas skeletal muscle cultures stained no more than normal serum controls. The absorbed antiserum also reacts preferentially with the cardiac myocyte as opposed to the cardiac fibroblast. Recent evidence now indicates that the cardiac fibroblast may also express cell surface antigens specific for its tissue type (Garrett and Conrad, 1979). Prior to our study, serological analyses of cardiac TABLE

As judged by the complement-dependent 51Crrelease assay and indirect immunofluorescence, lo-day chick embryonic cardiac myocytes exhibit both specific and shared antigens relative to skeletal muscle cell surfaces indicating a degree of serological complexity specific for cardiac differentiation. Characteristic of most lots of antisera, the specific antibodies were generally found at lower titers than the antibodies cross-reactive with skeletal muscle cells, presumably reflecting a weaker immunogenicity of the specific determinants. Specificity of the low dilution (l/25) anti-cardiac antiserum is supported by several lines of evidence. First, absorption of anti-cardiac antiserum with skeletal muscle cells completely removed lytic activity for skeletal muscle cultures but left intact a plateau of activity to-

3

EFFECTOFABSORBEDANTISERUMONPANELOF EMBRYONICCELLTYPES

DISCUSSION

Percentage Tissue type Heart Skeletal muscle Kidney Liver Lung Stomach Neural retina Cerebrum Optic tectum

Note. Cultures various dilution

Unabsorbed 95 98 97 92 92 91 93 94 94

“Cr released Absorbed 62 1 39 13 21 8 0 0 1

of %r-labeled lo-day chick embryonic cells from tissue types were assayed after 48 hr in culture with 10-l of unabsorbed and absorbed anti-cardiac antiserum.

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DEVELOPMENTAL BIOLOGY

muscle cell surfaces achieved only a limited degree of specificity. Thompson and Halbert (1971) clearly demonstrated that immunization across species lines generates cytotoxic antibody against cardiac cell surface antigens; but analysis of the tissue distribution of these antigens was not analyzed. Goldschneider and Moscona (1972) identified a class of mesodermal-specific antigens shared by cardiac and skeletal muscle cell surfaces but not present on endodermal or neural cell types; however, no tissue specificity for cardiac antigens was observed. Dewey et al. (1975) noted a particular class of antigens specific for “excitable membranes,” such as their anti-frog-heart antisera which showed immunofluorescent staining of only muscle and neuronal membranes. Lack of detection by previous investigators of cardiac-specific antigens not shared by skeletal muscle may reflect differences in sensitivity of the immunological methods employed. In comparison, we observed that the 51Crrelease assay was more sensitive than immunofluorescence microscopy for serological detection of these particular specific antigens; measurable release of isotope was still noticeable at dilutions of specific IgG antibodies where fluorescence was no longer observed.3 Other methodologies reflecting direct binding may detect additional surface antigens having different tissue reactivity. The antigens that we have detected by the 51Crrelease assay, and substantiated by immunofluorescence, apantigens” (Boyse pear to be genuine “differentiation and Old, 1969) preferentially expressed on cardiac cell surfaces, but also located to a less degree on other tissues. Some of the specific antigens are also present on kidney, which like muscle is of mesodermal origin. The crossreaction with endodermal organs may reflect a lower antigenic concentration on endodermal cells or the presence of subpopulations of mesodermally derived cell types. Thus, striated muscle cell-surface antigenicity diversifies as the mesoderm differentiates into cardiac and skeletal muscle cell types. Anti-cardiac antiserum demonstrates the strongest cross-reactivity with cardiac cell surfaces, whereas anti-skeletal muscle serum reacts preferentially with discrete stages of skeletal muscle differentiation (Friedlander and Fischman, 1977,1979). In contrast to this specification of striated muscle cell surfaces, other antigenic determinants remain expressed on both cell types (Friedlander and Fischman, 1977,1979; Goldschneider and Moscona, 1972). This pattern of surface differentiation is similar to the biochemical (Trowbridge et al., 1975) and antigenic (Boyse and Old, 1969) differences observed between two ontogenically related lymphocytes, T and B cells, in which some surface proteins are shared while others are tissue

VOLUME 81, 1981

specific. Distinctions in antigenic displays therefore may be important in the regulation of each tissue’s separate differentiative pathway. The absorbed antiserum’s reactivity against hearts from Day 4 to Day 15 of chick embryogenesis indicates that these myocyte-specific antigens are expressed during much or all of cardiac development. Similar titers observed from Day 4 to Day 15 suggest that the equivalent concentrations of surface antigens remain expressed during this developmental period; changes in titer with development can reflect corresponding changes in the number of antigenic sites on the cell surface (Moller, 1963). The uniform expression of these specific antigens is in contrast with other alterations that occur on the cardiac myocyte membrane and cell surface during these stages of cardiac development, including activation of fast sodium channels (Shigenobu and Sperelakis, 1971), innervation by sympathetic and parasympathetic neurons (Szepsenwol and Bron, 1936), alterations in the pharmacological specificity of the acetylcholine receptor (Roberts et al., 1965), and the appearance of insulin sensitivity of the glucose transport system (Guidotti et al., 1966). Such developmental changes in myocyte surface properties suggest that an active modification of surface chemistry occurs as cardiac differentiation proceeds. These changes may reflect the staging (loss or appearance) of qualitatively distinct cell-surface antigens, or merely represent a quantitative, conformational, or topographical rearrangement of preexisting sites. The identification of such possibilities is difficult because of the inherent heterogeneity of antibody-antigen reactions in rabbit anti-chick heart antiserum. Unequivocal analysis lies in the acquisition of monospecific antibodies, currently possible by use of lymphocyte-myeloma hybrids (Williams et al., 1977). Furthermore, the extreme unlikeliness that antibodies were raised against all the components of the cardiac membrane makes it important to devise new immunization protocols which will generate antibodies against a broader spectrum of surface antigens. Last, the possibility of changing surface displays during development requires that future studies use antisera prepared against hearts from several stages of cardiac differentiation. This work was supported by NIH Grants GM-07183, HL-13505, HL16637, and SCOR-IHD HL-17648 and was in partial completion of the degree of Doctor of Philosophy from the Department of Biology of the University of Chicago. We are grateful to Drs. Beatrice Garber, Frank Fitch, and Thomas Edgington for their useful comments and criticisms, and to Ms. Andrea Rothman for editorial advice. The authors extend thanks to Dr. Don Fischman for his support in initiating this project. Additional thanks are extended to Dr. Norman Wessels for use of his facilities for completion of some experiments.

HOLODY, ZAK, AND RABINOWITZ

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