Spatial distribution of Tissue-Specific Э antigens in the developing human heart and skeletal muscle. II. An immunohistochemical analysis of myosin heavy chain isoform expression patterns in the embryonic heartкод для вставкиСкачать
THE ANATOMICAL RECORD 229:355-368 (1991) Spatial Distribution of “Tissue-Specific” Antigens in the Developing Human Heart and Skeletal Muscle 11. AN IMMUNOHISTOCHEMICALANALYSIS OF MYOSIN HEAVY CHAIN ISOFORM EXPRESSION PATTERNS IN THE EMBRYONIC HEART A. WESSELS, J.L.M. VERMEULEN, Sz. VIRAGH, F. KALMAN, W.H. LAMERS, AND A.F.M. MOORMAN Department of Anatomy and Embryology, University of Amsterdam, Academic Medical Centre, Meibergdreef 15, 1105 A Z Amsterdam, The Netherlands (A.W., J.L.M.V., W.H.L., A.F.M.M.) and Department of Pathology, Postgraduate Medical School, Budapest 112, Hungary 1389 (S.V., F.K.) ABSTRACT The spatial distribution of a-and P-myosin heavy chain isoforms (MHCs) was investigated immunohistochemically in the embryonic human heart between the 4th and the 8th week of development. The development of the overall MHC isoform expression pattern can be outlined as follows: (1)In all stages examined, p-MHC is the predominant isoform in the ventricles and outflow tract (OFT), while a-MHC is the main isoform in the atria. In addition, a-MHC is also expressed in the ventricles at stage 14 and in the OFT from stage 14 to stage 19. This expression pattern is very reminiscent of that found in chicken and rat. (2) In the early embryonic stages the entire atrioventricular canal (AVC) wall expresses a-MHC whereas only the lower part expresses p-MHC. The separation of atria and ventricles by the fibrous annulus takes place a t the ventricular margin of the AVC wall. Hence, the P-MHC expressing part of the AVC wall, including the right atrioventricular ring bundle, is eventually incorporated in the atria. (3) In the late embryonic stages (approx. 8 weeks of development) areas of a-MHC reappear in the ventricular myocardium, in particular in the subendocardial region at the top of the interventricular septum. These coexpressing cells are topographically related to the developing ventricular conduction system. (4) In the sinoatrial junction of all hearts examined a-and P-MHC coexpressing cells are observed. In the older stages these cells are characteristically localized a t the periphery of the SA node. One of the proteins that plays a n important role in the contractile apparatus of the cardiomyocyte is myosin. The native molecule consists of one pair of heavy chains (MHCs) and two pairs of light chains (MLCs). Several MHC isoforms as well as MLC isoforms have been described. Combination of the different isoforms gives rise to the existence of different isoenzymes. A close correlation between the contractile properties of a muscle (e.g., the maximum velocity of contraction and the efficiency of force production) and its MHC isoform composition has been demonstrated for skeletal (Reiser et al., 1985, 1988) a s well a s cardiac muscle (Schwartz e t al., 1981). In the avian and mammalian heart two types of MHCs are expressed: a heart-specific MHC, designated a-MHC, and p-MHC that is also expressed in the slowtwitch skeletal muscle fibers (Hoh et al., 1979; Gorza et al., 1984; Sanders et al., 1984; De Groot et al., 1985). Myosin isoenzymes, and hence the MHC isoforms, are heterogeneously distributed over the myocardium. In the adult heart of the larger mammalian species including man, a-MHC predominates in the atria, Q 1991 WILEY-LISS, INC whereas p-MHC is the main MHC isoform in the ventricles (Lompre et al., 1981; Gorza e t al., 1984; Bouvagnet et al., 1984, 1985). Changes in the relative amounts of these isoforms have been described during development (Lompre et al., 1981; Cummins and Lambert, 1986; Everett, 1986; Hoffmann and Siegert, 1987; Bouvagnet et al., 1987), as a result of altering hormonal conditions (Morkin, 1979; Effron et al., 1987), a s a result of physical exercise (Belcastro e t al., 19881, after dietary manipulations (Morris et al., 1987), and under several pathological conditions (Gorza et al., 1984; Bouvagnet et al., 1985; Hoffmann and Siegert, 1987; Buttrick et al., 1986). The spatial distribution of MHC isoforms in the developing (i.e., embryonic/fetal) and adult heart has been investigated in several avian and mammalian Received March 27, 1990; accepted July 24, 1990. Address reprint requests to A.F.M. Moorman, Department of Anatomy and Embryology, University of Amsterdam, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. 356 A. WESSELS ET AL. species. One of the remarkable outcomes of these stud41 4 4MHC ies was the characteristic coexpression of both MHC a2 isoforms in the sinoatrial (SA) junction, in the atrioa3 ventricular (AV) junction, and in the outflow tract 44 (OFT) (Gorza et al., 1986; Sanders et al., 1986; De Jong e t al., 1987; De Groot et al., 1985, 1989). Interestingly, u5 these areas proved also to be characterized by relatively slow conduction of the cardiac impulse in com46 parison to areas in which only one of the two isoforms 47 is expressed, i.e., the developing atria and ventricles (Argiiello et al., 1986; De Jong et al., 1988). For the developing human heart, however, information on the spatial distribution of MHC isoforms is reFig. 1. Characterization of the antibodies by Western Blot analysis. stricted to those stages in which cardiogenesis has alprotein extracts of adult human left ventricle (M) and right ready been completed and is, in addition, primarily Total auricle (e-g) were loaded on 10%PAGE-SDS gels. After electrophorefocused on the conduction system (Bouvagnet et al., sis, protein patterns were visualized by Coomassie blue staining (b,e). 1984, 1985, 1987; Kuro-o et al., 1986). Data on the A prestained SDS molecular weight standard mixture (open triangles; spatial distribution of the MHC isoforms in the embry- M , 1, 180,000;2, 116,000; 3, 84,000; 4, 58,000; 5, 48,500, 6, 36,500; 7, 26,600) was transferred simultaneously to enable identification of anonic stages of the human heart do not exist. Such data, tibody-binding bands (a).To detect the specificity of the monoclonal combined with data of the older stages, are essential to antibodies, simultaneously run gels were blotted on nitrocellulose obtain a better insight into the relationship between (NC) sheets. The NC sheets were cut into small strips and incubated this distribution and the architecture and function of with the Mab 249-5A4 (c,D and Mab 169-ID5 (d,g). the developing human heart. With this idea in mind we have carried out immunohistochemical studies on prenatal (embryonic and fetal) and postnatal (neonatal and adult) human hearts. Medical School in Budapest. The studies were approved Preliminary results with the latter have been reported by the respective local medical-ethical committees. The recently (Wessels et al., 1988,1990~). In this paper the Carnegie stage of development of the embryos was esspatial distribution of the MHC isoforms in the embry- timated by comparison of the observed external landonic human heart from 4% weeks of development (Car- marks with literature data (Butler and Juurlink, 1987; O’Rahilly and Muller, 1987). No morphological abnornegie stage 14) onward will be discussed. malities were observed in the specimens described. MATERIALS AND METHODS From stage 20 of development onward isolated hearts were used; before this stage the embryos were fixed a s Tissue Sources and Preparation Twelve human embryos and fetuses from Carnegie a whole. Adult human muscle tissue was obtained at stage 14 of development onward were studied; six of autopsy at the Department of Pathology at the AMC. them are described in detail in this paper. They were Tissue specimens were fixed at room temperature in a obtained after legal abortions at the Academic Medical mixture of methano1:acetone:acetic acid:water [35:35: Centre of Amsterdam (AMC) and at the Postgraduate 5:25 (v/v)] for a minimum of 2 h, dehydrated in 2,2dimethoxypropane (Merck, Germany) or ethanol, embedded in Paraplast Plus (Monoject, Ireland), and cut into 5 Fm thick serial sections. A AF A0 AVC AVN DM His IAS IVC IVS LA LBB LSH LV Mi OFT RA RAVR RSH RV RVV SA SAN SLV svc vcs Abbreviations atrium (primitive) annulus fibrosus aorta atrioventricular canal atrioventricular node dorsal mesocardium bundle of His interatrial septum inferior vena cava interventricular septum left atrium left bundle branch left sinus horn left ventricle mitral valve outflow tract right atrium right atrioventricular ring right sinus horn right ventricle right venous valve sinoatrial junction sinoatrial node semilunar valves superior vena cava ventricular conduction system Monoclonal Antibody Production Myosin was isolated essentially according to the methods of Hoh et al. (1976). The monoclonal antibody Mab 249-5A4 was raised against myosin extracted from adult human atrium; Mab 169-ID5 was raised against chicken-heart myosin. The production of these antibodies has been described elsewhere (Wessels et al., 1988; de Groot et al., 1985, 1989). Immunization of BALB/c mice was performed by intraperitoneal injection of approximately 100 kg of antigen emulsified in Freunds’s complete adjuvant. After 6 months, the mice were boosted several times intravenously with 100 pg of antigen diluted in PBS. One week after the last booster, spleen lymphocytes were fused with NS1 myeloma cells, essentially according to the method of Fazekas de St. Groth and Scheidegger (1980). Hybridomas were screened by ELISA and recloned once or twice by limiting dilution. The specificity of the monoclonal antibodies present in the medium in which the hybridomas were cultured was examined on Western Blots and by immunohistochemical methods. MHC ISOFORMS IN EMBRYONIC HUMAN HEART 357 Fig. 2. Immunohistochemical characterization of the antibodies. Serial sections of adult human soleus muscle ia,b) and of human fetal heart a t approximately 17 weeks of development (c,d)were incubated with Mab 169-ID5 (a,c)and 249-5A4 ib,d). I, slow-twitch fibers; 11, fast-twitch fibers. and Schuell, Dassel Germany), using the Bio-Rad Minitransblot (1-2 h, 50 V). A prestained SDS molecTotal protein extracts of adult human atrium and ular weight standard mixture (SDS-7B, Sigma, St. ventricle were prepared according to Sweeney et al. Louis, MO) was transferred simultaneously to enable (1989). After electrophoresis of the extracts on 10% identification of antibody binding bands. NC sheets polyacrylamide gels in the presence of SDS according were first stained with amido black to demonstrate proto Laemmli (1970), the gels were blotted on nitrocellu- tein bands ( 3 min, 0.1% wiv amido black in 10% viv lose (NC) sheets (BAS 85, reinforced NC, Schleicher methanol, 10% viv acetic acid, 80% viv distilled water, Western Blotting 358 A. WESSELS ET AL. Fig. 3. MHC ISOFORMS IN EMBRYONIC HUMAN HEART Fig. 4. Myosin heavy chain expression at Carnegie stage 15.a shows a scanning electron micrograph of the dorsal aspect of a 35 day embryonic heart ( x 601, b,c,f, and g show immunohistochemical stainings of transverse serial sections of a 35-38 day embryonic heart, and 359 d and e show sagittal serial sections of a 35-36 day embryonic heart. (b,d, and 0 Incubation with anti-a-MHC; (c,e,and g) incubation with anti-P-MHC. f and g show the left AV junction and are mirror images of the areas encircled in b and c (b,c, X 30; d,e, x 40;f,g, x 125). with the respective antibodies, diluted in TEN-ST-BSA (RT, overnight). Primary antibody binding was detected using rabbit anti-mouse (RAM, noncommercial) immunoglobulin, goat anti-rabbit (GAR, noncommercial) immunoglobulin, and rabbit peroxidase-antiperoxidase (R-PAP, Nordic, The Netherlands) complex, respectively. All sera were diluted in TEN-ST-BSA. Between each incubation step, the strips were washed intensely in TEN-ST. After the last incubation the strips were rinsed for 10 min in 50 mM Tris-HC1 (pH Fig. 3. Myosin heavy chain expression at Carnegie stage 14.Immu7.6). The immunocomplex formed was visualized by innohistochemical stainings of transverse serial sections of a human embryo of 31-35 days of development at the level of the sinus horns cubation of the strips for approximately 5 min with 0.5 (a,d), the atrioventricular canal (b,e), and the outflow tract (c,D. mg/ml 3,3'-diaminobenzidine, 0.02% hydrogen perox(a-c) Incubation with anti-a-MHC; (d-h) incubation with anti@-MHC.Encircled areas in e are enlarged in g and h (a-f, X 45;g,h, ide in 50 mM Tris-HC1 (pH 7.6). The color reaction was stopped by rinsing the strips for 1 min in 1% HC1. x 170). followed by rinsing in a solution of 5% v/v methanol and 7.5%v/v acetic acid in water to remove background staining). After destaining the NC sheets in TEN-STBSA (50 mM Tris-HC1,5 mM EDTA, 150 mM NaC1, pH 7.4 with 0.1% w/v SDS, 1% Triton X-100, and 3% BSA) the sheets were rinsed in TEN-ST, cut, and incubated 360 A. WESSELS ET AL. Fig. 5. Myosin heavy chain expression a t Carnegie stage 17. a shows a scanning electron micrograph of the dorsal aspect of a 42-44 days embryonic heart ( x 70). b g show immunohistochemical stainings of transverse serial sections of a 42-44 day embryonic heart at the level of the sinoatrial junction (b,c,f,g)and the outflow-tract (d,e).(b,d, and 0 Incubation with anti-a-MHC; (c,e, and g ) incubation with anti-P-MHC. Small arrows in d and e indicate the area of coexpression around the developing semilunar valves in the OFT arrow in g points to the P-MHC expressing cells in the upstream part of the loosely arranged myocardium of the developing SA node (b,c, x 25; d-g, x 45). min. The immunocomplex formed was visualized by To detect the binding of the monoclonal antibodies incubation of the sections with 0.5 mg/ml 3,3’-diamiwith the respective antigens on paraplast sections, the nobenzidine, 0.02% hydrogen peroxide in 30 mM imiindirect unconjugated peroxidase-antiperoxidase tech- dazole, 1mM EDTA (pH 7.0). Sections were mounted in nique (PAP technique) was applied (Moorman et al., Entellan. Negative controls included incubations with1984; Wessels et al., 1990a,b). After deparaffination, out primary monoclonal antibodies and incubations the sections were treated with hydrogen peroxide (3% without one of the secondary antibodies (RAM or v/v in PBS, 30 min) to reduce endogenous peroxidase GAR). activity, followed by preincubation in TENG-T (10 mM Scanning Electron Microscopy (SEM) Tris, 5 mM EDTA, 150 mM NaCl, 0.25% gelatin, 0.05% For the sake of better spatial orientation SEM miTween-20, pH 8.0, 30 min) to reduce nonspecific binding. After incubation of the pretreated sections with crographs are added to the immunostained LM photothe monoclonal antibodies (RT, overnight), antibody graphs. For this purpose, embryonic human hearts binding was detected using rabbit anti-mouse (RAM) were fixed for several hours in freshly prepared formimmunoglobulin, goat anti-rabbit (GAR) immunoglob- aldehyde (4% in 0.1 M phosphate buffer, pH 7.2). The ulin, and rabbit peroxidase-antiperoxidase (R-PAP) specimens were then treated with 1% buffered OsO, for complex, respectively. Sera were diluted in PBS. All 1 h, dehydrated in a graded series of ethanol, criticalincubations were followed by three washes in PBS for 5 point dried, and eventually coated with 15 to 20 nm lmmunohistochemistry MHC ISOFORMS IN EMBRYONIC HUMAN HEART gold in a sputter coater. SEM was performed on a Philips 501 scanning electron microscope. RESULTS Characterization of the Antibodies (Figs. 1 and 2) The specificity of the monoclonal antibodies toward the human MHC isoforms was examined by Western Blot analysis. Figure 1 shows that in a total protein extract of adult human ventricle only Mab 169-ID5 binds specifically to a band of approx. 210 kDa characteristic for MHC (lane d), whereas with Mab 249-5A4 no reaction is observed (lane c). In a total protein extract of adult human atrium a reaction of both Mab 169-ID5 (lane g) and Mab 249-584 (lane f ) with MHC is observed. These results are in agreement with the described coexpression of a- and p-MHC in adult atrium and the predominance of P-MHC in the adult ventricle (cf. Bouvagnet et al., 1984, 1985). The immunohistochemical characterization of the antibodies is illustrated by the staining pattern on serial sections of adult human soleus muscle (Fig. 2a,b) and fetal human heart (Fig. 2c,d). In soleus muscle Mab 169-ID5 reacts with the majority of the muscle fibers, the slow-twitch, a-MHC-containing type I f i bers, and not with fast type I1 fibers (Fig. 2a). Mab 249-5A4 does not react with either of these fiber types (Fig. 2b). In the fetal heart, Mab 169-ID5 reacts strongly with all the ventricular myocytes and with a subpopulation of the atrial myocytes (Fig. 2c). Mab 249-5A4 binds exclusively to the myocytes of the atrium (Fig. 2d). In addition to the immunohistochemical characterization on paraplast sections, the specificity of both antibodies was also investigated by comparing the myofibrillar ATPase activity (see Staron e t al., 1983) with the immunohistochemical staining patterns on serial freeze sections of muscles of different species including rat and rabbit (A. Wessels, unpublished; Bredman et al., 1990). In these studies it was demonstrated that 169-ID5 reacts specifically with those fibers that, based on ATPase reactivity, could be identified as type I f i bers, whereas 249-5A4 did not show reaction with any of the muscle fibers types in the skeletal muscles. Therefore, taking the literature data into account, Mab 169-ID5 can be considered to be a specific monoclonal antibody for p-MHC and Mab 249-5A4 a specific monoclonal antibody for a-MHC. The Spatial Distribution of MHC lsoforms in the Developing Human Hearl Carnegie stage 14 (31-35 days) (Fig. 3) In the youngest specimen examined, a-MHC is expressed in nearly the whole heart, i.e., the primitive atrium, the embryonic left and right ventricle, and the outflow tract (OFT) (Fig. 3a,b,c). In addition, a-MHC expression is also observed in myocytes of the dorsal mesocardium (Fig. 3a). No expression is detected in the sinus horns (Fig. 3a). Expression of P-MHC is found in the OFT (Fig. 3f) and in the embryonic ventricles (Fig. 3d,e), showing a sharp boundary in the middle of the atrioventricular canal (AVC) wall (Fig. 3e,h). In addition, p-MHC-containing cells are observed in the sinoatrial (SA) junction and in the dorsolateral wall of the developing right atrium (Fig. 3e,g). No staining with anti-p-MHC is ob- 361 served in the sinus horns and in the myocytes of the dorsal mesocardium (Fig. 3d). Hence, in the youngest embryo available for examination, coexpression of both MHC isoforms is observed in the SA junction, in the lower part of the AVC wall, in the embryonic ventricles, and in the OFT. Carnegie stage 15 (35-38 days) (Fig. 4) In the two embryos examined of this stage the strong expression of a-MHC in the free walls of the developing ventricles, a s observed in stage 14 (e.g., Fig. 3b), has disappeared (Fig. 4b,d). Strong a-MHC expression is, however, still detected in the atria (Fig. 4b,d,f), the AVC wall (Fig. 4b,d,f), the OFT (Fig. 4d), and, to a lesser extent, in the ventricular trabeculae and in the ventricular wall just below the AV canal wall (Fig. 4b,d). Expression of a-MHC is now also detected in the sinus horns (Fig. 4d). As in the previous stages, anti-p-MHC reacts strongly with the developing ventricles. At the right aspect of the AVC wall (arrows in Fig. 4b and c) the so-called right atrioventricular ring (RAVR) tissue can be identified by its characteristic morphology (i.e., cylindrical orientation of the fibers and the compact structure). This ring tissue expresses p-MHC in addition to a-MHC. Compared with the previous stages the sharp boundary of p-MHC expression in the AVC wall is more pronounced (Fig. 4c,e,g) and a t the right AVC wall located at the atrial margin of the ring tissue. In the SA region the staining with anti-p-MHC has diminished and only a few cardiomyocytes in the loosely arranged mesenchyme of sinus horn origin at the transition of the superior vena cava (SVC) and right atrium are labeled (not shown). The reaction with the myocardium of the OFT is still very strong (Fig. 4e). Thus, areas with coexpression in this stage are located a t the SA junction, in the lower part of the AVC wall and in the OFT. Carnegie stage 17 (42-44 days) (Fig. 5) As in stage 15, a-MHC is mainly located in the atria (Fig. 5b,d,f) and p-MHC in the ventricles (Fig. 5c,e). Low concentrations of a-MHC outside the atria are detected in the ventricular myocardium adjacent to the AVC (Fig. 5b), in the ventricular trabeculae, and in the distal rim of the OFT (Fig. 5d). In addition to the strong p-MHC expression in the ventricles and OFT (Fig. 5c,e), p-MHC expression is still detected in some cells of the loosely arranged myocardium in the upstream part of the right sinus muscle (Fig. 5c,g) and in the atrial margin a t the AV transition just above the ingrowing fibrous annulus (Fig. 5c, see also Fig. 6 in Wessels et al., 1990b). Thus, the coexpressing areas in this stage are essentially the same as in stage 15. Carnegie stage 18-19 (44-51 days) (Fig. 6) The overall MHC expression pattern in this stage resembles that of the previous stage in most respects (Fig. 6b,c). In the parietal wall of the right AVC, the RAVR is easily recognizable by its compact structure, orientation of the fibers, and the expression of 6-MHC (open 362 A. WESSELS ET AL. arrows in Fig. 6d-i). Dorsally, at the myocardial junction of IAS and IVS, this ring tissue is continuous with compact myocardium that extends into the IVS. This relatively large area corresponds to the proximal part of the developing atrioventricular conduction system, i.e., the AV node and the His bundle (cf. Viragh et al., 1988). In this junctional tissue a strong expression of a-MHC is observed (Fig. 6d,f). The expression of pMHC, however, in particular in the central portion, is relatively weak compared to the adjacent myocardium of the IVS (Fig. 6e,g). As the compact myocardium is followed ventrally into the IVS the expression of aMHC decreases (Fig. 6h) whereas the relative amount of p-MHC increases (Fig. 6i). Thus, in this stage, in addition to the coexpression in OFT, SA junction, and in the lower part of the AVC wall, coexpression is also observed in the area in which the proximal part of the VCS is developing. Carnegie stage 23 (56-60 days) (Figs. 7 and 8) In this oldest embryonic stage, cardiogenesis is almost completed. The developing AV-conduction system is, in addition to the morphological features, now well characterized by its MHC expression. The AV node and the proximal part of the His bundle do react strongly with anti-a-MHC (Fig. 7a,b), whereas only some pMHC expressing cells are observed (Fig. 7f,g). More ventrally located sections show the distal part of the His bundle a t the bifurcation in the top of the IVS, where a strong coexpression of both MHC isoforms is observed (Fig. 7c,h). I n the right AVC wall the RAVR is much more recognizable by its characteristic morphology than in the previous stages (Figs. 7d,e and 8c,d). The RAVR expresses a-MHC, but only a few fibers still express PMHC (Fig. 8d). From the anterior portion of this ring, tracts of a- and p-MHC coexpressing cells extend into the ventricular myocardium of the developing tricuspid valves and papillary muscles (Fig. 7d,e). Although some p-MHC expressing cells are found more or less scattered throughout the atrial myocardium and in the SA junction, clusters of P-MHC expressing cells are found only in the IAS and in the ventral wall of the right atrium (encircled areas in Fig. 7f-j). Three-dimensional reconstruction of the serial sections reveals that these coexpressing cells form a tract running from the base of the interatrial septum, i.e., the AV nodal area, into the ventrallseptal wall of the right auricle. Whether this is a n area of specialized impulse conducting tissue or a n area with specific contractile properties remains to be elucidated. In the myocardium surrounding the developing semilunar valves of the aorta (Fig. 7h,i) and pulmonary trunk a remarkable strong p-MHC expression is observed. With anti-a-MHC, however, no staining in the OFT is detected anymore (Fig. 7c,d). Thus, in this stage coexpression is observed in the ventricles, where it correlates with the position of the developing ventricular conduction system, and in a tract of subendocardially located cells in the ventral portion of the right atrium. Some coexpressing cell are found in the SA nodal area, whereas the coexpression in the OFT around the developing semilunar valves has disappeared. DISCUSSION Spatial Distribution of the MHC lsoforms and the Functional Implication The results presented in this paper provide the first immunohistochemical information about the spatial distribution of the a- and p-MHC isoform in the embryonic human heart. In all the stages examined, all cardiomyocytes are labeled by either anti-a-MHC or anti-P-MHC, or by both. In rat heart, three different types of myosin molecules with different MHC composition can be distinguished: the aa- and PP-MHC homodimers and the ap-MHC heterodimer (Hoh et al., 1976, 1979). These isoforms can be separated by nondissociating PAGE electrophoresis (Hoh e t al., 1976). Thus far, in humans, both homodimers have been detected by this method (Buttrick et al., 1986). Although i t is not possible with the immunohistochemical techniques used in this study to distinguish between the coexistence of aa-MHC and PP-MHC homodimers and the presence of the up-MHC heterodimer in a cardiomyocyte labeled by both anti-a- and anti-p-MHC the data on the distribution of the MHC monomers therefore probably reflect the distribution of the aa- and PP-MHC homodimers. To settle this point other techniques, e.g., monoclonal antibody epitope mapping a s described by Dechesne et al. (1987a), would have to be applied. Although we could not investigate human hearts preceding stage 14, a comparison of our results on human heart development with related studies on the developing vertebrate heart reveals that the “overall” ontogenesis and spatial distribution of the MHC isoforms in circumscribed regions of the hearts of these species is similar, although some species-specific phenomena can be observed. The Sinoatrial Region In the sinoatrial junction of the youngest human embryonic hearts examined, high expression of a-MHC is detected in the compact atrial myocardium and in the adjacent loosely arranged sinus muscle, derived from the sinus venosus. A few cells in this area coexpress aand P-MHC. These cells are mainly localized in the sinus muscle at the SA junction, the area in which the sinoatrial node (SA node) will develop. As development progresses the relative amount of coexpressing cells in the atria rises. However, when the SA node becomes morphologically identifiable, p-MHC expressing cells are characteristically localized at its periphery. A similar distribution of MHC isoforms is also observed in the SA nodal area of fetal (Wessels e t al., 199Oc), neonatal (A. Wessels, in preparation), and adult human Fig. 6. Myosin heavy chain expression a t Carnegie stage 18-19. a shows a scanning electron micrograph of the dorsal aspect of a 49-52 day embryonic heart ( x 45). b-e show transverse serial sections of a 44-51 day embryonic heart at the level of the transition from interatrial and interventricular septum. (b,d,f, and h) Incubation with anti-a-MHC; (c,e,g, and i) incubation with anti-P-MHC. f and g are enlargements from b a n d c , respectively. Closed arrows in d-i point to the compact myocardium located in the top of the IVS; the open arrows indicate the coexpressing lateral part of the right atrioventricular ring (b,c, x 25; d-i, x 100). Fig. 6 Fig. 7. Myosin heavy chain expression a t Carnegie Stage 23. a-j show sections of a 56-60 day serial sectioned embryonic heart. ( a 4 Incubation with anti-a-MHC; Cf-j) the corresponding serial sections, incubated with anti-P-MHC. Boxed areas in d and i are enlarged in Fig. 8c and d. In f-j the p-MHC-positive cells in the medial wall of the right a t r i u d r i g h t auricle are encircled. Open arrows in h indicate the 6-MHC expressing, myocardial sleeve around the developing semilunar valves of the aorta (a-j, x 17). MHC ISOFORMS IN EMBRYONIC HUMAN HEART Fig. 8. Myosin heavy chain expression at Carnegie stage 23. a shows a scanning electron micrograph of the ventral aspect of an 8 week embryonic heart ( x 60). The ventral wall is cut off to expose the cavities of the left atrium and the left and right ventricle. b shows a dorsal aspect of a n 8 week embryonic heart; the arrow indicates the 365 localization of the tract of P-MHC-positive cells in the right atrium ( x 60). c and d show the compact structure of the myocardium in the atrial margin a t the right atrioventricular junction boxed in Fig. 7d and i. (c) Incubation with anti-a-MHC; (d) incubation with anti+MHC (c,d, X 122). The Atrioventricular Canal Wall and the Development of hearts (Kuro-o et al., 1986 and A. Wessels, unpublished the Atrioventricular Junction observations). The presence of a- and p-MHC coexpressing cells in the SA junction is a common feature In the developing human heart, p-MHC expression is among different species like chicken (De Groot e t al., 1987), rat (De Groot et al., 1989), and cow (Gorza et al., restricted to the myocardium of the lower part of the 1986), although the extent to which p-MHC is ex- AVC wall, the embryonic ventricles, and the OFT from pressed is species-dependent (De Groot et al., 1988). the earliest stage examined onward. In all these stages The characteristic p-MHC expression of cardiomyo- this expression shows a sharp boundary midway in the cytes flanking the SA node in the prenatal stages, a s myocardium of the AVC. Interestingly, the fibrous AV well as in the adult human heart, suggests that this junction, i.e., the sulcus tissue, develops at the ventricphenomenon is a feature of specialized cells in that ular margin of the AVC wall (Fig. 4c, and Wessels et area. Electron microscopic studies have demonstrated al., 1990b, Fig. 6f,g,h, Wessels et al., 1990d). This demtransitional cells that differ from their surrounding by onstrates that the entire myocardium of the AVC, intheir embryonic origin (Viragh and Challice, 19801, cluding the lower part that expresses @-MHC,is incortheir amount of myofilaments (Viragh and Challice, porated into the atrium. In fetal and neonatal human unpublished observations, Op’t Hof, 19861, and the rate hearts the remnants of the RAVR, that expresses pMHC in all embryonic stages, are still recognizable in of diastolic depolarization (Op’t Hof, 1986). 366 A. WESSELS ET AL. the myocardium of the right atrium, just above the fibrous annulus. This phenomenon is of interest, as the fate of the myocytes in the AVC wall has been a point of discussion for years (e.g., Viragh and Challice, 1977a). The Ventricular Conduction System The present study shows that the entire circumference of the lower part of the AVC shows strong coexpression in the early embryonic stages. Using morphological criteria the inner layer of the dorsal wall of the AVC was identified a s the myocardium in which the proximal part of the ventricular conduction system (VCS) develops (Viragh and Challice 1977a,b, 1982; Viragh et al., 1988). Hence, in these stages coexpression seems not strictly correlated with the development of the VCS. In older stages the proximal part of the VCS, i.e., the developing AV node, expresses predominantly a-MHC, whereas the developing His bundle is characterized by a strong coexpression. As development proceeds this coexpression gradually extends toward the distal parts, i.e., the bundle branches. These results are in agreement with those of Bouvagnet et al. (1987), who used immunofluorescence staining in human fetal hearts. Interestingly, in neonatal human hearts we have observed that the whole VCS, including the peripheral Purkinje system, is characterized by a strong MHC coexpression (Wessels et al., 199Oc),a phenomenon that is lost as the VCS matures further. Thus, after the disappearance of the a-MHC expression from the ventricular myocardium at stage 15, the local reexpression of a-MHC in the ventricles seems to be a good parameter for the differentiation of the VCS. Coexpression of atrial and ventricular isomyosins has also been reported in fibers of the VCS during lateembryonic and fetal stages of development in other species such as chicken (Zhang et al., 1984), rat (Dechesne et al., 19871, and cow (Komuro e t al., 1987). Previously, several authors have demonstrated that in the VCS of certain species, MHC isoforms are expressed that are not present in the ordinary myocytes of the force producing myocardium (Gonzalez-Sanchez and Bader, 1985; Gorza e t al., 1986, 1988). In a parallel study to the one presented in this paper, in which a panel of antibodies against different MHCs, including embryonic- and slow-tonic-MHC, was used we did not observe such a phenomenon (A. Wessels, unpublished observations). Apparently, the MHC expression in the developing VCS reveals a species-specific pattern. MHC lsoforms in the Developing Outflow Tract With respect to the expression of MHC isoforms, three phases in the developing OFT can be distinguished: I. Similar to the situation in chicken and rat (De Jong et al., 1987,1988; De Groot et al., 1989), coexpression of a- and P-MHC is observed in the myocardium of the OFT in early embryonic human heart. For the developing chicken heart it was demonstrated that in the coexpressing OFT and AV junction, the cardiac impulse is conducted relatively slowly (De Jong et al., 1988 and in preparation). Based on a detailed study of the contraction patterns of these young embryonic hearts (Arguello et al., 1986) i t was suggested that slow conduction in the relatively long OFT might be impor- tant in the prevention of backflow of blood during ventricular relaxation. In addition, in our study on the spatial distribution of CK isoforms in the developing human heart (Wessels et al., 1990b), we observed t h a t in the areas of MHC coexpression in the OFT, very small amounts of creatine kinase are expressed. In the same study it was demonstrated that in areas in which the cardiac impulse is known to be conducted very slowly (e.g., the AV junction) creatine kinase expression was also very low. These combined observations point to a slow conduction in the OFT of the early embryonic human heart and underline the hypothesis that the early OFT functions as a peristaltoid pump. 11. Strong single expression of P-MHC is observed in the myocardium around the developing semilunar valves in the late embryonic stages (i.e., approx. stage 23) and in the early fetal hearts (approx. 8-12 weeks of development, data not shown in this paper). In these cardiomyocytes we observed also a strong expression of creatine kinase (Wessels et al., 1990b). These data suggest that around the semilunar valves of aorta and pulmonary trunk myocardium with a potentially high contractile capacity is present. This myocardium might assist the immature valves in the prevention of backflow of blood during relaxation of the ventricles when the OFT can no longer function effectively as a peristaltoid pump due to its decreased length. A study in which two fetuses with aplasia of the semilunar valves t h a t died only after 12 weeks of development were described (Hartwig et al., 1990) indicates that prior to this developmental stage the absence of semilunar valves may be compatible with life. 111. 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