Ultrastructural and experimental evidence of myocardial cell differentiation into connective tissue cells in embryonic chick heart

Journal

of Molecular

and Cellular Cardiology

( 1978)

10, 307-3 15

Ultrastructural and Experimental Evidence of Myocardial Cell Differentiation into Connective Tissue Cells in Embryonic Chick Heart CARLOS

ARGUELLO, M. VICTORIA DE LA CRUZ AND CONCEPCION SatiNCHEZ Instituto National de Cardiologia, Debartment of Embryology, Mexico, D.F., Instituto Venezolano de Cardiologia and Department of Enfermedades Cardiovasculares, MSAS, (Received

14 February

1977,

Caracas,

Venezuela

accepted in revisedform

June 1977)

29

C. ARG~~ELLO, M. V. DE LA CRUZ and C. SANCHEZ. Ultrastructural and Experimental Evidence of Myocardial Cell Differentiation into Connective Tissue Cells in Embryonic Chick Heart. Journal of Molecular and Cellular Cardioloa (1978) 10,307-315. The embryonic cardiac cells originate from a very homogeneous population of myocytes which, by differentiation, will form the atria, the ventricles, the conuS and the truncus arteriosus. In the early stages of chick embryo development (3+ days), the wall of the truncus is formed by a layer of myocardial cells, and at about the 7th day it is formed by layers of fibroblasts, collagen, elastin and smooth muscle. The purpose of this investigation was to elucidate the ultrastructural events which occur during the development of the truncus, and to investigate if 5-bromodeoxyuridine (BrdU) could modify its differentiation. Our sequential ultrastructural observations of the myocardial layer of the truncus showed that the myocardial cells progressively acquire a fibroblastic phenotype. This was shown by the continuous diminution in the content of myofibrils and the increase in collagen, elastin and an amorphous granular material in the intercellular space. On the other hand, 5-bromodeoxyuridine prevented the “transformation” of the myocardium into fibroblast cells, and the truncus remained as a beating structure. From our results, we conclude that the myocardial cells of the truncus are able to differentiate into cells of a fibroblastic type. KEY

WORDS:

Fibroblast dine.

Embryonic chick heart; cells; Myocardial-fibroblast

Truncus; Myocardial cells; transformation; Ultrastructure;

Cell differentiation; 5-Bromodeoxyuri-

1. Introduction One of the distinguishing characteristics of developing myocardial cells is their ability to synthesize actin and myosin molecules [S, la]. These proteins, combined with tropomyosin and other proteins by an assembling process, give rise to the intracellular myofibrils, which, because of their biochemical properties make the myocardial cells contractile. In addition, it has been recently found that embryonic heart cells also synthesize several components of the extracellular matrix, such as sulfated glycosaminoglycan molecules [Is], glycoproteins [17] and a type I-like collagen [9]. 0022-2828/78/0401-0307

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(London)

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The biological significance of these findings is not well understood, although it is possible that some of the molecules of the extracellular matrix have a direct influence on the mechanisms of differentiation of the cells which form part of the adult heart [16]. In this report, we present ultrastructural and experimental evidence of the ability of the myocardial cells of the wall of the truncus to differentiate into cells of the connective tissue type. In the discussion, we analyze our findings, taking into consideration the possible role of the components of the extracellular matrix in the differentiation of the wall of the truncus.

2. Materials Preparation

and Methods

of the embryonic hearts for light and electron microscopic study

Embryonic chick hearts (white Leghorn) of 3, 3+, 4, 43, 4Q, 5, 5+, 52, 6, 6+ and 7 days were used. The age of the embryos was establishedaccording to Hamburger and Hamilton [8] and the degree of development of the hearts was determined following the description by De La Cruz et al. [4]. The hearts were removed from the embryos and washed several times with salt solution [25], and then fixed in glutraldehyde 2.5% in 0.1 M cacodylate buffer, pH 7.2, for 1 h at room temperature. The truncus was dissectedfrom the heart following the limits already found [.5l and then fixed for 1 h in 1% osmium tetroxide in cacodylate buffer. After fixation the truncus was dehydrated in increasing concentrations of ethanol and embedded in Epon. Transversal and longitudinal sectionsof 1 pm were done from proximal to distal portions of the truncus. Since no significant regional differences were observed in this study, only transversal sectionsare described. Thin sections were stained with toluidine blue. Ultrathin sections stained with uranyl acetate and lead citrate were observed with the electron microscope Carl ZeissEM9.

Treatment of the embryonic heart with 5-bromodeoxyuridine

(BrdU)

We studied the effect of BrdU on the development of the wall of the truncus, since this substance has been shown to inhibit the synthesisof specific cell products in a number of various cell types in differentiation [I, 11, 201. We injected 1.O pg of BrdU into the pericardial cavity of 30 chick embryos of 34 days, and 30 control embryos were injected with an equal amount of thymidine. Both groups of embryos were incubated until they reached the 6th day of development. The embryos were then removed from the egg and the truncus was dissected from the hearts and processedas above for light and electron microscopy study. Ten embryos of each group were used for structural studies.

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3. Results Development of the wall of the truncus

(a) The truncusof the heart of the chick embryo of 3+ to 4 days Since the truncus of the heart of the chick embryo of 33 days has a similar histologic structure to the truncus of 4 days, they will be described together. The truncus of 33 days [Plate 1(a)] is formed externally by a layer of several myocardial cells [Plate 1(b)]. These cells [Plate 1(c)J have abundant myofibrils in the cytoplasm which have different degreesof orientation and assembly. Some bundles of these myofibrils are inserted into the inner surface of developing desmosomesand intercalated discs.The rough endoplasmic reticulum is poorly developed, but there is a large quantity of polysomes and free ribosomes. Free cells of the mesenchyme surrounded by cardiac jelly and extracellular materials are found below the myocardium [Plate 1(b), me]. The lumen of the truncus is lined by a unicellular layer of endocardium [Plate 1(b), e]. In the chick embryo of 4 days, a new and distinct layer of cells is formed, the epicardium [Plate 1(b) ep]. The epicardium and myocardium are clearly separated by an intercellular space, but in some regions they are in intimate contact [Plate 1(b)]. Interestingly enough, in the intercellular space of the myocardium, bands of collagen-like fib& associatedwith an amorphous granular material are frequently observed. These are in contact with the myocardial cell membrane in some areas [Plate 1(d) arrows]. (b) Truncus of 4$ days At 4%days of truncus development [Plates 2(a), (b)], cells of the myocardium with a hybrid phenotype [Plate 2(c)] are found for the first time; that is, they can be recognized as myocardial cells becausethey show myofibrils and developing intercalated discs. They also resemble fibroblasts becauseof the well developed endoplasmic reticulum [Plate 2(c), er]. The degree of organization of the myofibrils seen in the cytoplasm seemsto decrease; whereas in the intercellular space of the myocardium the extracellular granular material associated with collagen-like fibrils seemsto increase [Plate 2(d)]. (c) Truncusof 5 to 54 days From day 5 to 5s the myocardial layer of the truncus begins to change into cells with a fibroblast phenotype [Plates 3(b) and 4(b)]. These cells progressively acquire a circular arrangement around the truncus [Plate 4(b)], and start the synthesis of extracellular components characteristic of connective tissue [Plate 4(c)]. At the 5th day of development, the ridges of the truncus have already fused, causing the septation of the developing aorta and pulmonary arteries [Plate 3 (b)] . Since this septation is internal, somehistological structures of the external wall of the truncus are common to both arteries. However, one of the arteries with early developing semilunar valves has a wall composed predominantly of myocardial

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cells [Plate 3(c)], whereas the other shows only fibroblast-like cells [Plate 3(d)]. This circumstance makes it easy to understand what actually happens to the myocardial cells during the development of the wall of the truncus. These cells have very conspicuous intercellular spaces, giving the impression that they are separating from each other [Plate 3(c)] and in some areas from the myocardial layer. Closer observation of the myocardial cells shows that they remain in contact by means of the apposition of unspecialized regions of the plasma membrane, as well as by desmosomes [Plate 3 (e)]. The cytoplasmic content of myofibrils is well represented in some cells [Plate 3(e)], while in others the endoplasmic reticulum predominates, with the concomitant diminution of myofibrils. Furthermore, the myocardial cells acquire a stellate appearance quite similar to that of the mesenchyme cells [Plate 3 (c) arrows]. We already noted our belief, based on the characteristics of the myocardial cells, that these cells actually are transformed into cells with a mensenchymal phenotype, and then are transformed in fibroblast-like cells. We found evidence for this when we directed our attention to the cells with a mensenchymal phenotype, closer to the myocardial layer [Plate 3(c)]. Some single cells or small groups of them [Plate 3(f)], h a d small bundles of myofibrils associated with Z-bands in the cytoplasm [Plate 3(f)]. I n addition, the intracellular structures of these cells are similar to the myocardial cells shown in Plate 3(e). In the embryonic heart of 54 days [Plate 4(a), (b)], the wall of the truncus is formed by the epicardium and the endocardium. Between them there is a thick layer of cells with a fibroblast phenotype [lo]. Th ese cells are rich in free ribosomes and the rough endoplasmic reticulum is poorly developed [Plate 4(c)]. In the periphery of the fibroblasts there are bundles of fibrils probably related to the process of fibrogenesis. The amorphous granular material and the collagen fibrils present in the intercellular space have the same orientation as the cells. After the 6th day [Plate 5(a)], the fibroblast-like cells of the truncus [Plate 5(b)] begin to show a more defined circular pattern around this structure. The number and organization of the collagen fibrils increase and the elastic fibrils are identified for the first time in the intercellular space [Plate 5 (c), (d)]. Treatment

with 5-bromodeoxyuridine

(BrdU)

Although the observations already described support the view that the myocardial cells of the truncus are transformed into cells with a fibroblast phenotype, more direct evidence was desirable. In order to obtain this, we tried to prevent the transformation of myocardial cells into cells of connective tissue by means of BrdU. This substanceinterferes with the synthesisof new molecules, such as collagen and acid mucopolysaccharides of the extracellular matrix [13]. The embryos of 39 days, which were left under the action of BrdU for 23 days reached the 6th day of development. At this time the truncus remains qualitatively unchanged [Plate 6(a)]. The cells of the epicardium, however, continued dividing and a layer of approxi-

PLATE 1. (a) A diagram of the heart of the chick embryo of 4 days is shown. The region under the shaded area corresponds to the truncus. [In Plates 2(a), 3(a), 4(a) and 5(a) it will be the same.] Truncus (T); conus (C) ; ventricle (V); auricle (A). (b) Light micrograph of a cross-section of the truncus. On the outermost region, the epicardium (ep) and iyocardium (m) are observed. Intercellular spaces are frequent in the myocardium (arrows). Further down, mesenchymal cells (me) and the endocardium (e) are seen. x 300. (c) Electron micrograph of the myocardial cells showing the arrangement of myofibrils (mf) in the cytoplasm and the developing intercalated discs (id). x 18 000. (d) In the intercellular space of the myocardium, bands of collagen-like fibrils (c) associated with an amorphous granular material (am) are frequently observed. In some areas the amorphous x 20 000. granular material is in contact with the myocardial cell membrane (arrows). PLATE 2. (a) Diagram of the heart of the chick embryo of 43 days. (b) In this cross-section of the truncus, the myocardial cells (m) are very well identified. x 800. (c) A cell of the myocardium with a hybrid phenotype is shown. This cell has a very well developed endoplasmic reticulum (er) as well as myofibrils and intercalated discs. x 18 000. (d) In this electron micrograph collagen-like fibrils (c) and amorphous granular materials (am) are abundantly represented in the intercellular space. x 18 000. PLATE 3. (a) Diagram of the heart of the chick embryo of 5 days. (b) At this stage, the ridges of the truncus have already fused forming the internal septation of the arteries (P) . The wall of the truncus, which leads to development of the aorta (AO) and pulmonary pulmonary artery is composed predominantly of myocardial cells (m), whereas the aorta is composed of fibroblast-like cells (f). x 150. (c) and (d) are photographs of the walls of the pulmonary artery and the aorta, respectively. (c) The intercellular spaces of the myocardium are very conspicuous (arrows), giving the impression that the cells are separating from each other. x 800. (d) In this micrograph only fibroblast-like cells are observed (f). x 800. (e) This is an electron micrograph of a myocardial cell of the pulmonary artery. This cell remains in contact with other cells by the apposition of the plasma membrane (arrows) and desmosomes (d). The content of myofibrils (mf) in some cells is very abundant. x 16 000. (f) This is an electron micrograph of two cells laying very close to the myocardium layer shown in Plate 3 (c) (arrowhead), These cells present small bundles of myofibrils associated with Z-bands in the cytoplasm (circles). x 16 000. The insets are enlargements of the regions encircled. x 20 000. PLATE 4. (a) Diagram of the heart of the chick embryo of 5f days. (b) In this light micrograph, the wall of the truncus is made up of the epicardium (e), cells with a fibroblast phenotype (f) and the endocardium. x 800. (c) Under the electron microscope bundles of fibrils (fi) are seen in the periphery of the fibroblast. The cytoplasm is rich in free ribosomes (ri). In the intercellular space the collagen fibrils (c) and the amorphous materials have the same orientation as the fibroblasts. x 5700. PLATE 5. (a) Diagram of the heart of the chick embryo of 6 days. (b) This is a light micrograph of the wall of the truncus, in which the fibroblasts show a more x 800. regular pattern around this structure. cc) In this electron micrograph the elastin fibrils (el) are identified for the first time in the intercellular space of fibroblasts. x 18 000. (d) Observe how the collagen fibrils (c) are organized in bundles very close to the cell membrane. x 20 000. PLATE 6. (a) and (b) are two cross-sections of the truncus of chick embryos of 6 days. (a) A section of the truncus treated with 1 pg of 5-bromodeoxyuridine. (b) A section of the truncus of a control treated with 1 pg of thymidine. x 800. (c) This electron micrograph shows several myocardial cells after treatment with 5-bromodeoxyuridine. Observe the abundance of myofibrils in the cytoplasm. x 12 000.

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mately three cells was formed. Similarly, it was observed that the myocardium became thicker [Plate 6(a)] and the cells showed an increase in the number of myofibrils [Plate 6(c)]. This is more evident when the content of myofibrils of these cells is compared with those of embryos of 3$ days [Plate 1 (c)l. The mesenchymal cells have a stellate appearance. These differ from those of 3-4 days, which are rounded [Plate 1 (b)]. On the other hand, the controls of 6 days have lost the myocardial layer of the truncus and the fibroblast-like cells are the main constituents of this structure [Plate 6(b)]. Wh en observed under the electron microscope, they are similar to the one shown in Plate 5(c). 4. Discussion In the early stages of development of the chick embryo (10 to 13), the heart is formed by the myocardium and the endocardium [14’J, the myocardium consisting of a very homogeneous population of myocytes. Later, at about stage 17, two new components can be identified: the epicardium, the cells of which partially cover the myocardium, and the cells of the endocardial cushions of the conus and truncus. It has been suggested that the epicardium is derived from cells completely different from those of the myocardium [15]. It has also been suggested that the epicardial cells migrate from the septum transversum to the external surface of the myocardium [.%I. Contrary to these views, there is the hypothesis that the epicardium is formed “in situ” by “dedifferentiation” of the myocardial layer [21]. If the epicardium is going to develop “in situ” from the underlying myocytes, one would expect to find cells with a transitory phenotype. Manasek [15] was unable to find such transitory cells, ahhough he mentioned that the outer cells of the myocardium contained fewer myofibrils than did the deeper cells. In the light of our present findings, we believe that if a more sequential study were conducted, the probability of seeing cells with an intermediate morphology would increase. The same problem occurred in elucidating the process of the differentiation of the wall of the truncus. For instance, we found that from day 5 to 53 we could not observe any transitory steps. In order to observe such transition, we had to include embryos of 5* days. Consequently, we believe that if future work is done in this direction, it would be desirable to study the developmental events of the heart in periods of even less than 6 h. The structural organization of the endocardial cushions of the cone-truncus is more complex. There are free cells similar to the mesenchyme (whose origin is probably from the endocardium) and the accumulation of extracellular materials rich in acid glycosaminoglycan molecules [7]. Some of these molecules are synthesized by the mesenchyme and others by the myocardial cells. Which molecules are synthesized by each cell is unknown, but it seems that the myocytes contribute to a lesser degree. It has been suggested [16’j that the acid glycosaminoglycan molecules of the

312

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AL.

extracellular matrix interact with cations such as Na+, and in this way regulate the ionic milieu. On the other hand, it also has been suggested [l6] that the components of the matrix could stimulate the proliferation of the endocardial cells and give rise to endocardial cushions. We would like to propose that the anionic character of the molecules of the extracellular matrix could induce the differentiation of the myocytes of the wall of the truncus into cells of fibroblast lineage. Our sequential structural study of the truncus demonstrated that the myocardial layer is transformed into connective tissue cells. At about the 3rd day of the chick embryo development, the truncus is composed of the myocardium, the extracellular matrix of the endocardial cushions, the free cells of the mesenchyme and the endocardium. As far as we know, there is no reason to believe that the mesenchyme cells and the endocardium have any direct influence on the differentiation of the myocardium. Therefore, the molecules of the extracellular matrix remain as the single candidate. Several experimental studies [Z, 13, 19, 23, 241 give support to the idea that the acid mucopolysaccharides play a very important role in the control of cell proliferation and cell differentiation. For example, Nevo and Dorfman [19] have shown that various polyanionic molecules stimulate the synthesis of chondroitin sulfate during the differentiation of chondrocytes cultured in suspension. In other cases [24] a conditioned culture medium containing a glycoprotein with sulfhydryl and disulfide groups produces an increase in the incorporation of [s%] 04 into hyaluronic acid by chondrocytes. With respect to the mechanism of action of the acid mucopolysaccharides on the target cells, there is general agreement that they could interact directly with the cell surface [13, 241. Unfortunately, most of this evidence comes from experiments done in culture and, as pointed out by Manasek [16], it is difficult to know if they actually represent normal situations “in ~itzJ’. In our “in vz’vo” system, we observed [Plates 1(b), (d) and 2(d)], that one of the events prior to myocyte “transformation” is the appearance of an amorphous granular material associated with collagen-like fib& in the intercellular space. Since this extracellular material is intimately associated with the myocytes, it is possible that they were responsible for its synthesis. On the other hand, it is difficult to believe that these extracellular materials result from a diffusion process from the endocardial cushions to the myocardial layer. It has been beautifully demonstrated by Manasek [17] that the myocytes can synthesize collagen I-like molecules and glycosaminoglycan substances of the extracellular matrix, which makes our suggestion more feasible. The question is whether the amorphous granular materials and the collagen-like fibrils of the intercellular space of the myocardium have anything to do with its further differentiation. In the event that the extracellular matrix molecules have any influence over the myocytes, we must assume that this effect is the stimulation of

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the synthesis of more extracellular materials and the concomitant reduction in the synthesis of actin and myosin molecules. In fact, our observations show that the myofibrils decrease to the point that they are difficult to identify [Plate 3(f)]. Therefore, there is a stage in the development of the truncus in which it is impossible to ascertain which cells with a fibroblast phenotype come from myocytes,and which from mensenchyme cells of the endocardial cushions. Our results from the treatment of chick embryo hearts with 5-BrdU showed that the myocytes of the wall of the truncus were prevented from being transformed into fibroblast cells. However 5-BrdU did not inhibit the formation of new myofibrils [Plate 5(c)], nor did it interfere with cellular division, as shown by the increase in thickness of the myocardial layer and the epicardium. All these findings are in agreement with the experimental evidence of Chacko and Joseph [3].They showed that 5-BrdU did not prevent the growth and differentiation of cardiac cells, except when cardiac myogenic precursor cells were used. They thought that 5-BrdU did not interfere with muscle differentiation, because mRNA specific for cardiac muscle was already synthesized. How can we explain the inhibitory effect of 5-BrdU on the “transformation” of myocytes into fibroblasts ? We can offer one possible explanation: that 5-BrdU was incorporated into the DNA of myocytes and produced changes in the genes related to the synthesis of collagen and glycosaminoglycan molecules. If this were the case, then the myocytes would be blocked in their transformation to the fibroblast phenotype, and would retain the muscle characteristics. This possibility is based on the evidence presented by Levitt and Dorfman [12], Abbott and Holtzer [I], in which they showed that 5-BrdU inhibited the synthesis of specific molecules of the extracellular matrix of chondrocytes. It is clear that more experimental work should be done in order to understand which are the normal interactions established between the myocytes and the extracellular matrix, as well as to know which mechanisms are involved in the control of their differentiation. From the evidence presented in this work we arrive at the conclusion that the myocytes of the wall of the truncus are able to differentiate “in viva” into cells with a fibroblast phenotype. This property of the myocytes is not restricted to the truncus only. Preliminary observations indicate that the same thing happens to the myocytes of the wall of the atrio-ventricular canal, which are also transformed into connective tissue cells [22]. It is very interesting to point out that both regions are quite similar in structure, therefore we suspect that the same developmental events occur in the wall of the atrio-ventricular canal. REFERENCES 1.

ABBOTT,

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PATTEN, B. M. The development of the heart. In The Pathology of the Heart, S. E. Gould, Ed. Springfield, Illinois: Chas. C. Thomas (1953). PATTEN, B. M. The development of the sinoventricular conduction system. University of Michigan Medical Bulletin 22, l-2 1 (1956). REGELSON, W. The growth-regulating activity of polyanions : a theoretical discussion of their place in the intercelIuIar environment and their role in cell physiology. Advances in Cancer Research 11, 223-304 (1968). SOLURSH, M., MEIER, S. & VAEREWYCK, S. Modulation of extracellular matrix production by conditioned medium. American