/. Embryol. exp. Morph. Vol. 21, 2, pp. 271-84, April 1969
271
Printed in Great Britain
Myocardial cell death in the embryonic
chick ventricle
By FRANCIS J. MANASEK 1
From the Division of Cardiology, Children's Hospital Medical Center,
Boston, Mass., 02115, U.S.A.
The degeneration and death of cells is a normal occurrence in renewing cell
systems in mature animals. Cell death is also a frequent event during embryonic
development and appears to be a normal, and perhaps necessary constituent
process of morphogenesis (see Glucksmann (1951) and Saunders (1966) for
reviews). The events of cell degeneration and death during ontogenesis have been
observed in many developing tissues, even those which have a relatively static
cell population in the adult, such as the nervous system (Hamburger & LeviMontalcini, 1949).
The widespread occurrence of this phenomenon and its apparent relationship
to morphogenesis in some developing organs has led to the suggestion that, in
some instances, cell death may be a regulatory mechanism for controlling
organogenesis (Saunders, Gasseling & Saunders, 1962), perhaps by compensating
for overproduction of cells or by removing cells which cannot adapt to a
changing environment (Glucksmann, 1951).
The degenerative changes exhibited by dying cells have been extensively
studied with light-microscope techniques during normal ontogeny (Bessis, 1964;
Glucksmann, 1965) and under experimental conditions (Saunders et al. 1962).
The electron-microscope investigation of Bellairs (1961) described cell death
occurring in relatively undifferentiated cells of the early chick embryo, and
Behnke (1963) characterized some aspects of this process in developing intestinal
epithelium. Although Rumery & Blandau (1964) described the light-microscope
appearance of degenerating embryonic heart cells in culture, little information
has been available concerning cell death in the intact heart. The present paper
reports on a series of electron-microscope observations on dying cardiac muscle
cells in the intact embryonic ventricular myocardium at a time when the cells
have already formed myofibrils and have the characteristics of the differentiated
state.
1
Author's address: Department of Embryology, Carnegie Institution of Washington,
115 W. University Parkway, Baltimore, Md., 21210, U.S.A.
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F. J. MANASEK
MATERIALS AND METHODS
Fertile white leghorn eggs were incubated at 38 °C for 7 days. Eggs were
opened and embryos removed and placed in cold (0 °C) glutaraldehydeformaldehyde fixative (Karnovsky, 1965) buffered at pH 7-6 with 0-2 M cacodylate buffer. Hearts were rapidly dissected free and placed in fresh fixative for
a period of 15 min, briefly rinsed in 0-2 M cacodylate buffer, pH 7-6, and transferred to 1 % OsO4 for an additional 2 h. Some specimens were block stained
with uranyl acetate (Trelstad, Hay & Revel, 1967) prior to dehydration.
Although this procedure enhanced the contrast of membrane and cytoplasmic
filaments, it adversely affected glycogen and destroyed its characteristic particulate appearance.
Entire hearts were embedded in Araldite and were sectioned with glass or
diamond knives. Sections were placed on uncoated copper grids, stained with
lead citrate (Venable & Coggeshall, 1965) and examined in an RCA EMU-3F
microscope, operated at 50 kV.
RESULTS AND DISCUSSION
During the course of an electron-microscope study of the embryonic chick
ventricular myocardium, few examples of dying cells were seen in specimens
younger than about 4 days of incubation. Thereafter, occasional examples of
dying cells were seen scattered throughout the ventricular wall.
Although it has been suggested (Glucksmann, 1965) that embryonic cell
death may reflect an inability of an immature cell to differentiate, it is interesting
to note that myocardial cell degeneration occurs after cells have already
synthesized large amounts of contractile proteins. Thus, myoblast-myocyte
transformation which occurs prior to Hamburger & Hamilton (1952) stage 12
(Manasek, 19686) does not appear to resulti n a wave of degenerations. The cells
present in later developmental stages appear to have differentiated successfully,
suggesting that in this organ, cell death is not the immediate result of an intrinsic
Fig. 1. This dying cell, situated in the outer layer of the ventricular myocardium,
exhibits marked cytoplasmic as well as nuclear changes. The cytoplasm is largely
comprised of a dense matrix containing scattered clumps of free ribosomes (inset).
A large pool of glycogen (G) has become sequestered in one region of the cytoplasm. Note the wide perinuclear cisterna (P) surrounding the pyknotic nucleus.
A nucleolus (/?) can be seen in the dense karyoplasm, as well as clumps of chromatin.
A lipid droplet (/) is close to the nucleus. The entire cell appears to be breaking
free from its normal neighbors and the remnants of an adhering junction (arrow,
inset) are visible. The region at the top of the plate shows the developing subepicardial space which contains mesenchymal cell processes and developing collagen
bundles (C). This and all subsequent micrographs are of ventricular cells from
7-day chick embryos. The scale lines in the lower right on all the illustrations
represent 1/t.
Myocardial cell death
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F. J. MANASEK
Fig. 2. Although this dead cell is still surrounded by normal cardiac muscle cells, it
has rounded up and no longer demonstrates any junctions with its neighbors.
Note the disrupted myofilaments which are scattered throughout the dense cytoplasmic matrix of the dead cell. Compare these scattered filaments to the tangential ly
sectioned myofibrils in the normal cell at the upper right. This specimen was block
stained with uranyl acetate, a preparative technique which enhances filament and
membrane contrast, but which disrupts the normal appearance of glycogen pools,
causing them to appear as seen in the center of the cell (G).
Myocardial cell death
275
Fig. 3. The pleomorphic nucleus of this dead cell shows two distinct clumps of
chromatin (C) and a widened perinuclear cisterna. The beaded structure (arrow)
seen in the perinuclear cisterna may have been produced by disruption of the
membranes of the nuclear envelope. Dense mitochondria (m) are seen in the
cytoplasm and some membranous whorls are present. A few scattered filaments
are visible (F) as well as lipid droplets (£). Specimen block stained with uranyl
acetate.
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F. J. MANASEK
Fig. 4. Whereas the cells in the previous plates were still closely surrounded by
adjacent developing cardiac muscle cells, this example is seen situated in one of the
intercellular spaces which characterize the developing ventricular myocardium.
Nuclear chromatin has clumped on one side of the nucleus, which is surrounded
by a deformed perinuclear cisterna. Two pools of glycogen (G) are still intact in the
cytoplasm, and several membranous whorls (arrows) are present. Specimen was
block stained with uranyl acetate.
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Fig. 5. The degenerating cell in this plate is being engulfed by a phagocyte (M). The
large accumulation of glycogen present serves to identify the cell as myocardial.
Although this material was not block stained with uranium, a filament is visible
{MF). Vacuoles, present in large numbers in this specimen, are occasionally present
in degenerating cardiac muscle cells, but are not a universal finding. Compare the
size and density of the degenerating mitochondria to those in the normal adjacent
muscle cells. Mitochondrial changes during cardiac muscle cell death are variable.
They sometimes appear to develop an increased matrix density.
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F. J. MANASEK
inability of some myocardial cells to undergo differentiation. However, the
possibility that a small number of myocardial cells may be unable to continue
their development, and consequently die, is not ruled out.
Although the embryonic myocardium, derived from splanchnic mesoderm, is
an epithelial tissue and maintains this type of organization during formation of
the functional tubular heart (Manasek, 19686), neither mitotic nor degenerative
events were limited to specific layers. Although mitotic figures were often
observed in the 7-day-old ventricular myocardium (Manasek, 1968#) it was
impossible to ascertain whether the degenerative events reported in this paper
resulted from aborted divisions. Cell death was an extremely rare phenomenon
in developing ventricular trabeculae but there did not appear to be any specific
distribution of dying cells in the remainder of the ventricular wall. This essentially
random distribution of degenerating myocardial cells strongly suggests that cell
death does not play a readily recognizable role in determining the adult gross
structure of this organ.
The earliest morphological changes indicative of myocardial cell degeneration
are similar to those seen in other embryonic cells. The nucleus becomes dense
and pleomorphic (Glucksmann, 1951). Condensing chromatin often forms dense
patches around the nuclear periphery (Bellairs, 1961; Saunders & Fallon, 1966)
and within the nucleoplasm (Fig. 1). During these early stages of nuclear
condensation, the nucleolus remains visible. In addition to these nucleoplasmic
changes, there is also a widening of the perinuclear cisterna (Fig. 1).
As degeneration proceeds, normal cell shape is dramatically altered. Dying
myocardial cells gradually assume a more rounded shape and lose their connexions with adjacent cells. Cardiac muscle cells are normally closely bound to
their neighbors by several types of intercellular junctions, notably intercalated
discs and adhering junctions such as desmosomes. Breakdown of these sites of
cell attachment invariably precedes rounding up of the dying cell. Once the cell
has rounded it is completely devoid of junctions with neighboring muscle cells
(Figs. 2, 3). The loss of cell attachments probably facilitates not only rounding
of cells but also their eventual expulsion from the surrounding tissue mass
(Fig. 4).
The loss of normal cell shape is accompanied by a breakdown of cytoarchitecture and an increase in matrix density (Fig. 1). This apparent condensation produces a coarse, granular cytoplasmic matrix containing discrete particles
Fig. 6. In this plate, a degenerating myocardial cell characterized by a prominent
central pool of glycogen and a very dense cytoplasmic matrix has been engulfed by
a phagocyte. This specimen was found in the developing subepicardial space
surrounding the ventricle of a 7-day-old chick embryo. Its position, several microns
away from the outer myocardial cell layer, strongly suggests that at least some of
the ingested dead cells are removed from the myocardial wall. This specimen was
block stained with uranyl acetate, which accounts for the appearance of the glycogen.
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F. J. MANASEK
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281
(Figs. 2, 3), which probably represent clumps of free ribosomes (Bellairs, 1961).
Disruption of myofibrils occurs throughout the cytoplasm (Fig. 1) and appropriate techniques of tissue preparation reveal only scattered myofilaments
(Figs. 2, 3). The large pools of glycogen which are a characteristic of developing
ventricular muscle remain intact (Figs. 1, 2, 4-7).
The continued presence of scattered cytoplasmic myofilaments (Weber, 1964)
serves as a marker to identify the degenerating cells as muscle cells (Figs. 2-5).
Presence of large amounts of glycogen also serves the same purpose since, in the
developing heart, large pools of glycogen are found only in cardiac muscle cells
(Manasek, 1968 c), and not in epicardium, endocardium or other non-myocardial
elements of the organ.
Lipid droplets remain recognizable (Figs. 2-4), but no unusual accumulations
of lipid (Glucksmann, 1951) were noted in degenerating ventricular myocardial
cells.
In an earlier light-microscope study of cultured embryonic heart cells,
Rumery & Blandau (1964) were able to identify dying muscle cells. Degenerating
cultured cells appear to undergo a somewhat different sequence of events than
those described in the present paper and these workers ascertained that mitochondrial changes, in the form of vesiculation, appear first. Comparable early
mitochondrial changes were never observed in the present study. Indeed, it is
difficult to assign any degenerative sequence to mitochondria. In some dying
cells these organelles appear swollen and have a relatively light matrix density
when compared with normal mitochondria of neighboring cells (Figs. 1, 5). On
the other hand, mitochondria often appear to contain a matrix of greatly
increased density (Figs. 3, 4) which makes them almost indistinguishable from
the surrounding dense cytoplasm. Whereas Rumery & Blandau noted that
myofibrils remained intact even after disintegration of cultured cells, the present
observations demonstrate a complete early disruption of myofibrillar structure
(Fig. 1) although scattered myofilaments remain recognizable (Fig. 2). Indeed,
the early disruption of myofibrillar organization seen in dying myocardial
muscle appears similar to that observed in intact Xenopus skeletal muscle by
Weber (1964). The differences between the sequence of degenerative events
observed in intact specimens and those observed by Rumery & Blandau in
cultured cells may be related to different causes of death.
Fig. 7. This plate contains three different cell types. The endothelial cells {E) in the
lower portion are part of a developing coronary blood vessel. The vessel is partially
collapsed and the lumen (L) is quite narrow. The absence of particulate glycogen in
this cell type is striking when the cytoplasm of the developing vascular endothelium
is compared to the normal, developing cardiac muscle cell in the upper right.
A phagocyte containing an ingested dead cell is seen in the perivascular space, and
a portion of its nucleus (/?) can be seen in this section. The dead cell ingested by this
phagocyte has undergone a considerable degree of lysis. Its nucleus is extremely
dense and very little recognizable cytoplasmic structure, with the exception of a
pool of glycogen (C), remains.
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F. J. MANASEK
Following the rounding up of the dying cell, nuclear changes become more
profound. Most of the chromatin becomes localized in a granular dense patch
(Figs. 3, 4). However, scattered dense particles persist throughout the remainder
of the condensed nucleoplasm (Fig. 5).
The foregoing degenerative changes all occur prior to the ingestion of the
dying cells by phagocytes, confirming the suggestion that phagocytes may not
be the direct cause of cell death (Saunders, 1966). Although Saunders & Fallon
(1966) have reported difficulty in recognizing dying cells in the posterior necrotic
zone of the chick limb bud prior to their ingestion by phagocytes, the present
study shows that extensive necrotic changes occur in myocardial cells before
they are phagocytized. These differences may, in part, be related to differences
in cell type. Degree of development exhibited by a cell prior to degeneration
does not appear to determine the extent of degeneration occurring prior to
phagocytosis, since although the myocardial cells studied in the present investigation were well-developed, extensive degeneration in the early blastoderm
(Bellairs, 1961) appeared to occur also without the intervention of phagocytes.
Rounded degenerating cells are expelled from surrounding normal tissue into
extracellular space (Holtfreter, 1945) as observed in the present study (Fig. 4).
It is suggested that the expulsion of dead cells from the normal tissue mass may
facilitate their ingestion by phagocytes (Fig. 5). Although this event may occur
within the intercellular space of the myocardial muscle mass, phagocytes
bearing ingested dead cells are often found in the subepicardial space (Plate 6)
many microns away from the outer surface of the myocardium. Such observations suggest that the dead cells may be removed from the myocardium and
may account for the rarity with which they are seen.
Following ingestion by phagocytes, dead myocardial cells may still retain
recognizable organelles such as mitochondria (Fig. 5) and continue to exhibit
identifiable glycogen (Figs. 5-7). Degeneration continues and is finally characterized by the production of a dense mass containing no recognizable filaments
or organelles (Fig. 7). Nuclear condensation during this final phase of degeneration results in a uniformly electron-opaque structure. However, even at this
advanced stage of degeneration, a significant amount of glycogen remains
present in the ingested mass (Fig. 7). The particles comprising this pool of
glycogen do not differ from those in adjacent normal cells, suggesting that this
cytoplasmic inclusion is particularly resistant to breakdown, both by the dying
cell and by the phagocyte ingesting it.
SUMMARY
1. Myocardial cell death in the embryonic chick heart is a rare event and was
not observed before about the fourth day of development.
2. The electron-microscope reveals extensive nuclear and cytoplasmic changes
occurring while the dying cell is still surrounded by normal tissue.
Myocardial cell death
283
3. Following loss of intercellular junctions, the degenerating cells round up
and are expelled into the large intercellular spaces characteristic of the 7-dayold myocardium.
4. Disrupted myofibrils and large pools of glycogen remain visible in the
increasingly electron dense cytoplasm of the degenerating cells.
5. Further degeneration occurs following ingestion by phagocytes. Phagocytes bearing engulfed dead muscle cells have been observed both within the
myocardium and in the subepicardial space, suggesting that some dead cells
may be removed from the developing organ.
RESUME
Mort des cellules myocardiques dans le ventricule de Vembryon de Poulet
1. La mort des cellules du myocarde dans le coeur embryonnaire du poulet
est un evenement rare et n'a pas ete observee avant le 4e jour du developpement.
2. Le microscope electronique revele des modifications nucleaires et cytoplasm iques etendues qui surviennent alors que la cellule mourante est entouree
de tissue normal.
3. A la suite de la perte des jonctions intercellulaires, les cellules en degenerescence s'arrondissent et sont expulsees dans les grands espaces intercellulaires caracteristiques du myocarde de 7 jours.
4. Des myofibrilles rompues et de grands amas de glycogene restent visibles
dans le cytoplasme des cellules en degenerescence, dont Fopacite aux electrons
s'accroit.
5. La degenerescence se poursuit apres ingestion par des macrophages. On a
observe de tels macrophages, transportant des cellules musculaires mortes
phagocytees, a la fois a Pinterieur du myocarde et dans l'espace sous-epicardique
ce qui permet de penser que quelques cellules mortes peuvent etre eliminees de
l'organe en cours de developpement.
I would like to thank Dr James D. Ebert for making facilities at the Carnegie Institution
available to me. Thanks are also due to Dr Robert L. DeHaan for his careful reading of the
manuscript. This work was begun in the Department of Anatomy, Harvard Medical School,
Boston, Mass. under support from United States Public Health Training Grant GM 00406.
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