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Intercalated discs in heart muscle studied with the electron microscope.

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INTERCALATED DISCS I N HEART MUSCLE
STUDIED 1 1 7 1 ~THE
~
ELECTRON
hlICROSCOPE a
V. L. VAN BREEMEN?
Department of Anatomy, University of California School
of Xedicine, Los Angelrs
EIGHT FIGURES
I n a review of the literature concerning intercalated discs
found in heart muscle, variance of interpretation is apparent.
According to many authors, cardiac muscle represents a
syncytial, multinucleated mass of reticularly arranged protoplasmic bars in which contractile fibrils pass independently
of cell territories. Present opinion of the discs largely centers around the ideas that they are intracellular and may
be explainable as modified irreversible contraction bands.
This study with the electron microscope was undertaken in
the hope that the problem could be further clarified.
MATERIAL AND METHODS
Papillary muscle was used in this study since it has a large
number of intercalated discs. Hearts were removed from
dogs and pieces ( I m m " or smaller) were cut from the papillary muscle arid fixed in osmic acid buffered to pH 7.4 with
veronal and acetate buffer according to the method of Palade
('52). After two hours' fixation, without being washed in
water, the tissue was placed in 50% ethanol f o r one hour
and was subsequently dehydrated in 70Cj0, 95% and 100%
This investigation was supported in part by a research grant froin the Satioiial
Institutes of Health, Public Health Service.
Appreciation is expressed t o the Radioisotope Unit of the Veterans Administration Hospital, Los Angeles, California, f o r the use of its electron microscope.
Present address : Department of Anatomy, University of Colorado Medie:il
Center, Denver.
49
50
V. L. VAN BEEEMEN
ethanol (one hour in each). This was followed by an hour
in a 50-50% mixture of ethanol and n-butyl methacrylate
(with 2% catalyst), after which the tissue was left overnight
in methacrylate (with catalyst) in a refrigerator, the low
temperature inhibiting polymerization. This seemed to achieve
adequate infiltration of the tissue by the methacrylate. Then
the tissue was placed in fresh plastic in gelatine capsules
and left in an oven a t 50°C. until polymerization took place.
Sectioning was done with a Spencer microtome (no. 820)
equipped with the following: a Pease and Baker ( ’48) thin
section adapter; a Spencer razor blade holder; a “Valet”
(Gillette) razor blade ; and a section-collecting well, attached
to the blade holder and filled with a mixture of acetone (5lo%), alcohol (30-40%) and water (the other 50-65%) ; this
mixture was varied to produce proper surface tension for
floating the sections. The microtome was set for sections
0.03 p in thicliness.
Electron microscope specimen grids (3 00 and 200 mesh)
~ v e wprepared with very thin parlodion membranes on which
the sections were mounted. The methacrylate was not extracted from the tissue, except as sublimation occurred in
slight amount in the microscope during observation. I t was
believed that tlie plastic added needed support to the fine
structures in the thin sections.
The material was examined in an R.C.A electron microscope model EMU, with a 2 or 3 mil aperture in the objective
lens. Original magnification of the micrographs (2,000 X,
3,100 X or 6,000 x ) ; each figure is marked with a micron
scale.
OBSERVATIONS
The successive figures illustrate several types of discs described in the literature; figures 1, 2 and 6, the plate type;
figures 3 and 7, the step variety; and figures 4 and 5, irregular discs. I n all of these cases it may be pointed out that the
intercalated discs form real lines of division and not lines of
structural and functional unit such as the cross-striations
INTERCALSTED DISCS
51
(the Z, A and I bands). The myofilaments do not pass through
the intercalated discs; therefore, in a strict sense, the myofibrils are not continuous through the disc.
The character of the disc is distinct from that of the Z and
A bands and is very different from that of a contracted sarconiere. As is shown in figure 1the disc has much the appearance of the sarcolemma and in fact seems to be continuous
with it. The disc in this figure (unfortunately limited to the
two dimensions of a thin section) appears as a simple irregular line transecting a muscle fiber, o r more probably interposed between the adjoining ends of two fibers, being continuous with the sarcolemma on each side. This figure also
illustrates the well known occurrence of a contraction wave
on only one side of a disc. The continuity of the sarcolemma
and intercalated disc is also apparent in figures 2, 5 and 6.
The sarcolemma in figure 6 seems to have a separate connection to the disc from each side; this separate origin of
the disc is further indicated in figure 5 where a double wall
is seen connecting tu7o portions of the irregular disc. This
suggests a probable interpretation of figures 7 and 8 in which
the discs are characterized by two parallel lines passing between the ends of the fibers in a zigzag course; presumably
these parallel structures are derived from the sarcolemma
or are sarcolemma, probably being closely knit walls of adjoining muscle cells reinf orced with fine collagenous fibrils.
I)ISCl’SSIOK
I n referring t o heart muscle Cowdry (’50) speaks of “cardiac muscle cells” indicating that “the width of the cells is
evident but their ends are so ill-defined that it has been said
that they form a syncytiuni. . . . But there exist, at what are
believed to be the surfaces of contact between cells in series,
peculiar differentiations, known as intercalated discs. . . .? ,
There are a number of reasons why the intercalated discs
might mark the junction of cells. There is often a conspicuous difference in the amount of contraction on opposite sides
of the disc (D’Ancona, ’29). I n pathological myocardial seg-
52
V. L. \’AS HREEMEX
nienta tion and with experimental mechanical stress the fibers
separate at the discs (Jordan and Bardin, ’ 3 3 ; Saphir and
Karsner, ’24). They appear at about the time of change
from “cells to fibers” and increase in number and complexity
with growth and activity of the heart (Witte, ’19; Cohn, ’28).
The microdissection experiments of de RQnyi ( ’45) indicate
the discs to be of different material from the muscle film
itself and possibly give more evidence that the discs appear
at cell junctions. According to these studies the discs showed
resistance to pull, whether applied in longitudinal or transverse direction. When saline was injected under low pressure
into the fiber it was prevented hy the disc from passing from
the injected segment to the succeeding one, but under greater
pressure the disc broke allowing the saline to pass through.
It is interesting that, in spite of his experimental evidence,
de RBnyi agreed with many of the authors referred to here,
concluding that no cell membranes cross the fibers at any
location.
Hogue (’47) studied intercalated discs in tissue cultures,
observing that the substance of the discs seemed different
from material of the myofibrils on either side of the discs,
indicated by their different refractility, the discs appearing
as white or colorless lines. Furthermore, the fact that there
were slight constrictions at the site of the discs when cells
contracted indicated that the cells were not as plastic at these
points as elsewhere. Hogue stated that the discs appeared
only after the tissues were grown for several weeks in test
tube cultures and remarked that “this is a good example of
differentiation of cells in tissue cultures as they grow older.’’
Could it be rather that the cell junctions were not apparent
until they had been invaded by collagenous fibrils, after which
they could be demonstrated by their differential refractility
or characteristic staining? D’Ancona (’29) and others have
used the Hortega technique for neuroglia to demonstrate
the discs. Similar silver staining techniques have also been
used to demonstrate the fine collagenous reticular fibrils which
surround the muscle fibers. The fibrils of the sarcolemma are
INTERCALATED DISCS
53
continuous with the reticular fibrils of the endomysium (Feriibndez Ballas, ’44; Fenn, ’45; Draper and Hodge, ’49). Jordan and Banks (’17) considered that the precipitation of
silver nitrate in the discs was due to the modification of the
discs by the infiltration of intercellular tissue fluid.
Heart muscle can readily be grown in tissue culture (Lewis,
’20; Hogue, ’47), which shows that an apparent syncytium
can break down into separate cells. Maximow and Bloom
(’48) refer to work by the Lewises as follows:
“ I t has been clearly showii in tissue culture that two heart
muscle cells, which are not completely separated by a distinct
cell membrane and which seem to have a partially continuous
protoplasm and common fibrils, may beat with independent
rhythms. This observation offers a fairly strong argument
in favor of the view that the cardiac muscle cells are independent cells. . . . Although the cardiac muscle cells may have
a certain degree of morphological continuity, they are obviously discontinuous functionally. ”
The interpretation in the present study has an historical
background and is supported in other observations, as previously discussed. The most significant suggestions drawn
from the electron micrographs are that the discs are distinct
from the Z, A and contraction bands; that the intercalated
discs are continuous with the sarcolemma; that the discs form
real divisional lines between cardiac muscle fibers ; that the
myofibrils are not continuous across the discs; that the disc
is not intracellular; that the disc is somewhat like a “corrugated” wall between fibers and may be shown to be made up
of two laminated layers (probably derived from two cell
walls).
The electron microscope studies of Draper and Hodge (’49)
have demonstrated that the sarcolemma is a complex sheath
made up of collagenous fibrils in layers over a basic membrane. This agrees with Fenn (’45) who considered the sarcolemma to be composed of “interlacing collagenous birefringent fibers embedded in a thick colloidal matrix.” A
logical corollary to this, drawn from the interpretations in
54
V. L. VAN BREEMEN
the present study, would be that the intercalated discs are
structures produced by collagenous invasion a t cell wall junctions. This could explain the late appearance of the discs in
embryonic development ; the tightly-fit end walls of the myoblasts would not be recognized, while the lateral walls would
soon become thickened with collagen and have the characteristic appearance of the sarcolenima; later with the stress of
actirity collagen would invade the closely-fit end walls of the
fibers and gradually build up the structure of the intercalated
disc. It may be concluded from their transverse orientation
and drawn out “corrugated” appearance that the discs were
formed and function under stress. Interesting in this regard,
IYitte’s ( ’19) theory considered the discs as strengthening
bands in the muscle fibers. Xarceau (’04) suggested that
the discs divide the cardiac fibers into successive segments
“in the fashion of small tendons.” However, it may be noted
that Xareeau nevertheless considered heart muscle to be a
syncytium.
Present observations of other muscle structure agree with
the electron microscope studies of cardiac muscle by Beams
Oures.
et al. ( ’49). Various structures are labeled in the fig
Other electron microscope studies of striated muscle were
made by Hall, Jakus and Schmitt (’46) and Draper and
Hodge (’49). For reviews of the literature on the intercalated
disc refer to Saphir and Karsner ( ‘24) and Jordan ( ’33).
HCJfJIART
In order to study intercalated discs, papillary muscle from
dog hearts was fixed in acetate-veronal buffered (pH 7.4)
osmium tetroside, embedded in n-butyl methacrylate and
sectioned f o r observation with the electron microscope. The
discussion draws from the literature a number of reasons
wh>- the intercalated discs might mark the junction of cells.
The most significant points suggested by the electron micrographs in this study are that the intercalated discs are continuous with the sarcolemma; that they form real divisional
lines between cardiac muscle fibers; that the myofibrils arc
INTERCALATED DISCS
55
not continuous through the discs; that the discs are not intracellular; that they are somewhat like a “corrugated” wall
between muscle fibers and can be shown t o be made up of two
laminated layers which may be derived from closely knit cell
walls.
L I T E R B T U R E CITED
BEAMS,H. W., T. C. EVANS,C. D. JANNEY
AND W. W. BAKER 1949 Electron
microscope studies on the structure of cardiac muscle. Anat. Rec., 105:
59-82.
COHN, A. E. 1932 Cardiac muscle. I n Special Cytology. P a u l B. Hoeber, Kew
York, 2 : 1127-1172.
COWDRY,E. V. 1950 A Textbook of Histology. Lea and Febiger, Philadelphia,
4 : 461.
D’ANCONA,U. 1929 Contributo a una revisioue delle nostre conoscenze sulla
morfologia della fibra muscolare striata. Protoplasma, 1 0 : 177.
DE R ~ N Y G
I ,. S. 1945 The nature of iutercalated disks of cardiac muscle studied
by the micro-dissection method. Am. J . Med. Sci., $09: 270-271.
DRAPER, M. H., AND A. J. HODGE1949 Studies on muscle with the electron
microscope. I. The ultrastructure of toad striated muscle. Aust. J.
Exp. Biol. Med. Sci., 17(6) : 465-503.
FENN,
W. 0. 1945 Contractility. Section 7, chapter 33 i n Hober’s Physical
Chemistry of Cells and Tissues. Blakiston Company, Philadelphia,
pp. 455456.
FERNhNDEZ BALLAS,W. 1944 El probleuia de la naturaleza y origen del sarcolema en las fibras musculares cstriadas. P a r t e I. Comunicaciirn preliminar. Biol6gica, 2 : 5-25.
HALL, C. E., M. A. JAKUS
AND F. 0. ScImIi*T 1946 An investigation of cross
striations and myosin filaments in muscle. Biol. Bull., 90: 32-50.
HOGUE,
111. J. 1947 Intercalated disks i n tissue cultures. Anat. Rec., 99: 157-162.
JORDAN,
H. E. 1933 The structural changes in striped muscle during contraction.
Physiol. Rev., 13: 301-324.
JORDAN,
H. E., .4ND J. B. RANKS 1917 .I study of the intercalated discs of the
heart of the beef. Am. J . Anat., I S : 285-339.
JORDAN,
H. E., A N D J. B 4 R D 1 N 1913 The relation of the intercalated discs t o
the so-called “segmentation” and “fragmentation” of heart muscle.
Anat. Anz., 4 3 : 612-617.
LEWIS,M. R. 1920 Muscular contraction i n tissue culture. Contributions t o
Embryology no. 35. Carnegie Inst., 9: 193.
MARCEAU,F. 1904 Recherches sur la structure e t le dkveloppement compares
des fibres cardiaques d a m la sBrie des vertbbres. Ann. des Sci. Nat.
Series 8. Zoologie, 19: 191-363.
MAXIMOW, A. A,, AKD W. BLOOM 1948 A Textbook of Histology. W. B.
Saunders, Philadelphia, 5: 172-174.
PEASE,
D. C., BND R. E. BAKER 1948 Sectioning techniques f o r electron microscopy using a conventional microtome. Proc. SOC.Exp. Biol and hled.,
6 7 : 470-47-1.
56
V. L. V A N BREEMEN
SAPHIR,
O., AND H. T. KARSNER 1924 An :rnatoinical and experimental study
of segmentation of the myocardiuni and its relation to the intercalated
discs. J. Med. Res., 4 4 : 539-556.
WITTE, L. 1919 Histogenesis of the heart muscle of the pig i n relation to the
appearance and development of tlie intercalated discs. *4m. J. Anat.,
95: 333-3.47.
PLATE 1
EXPLANATIOX OF FIGURES
Scale indicates one micron
1 Papillary muscle fibers, separated by a simple intercalated disc ( I D ) . The
intercalated disc seeins to be continuous with the sarcoleinma (8). 9 contraction
wave is on one side of the disc. X 3,600.
2 Papillary muscle with plate type intercn1:tted disc ( I D ) connecting to the
sarcolemma (S). There are many mitorhoiitlria (Mit) in heart muscle. A fibrohlast (F) occurs in the line of the sarcoleinma. X 3,000.
INTERCALATED 1)ISCS
V. L. V A S B R E E M l i N
57
PLATE 2
EXPIANATION OF PIGURES
Ncvilr. indknlw o w
ttiicroii
3 Step type of interealtltc.cl clisc (11)) ; tlir sneceuuiw steps :we npptireiitly
coiiiwtrxl. A nucleus (N) :ipl)eiirs iii tlie upper left of the figure, with iiiitoch.mlhi ( Y i t ) clustered liest it and scattered tlrroiigli the myofibrils. Ail hl
I)anil (31) ant1 eontrtictioii Imiil (on) are I:ilwlecl. X 6,000.
4 A n irregullir iirtc~rcrluteildi8c
iii
pipi!lary iiiuwle.
x 6,500.
I'L.\'I-l.:
2
ESPI,;\N:\TION
OF FIGURES
Sccilc indicutcs one niicroii
5 Papillary muscle wit11 an irregular iiiterc:ilated disc. Tlic sarcolrinnin (S)
is double at some points. X f!,OOO.
ti Platc type of disc. Tlic s:ircoleiiiiii:i (S) again continues to the disc :is n
double wall. Two mitocliondri:r ( M i t ) : i i i d :i contraction I):ind ( ( : I i ) :ire labeled.
x 19,500.
60
61
PLATE 4
EXPLSNAlION O F FIGURES
Sccile intlicotcs m e ?riicron
7 Papillayy iiiuscle and step t y p e intercalated disc with ;I “eourugated”
double mall. X 18,000.
8 P l a t e t y p e of disc (ID) also tlenio~istr:iting ;L “corlugated”
possibly derived f r o m closely knit cell walls. X 30,000.
62
double wall
PLATE 4
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