Download Myocardial Lysosomes in Experimental Atrial Septal Defects

Myocardial Lysosomes in Experimental
Atrial Septal Defects
By Charles A. Kottmeier, M.D., and Myron W. Wheat, Jr., M.D.
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ABSTRACT
Preliminary observations in patients undergoing open-heart surgery have
suggested that the lysosomes in the right atrium are increased in patients with
atrial septal defects. This increase in lysosomes per myocardial cell appears to
be directly related to the size of the left to right shunt. The following experiments were performed to see if similar findings could be produced in an experimental model. Atrial septal defects were produced in seven dogs. The biopsy
specimens were prepared for ultrastructural examination and viewed with an
electron microscope. Lysosomes were counted in representative sections and an
estimate of the lysosomes per square micron of heart tissue obtained. The
number of lysosomes per square micron of myocardium increased significantly
following the creation of atrial septal defects in dogs, with the most marked
increase occurring in the right ventricle. Several small atrial septal defects
closed spontaneously, as proved by cardiac catheterization, and in these animals
the elevated lysosome counts present initially returned toward normal after
obliteration of the left to right shunt at the atrial level. Control dogs showed no
significant increase in myocardial lysosomes over the 18-month period despite
repeated thoracotomies and myocardial biopsy. These studies add further
evidence to support the role of the lysosome as an important intracellular
organelle which is related to cellular stress.
ADDITIONAL KEY WORDS
chronically stressed myocardium
lysosomal activity
dog
• Since 1955, when de Duve postulated the
existence of the lysosome (1, 2), there have
been many attempts to characterize the activity of this small intracellular organelle (3).
Many of the enzymes contained in the lysosome are well known (3, 4). Lysosomal activity in liver and kidney has been investigated
following the administration of toxic substances (5-8), and this research has led to
the suggestion that one function of the lysoFrom the Division of Thoracic and Cardiovascular
Surgery, College of Medicine, University of Florida,
Gainesville, Florida 32601.
This work was supported by a grant from the John
A. Hartford Foundation and supported in part by
Public Health Service Cardiovascular Training Grant
2T1-HE-5493 from the National Heart Institute.
This paper was presented in part to the 39th
Scientific Sessions of the American Heart Association,
October 22, 1966, New York, New York. (Kottmeier,
C. A., Wheat, M. W., Jr.; Myocardial lysosomes in
experimental atrial septal defects, (abstr.) Circulation
34(4), (Suppl. Ill), 148, 1966.
Accepted for publication May 18, 1967.
Circulation Research, Vol. XXI, July 1967
left to right shunt at the atrial level
intracellular organelle
some may be to rid the cell of foreign material and debris.
Preliminary observations in patients undergoing open-heart surgery have suggested that
the number of lysosomes in the right atrium
is increased in patients with atrial septal defects (9, 10). This increase in lysosomes per
myocardial cell appears to be directly related
to the size of the left to right shunt and therefore is related to the amount of strain on the
right side of the heart. In addition, the lysosomes in the atrial muscle are found to be increased in chronically diseased or stressed
hearts in certain types of acquired heart disease such as mitral stenosis (9, 10). If lysosomes are an integral part of the intracellular
mechanism for the elimination of debris and
autolytic products, an increased number of
lysosomes should be produced in the chronically stressed myocardium in an experimental model. Experimental evidence of increased
lysosomal activity in stressed myocardium
17
18
KOTTMEIER, WHEAT
would confirm the previous observations in
patients and further support the role of the
lysosome as an integral part of the intracellular metabolic process.
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cardial biopsies was performed but no atrial septal defect was created. At intervals of 1, 3, 6,
12, and 18 months later, repeat biopsies were
taken by open thoracotomy.
The tissues obtained by biopsy were immediately diced in 3% glutaraldehyde buffered to pH
7.4 with 0.2M sodium cacodylate buffer with
sucrose (12) and fixed at 0 to 4°C for 1 hr.
They were then rinsed and stored in sodium
cacodylate buffer (pH 7.4). When further processing was done, the specimens were postfixed
in phosphate buffered osmium (OsO4) tetroxide
(pH 7.4) (13) at 0 to 4°C for 1 hr. Dehydration
was carried out in graded concentrations of
ethyl alcohol and embedding was performed in
a 7:3 mixture of Epon 812 or Araldite 502 (14).
The tissues were sectioned on Porter-Blum
MT-1 and MT-2 ultramicrotomes using diamond
knives and mounted on 300 mesh copper grids
with colloidin film and carbon coating. The
grids were stained with uranyl acetate and lead
citrate (15, 16) and viewed in an Akashi Tronscope TRS50E1 or an Hitachi Hu 11-B electron
microscope.
Lysosomes were counted on a morphological
basis after having first established that organelles
of this morphological description in both human
Methods and Materials
Mongrel dogs, 12 to 20 kg, were anesthetized
with pentobarbital, placed on a positive pressure
ventilator, and myocardial biopsies of the walls
of the four heart chambers were taken through
an open thoracotomy. Atrial wall biopsy sites
were from the base of each appendage. Ventricular biopsies were taken approximately 2.0 cm
from the interventricular septum midway between the apex and great vessels. After the control biopsies, an atrial septal defect was created
by the closed technique of Blalock-Hanlon (11)
in 4 dogs or by direct, open visualization and
removal of a large segment of the interatrial
septum using inflow occlusion in 2 dogs. The
period of inflow occlusion was never longer than
70 sec. At the time of surgery, the atrial septal
segments removed were 1.0 to 1.5 cm diam in
the Blalock-Hanlon procedures, and up to 2.5
cm diam in the inflow occlusion procedures. In
2 control animals, open thoracotomy with myo-
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FIGURE 1
Changes in lysosomes in right atrium and right and left ventricles in dogs with Blalock-Hanlon
atrial septal defects which closed, Blalock-Hanlon atrial septal defects which remained open
(upper graphs), and large, 2.0 to 2.5 cm atrial septal defects which remained open (lower
graphs), as compared to controls.
Circulation Research, Vol. XXI, July 1967
MYOCARDIAL LYSOSOMES
19
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and canine myocardium contained acid phosphatase. The method for the localization of acid
phosphatase was the same as previously reported
in studies on patient material (9).
In the present studies, lysosomes were counted
on the fluorescent screen at magnifications of
from 6,000 to 8,000x, and verified with occasional representative electron micrographs (see
figures). The number of lysosomes in each grid
square was counted. Only one square of serial
sections was counted to avoid counting the
same lysosome twice. Each grid square is 50 jit2.
Only grid squares containing exclusively muscle
cells were counted. Twenty-five to 50 grid
squares from one or more blocks of tissue were
counted to constitute a representative sampling
of each specimen and the average number of
lysosomes per 100 tt3 in each group were plotted
against the time after initial biopsy and creation
of the atrial septal defect.
In the 4 dogs with Blalock-Hanlon atrial septa! defects, right heart catheterizations were performed after the 6-month interval biopsy. Dye
dilution curves (17) using indocyanine green
(Cardio-green) were obtained with the injection sites in the pulmonary artery, right ven-
tricle, high, middle, and low right atrium and
the superior and inferior venae cavae. A retrograde femoral arterial catheter for sampling was
passed well up into the aorta. Location of the
catheter on the right side of the heart was determined by pressure tracing and fluoroscopy.
No cardiac catheterizations were performed on
control dogs or those with defects created by
the inflow occlusion technique. Dogs which died
either during or after later biopsies were necropsied and the defects were examined to correlate
the anatomy with the experimental data.
The data were analyzed using the Dunnett
multiple comparison method which seems to be
the most reliable and accurate analysis method
available for our experiments (18).
Results
In the 2 dogs with atrial septal defects created by the Blalock-Hanlon technique, there
was a significant (P = .O5), greater than twofold increase in lysosomes in only the right
ventricle at the 1-month biopsy. Lysosome
counts from biopsies at 3, 6, and 12 months
FIGURE 2
Electron micrograph of control biopsy from the right ventricle showing normal architecture of
myocardial cell with S = sarcomere, M — mitochondria, EG z= ergastoplasm and Golgi apparatus, G = glycogen granules, L = lysosomes. Original magnification X 8,000.
Circulation Research, Vol. XXI, July 1967
20
KOTTMEIER, WHEAT
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after the initial procedure showed no statistically significant difference from control
counts. After 6 months cardiac catheterization
in these 2 dogs showed no left to right shunts,
indicating that the atrial septal defects had
healed. There were no statistically significant
changes in the lysosome counts in biopsies
obtained from the other chambers in these
animals (Fig. 1).
In 2 dogs with Blalock-Hanlon defects,
which had 10 to 12% left to right shunts at
the atrial level after the 6-month biopsy documented by dye curves, the myocardial lysosomes showed a three to fourfold persistent
increase in the right atrium, and right and left
ventricles (Fig. 1). In the right atrium, all
changes were statistically significant (P =
.01) at the 1-month and subsequent biopsies.
In the right ventricle, the change in lysosomes
was significant (P = .O5) at the 1-month interval, and subsequent biopsies showed
changes of greater significance (P = .01) at
the 3-, 6-, and 12-month intervals. In the left
ventricle, changes in lysosomes were significant (P = .05) in all interval biopsies.
A fifth dog was initially included in this
study but died of ventricular fibrillation as
the incision was being closed after the 3month interval biopsy. Necropsy revealed
definite partial closure of the septal defect,
with a 3 mm margin of scar tissue extending
from the margins of the resected septum. The
original defect in this animal was 1.0 cm
diam.
In the 2 animals having 2.0 to 2.5 cm defects created by the open inflow occlusion
technique, the number of lysosomes seen was
as much as six times that seen in the control
FIGURE 3
Electron micrograph of a 3-month biopsy from the right ventricle showing oacuolated lysosomes
in clumps dispersed between mitochondria. EC = ergastoplasm, others as in Figure 2. Original
magnification X 9,000.
Circulation Research, Vol. XXI, July 1967
21
MYOCARDIAL LYSOSOMES
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(Fig. 1). These elevations in lysosome
counts persisted through the 18-month period
after the initial procedure. The most marked
increases in lysosomes were in the ventricles
where they ranged as high as 20 lysosomes
per 100 /A2 compared to the control levels of
3 lysosomes per 100 p? (Fig. 1). In the right
atrium the changes in the number of lysosomes were significant (P = .01) for all experimental biopsies as were the changes significant in the right ventricle (P = .01). The
changes in lysosomes in the left ventricle at
the 1-month biopsy were less but still statistically significant ( P = .05). There were more
significant differences in subsequent biopsies
(P = .01) at 3, 6, and 18 months after the
initial procedure.
Control animals showed no increases in
the number of lysosomes for 18 months despite repeated thoracotomies and biopsies.
In our studies of the lysosome, their appearance has been strikingly similar from
sample to sample (see Figs. 4, 5, and 6).
They are usually oval or elongated oval, from
4 to 12 (Jb average diam, and are bounded
by a unit membrane. They contain a coarsely
granular, moderately dense osmiophilic material which surrounds one or more irregular, less dense areas which are frequently
near the center of the lysosome. The lysosomes also contain relatively large globules
of medium osmiophilia which are surrounded
by a well defined dense membrane and contain clear, sharply bounded spaces within
fit
$
fcSSE
FIGURE 4
Electron micrograph of a 6-month biopsy from the right ventricle showing a small clump of
densely vacuolated lysosomes. Mf = myojxbrils, others as in Figure 2. Original magnification
X 13,000.
Circulation Research, Vol. XXI, July 1967
22
KOTTMEIER, WHEAT
them. The large globules of medium osmiophilia are most often adjacent to the outer
wall of the main body of the lysosomes and
frequently show some concavity along one
edge.
In the present studies not only the quantity
of lysosomes changed but there appeared to
be an additional qualitative change. The
structure of the lysosomes changed with the
persistence of the left to right shunt. Initially, lysosomes were seen in small numbers,
and were small in size with small and infrequent vacuoles (Fig. 2). During later stages,
following the creation of atrial septal defects,
the lysosomes increased in over-all size, in the
numbers of vacuoles within each lysosome
and in the size and complexity of the vacuoles themselves (Figs. 3 and 4). Clumps of
lysosomes were frequently seen in biopsies
from the hearts with larger defects (Figs. 3
and 5). These clumps of lysosomes contained
many large lysosomes as well as what appeared to be small developing "primary" or
"prelysosome" forms (Fig. 5, PL). These
lysosomes have the appearance of the organelles described by Jamieson and Palade
(19) as occurring most frequently in the
atrial tissue of smaller animals, such as the
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FIGURE 5
Electron micrograph of a 12-month biopsy from the right ventricle showing multiple clumps
of vacuolated and nonvacuolated lysosomes found consistently in biopsies taken at this interval
after creation of a large atrial septal defect. Small prehjsosome or primary lysosome forms are
seen; they were shown not to be portions of larger lysosomes by evaluating serial sections. PL =
primary lysosomes, others as in Figure 2. Original magnification X 5,000.
Circulation Research, Vol. XXI, July 1967
MYOCARDIAL LYSOSOMES
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rat and dog. However, they were present in
both the atrial and ventricular muscle of our
animals and therefore are not specifically related to one heart chamber or the other. The
distribution of these clumps of lysosomes did
not follow a specific pattern but many were
noted near the nuclear poles. There seemed
to be no orientation or localization of lysosomes specifically related to intercalated discs.
In control specimens, lysosomes were fairly
evenly dispersed throughout the sarcoplasm
of the cell, interspersed among the myofibrils
with the mitochondria and other intercellular
organelles. The lysosomes seemed to be more
prominent at the nuclear poles than any other single area. In all representative electron
micrographs taken, there appears to be a
constant anatomical association of lysosomes
with ergastoplasm and Golgi apparatus. This
would be expected if NovikofFs postulation of
the origin of lysosomes from the Golgi apparatus is correct (20).
Discussion
In this series of experiments, each experimental dog served as his own control in addition to two animals that were run as strict
parallel controls. It was not the original intent of the experiment to have spontaneous
closure of some of the defects. However, having two defects close spontaneously showed
that the stress of a left to right shunt produced an increase in myocardial lysosomes
which was followed by a return toward control levels once the stress was no longer present (Fig. 1). The opposite effect was also
seen in that persistence of the atrial septal
defect with the continued stress of a left to
right shunt produced a continued elevation in
the number of myocardial lysosomes and,
presumably, a sustained increase in myocardial lysosomal activity.
Both the right and left ventricles had an
increased number of lysosomes during prolonged stress. At first it would seem unexpected to find increased numbers of lysosomes in the left ventricle since the left to
right shunt in atrial septal defects bypasses
the left ventricle. However, although the
heart does have a right and a left side, it
Circulation Research, Vol. XXI, July 1967
23
functions as an over-all, single organ. A given
hemodynamic lesion may affect one side more
than the other, but in a chronic situation,
particularly, intracellular manifestations of
increased work by any part of the heart
should be present to some extent in all myocardial cells.
In these experiments, chronic stress to the
myocardium was applied by surgically creating left to right shunts of varying degrees.
The results indicate that there is a positive
correlation between the size and persistence
of the left to right shunt and the number of
lysosomes. The morphologic changes in the
lysosomes seen in the more chronically
stressed myocardium suggest increased enzymatic activity, perhaps as a response to
increased metabolic products and autolytic
debris within the myocardial cells. The
mechanisms of this phenomenon are not explained here, but the lysosome, by its definite
quantitative as well as qualitative change, is
undoubtedly involved in the intracellular response to stress, in this instance primarily
metabolic. The response and activity of the
lysosome to foreign body and bacterial intracellular invasion has been previously well
documented (5-8). The present studies confirm our earlier findings obtained in human
myocardium chronically stressed by congenital and acquired heart disease (9, 10). The
right atrium was the only myocardial tissue
previously evaluated in the human studies.
However, on the basis of the results in these
experiments, it appears that an increase in
the number of myocardial lysosomes can be
expected in those cells subjected to increased
metabolic demands. The greatest change in
the number of lysosomes in these experiments was in the chamber subjected to the
greatest stress, the right ventricle. How close
the correlation is between the degree of stress
and the elevation in lysosome count cannot
be answered from our present data. Although
a lysosomal/ myocardial index as a possible
indicator of the degree of severity of heart
disease would be a helpful tool, we cannot
substantiate more than a general relationship
at this time.
KOTTMEIER, WHEAT
24
Acknowledgment
11. BLALOCK, A., AND HANLON,
This is to acknowledge the technical assistance of
Mr. James Smith, Mrs. Mary Taylor and Mrs. Marcia
Degenhardt.
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Circulation Research, Vol. XXI, July 1967
Myocardial Lysosomes in Experimental Atrial Septal Defects
CHARLES A. KOTTMEIER and MYRON W. WHEAT, Jr.
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Circ Res. 1967;21:17-24
doi: 10.1161/01.RES.21.1.17
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