Download Ventricular Nuclei-DNA Relationships with Myocardial Growth and

Ventricular Nuclei-DNA Relationships with
Myocardial Growth and Hypertrophy
in the Rat
By Arthur F. Grimm, D.D.S., Ph.D., Luis de la Torre, Ph.D., and
Michael La Porra, Jr., D.D.S.
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ABSTRACT
Rat ventricles, ranging from 306 to 1999 mg, were obtained from normal
animals of different ages and from animals subjected to chronic aortic
constriction. The concentration of DNA in the paired ventricles (right and
left) was found to be closely related to the concentration of nuclei in the left
ventricular papillary muscles. Muscle nuclei represented only 10 to 15% of this
population of nuclei. In young animals (phase 1), the total ventricular content
of muscle nuclei, nonmuscle nuclei, and of DNA were increasing with
ventricular growth. In the adult rat (phase 2), the total ventricular content of
DNA and of both muscle and nonmuscle nuclei remained relatively constant
with ventricular growth. In the enlarged hearts (phase 3), there was a further
increase in total ventricular DNA but there was no further increase in the total
number of muscle nuclei. Spectrophotometric studies (Feulgen stain), showed
that 88% of the muscle nuclei belonged to a single ploidy class (probably
diploid). No relation could be demonstrated between the extent of nuclear
polyploidy and the weight of the ventricles. It was concluded that polyploidy
was not a significant factor in the increased total DNA of phase 3. The
increased ventricular DNA of phase 3 was explained by the proliferation of the
nonmuscle nuclei.
ADDITIONAL KEY WORDS
myocardial nuclei
nuclear volume
nuclear activity
nuclear ploidy
Feulgen spectrophotometry
aortic constriction
papillary muscle
myocardial hypertrophy
myocardial postnatal growth and development
• Shortly after birth, mitotic activity of
cardiac muscle cells appears to become rare or
even absent (1-6). This observation has led to
examinations of the myocardial DNA relationships during cardiac growth and enlargement
(7-13).
In an earlier study (7), the DNA, RNA,
actomyosin, and total protein concentrations
were determined for a wide range of rat
paired (right and left) ventricular weights.
Three phases of growth were described: In
phase 1 (hearts from young animals with
From the Departments of Histology and Physiology, Colleges of Dentistry and of Medicine, University
of Illinois, Chicago, Illinois 60680.
This study was partially supported by U. S. Public
Health Service Grant DE-02417. Dr. Grimm is
recipient of a U. S. Public Health Service Career
Research Development Award (5K3-DE-21, 938).
Received May 26, 1969. Accepted for publication
November 4, 1969.
Circulation Research, Vol. XXVI, January 1970
ventricular weights of less than 550 mg),
DNA concentrations remained relatively constant with increasing ventricular weights. The
increasing total DNA in the paired ventricles
(right and left) suggested continuing mitotic
activity. This conclusion is supported by the
histologic demonstration of both DNA labeling (with 3H-thymidine and autoradiography) and of mitotic division in young
animals (1, 2, 4-6). In phase 2 (ventricular
weights were between 550 and 1000 mg), total
DNA per the paired ventricles remained
relatively constant; with ventricular growth
there was a progressive decrease in DNA
concentrations. This finding can be explained
by the reduced rate of cardiac mitotic activity
in older animals (1, 2, 5, 10). This hypertrophic phase probably includes the range of
weights of normal adult rat paired ventricles
(a reexamination of this data suggests that the
45
46
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upper limits of phase 2 should be expanded to
include ventricular weights between 550 and
1200 mg). In phase 3 (ventricular weights
exceeding 1200 mg), DNA concentrations
remained relatively constant as the total DNA
in the paired ventricles, again increased.
Several reasonable possibilities exist to explain
this increase: (a) there may have been
mobilization and entrapment of nucleated
cells from the circulation; (b) there may have
been an increase in the amount of DNA per
nucleus (an increase in the state of ploidy).
Kompmann et al. (14), from cytophotometric
measurements of nuclear DNA, have described significant degrees of nuclear polyploidization in normal and hypertrophied
human hearts; (c) there may have been
increased mitotic activity of the cardiac
muscle cells or any of the other ventricular cell
types. Indeed, proliferation of the muscle
nuclei (15) and of the interstitial cells (16) of
the heart has been described during the
development of cardiac hypertrophy after
experimental aortic stenosis.
The present histologic investigation was
made to determine the actual cellular mechanisms responsible for the described DNA
relationships, using some of the experimental
material from the previous study (7).
Methods
Sprague-Dawley male albino rats were used in
these studies. As previously described (7), two
experimental approaches were used to obtain a
wide range of ventricular weights. In the first
approach, hearts were obtained from normal
animals of different ages and body weights; in the
second, hearts were obtained from animals widi a
subdiaphragmatic-suprarenal aortic constriction
and from their sham-operated litter mates examined 5 to 7 months postoperatively. The aortic
constriction was produced by: (a) placing a rod
1.1 mm in diameter next to the aorta of rats
weighing 150 to 250 g, (b) completely occluding
the descending aorta with a subdiaphragmaticsuprarenal ligature about the rod and the aorta,
(c) removing the rod. This procedure, which
should produce an aortic constriction approximately equal to the diameter of the rod, resulted
in about 50% mortality after surgery.
The three experimental groups (A, normal
animals of different ages, body weights, and
GRIMM, DE LA TORRE, LA PORTA
ventricular weights; B, sham-operated animals; C,
animals with aortic constriction) were further
subdivided into groups 1 and 2 on the basis of
ventricular weights. The specimens were selected
so that some of the larger ventricles of group A,
subgroup 2, were similar in weight to the smaller
ventricles of group B, subgroup 1, and some of
the larger ventricles of group B, subgroup 2, were
similar in weight to the smaller ventricles of
group C, subgroup 1. Since there were no
apparent experimental differences
between
groups in the regions of overlap, the results are
treated as a continuum.
DNA concentrations were determined in 200 to
400 mg of the paired ventricular muscle using
essentially the Schmidt-Thannhauser method as
described by Korn (17). The total DNA in paired
ventricles was calculated from the DNA concentrations and the ventricular weights. At the
termination of the experiment, a single left
ventricular papillary muscle was tied in situ to an
applicator stick, freed from the ventricle, and
fixed with 10% formalin for 2 hours. Following
standard dehydration and paraffin embedding
procedures, sections 15/JL thick were cut with a
Leitz No. 1300 large-base sledge microtome.
Thirty papillary muscles were selected from
specimens of the preceding study (7) to provide
specimens distributed relatively evenly according
to their ventricular weights. Feulgen stained
sections were prepared from this group of 30
muscles using essentially the method described by
Leuchtenberger (18). Each of these muscles was
derived from a separate animal. An hydrolysis
time of 8 minutes was chosen as the optimum for
this material following preliminary studies. The
nuclear volume and DNA content were determined for 30 muscle nuclei from each of the
muscle specimens using essentially the microspectrophotometric techniques described by Swift and
Rasch (19).
Subsequently the sections from 27 of these
muscles, together with sections from an additional
12 papillary muscles from the original study (7)
were counterstained with fast green. A nuclear
count of both total nuclei and cardiac muscle
nuclei was made for a microscopic field of 37,000
jU,2 using a net micrometer eyepiece and a
magnification of 450X. Six fields were measured
for each muscle specimen. Since the histologic
sections had a constant thickness of 15/JL, the
nuclear count per area is more correctly a nuclear
count per volume: 15;u. X 37,000 \i? — 555,000
/i,3/field; 6 fields = 3.3 X 106 ^ 3 , or weight of
muscle. The ventricular nuclear content can be
estimated from the nuclear concentration per
section volume if one knows the tissue density per
unit volume. Since histologic preparation reduces
cardiac tissue volumes by approximately 50%
Circulation Research. Vol. XXVI. January 1970
47
VENTRICULAR NUCLEI-DNA RELATIONSHIPS
TABLE 1
Ventricular DNA and Papillary Muscle Nuclei
AH nuclei
DNA
Group
No. of Ventricular
animals
wt (mg)
Terminal
body wt Concentration
(g)
(jig/tng)
Calc.
ventricular
total Gig)
Muscle nuclei
Calc.
Calc.s no.
Concn
(10 )
(no./fleld) no. (10«)
Vol.
A
1
6
378 =4=31
116
2
6
647 ± 4 3
234 ± 2 4
=fc 1 1
1.45 ± 0.15
550
1894 ± 143
107
13.6 ± 1.5
152 ± 31
1.08 ±0.16
S30
1361 ± 165
130
20.2 ± 2.5
158 ± 14
(4)
(5)
B
1
7
782 ± 4 0
316 ± 3 0
1.07 ± 0.15
820 (5)
1038 ± 91
121
18.6 ± 1.7
258 ± 47
1140(5)
871 ± 57
143
18.8 ± 1.5
184 ± 15
980
924 ± 103
175
21.9 ± 1.6
169 ± 28
1128
1029 ± 107
219
24.8 ± 3.9
205 ± 29
(3)
1757
532
(4)
(5)
2
7
1084 ± 30
478 ± 14
0.85 ± 0.10
(5)
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C
1
6
124S ± 19
.546 ± 12
0.78 ± 0.06
(4)
(•5)
2
7
1425 ± 4 3
549 ± 7
0.79 ± 0.11
(6)
Extremely
large
heart
1999
18.6
See text for description of group classification. Values are means ± sic. Number in parentheses is number of specimens when less than that of number of animals.
(unpublished observations), a correction factor of
2 was used in the following calculation:
constant. These results are identical with those
previously described (7). The concentration
_
,
,
i
r
i • (nuclear count per section area) (ventricular wt in me)
Estimated total number or nuclei =
—
(2) (3.3 X 10-3)
A computer analysis was used to determine the
of the total nuclei, which also fell during
population distribution of the DNA content per
phases 1 and 2, was relatively constant from
nucleus. This employed a multiple gaussian curve
the middle of phase 2 through phase 3. The
decomposition program by the gaussian transformnuclear concentration is expressed as numbers
least squares method.
Results
DNA AND NUMBERS OF NUCLEI
Table 1 presents the mean values and the SE
of the DNA concentrations in the ventricles
and the concentrations of nuclei in the left
ventricular papillary muscles. Both determinations were carried out on material from the
same hearts. The DNA concentrations and the
total nuclear concentrations are presented in
Figure 1. DNA concentrations fell from a high
level in phase 1 to a much lower value in
phase 2. From the end of phase 2 through
phase 3, DNA concentrations were relatively
Circulation Research, Vol. XXVI, January 1970
of nuclei counted per standard field (0.22 X
10" JJL1). In the group with the largest
ventricles (group C-2), there may be a
suggestion of an increase in the total nuclear
concentration. However, this increase only
approaches the 5% significance level. The
largest of the ventricles (1999 mg) substantially exceeded the size of any other specimen
in group C-2 and was consequently treated
independently. Since this very large ventricle
had an even greater concentration of total
nuclei, the increase in nuclear concentration
with the very large ventricular weights (group
C-2 and the 1999-mg specimen) may be more
GRIMM, DE LA TORRE, LA PORTA
48
1300
1200
1100
1000
900
800
700
600
• 500
400
300
200
100
0
220
200
I '80
|
10
2000 •
1900
1800
-^
o
x
•09
17001600
1500 -
08
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Si 1400
5 1300
f 1200
z
E
07
140
si2°
=
too
i
so
*
60
40
20
0
2
4
6
8
10
Ventricular Weight
06
1100 •
1000900 •
BOO
700
0.5
4
6
8
10
Ventricular Weight
12
14
16
mg(xlOO)
12
14
16
mgUIOOJ
FIGURE 2
Relationships between ventricular weight and total
ventricular DNA, total ventricular total nuclei, and
total ventricular muscle nuclei. • = normal animals;
A = sham-operated animals; A = animals with aortic
constriction.
FIGURE 1
Relationships between ventricular weight and concentrations of ventricular DNA and papillary muscle
total nulclei. • = normal animals; a = sham-operated
animals; A = animals with aortic constriction.
than a coincidental occurrence. Particular
attention should be directed to the almost
identical shape of the ventricular DNA
concentration curve and the nuclear concentration curve. It would appear that the
histologically determined nuclear concentration in the left ventricular papillary muscle
closely reflected the DNA concentration of the
paired ventricles.
The total DNA and the total number of
nuclei for the paired ventricles are presented
in Table 1 and Figure 2. As previously
described, phase 1 was characterized by an
increase in the total ventricular DNA, phase 2
by a relatively constant level of ventricular
DNA, and phase 3 by a renewed synthesis of
DNA. The shape of the curve for the total
number of nuclei for the paired ventricles
appears almost identical with the total DNA
curve.
Figure 2 also presents the relationship
between the ventricular weights and the
number of muscle nuclei. In phase 1, both
muscle and nonmuscle nuclei are probably
increasing in total number. The increase in the
number of muscle nuclei from phase 1 to
phase 2 was statistically significant (A-l vs. A2, P<0.05; A-l vs. A-2, B-l, B-2, P<0.01).
However, in ventricles greater than 550 mg,
there was no statistically significant further
increase in the total number of muscle nuclei.
The number of muscle nuclei was found to
represent approximately 10 to 15% of the total
number of nuclei. (For reasons that are not
clear, this value is less than that described by
Morkin and Ashford [16] who reported that
muscle nuclei compose approximately 25% of
the total nuclei in 200 g female rats.)
The absolute number of nonmuscle nuclei
also increased in phase 1, was relatively
constant in phase 2 but, in contrast to the
muscle nuclei, markedly increased in phase 3.
The increase in total nuclei in phase 3 is
primarily due to the increased number of
nonmuscle nuclei.
MUSCLE NUCLEI: DNA
NUCLEAR VOLUME
CONTENT
AND
Figure 3 presents the frequency distribution
of the DNA contents in muscle nuclei
expressed in arbitrary units. By the computer
analysis, the skewed curve here can be best
Circulation Research. Vol. XXVI, January 1970
VENTRICULAR NUCLEI-DNA RELATIONSHIPS
49
TABLE 2
Muscle Nuclei: DNA and Nuclear Volume
Nuclear DNA content
(arbitrary units)
No. of nuclei
<
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!
4
6 8 10 12 14 16 18 20 22 24 26 28 30 32
Nuclear DNA Content (arbitrary units)
FIGURE 3
Frequency distribution of the DNA contents of the
900 muscle nuclei. Solid line drawn from measured
points; dashed line, shape of second curve (mean
value 2 x mean of first curve); hatched and stippled
areas, contribution of second curve.
755
77
Mean
vol. G>>)
168
327
<=14.0
P<0.001
ploid nuclei. Indeed in the predominantly
tetraploid nuclei, the mean muscle nuclear
volumes are approximately twice that of the
diploid nuclei.
As seen in Table 1, mean muscle nuclear
volume increased during phase 2, an increase
which was not related to the degree of nuclear
ploidy. Accordingly, a statistical comparison
was made between the volumes of the muscle
nuclei from ventricles greater and less than
900 mg. Table 3 shows that this increase
(approximately 25%) in mean nuclear volume
was highly significant.
Discussion
explained by the presence of a second curve of
low magnitude whose peak mean value occurs
at twice that of the primary curve. The dashed
lines represent such a curve; the hatched areas
represent the contribution of the second curve.
On this basis, 88% of the nuclei are contained
within a single ploidy class which will be
assumed to be the diploid class (2n), and an
additional 11% were within the tetraploid class
(4n). It can be calculated that the presence of
higher ploidy classes (above 2n) only increases the mean DNA per nucleus to about
110 to 115% of the diploid value. A relation
between ventricular weight and the degree of
polyploidy could not be demonstrated.
Table 2 presents the relation between the
amount of DNA (degree of ploidy) in a
muscle nucleus and the volume of that muscle
nucleus. Values are selected from those nuclei
which could be segregated into distinct ploidy
classes: nuclei with DNA contents below 8
arbitrary units are compared with those above
12; values in the overlap region of the two
curves of Figure 3 were not included in the
analysis. There is a statistically highly significant increased nuclear volume in the polyCirculation Research, Vol. XXVI, January 1970
In phase 1, the increasing total DNA in
paired ventricles (7) is accompanied by an
increasing total number of both muscle and
nonmuscle nuclei. This is almost certainly the
result of continuing mitotic activity in the
young animal, a conclusion supported by the
demonstration of mitotic figures in both hearts
of infants and young rats (2, 6, 20).
In phase 2, which probably includes the
range of ventricular weights that occur during
the period of normal adult life in the rat, there
are relatively stable levels of both total nuclei
and total DNA over a wide range of
ventricular weights. Phase 2 is a period of
almost pure hypertrophy. During this phase,
the constant number of muscle nuclei is being
diluted by the increasing muscle cytoplasmic
volumes. Since it is probable that the increased cytoplasmic volumes of the muscle
TABLE 3
Muscle Nuclei: Ventricular Weight and Nuclear Volume
Wt. of paired ventricle
(mg)
No. of nuclei
330
<900
480
>900
Mean
vol. <jt)
159
202
i = 5.69
P<0.001
50
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cells do impose increased functional demands
on the constant number of muscle nuclei, the
larger muscle nuclear volumes found in the
larger ventricles (>900 mg) may simply
reflect this elevated nuclear activity. This is
not unlikely since nuclei of active cells are
often larger than those of similar inactive cells
(21). The slight rise in total DNA and total
nuclei in phase 2 appears to be due to a slow
continuing proliferation of nonmuscle nuclei.
In phase 3, there were increasing amounts
of DNA. However, after phase 1, the number
of muscle nuclei remained relatively constant.
Previous studies, which have described the
apparent absence of mitotic activity in cardiac
muscle nuclei and their dilution with growth,
have also concluded that there is a relatively
constant number of muscle nuclei in the adult
growing or enlarged heart (1, 3, 10).
In contrast to these conclusions, there have
been reports of muscle nuclear labeling in the
heart subsequent to the administration of
tritiated thymidine. These results have been
supported by reports of apparent labeled
mitotic figures in muscle nuclei. The usual
interpretation is that these findings demonstrate either continuing mitotic activity or else
the process of polyploidization (22, 23).
However, Walker and Adrian (1), who
studied and counted only labeled nuclei cut in
cross section, found no labeled muscle nuclei
in 21- and 30-day-old mice. They concluded
that reports of labeled muscle nuclei are based
on the presence of nonmuscle nuclei superimposed on the muscle fibers. Another explanation has been proposed by Pelc (24, 25) who
has concluded that there is a regular renewal
of some or all of the DNA in nondividing
nuclei (metabolic DNA). Either of the latter
may provide the explanation for the reported
presence of labeled nuclei in the heart when
muscle nuclear multiplication is absent.
In the present study, there was no real
evidence for a significant degree of polyploidy; only about 10% of the muscle nuclei
were polyploid. This finding is similar to that
reported for the normal and hypertrophied
human heart by Capers (26), and for the
growing mouse (10). Furthermore, since no
GRIMM, DE LA TORRE, LA PORTA
relation could be demonstrated between ventricular weights and the number of polyploid
nuclei, it is concluded that there is no
significant role for polyploidy in the increasing
total DNA of phase 3. The increasing total
ventricular DNA levels of phase 3 are due to
the proliferation of nonmuscle nuclei. A
similar conclusion has been drawn by Morkin
and Ashford (16) and by Grove et al. (27). In
histologic studies, these authors have described a proliferation of nonmuscle nuclei in
hypertrophied rat hearts.
Since polyploidy was not an important
factor in the present studies, the ventricular
DNA should be directly related to the number
of ventricular nuclei. Petersen and Baserga
(10) using a nuclear DNA content of
7 X 10"12 g demonstrated such a relationship in
the mouse. Using the same nuclear DNA
content per nucleus and multiplying it by the
number of nuclei (121 X 10° at a ventricular
weight of 782 mg) the ventricular DNA
should be equal to 847 fig, a value in very
good agreement with the 820 ju,g actually
obtained. Indeed for the six groups, this
calculated DNA [Number of nuclei X (7 X
10~12)] was 116 ±19% of the measured
value.
In contrast to these findings, Kompmann et
al. (14) have described a very substantial
amount of polyploidy in the muscle nuclei of
human hypertrophied hearts. This very interesting report, if confirmed, may demonstrate a
species difference between the rat and man
either in their capacity for polyploidy or in the
extent of possible hypertrophy. However, it
should be restated that in the present study no
relation could be demonstrated between the
ventricular weight and the degree of ploidy.
A significant relation was found between
the degree of muscle nuclear ploidy and the
muscle nuclear volumes in the present study.
Similar increases in nuclear volumes with
polyploidy have been reported for other
tissues (28,29).
In the largest hearts, there may be evidence
for an additional change in the nuclear DNA
pattern with growth. The group of the largest
hearts and the observations in the one
Circulation Research, Vol. XXVI, January 1970
51
VENTRICULAR NUCLEI-DNA RELATIONSHIPS
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extremely large heart may suggest that, with
very large hearts, nuclear proliferation may
increase to a point where nuclear concentrations could be similar to levels found in the
young small heart. This may provide an
explanation for the findings of Korecky and
French (30) who produced extremely large
hearts by iron deficiency anemias and found
essentially normal concentrations of ventricular DNA. The site of this proliferation should
be of particular interest since the present
study would suggest that the predominant
response is a massive proliferation of nonmuscle nuclei. The question remains as to the type
or types of nonmuscle nuclei which do
proliferate in phase 3. The possible proliferation of the blood vessels in hypertrophy (3,
31) suggests that there may be a proliferation
of the endothelial and allied tissue nuclei.
Recently, Buccino et al. have reported an
increased collagen content in cat hearts
hypertrophied by means of a pulmonary
artery constriction (32). This report would
suggest that there may also be proliferation of
the cardiac fibroblasts. Proliferation of both
connective tissue and endothelial cells has
been described during the acute phases of
cardiac hypertrophy (16). Further investigations appear to be required to determine the
type or types of proliferating nonmuscle
nuclei in phase 3.
The dilution of muscle nuclei is a striking
feature of this type of study. Since polyploidy
is apparently not a significant factor, this
muscle nuclear dilution also represents a
dilution of the nuclear material (DNA) of the
muscle cells. The ventricular concentration of
muscle nuclei fell from a value of 36 X 103/mg
at a ventricular weight of 378 mg to a value of
17 X 103/mg at a ventricular weight of 1425
mg (and a value of 9 X 103/mg at the 1999
ventricular weight). Though this extreme twoto fourfold dilution of the muscle nuclear
material might be expected to have functional
significance, it must be emphasized that
experimental evidence which can ascribe
functional changes in the myocardium to this
nuclear dilution is presently lacking.
Circulation Research, Vol. XXVI,
January 1970
Acknowledgment
The authors express their gratitude to Mr. K. V.
Katele and Mr. R. Kubota for technical assistance, and
to Dr. N. Shioura, Research Resources Laboratory,
University of Illinois, for his assistance in the
computer analysis of the data.
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Circulation Research, Vol. XXVI, January 197Q
Ventricular Nuclei-DNA Relationships with Myocardial Growth and Hypertrophy in the
Rat
ARTHUR F. GRIMM, LUIS DE LA TORRE and MICHAEL LA PORTA, Jr.
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
Circ Res. 1970;26:45-52
doi: 10.1161/01.RES.26.1.45
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