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1159
Myocyte Mitotic Division in the Aging
Mammalian Rat Heart
Piero Anversa, Debra Fitzpatrick, Sholey Argani, and Joseph M. Capasso
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To determine whether myocyte mitotic division occurs in the adult mammalian heart and
whether this cellular process is affected by aging, we measured the percentage of myocyte nuclei
showing metaphase chromosomes in myocytes isolated from the left and right ventricles of rats
at 8-12, 19-24, and 28-32 months after birth. Metaphase chromosomes were found at all ages
in both ventricles. However, from 8-12 to 28-32 months, the fraction of nuclei exhibiting
metaphase chromosomes increased 6.3-fold and 2.3-fold in the left and right ventricles,
respectively. Thus, myocyte cellular hyperplasia is present in the adult and aging myocardium
as a compensatory mechanism to regenerate tissue mass and recover function, which are lost
with the progression of life and senescence. (Circulation Research 1991;69:1159-1164)
lthough it is generally believed that differentiated cardiac myocytes are unable to synthesize DNA and undergo cellular mitotic
division, recent work has shown that the long-term
effects of pressure overload on the left' and right2'3
ventricles of the adult rat heart lead to hyperplasia of
myocyte nuclei. Moreover, myocyte cellular hyperplasia appears to occur in the right myocardium of
the senescent rat heart4 in association with the
development of severe myocardial dysfunction and
failure.45 However, the aging process of the heart
also is characterized by a significant loss of myocytes,4'6 which complicates the analysis of cell proliferation because these two opposite events tend to
obfuscate each other. Proliferation of myocytes may
initiate early in life, but this cellular process may not
be apparent because of the concomitant presence of
cell death.4 On this basis, the possibility can be raised
that the potential for myocyte cellular hyperplasia is
preserved in the adult mammalian heart and it may
represent a significant growth reserve mechanism for
tissue regeneration and recovery of function in different forms of heart disease. Therefore, the frequency of metaphase chromosomes was measured in
myocyte nuclei prepared from the myocardium of
rats at 8-12, 19-24, and 28-32 months of age to
provide a direct documentation of myocyte mitotic
division with aging. These time periods were selected
A
From the Department of Medicine, New York Medical College,
Valhalla, N.Y.
Supported by National Institutes of Health grants HL-38132,
HL-39902, HL-40561 and a Grant-in-Aid from the Westchester
Heart Association.
Address for reprints: Piero Anversa, MD, Department of Medicine, New York Medical College, Valhalla, NY 10595.
Received December 13, 1990; accepted June 7, 1991.
because they correspond to fully mature adult, aged,
and senescent animals in this experimental model.4'5
Materials and Methods
Fischer 344 rats were heparinized and killed by
decapitation. Animal care was in accordance with
institutional guidelines. Hearts were excised, and
myocytes from the left and right ventricular myocardium were enzymatically dissociated.4'7 The cell isolation technique consisted of three main steps. 1)
Low calcium perfusion (0.3 mM CaCl2): Blood washout and collagenase (selected type II, Worthington
Biochemical Corp., Freehold, N.J.) perfusion of the
heart was carried out at 32°C with HEPES-minimum
essential medium gassed with 85% 02-15% N2. 2)
Mechanical tissue dissociation: After perfusion, the
left and right ventricles were separated and minced.
Tissues subsequently were shaken in resuspension
medium containing creatine, collagenase, and 0.3
mM CaCl2. Supernatant cell suspensions were
washed and resuspended in resuspension medium. 3)
Separation of intact cells: Intact cardiac cells were
enriched by centrifugation through Percoll (Pharmacia LKB Biotechnology, Uppsala, Sweden). Approximately 106 cells were suspended in 10 ml isotonic
Percoll (final concentration, 41% in resuspension
medium) and centrifuged for 10 minutes at 34g.
Intact cells were recovered from the pellet and
washed, and smears were made to control the purity
of the preparation. Rectangular, trypan blue-excluding cells constituted nearly 80% of all myocytes. The
number of viable myocytes obtained from each ventricle decreased as a function of age. At 8-12, 19-24,
and 28-32 months, the average number of myocytes
obtained from the left ventricle was 3.2, 1.3, and 1.0
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Circulation Research Vol 69, No 4 October 1991
N
cm%
1.
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FIGURE 1. Examples of metaphase chromosomes prepared from the left (left panel) and right (right panel) ventricles
Fischer 344 ra t h earts. Left panel: Giemsa staining, x 1, 800; right pa nel: fluorescent, x 600.
million. Corresponding values for the right ventricle
were 1.3, 0.52, and 0.60 million.
Cells were treated with hypotonic buffer (0.01 M
HEPES and 1.5 mM MgC, pH 7.4) for 5 minutes,
and then lysis buffer (3cc glacial acetic acid and 5t
ethylhexadecyldimethyl ammonium bromide in water) was added and tubes were shaken everv 2
minutes for 10 minutes.7 By this procedure, myocyte
nuclei were fully dissociated from the myocyte cytoplasm. For the visualization of metaphase chromosomes, nuclei were stained with propidium iodide,
and chromosomes were made apparent by the splash
technique." Subsequently, the percentage of nuclei
exhibiting metaphase chromosomes was determined
at x400 with an epifluorescent microscope (Nikon,
Tokyo, Japan) by counting an average of 500 nuclei
per ventricle in each animal. Chromosomes also were
stained with Giemsa and photographed with a regular light microscope.
The extent of nonmyocyte cells present in each
preparation in each ventricle was determined by
preparing smears of the isolated myocytes and staining them with hematoxylin and eosin. We assessed
the contribution of interstitial cells by counting 500
cells in each ventricle and then computing from these
counts the respective fractions of myocytes and nonmyocytes encountered.
On the assumption that under an extreme situation
100%c of nonmyocyte cells proliferate with a 20-hour
cell cycle and that mitosis corresponds to approximately 2 hours, it can be predicted that at any point
in time 10% of the cells will be somewhere in mitosis.
Because metaphase is one of the four phases of
mitosis, at most 2-36c of the contaminating cells will
be in metaphase. This calculation, in combination
with the estimation of nonmyocytes present in each
cell isolation, was used to correct the percentages of
of aging
nuclei exhibiting metaphase chromosomes that were
attributed to the myocyte population.
Data were collected blindly, and the code was
broken at the end of the experiment. Results are
presented as mcan+SEM. Comparisons among the
three age groups were performed with a multiple
analysis of variance and Scheffe's method.5 Comparisons between values of the left and right ventricles at
each age interval were performed with a paired
Student's t test. Values of p<0.05 were considered
significant.
The magnitude of counts used in this investigation
was selected on the basis of the principle of Poisson
statistics.9 With 500 nuclear images counted per
ventricle, sampling error in each determination was
4.0%, which is significantly lower than biological
variability, assumed to be at least 1OCtc.10 Because
biological variation, that is, difference among animals, is the major determinant of standard error,11 it
seemed justified to keep sampling error within 5.Otc.
Moreover, sampling error in each group of animals in
each determination was 2.2cc or less.
Results
Five animals were analyzed at 8-12 months of age
(body weight, 390+27 g) and four animals each at
19-24 (body weight. 428+20 g) and 28-32 (body
weight. 320t23 g) months of age. Heart weight was
898+25, l.007+58, and 1,224+72 mg, respectively.
Figure 1 shows metaphase chromosomes in nuclei
prepared from the left and right ventricular myocardium of Fischer 344 rats from adulthood to senescence. By counting the fraction of nuclei showing
metaphase chromosomes, we obtained the results
illustrated in Figure 2. Whereas values of 0.396c and
1.376c were found in the left and right ventricles at the
younger age interval, greater percentages were mea-
Anversa et al Myocyte Cellular Hyperplasia
4.0 r A
Left Ventricle
3.5
a)
Pe
0
3.0
2.5
:S
a)
2.0
0
0
1.5
1.0
0.5
0.0
a)
0
0.
m
0
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Age (months)
FIGURE 2. Effects of aging on the fraction of myocyte nuclei
exhibiting metaphase chromosomes. Results are presented as
mean ±SEM. *Statistically different from value measured at
8-12 months, p<O.05.
sured at 19-24 months in both ventricles. In the left
side, a 4.9-fold increase (p<0.03) occurred, whereas
in the right side, only a 1.35-fold augmentation was
seen. Moreover, this latter change was not statistically
significant (p<0.6). Thus, the fraction of nuclei exhibiting metaphase chromosomes increased 263% more
in the left than in the right myocardium. At 28-32
months of age, the percentages of nuclei with metaphase chromosomes were higher than those found in
the other two age periods. Throughout the entire age
interval examined, from 8-12 to 28-32 months, these
values increased by 6.3-fold (p<0.005) in the left and
2.3-fold (p<0.03) in the right ventricle. It should be
pointed out that these determinations have all been
corrected by their respective amounts of contamination represented by the fraction of nonmyocyte cells
found to be present after Percoll separation. Interstitial cells were seen to comprise 0.87±0.09%,
1.37±0.10%, and 1.43±0.11% of the myocyte preparations obtained from the left ventricle from rats at
8-12, 19-24, and 28-32 months of age, respectively.
Corresponding values in the right ventricle were
1.50±0.20%, 1.63±0.40, and 1.75±0.31%.
When comparisons between the two ventricles were
made, it was noted that nuclei showing metaphase
chromosomes represented a larger percentage in the
right than in the left myocardium at 8-12 months of
age. The right myocardium possessed 3.5-fold
(p<0.004) more nuclei with metaphase chromosomes
than the left myocardium. However, this difference was
no longer apparent at 19-24 and 28-32 months.
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Discussion
The results of this study indicate that ventricular
myocytes of the mammalian rat heart retain their
capacity to multiply by mitotic division in adulthood
and senescence. This possibility was demonstrated
here by the identification of metaphase chromosomes
in myocyte nuclei. Moreover, the ability of myocytes
to proliferate was found to be present in both the left
and right ventricular myocardium. However, differences were documented in the extent of this cellular
growth process as a function of age and between the
two ventricles. Whereas at 8-12 months relatively
lower percentages of myocyte nuclei showing metaphase chromosomes were detected in the ventricles,
these fractions consistently increased at 19-24 and
28-32 months. In addition, the degree of myocyte
nuclear mitotic division was found to be greater in
the right myocardium at 8-12 months, whereas similar values were seen in both ventricles at 19-24 and
28-32 months.
Although it is widely assumed that no division of
muscle nuclei and cells occurs once cell division has
ceased, shortly after birth in the mammalian myocardium,12 it has been demonstrated in humans13-15 and
animal experimentation16-18 that myocyte nuclei retain their capacity to endoreplicate their DNA with
ploidy formation.19 Moreover, myocyte nuclear hyperplasia repeatedly has been documented in the
heart of patients who died with severe degrees of
cardiac hypertrophy.20-22 Recently, multiplications of
myocyte nuclei in the left' and right2 myocardium in
association with proliferation of right ventricular
myocytes3,4 have been shown after prolonged pressure overload3 and aging.4 However, mitosis has
never been seen in myocytes of hypertrophying adult
heart, and questions have been raised concerning the
validity and the conclusions drawn from these quantitative evaluations.1-4,20-22
A number of studies addressing the temporal
relation between postnatal myocardial growth and
myocyte proliferation in the mammalian heart have
documented that DNA synthesis in myocyte nuclei is
present in nearly 20% of cells in the rat heart at birth
but decreases rapidly in the following 2-week period.23 A similar fast decline in the ability of myocytes
to synthesize DNA has been shown by Claycomb,24
and the disappearance of DNA polymerase activity in
these cells by day 17 after birth has been postulated
to represent the final event in the cessation of
myocyte proliferation. Consistent with these observations, the amount of mitotic activity in myocytes is
markedly reduced early after birth, becoming almost
undetectable in the heart of rats at 1 month of
age.25-27 The results obtained here in the ventricular
myocardium of aging Fischer 344 rats are clearly at
variance with these previous findings. This discrepancy is difficult to explain and may reflect not only
strain differences but also the precocious effects of
aging on the Fischer heart, which led to an abnormal
loading state of the ventricular myocardium during
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Circulation Research Vol 69, No 4 October 1991
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4-12 months of age.5 The hemodynamic profile,4-6
mechanical characteristics,28-30 and biochemical
properties31,32 of the heart vary among strains of rats,
and these factors all may be responsible for their
distinct life span33'34 and the variability in the myocyte capacity to proliferate at an earlier or later time
period of postnatal life. Importantly, myocyte hyperplasia was not documented in Sprague-Dawley rats
up to 19-21 months of age.6
Although the current results are necessarily restricted to the Fischer 344 rat model, they provide
not only a direct visualization of mitotic division in
myocytes but also a numerical estimate of this process in the aging myocardium. These data significantly extend the previous morphometric findings,4
which were limited in the characterization of the
dynamic state of the tissue in terms of whether
nuclear and cellular divisions were occurring at the
time of observation. Additionally, stereological measurements may fail in the recognition of myocyte
proliferation when loss of cells simultaneously occurs.4 Because of the opposite effects of myocyte
death and cell proliferation on the total number of
myocytes in the ventricular myocardium,4 quantitative morphology was unable to demonstrate myocyte
division in the aging left ventricle.
It should be recognized that nuclear mitotic division does not necessarily imply cytokinesis. At all
stages of growth, cardiac myocytes in the rat heart
are composed of mononucleated and binucleated
cells, and their proportion in the myocardium
changes with maturation and aging.4 This phenomenon complicates the interpretation of nuclear proliferation in terms of cell proliferation, because the
increase in number of nuclei merely may reflect an
augmentation in the frequency of nuclei per cell
without true cellular multiplication.1'2 However, the
fraction of mononucleated cells increases from 4 to
29 months in the left and right ventricles of Fischer
344 rats, whereas the percentage of binucleated cells
decreases.4 Thus, the phenomenon of myocyte nuclear mitotic division demonstrated here corresponds
to the actual addition of newly formed myocytes.
Moreover, with the assumption that the contaminating nonmyocyte nuclei in the preparation were undergoing rapid mitotic division, the magnitudes of
myocyte proliferation claimed here may represent
only minimal indexes of the real extent of this cellular
growth process in the myocardium.
The mechanism responsible for myocyte proliferation currently is unknown. However, the phenomenon of myocyte loss that accompanies the evolution
of life in rats4,6 can be expected to generate a greater
work load on the remaining myocytes proportional to
the amount of myocyte loss. A similar phenomenon
has been observed previously in experimental myocardial infarction.35 The condition of mechanical
overloading on the remaining cells may represent the
physical stimulus for myocyte growth, which is enhanced further by the deterioration of cardiac pump
function with aging.5 Although the increase in sys-
tolic and diastolic wall and cell stress5 may be implicated in the fundamental processes of cellular hypertrophy and hyperplasia,4 the pathway by which
external signals are transmitted from membrane to
nucleus still is unknown. Accumulating evidence
favors a role for the a1-adrenoreceptor as a molecular mediator of myocyte hypertrophy, hyperplasia,
and gene expression.36-38 In cardiac myocytes, occupation of surface a1-adrenoreceptors by an agonist
stimulates hydrolysis of phosphoinositides in a dosedependent, highly specific manner.39,40 Because many
known mitogens stimulate this effector pathway, it
has been suggested that intermediates of phosphoinositide hydrolysis may be involved in transducing
signals to the nucleus that alter transcription and
mitosis.41 Although the precise sequence of molecular events has not been clearly identified, it has been
demonstrated that stimulation of a1-adrenoreceptors
can induce the expression of growth-related factors42
and selectively increase the transcription of sarcomeric actin isogenes during myocyte hypertrophy.37'40
Whether these mediators may interact with other
intracellular signals to elicit a hyperplastic response
of myocytes is an important unknown question.
In the last two decades, a number of methodologies
have been described for the evaluation of myocyte
cellular hyperplasia in the ventricular myocardium.
Autoradiographic analysis of [3H]thymidine-labeled tissue has been used to establish whether DNA synthesis
in myocytes occurs during postnatal development23 and
after the imposition of an overload.17 However, limitations of this technique include the inability to demonstrate nuclear division or cytokinesis. Moreover, it is
impossible by this approach to distinguish whether
DNA synthesis in nuclei is due to nuclear hyperplasia,
ploidy formation, or DNA repair. Recently, the questionable reliability of thymidine labeling as an index of
cell proliferation has been emphasized.43 Flow cytometric determinations also have been used to characterize changes in DNA of myocyte nuclei.14'15 Although
this methodology offers the unique possibility of evaluating whether DNA content in myocyte nuclei is increased and in which phase of the cell cycle the nuclei
are, two significant problems with this technique have
to be considered, particularly in the case of binucleated
cells. If the myocyte cytoplasm is not fully destroyed
before measurements, two adjacent diploid nuclei of a
binucleated cell may be evaluated together, yielding a
false mitotic division count. Therefore, pure preparations of myocytes have to be obtained and the nuclei
subsequently separated before flow cytometric readings. Moreover, and most importantly, flow cytometry
does not allow the distinction between nuclei in the G2
phase and nuclei in the mitotic phase.
So far, the quantitative assessment of myocyte
nuclei,2-4,6,35 combined with the evaluation of the
distribution of nuclei in the myocyte cell population
after enzymatic dissociation of these cells,4 appears
to be the only approach that can demonstrate myocyte cellular hyperplasia in the myocardium. To the
Anversa et al Myocyte Cellular Hyperplasia
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best of our knowledge, no other technique can unequivocally document myocyte proliferation. However, morphometric estimations can characterize and
identify a phenomenon between two points in time,
but they provide no information on the dynamic state
of the tissue or cells at the moment of observation.
Moreover, the concomitant presence of myocyte loss
can obfuscate the detection of myocyte hyperplasia
by this methodology.4 On the basis of the discussion
above and the previous quantitative results,4 the
fraction of nuclei exhibiting metaphase chromosomes
was evaluated not only to show that myocyte cellular
hyperplasia occurs but also to demonstrate that the
increase in myocyte number occurs through mitotic
division. Thus, the visualization of chromosomes in
nuclei appeared to be the preferable technique to
answer the questions raised.
Having indicated the rationale for selecting the
methodology used in the current investigation, we
must acknowledge several limitations. The myocyte
isolation procedure may preferentially dissociate myocytes undergoing cell division, yielding a high percentage of mitotic counts. Moreover, the number of
viable myocytes collected after collagenase digestion
decreased as a function of age, and this phenomenon
may have influenced the accumulated results. Although it is not apparent why smaller fractions of
myocytes are obtained with aging and senescence,
the possibility may be raised that the continuous
accumulation of collagen in the myocardium with the
progression of life in the Fischer 344 rat4,5,28 may
interfere with the isolation procedure. In addition,
the increase of collagen is associated with a corresponding decrease in the percent of myocytes in the
tissue.4 A similar reduction in the yields of myocyte
isolation also has been found in Wistar rats.44 A
second potential problem concerns the impossibility
of maintaining the integrity of the cells, because
chromosomes can be identified only in lysed preparations of nuclei. Thus, correction for eventual contaminations from nonmyocyte nuclei have to be performed for quantitative data analysis. Finally, it is not
possible with this technique to establish for how long
metaphase chromosomes have been present in nuclei
and whether a block exists in this phase of mitosis.
All these factors may have contributed to an abnormal increase in the relative percentages of nuclei
exhibiting metaphase chromosomes without necessarily indicating a corresponding level of myocyte
cellular mitotic division.
In spite of these limitations, the results of the
present study indicate that the perennial question of
whether adult cardiac myocytes can proliferate by
mitotic division can be answered positively. Moreover, this cellular growth process occurs in left and
right ventricular myocytes and appears to parallel the
alterations in the loading state of the myocardium
accompanying age and senescence. However, the
actual magnitude of this phenomenon still is unclear.
Although these observations challenge the longstanding belief that myocyte mitotic division does not
1163
occur in the adult myocardium, the complexity of the
problem and the potential clinical importance of
these findings require further investigation. This is
currently in progress in our laboratory.
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KEY WORDS
* ventricles
metaphase chromosomes * aging * myocytes
Myocyte mitotic division in the aging mammalian rat heart.
P Anversa, D Fitzpatrick, S Argani and J M Capasso
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Circ Res. 1991;69:1159-1164
doi: 10.1161/01.RES.69.4.1159
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