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Point /Counterpoint
Ventricular Myocytes Are Not Terminally Differentiated in
the Adult Mammalian Heart
Piero Anversa, Jan Kajstura
condition of terminal differentiation, duplicate DNA, and
increase in number, regardless of the disease process and the
magnitude of myocardial mass. It is rather surprising that in
vivo documentation of the replicative capacity of fully
differentiated ventricular myocytes21 is questioned in view of
the absence of mitosis in artificial in vitro preparations mostly
restricted to neonatal myocytes.22–24 The logical expectation
would be the opposite. This article will review available
information obtained in vivo in animal models and humans
favoring and opposing the belief that myocyte cellular hyperplasia is a significant component of the growth mechanisms
of the overloaded heart.
Terminally Differentiated Cells
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Primary cultures of various cell types contain dividing and
nondividing cells.1,2 However, nondividing cells can rest in
the G0 phase and reenter the cell cycle on stimulation3–5 or
become terminally differentiated and die without dividing.6
This third category of cells is not dormant in G0 and cannot
reenter the cell cycle.6 For example, such a phenomenon is
present in vivo at the tip of the villi of the small intestine,7 in
the auditory hair cells of the ear,8 and in the upper layers of
the epidermis.9 In contrast, hepatocytes in vivo are in a G0
state,10 –12 and after partial hepatectomy or severe injury, liver
regeneration is accomplished by proliferation of mature
hepatocytes as well as biliary epithelial cells and fenestrated
endothelial cells.12 The reconstitution of liver mass is not
dependent on a reserve of stem cells, which condition
regeneration of bone marrow13 and skin.14 Multipotential cells
have also been identified in the central nervous system, but
their ability to grow, differentiate, and ultimately survive has
been problematic.15 In skeletal muscle, satellite cells can be
stimulated to proliferate and develop into mature myocytes,
representing an additional form of tissue regeneration.16 –18
These mechanisms of cellular growth have not been considered possible in adult cardiac myocytes, and the concept was
advanced that no proliferation of ventricular muscle cells
occurs once cell division has ceased, shortly after birth in the
mammalian heart.19
The dogma was introduced that adult cardiac myocytes are
terminally differentiated cells and, therefore, cannot be recalled into the cell cycle.20,21 These cells are not in G0, cannot
be triggered into the proliferative phase, but can perform their
physiological functions, undergo cellular hypertrophy, and
ultimately die. On this basis, in vitro preparations of neonatal
cardiac myocytes22–24 and skeletal muscle cell lines25 have
been used for the analysis of the molecular control of the cell
cycle in an attempt to identify the key factors of terminal
differentiation of myocytes. However, we will challenge the
premise that myocytes cannot reenter the cell cycle, synthesize DNA, undergo nuclear mitotic division, and proliferate
in vivo in the adult heart. To the best of our knowledge, there
is not a single piece of evidence that this cell population is
terminally differentiated in vivo. Such a contention requires
the demonstration that after mitosis these cells have entered a
physiological state from which they cannot be rescued by an
appropriate stimulus and that they are not able to leave this
History of the Terminally
Differentiated Myocyte
Numerous studies of the human heart from 1850 to 1911 held
the view that myocardial hypertrophy was the consequence of
hyperplasia and hypertrophy of existing myocytes.26 –35 However, subsequent reports from 1921 to 1925 have questioned
the ability of myocytes to proliferate, suggesting that the
increase in cardiac muscle mass in the pathological heart was
the result of pure cellular hypertrophy.36 –38 The concept that
myocytes cannot divide originated from the difficulty of
identifying mitotic figures in the cells and not from any
estimation of myocyte cell volume and number.38 Such a
conviction has gained support from the autoradiographic
analysis of [3H]thymidine incorporation in hearts of animals
during postnatal development39 and after conditions of overload.40 – 42 DNA synthesis in myocyte nuclei either was not
detected or was found to be essentially negligible. Although
we will subsequently provide evidence to the contrary, the
lack of DNA replication in cardiac myocytes experimentally
with physiological growth and cardiac hypertrophy and the
inability to recognize mitoses in the enlarged human heart led
to the conclusion that this cell population is not in a G0 state,
cannot reenter the cell cycle, and is, therefore, terminally
differentiated.
There are 4 issues that must be emphasized: (1) Results in
animals of different species are not directly applicable to the
human heart. (2) Postnatal myocardial growth is a slow
physiological process that involves several maturational
changes in myocytes, including differentiation and
multinucleation; myocardial hypertrophy in the adult is accomplished by the growth response of mature myocytes,
Received September 25, 1997; accepted April 8, 1998.
From the Department of Medicine, New York Medical College, Valhalla.
Reprint requests to Piero Anversa, MD, Department of Medicine, Vosburgh Pavilion, Room 302A, New York Medical College, Valhalla, NY 10595.
(Circ Res. 1998;83:1-14.)
© 1998 American Heart Association, Inc.
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Myocyte Proliferation
which is influenced by the nature and degree of the overload.
(3) [3H]Thymidine labeling cannot be equated with an increase in cell number. (4) The presence or absence of cell
proliferation can be determined only by counting the number
of cells. We will first summarize observations concerning the
changes in number and size of ventricular myocytes from
birth to adulthood in humans and animals and subsequently
relate how aging and pathological loads affect these cellular
characteristics. Finally, evidence supporting the role of insulin-like growth factor-1 (IGF-1) in stimulating adult ventricular myocytes to reenter the cell cycle and ultimately divide
will be discussed.
Postnatal Myocardial Growth
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The expansion of cardiac mass postnatally is accomplished
by increases in myocyte size and number.19,43 However, the
cellular changes taking place in the human heart have not
been carefully defined. There is no information on myocyte
volume at birth in the 2 ventricles, although the claim has
been made that myocyte diameter is comparable shortly after
birth in the left and right sides of the heart44,45 but that later in
life left ventricular myocytes are slightly larger than right
ventricular myocytes.46,47 The total number of ventricular
myocytes at birth is '13109,48,49 but this parameter is
extremely variable.50,51 Recently, myocyte number has been
measured in the male and female human heart at 20 years of
age. The male left ventricle contains 5.83109 myocytes, and
the right ventricular free wall possesses 2.03109 muscle
cells.46,47 Corresponding values in the female heart are
4.53109 and 1.43109 myocytes.47 These data suggest that a
significant increase in myocyte number occurs in both ventricles with maturation in men and women. However, whether
the process of cell proliferation persists in this 20-year period
or is completed by 5 months after birth51,52 or earlier53 remains
to be determined. The lack of knowledge involving the
cellular mechanisms implicated in the growth of the heart
during physiological cardiac hypertrophy is part of the
uncertainty concerning whether ventricular myocytes in humans actually become terminally differentiated rapidly after
birth. Cells are believed to be retained throughout adult life
and not to be replaced if they die. Understanding why such a
condition should exist in the heart is rather complex, since
myocytes are continuously lost as a function of aging
alone43,46,47,54 and in combination with various pathological
states.54 –58 Myocyte volume markedly increases postnatally,52,59 but further hypertrophy is limited in adulthood.60 As
stated in Molecular Biology of the Cell,61 “. . . it is difficult to
give any reason at all” why heart muscle cells should be
permanent and irreplaceable.
Similar problems exist in understanding the role of myocyte hypertrophy and hyperplasia in the developing heart of
the rat, the animal model most extensively investigated.43,62– 67
More limited studies have also been performed in mice.39,68,69
Two essential processes occur in the prenatal period: myocyte
mitotic division and structural differentiation of the myocyte
cytoplasm, involving the synthesis and organization of myofibrils and other cytoplasmic components.19,70 –72 In an attempt
to evaluate the contribution of myocyte proliferation prenatally and postnatally, the extent of DNA synthesis in myo-
cytes has recently been measured in rats73 and mice.69 However, the behavior of the rat heart differs from that of the
mouse heart. Bromodeoxyuridine (BrdU) labeling of left
ventricular myocytes involves nearly 17% of cells in the fetal
rat heart at the end of gestation, decreasing to 13% one day
after birth. In the adult rat heart, values of 0.2%,73 0.45%,74
0.9%,75 and 2%76 have been reported. Conversely, in the
mouse, DNA synthesis is markedly reduced at birth but
increases to '10% at neonatal days 4 and 5. DNA replication
disappears at day 10 and remains absent later in life.69
However, Petersen and Baserga39 demonstrated in 1965 that
DNA synthesis occurs in myocyte nuclei of the mouse heart,
and a thymidine labeling index of 2.2% was found at 32 days
of age. Lower percentages of myocyte nuclei labeling and
earlier disappearance of DNA replication also have been
described.77,78 The inability of myocytes to synthesize DNA
may not apply to all strains of mice; a mitotic image in a
myocyte of the left ventricle of a normal FVB/N mouse at 2.5
months of age is illustrated by confocal microscopy in Figure
1. Mitosis in myocytes has been detected in rats at 2 months
of age as well.79 Although differences exist in the literature,
the apparent contrasting observations in rats and mice have
suggested opposite conclusions: myocytes maintain low levels of DNA replication in the normal adult rat heart,73–76
whereas this capacity is excluded in the adult mouse myocardium. In the latter case, it is proposed that myocyte
proliferation is completed prenatally, and postnatal DNA
synthesis reflects binucleation only.69
A few comments have to be made in an attempt to explain
the discrepancy in magnitude and timing of DNA labeling
between the rat and mouse heart. In different species, including humans, the mammalian heart varies dramatically in
weight, although myocyte cell volume remains relatively
constant.46,47,68,80,81 Similarly, in individual species, the greater
muscle mass of the left ventricle with respect to the right
ventricle is only minimally influenced by myocyte
size.46,47,53,80 Myocyte cell number is the critical factor responsible for the increases in cardiac weight in larger animals. On
this basis, it is reasonable to assume that heavier hearts with
several-fold higher numbers of myocytes may undergo DNA
replication and myocyte proliferation for longer periods of
time after birth. The persistence of thymidine labeling in
myocyte nuclei postnatally in the rat heart has been observed
in several other studies,82– 86 indicating that mice and rats may
not be identical in this cellular aspect. Moreover, caution
should be exercised in the interpretation of BrdU and thymidine labeling data as indicators of the terminal differentiation
of myocytes. An example is given below.
Recently, the suggestion has been made that DNA replication in myocytes at day 4 and later in rats is coupled with
binucleation exclusively, in the absence of cell proliferation.87
This conclusion was reached since a single injection of BrdU
at day 4, when most of the cells are mononucleated, resulted
in the same percentage of positively labeled myocytes at 2
hours and up to 8 days after BrdU administration. Myocyte
binucleation has been claimed to be completed by day 12.87
However, the BrdU labeling index in the manner used has
little to do with either binucleation or lack of cell division. Let
us assume that 10 myocytes are present in the heart and that
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Figure 1. Nuclear mitotic division in a myocyte nucleus from
the left ventricle of a normal adult mouse heart (A to C). The
nuclear membrane is lost, and cross sections of metaphase
chromosomes (arrows) are illustrated by blue fluorescence of
propidium iodide (A). Myocytes are recognized by the red fluorescence of a-sarcomeric actin antibody labeling of the cytoplasm (B). These two images are combined to demonstrate the
localization of mitosis in a myocyte (C). Original magnification
(by confocal microscopy) 32000.
2 mononucleated cells incorporate BrdU, ie, 20%. If these 2
myocytes become binucleated as do the remaining 8 cells, 2
myocytes (4 nuclei) out of 10 (20 nuclei) will be labeled, ie,
20% of the population. Consistent with the authors’ contention, BrdU staining was associated with binucleation without
cell proliferation. Let us now consider that these 10 binucleated myocytes in which 2 are labeled by BrdU all divide,
generating 20 cells (40 nuclei). As a consequence, 4 myo-
July 13, 1998
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cytes (8 nuclei) will be labeled, ie, 20%; this condition
reflects a 2-fold increase in cell number in spite of an
identical percentage of BrdU labeling. Let us now take into
account a more complex situation: a population of 20 cells, 10
mononucleated and 10 binucleated in which 2 in each group
are BrdU-labeled. If 50% of each cell category enter the cell
cycle and mononucleated cells become binucleated and
binucleated cells form new binucleated myocytes, the results
are as follows: 5 mononucleated myocytes with 1 stained by
BrdU and 20 binucleated myocytes with 4 stained by BrdU.
Again, 5 BrdU-labeled cells out of 25 equals 20% of labeling
in the presence of a 25% myocyte proliferation. It is relevant
to emphasize that these calculations correctly consider that
BrdU-labeled myocytes behave in a manner identical to
unstained cells. Importantly, the claim has been made that the
consistency in the fraction of myocytes labeled by BrdU over
time is a demonstration of the lack of cell division, whereas
an increase in labeling index would be indicative of cell
multiplication.87 The latter implies that BrdU, per se, can
trigger a cell to reenter the cell cycle and undergo cytokinesis.
As stated in Baserga’s book in 1985,6 “cell proliferation
means that cells are dividing, and the only way to determine
the number of cells . . . is to count them.” Additionally, DNA
synthesis in myocyte nuclei in the rat heart does not end at
day 12, when binucleation is completed. Results from our
laboratory in the same strain of Sprague-Dawley rats have
shown a 0.52% labeling index at 3 weeks,73 and values of
1.5% and 0.58% have been reported from Clubb and Bishop67
for the left and right ventricles of Fischer 344 rats at the
identical age.
Quantitative analyses of the changes in myocyte number in
the heart of Wistar and Sprague-Dawley rats with maturation
are rare; the results are variable; and the differences are, at
times, difficult to explain.86 –92 However, the major increases
in cell number appear to occur in the early postnatal period,
a phenomenon that may also apply to the mouse heart.68,93 The
contradictory observations may reflect, at least in part, the
limitations inherent in the methodologies used and instrumentation available at the time of these investigations. Additionally, not a single experiment examined the changes in
myocyte cell volume and number at different time points
during postnatal development and in the adult heart simultaneously. The number of myocytes in the right ventricle
achieves an adult value almost immediately after birth,88 and
numerous studies have examined the entire heart, neglecting
the possibility of regional differences in myocyte proliferation. The deficiencies in the in situ morphometric techniques
involve several factors: (1) The presence of multinucleated
myocytes could not be detected in histological sections,
affecting the recognition of the various cell populations in
situ.94 (2) Neither the percentage of myocardium occupied by
mononucleated and multinucleated myocytes nor the relative
contribution of each cell category to the aggregate myocyte
mass could be determined.81 (3) In situ determinations of cell
volume were restricted to myocyte volume per nucleus, and
cell number was based on the measurement of the total
number of nuclei in the ventricle.94 (4) The enzymatic
dissociation of myocytes, which allows evaluation of the
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Myocyte Proliferation
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percentages of mononucleated and multinucleated cells,64 was
not used in early studies.88
Problems also exist in the assessment of myocyte volume
of isolated cells by Coulter counter from which cell number
was calculated: (1) A cell suspension is run through the
machine, and the distribution curve generated by the computer requires that an arbitrary gate be introduced by the
observer to establish what will be considered a reliable value
of cell volume. This approach results in the exclusion of small
and large myocytes, introducing a bias factor, defeating the
principle of morphometry. (2) Fragments of myocytes, comparable in volume to an assumed acceptable cell size, are
included in the estimation of this parameter. Aggregates of
interstitial cells are commonly present at this stage, further
weakening the validity of this technique. (3) Coulter counter
data do not provide information regarding the volume of
mononucleated and multinucleated cells, which is critical for
assessment of the total number of myocytes in the ventricles.
In summary, whether an increase in cell number occurs in
the myocardium in the later phases of postnatal development
remains to be determined. Future studies applying morphometric methodologies in situ in combination with myocyte
isolation and confocal microscopy68,81,95 will enable us to
establish whether cell division is a critical aspect of the
maturation of the heart. However, the question whether
myocytes become terminally differentiated shortly after birth,
ie, cannot reenter the cell cycle, may be negatively answered.
DNA synthesis and nuclear mitotic division persist in the
adult mammalian heart. The apparent discrepancies between
the ability of myocytes to divide and the quantitative results
may reflect ongoing cell dropout with aging and the possibility of a slow continuing turnover of old dying myocytes
and new cells.
Aging of the Myocardium
According to the dogma, the number of muscle cells in the
mammalian heart is defined at birth, and in the absence of
cardiac disease, these myocytes persist throughout the life of
an individual or animal. Men and women of 100 years of age
and older are present in our society, and nobody dies without
a heart. The inevitable implication of the dogma is that
myocytes are immortal. Alternatively, myocytes may not live
indefinitely, they may have a predetermined life span, and the
heart may be characterized by ongoing cell loss and cell
regeneration during the entire life of a human being or an
animal. We will summarize available information supporting
the concept that myocyte death occurs with aging; more
complex is recognizing whether cell renewal accompanies the
progression of life in the normal heart.
In the last 25 years, numerous investigations have been
performed to identify the pathophysiological bases responsible for the diminished ability of the aged heart to sustain
increases in pressure and volume loads.58,96 –100 Moreover, the
adaptation of the coronary vasculature and microvasculature
with aging has been characterized functionally and structurally.43,54,96,101–103 Effort has also been made to identify the
molecular mechanisms implicated in the limitations of the
aged heart to adapt to stressful conditions.104,105 However, to
the best of our knowledge, there are only 2 studies in which
the alterations in the number of ventricular myocytes with
aging alone have been measured in the male46,47 and female47
human heart. In the male heart, from 17 to 89 years of age, the
absolute number of myocytes decreases by 45 million per
year in the left ventricle and by 19 million per year in the right
ventricle. In contrast, the number of myocytes in the left and
right ventricles remains essentially constant in women from
20 to 95 years of age. Myocyte loss in the male heart is
associated with hypertrophy of the remaining cells. Cell
volume is not altered in women. Importantly, the etiology of
myocyte death in the aging human heart remains to be
determined.
The bases for the differential effects of aging on ventricular
myocytes in men and women are currently unknown. Although
cardiovascular events in women increase after menopause,100,106
sex hormones do not appear to influence myocyte loss with
aging. The number of myocytes in the myocardium does not
decrease in women from 55 to 95 years.47 It is surprising that no
information is available concerning the influence of gender on
the volume composition of the myocardium and quantitative
structural properties of the capillary network controlling tissue
oxygenation as a function of aging. However, apoptosis of
myocytes occurs in both men and women under pathological
conditions,107–110 suggesting that the female heart is not protected
from this form of cell death. Similarly, cell necrosis is present in
women with ischemic cardiomyopathy.111,112 It is unlikely that
cell death is not a component of the aging female heart.
Conversely, the hypothesis may be advanced that the regenerative capacity of myocytes is greater in women, maintaining the
number of myocytes constant. Additionally, when normal aging
is not distinguished from the superimposition of hypertension,
valvular disorders, diabetes, amyloidosis, and ischemic heart
disease, which all increase in the elderly,113 no difference is
detected between the sexes, and cardiac weights increase up to
110 years of age.114 The inclusion of myocardial hypertrophy of
various origin in the study of the aging heart has indicated that
the number of myocytes does not change in the left and right
ventricles up to 90 years.51,115 These apparent contradictory
observations are difficult to explain. However, the suggestion
can be made that myocyte proliferation may occur in the
hypertrophied ventricles,116 masking the phenomenon of cell loss.
In view of the difficulty in identifying the mechanisms of
myocyte loss and the potential of cell proliferation in the
human heart with age, these cellular processes have been
studied in aging male Fischer 344 rats. In this strain and sex,
cell death has been detected as early as 3 months after birth
in the left and right ventricles. However, myocyte necrosis
and apoptosis are both present in the left ventricle, whereas
myocyte necrosis exclusively affects the right ventricle.117
This distinction in the forms of cell death between the
ventricles persists throughout life. Myocyte death increases
with age, but a reduction in the total number of myocytes of
nearly 19% occurs biventricularly only at 12 months.118
Myocyte number in the left ventricle does not change from 12
to 29 months, and a 1.8-fold increase takes place in the right
ventricle during this interval. The preservation of the aggregate number of muscle cells in the left and the increase in the
right ventricle with age occur in spite of the concurrent loss
of myocytes in the entire heart.117 Myocyte death and regen-
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Figure 2. A and B, DNA synthesis in a myocyte
nucleus from the left ventricle of a male
4-month-old Fischer 344 rat. Bromodeoxyuridine labeling of the nucleus is shown by green
fluorescence (A), and the localization of the bromodeoxyuridine-stained nucleus in the myocyte
is illustrated by a combination of phase-contrast microscopy and bisbenzimide fluorescence (B). Arrows indicate the labeled nucleus.
Small fluorescence dots in panel A correspond
to lipofuscin. C and D, Nuclear mitotic division
in a myocyte nucleus from the left ventricle of a
male 4-month-old Fischer 344 rat. Metaphase
chromosomes (arrows) are shown by bisbenzimide fluorescence (C), and myocytes are recognized by the red fluorescence of a-sarcomeric actin labeling of the cytoplasm (D).
Arrowheads indicate nondividing myocyte
nuclei. Original magnification 31200 (A and B)
and 31400 (C and D).
eration are easily identified in adult rats at 3 to 4 months,
since not only are apoptosis and necrosis present, but BrdU
labeling and nuclear mitotic division are also observed in
these cells (Figure 2). Ventricular dysfunction and failure in
this model become apparent at 24 to 29 months,97,98,117,118
minimizing the role of hemodynamic factors in cell death and
proliferation in young animals. Thus, adult myocytes are not
immortal and may have a predetermined life span, and new
cells can be made by mitotic division. However, whether old
cells are more predisposed to die cannot be established at
present. There are no markers of myocyte aging, and the
acquisition of tools capable of discriminating senescent from
young cells99,119 may be a major challenge of future research.
The combination of cell death, DNA replication, and
myocyte nuclear mitotic division is not restricted to Fischer
344 rats. BrdU labeling of myocyte nuclei73–75 and mitotic
images are characteristic aspects of male Sprague-Dawley
rats as well.79 Mitoses in adult myocytes79 and cell loss80 have
been reported in this strain, suggesting that a balance between
cell growth and cell death may be present in adult animals. In
this model, as in the Fischer rat, myocyte cell loss precedes
the occurrence of ventricular dysfunction.80 These experimental findings and the results in humans are consistent with the
contention that myocyte cell death is a typical feature of the
detrimental effect of aging on the male mammalian heart.
Additionally, a slow rate of myocyte turnover may be
implemented in the rat heart, but no information is available
to indicate that a similar phenomenon occurs in humans.
Whether sex differences are important variables that influence the susceptibility of myocytes to die and regenerate in
adulthood and aging is currently unknown.
Although heart failure is a disease of the elderly, with 3.2
million patients older than 65 years of age,120 the results
discussed above46,47 do not support the concept that aging, per
se, leads to an impairment in pump performance. In the
population examined, stringent criteria for inclusion were
followed, excluding all hearts with any form of functional or
histologically detectable myocardial damage.46,47 However,
whether heart failure in an aged individual is always attributable to a secondary disease of the myocardium and coronary vasculature or whether, at times, aging may be regarded
as the primary cause of the abnormality in cardiac hemodynamics remains to be determined clinically. This is a relevant
issue, since heart failure is the major cause of death in the
elderly.58 Moreover, severe ventricular dysfunction develops
in Fischer 344 rats late in life97,98,117,118; this deleterious impact
of aging in the animal model is characterized by a high level
of mitosis and myocyte proliferation.80,121 Flow cytometric
analyses of DNA content in myocyte nuclei have been
consistent with these results.122 Preliminary observations by
confocal microscopy of BrdU-labeled myocyte nuclei in the
tissue and mitoses in isolated myocyte preparations confirm
active DNA replication (Figure 3) and nuclear mitotic division (Figure 4) in aged animals. Conversely, it is not clear
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Myocyte Proliferation
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Figure 3. DNA synthesis in myocyte nuclei from the left ventricle of a male 27-month-old Fischer 344 rat. Bromodeoxyuridine labeling
of nuclei (arrows) is shown by the green fluorescence (A and D), and myocytes are identified by the red fluorescence of a-sarcomeric
actin staining of the cytoplasm (B and E). These images are combined in panels C and F. Original magnification (by confocal microscopy) 32000 (A to C) and 31400 (D to F).
whether these findings in male Fischer 344 rats are actually
applicable to the aged, senescent, failing human heart.
Heart Failure
The possibility that cardiac hypertrophy in humans is the
result of myocyte hypertrophy and hyperplasia was advanced
.40 years ago by Linzbach.45,60 The critical heart weight
theory introduced by this author in the late 1940s and early
1950s has suggested that, in the presence of a cardiac weight
of $500 g, myocyte proliferation begins and that this cellular
process constitutes the prevailing mechanism of additional
growth of the heart.116 These findings were restricted to the
left ventricle, but subsequent observations have demonstrated
that a similar response occurs in the right ventricle.123 The
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Figure 4. Metaphase chromosomes (arrows) in a binucleated myocyte are shown separately by blue fluorescence in panels A and D
and in combination in panel G. Myocytes are recognized by the red fluorescence of a-sarcomeric actin labeling of the myofibrils
located at the periphery of the cell. The empty area in the center corresponds to undifferentiated cytoplasm (B, E, and H). Arrows in
these 3 panels point to the localization of metaphase chromosomes. These images are combined in panels C, F, and I. The 2 nuclei
undergoing mitosis were 11 mm apart. Original magnification (by confocal microscopy) 31400.
existence of myocyte proliferation in the human heart was
challenged in 1972,51,115 but several more recent studies have
confirmed Linzbach’s results.50,124 –130 However, the morphometric data supporting cellular hyperplasia have been based
mostly on the assumption that myocytes are mononucleated
cells and that an increase in the total number of myocyte
nuclei in the ventricle corresponds to an identical increase in
the aggregate number of cells in the heart. Different proportions of mononucleated and multinucleated myocytes may be
present in the myocardium during normal and pathological
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Myocyte Proliferation
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states, and this raises the question of the distinction between
nuclear hyperplasia without cytokinesis, ie, increased number
of nuclei per cell, and cellular hyperplasia, ie, increased
number of myocytes. This critical issue has recently been
addressed by examining the distribution of nuclei in myocytes isolated from 72 normal and 176 pathological hearts.130
Aging, cardiac hypertrophy, and ischemic cardiomyopathy do
not change the fraction of mononucleated and multinucleated
myocytes in the ventricular myocardium.
The documentation that conditions of overload, aging, and
ischemic heart disease do not alter the relative proportion of
the various myocyte populations130 strongly suggests that the
demonstrations of myocyte cellular hyperplasia obtained
initially by Linzbach45,60 and subsequently by other investigators50,124 –130 are valid. Additionally, mononucleated cells
represent 3/4 of the myocytes of the human heart, differing
significantly from other mammals, such as mice,68 rats,64 and
dogs.81 Whether this cellular characteristic may influence the
ability of myocytes to reenter the cell cycle and ultimately
divide is currently unknown. Similarly, it is not clear whether
the massive cardiac hypertrophy that can be achieved in the
human heart116,125,130 is related, at least in part, to the high
percentage of mononucleated myocytes. Heart weight in
humans may increase 3-fold, reaching values of $1000
g.60,116,125,130 Decompensated eccentric hypertrophy in humans
is characterized by increases in myocyte number that vary
from 20% to .100%.60,116,123–130 This phenomenon does not
seem to be affected by aging,129 although difficulties exist in
establishing whether myocyte proliferation in the aged pathological heart occurred at a younger age. In an analysis of 7112
human hearts from birth to 110 years of age, Linzbach114 has
shown that extreme forms of cardiac hypertrophy are detectable up to the ninth decade of life, and heart weights of 500
and 600 g are present in patients at 100 years and older.
The morphometric results in the human heart have been
questioned repeatedly, and the ability of adult myocytes to
undergo hyperplasia has been a matter of controversy for
several decades and still persists in the scientific community. The bases for the opposition to these quantitative
findings are difficult to understand because the issue of the
existence or absence of an increase in the number of
myocytes in the hypertrophied decompensated human
heart can be addressed only by morphometric methodologies. There is no alternative technique that can be used.
Moreover, scar formation and myocyte loss are typical
aspects of cardiac failure, suggesting that the measurements of myocytes in the pathological heart are underestimations of the actual number of cells that has been
formed in the course of the disease. Cell death by apoptosis
is present in the terminal phases of heart failure,108,110 and
this ongoing process, distinct from myocyte necrosis and
tissue fibrosis,131 further affects the aggregate number of
cells in the ventricle. This may not be the case when
cardiac hypertrophy is in its compensated stage.132
We will now consider the determinant factor on which the
dogma that ventricular myocytes are terminally differentiated
cells was based in 1925.38 As indicated earlier, this consisted
of the inability to detect mitotic figures in myocytes of
hypertrophied hearts. However, in the last few years, such
documentation has been obtained in patients affected by acute
and chronic intractable cardiac failure,21,133 and a new example is illustrated in Figure 5. For the first time, a mitotic index
has been measured.133 The demonstration that 11 myocyte
nuclei per 1 million cells, or 59 000 nuclei in the entire left
ventricle, exhibit mitotic images in the failing heart may
explain why difficulties exist in identifying mitoses with
routine microscopic sampling. Moreover, in most cell systems, the 4 phases of mitosis are completed in 1 hour.6 Since
there is no logical reason to assume that the duration of
mitosis is different in myocytes, the chances of its detection
in this cell population are extremely limited. The impossibility of seeing prophase in tissue sections may reduce further
our time of recognition of mitosis. However, the low frequency of mitosis should not suggest that this cellular process
is of minor importance in the generation of new muscle cells:
in a 45-year-old man with 5.23109 nuclei in the left ventricle,47 a mitotic index of 11 nuclei per 106 and a mitotic time
of 1 hour will generate 10% new myocytes in 1 year and
almost double, 1.96-fold, the entire number of ventricular
myocytes over a period of 10 years. These calculations
assume that myocytes divide only once and are consistent
with quantitative results in the severely hypertrophied human
heart.60,116,124,125
Experimentally, results concerning the terminal and nonterminal differentiation of ventricular myocytes in the adult
heart have been contradictory. Initial observations in the late
1960s and early 1970s challenged not only the existence of
myocyte proliferation but also the sequence of events postulated by Linzbach45,60,116 in which myocyte hyperplasia was
regarded as a late response of the myocardium preceded by
cellular hypertrophy. This is because short-term cardiac
hypertrophy in the rat is characterized by enlargement of
preexisting myocytes with little or no DNA synthesis in
myocyte nuclei, possibly representing polyploid cells.40 – 42
However, these studies neglected the role of time and the
condition of the overload. It is rather surprising that no
functional measurements were obtained in these and more
recent investigations to establish whether ventricular dysfunction and failure were present in the animal models. In the
absence of these criteria, comparisons with the decompensated human heart are impossible.
The magnitude of the hemodynamic stress may be the
most critical variable in the initiation of myocyte hypertrophy or hyperplasia in the pathological heart. After a
gradual and moderate increase in workload on the heart,
myocyte cellular hypertrophy predominates,41,42,65,134 –136
whereas a severe increase in ventricular loading, acute and
chronic in nature, may engender DNA synthesis, mitosis,
and myocyte proliferation.83,86,137–139 DNA replication and
mitotic divisions in myocytes are apparent in the decompensated postinfarcted rat heart (Figure 6). Moreover,
quantitative analysis in rats86,118 and dogs81,139 has documented that the number of nuclei per cell does not change
in the failing heart, indicating that increases in the number
of myocyte nuclei in the ventricle correspond to identical
increases in myocyte number. Similarly, nuclear mitotic
divisions reflect the generation of new myocytes. These
conclusions are in contrast with observations in the mouse
Anversa and Kajstura
July 13, 1998
9
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Figure 5. Mitotic figure in a left ventricular
myocyte of a patient affected by ischemic cardiomyopathy. Metaphase chromosomes
(arrows) are illustrated by blue fluorescence (A).
Myocytes are recognized by the red fluorescence of a-sarcomeric actin antibody labeling
of the cytoplasm (B). These 2 images are combined to demonstrate the localization of mitosis
in a myocyte (C). Original magnification (by
confocal microscopy) 31400.
heart, in which isoproterenol-induced cardiac hypertrophy
did not show DNA replication in myocyte nuclei.140 This
apparent discrepancy may be due to the preservation of
function in this model. Conversely, BrdU labeling of
myocyte nuclei is present in the surviving myocardium
after infarction and ventricular failure in the mouse (not
shown). In this regard, cyclin D1 overexpression in myocytes in vivo is coupled with DNA synthesis and
multinucleation.141 Whether these transgenic mice are
characterized by myocyte proliferation remains to be
determined.
Increases in the number of myocytes in the overloaded
rat heart vary from 20% to 45%.86,89,118 These values are
lower than those detected in humans but may be influenced
by the simultaneous occurrence of myocardial damage and
cell death. Myocyte loss complicates the estimation of the
real number of newly formed myocytes in the ventricle;
myocyte loss results in an underestimation of myocyte
cellular hyperplasia, whereas myocyte hyperplasia leads to
an underestimation of the magnitude of myocyte death in
the myocardium. Thus, myocyte proliferation may be
obscured by myocyte loss, and the measurement of cell
number may not provide evidence of cell regeneration.
This seems to be the case in the failing left ventricle of rats
after coronary artery constriction86 and of dogs after
ventricular pacing.81,139 Prolonged systemic hypertension in
rats is associated with cellular hyperplasia142,143 in spite of
diffuse tissue injury.143
10
Myocyte Proliferation
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Figure 6. Nuclear mitotic divisions in myocytes located in
the region adjacent to (A and
B) and distant from (C and D)
the infarcted portion of the left
ventricle in rats 7 days after
coronary artery occlusion. Late
prophase with the appearance
of chromosomes and loss of
nuclear membrane is apparent
in panels A and B. Metaphase
chromosomes are evident in
panels C and D. Mitoses are
shown by blue fluorescence,
and myocytes are identified by
the red fluorescence of a-sarcomeric actin antibody labeling
of the cytoplasm. Original
magnification (by confocal
microscopy) 31400.
IGF-1 and Myocyte Growth
Several studies from our laboratory have raised the possibility
that the reentry of myocytes into the cell cycle occurs in the
adult rat heart in the presence of cardiac failure.86,118,143 Such
a response of the overloaded heart is characterized by the
stimulation of IGF-1 and IGF-1 receptors in the cells, which
precedes the expression of late growth-related genes, DNA
replication, myocyte nuclear mitotic division, and cell division.86,137,138,144 Moreover, the quantity of cyclins E, A, and B
and cyclin-dependent kinases, cdk2 and cdc2, is enhanced in
combination with their associated histone H1 kinase activity.145 These latter observations are consistent with the con-
tention that myocytes can synthesize nuclear proteins, modulating the progression of these cells through the cell cycle
and mitosis. Conversely, the suggestion has been made that
cyclin A disappears during early postnatal life in the rat,
becoming undetectable by day 14 after birth.146 Such a
phenomenon has been implicated in the permanent withdrawal of myocytes from the cell cycle. The discrepancy
between these results is difficult to explain, because the lack
of this cyclin has been claimed after analyzing tissue samples
that contain fibroblasts and endothelial cells that actively
proliferate with maturation.73 Additionally, numerous reports
have shown the persistence of low levels of DNA replication
Anversa and Kajstura
July 13, 1998
11
of myocyte volume and number or of indices of cell proliferation. Importantly, mice overexpressing IGF-1 characteristically show an attenuation in ventricular dilation and myocardial loading after infarction,154 but whether this beneficial
effect is mediated by the increased number of ventricular
myocytes and/or reduction of myocyte death in the surviving
myocardium remains to be determined.
Acknowledgments
Figure 7. Effects of postnatal maturation on the aggregate number of myocytes in the heart of wild-type (open bars) and IGF-1
transgenic (solid bars) mice. Results are presented as
mean6SD. d indicates days of age. *P,0.05 vs corresponding
value in wild-type mice.
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in adult myocytes.73–75 Attenuation of the IGF-1–IGF-1 receptor autocrine system in the cells appears to be coupled with
the decline in myocyte proliferation with cardiac
development.73,147
The in vivo results suggesting a potential link between
IGF-1 and multiplication of myocytes have not been paralleled by similar observations in vitro. Culture studies of adult
myocytes have documented that IGF-1 activates DNA synthesis148,149 and the expression of structural proteins, which are
consonant with a hyperplastic and hypertrophic response of
these cells.148 Moreover, IGF-1 increases the formation of
myofibrils only in long-term cultures of matured myocytes.150
When myocytes surviving an acute myocardial infarction are
stimulated by IGF-1 in vitro, the quantities of cyclin D1, E, A,
and B are upregulated, and this adaptation may reflect the
increase in IGF-1 receptors on the cells.149 Cyclin D1, E, and
A kinase activity also increases, but cyclin B kinase activity
is not enhanced by IGF-1. Concurrently, DNA replication in
myocyte nuclei is detected, whereas mitotic division is not
observed. Several phenomena may be involved in this block
of the cell cycle in vitro. Myocardial loading may be critical
for the progression of myocytes through mitosis, and mechanical forces are abolished in culture. Alternatively, other
growth factors may be implicated in combination with IGF-1
in the regulation of mitosis in this cell population.
To characterize more directly the growth-promoting effect
of IGF-1 on myocytes in vivo, transgenic mice have been
obtained in which the cDNA for the human IGF-1B was
placed under the control of a rat a-myosin heavy chain
promoter.68 In mice heterozygous for the transgene, the
aggregate number of myocytes in the heart is identical to that
in nontransgenic littermates at birth but increases 21%, 31%,
and 55% at 45, 75, and 210 days of age (Figure 7). This
increase in cell number involves mostly binucleated myocytes. By use of confocal microscopy, the average volume of
mononucleated, binucleated, trinucleated, and tetranucleated
myocytes has been measured for the first time. Overexpression of IGF-1 appears to have no influence on cell volume,
further suggesting that this growth factor sustains cell division in myocytes of the adult heart. These results are in
contrast with the contention that IGF-1 induces only cellular
hypertrophy in vivo in the overloaded heart.151–153 This conclusion has been reached in the absence of any measurements
This study was supported by grants HL-38132, HL-39902, HL43023, and AG-15746 from the National Institutes of Health and by
a Grant-in-Aid from the American Heart Association (No. 950321).
The expert technical assistance of Maria Feliciano is
greatly appreciated.
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KEY WORDS: cell proliferation
n cell cycle
n cell
death
n insulin-like
growth factor-1
Ventricular Myocytes Are Not Terminally Differentiated in the Adult Mammalian Heart
Piero Anversa and Jan Kajstura
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Circ Res. 1998;83:1-14
doi: 10.1161/01.RES.83.1.1
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