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The Influence of Naturally Occurring Differences in Birthweight on Ventricular Cardiomyocyte Number in Sheep.

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THE ANATOMICAL RECORD 292:29–37 (2009)
The Influence of Naturally Occurring
Differences in Birthweight on
Ventricular Cardiomyocyte
Number in Sheep
VICTORIA STACY, ROBERT DE MATTEO, NADINE BREW, FOULA SOZO,
MEGAN E. PROBYN, RICHARD HARDING,* AND M. JANE BLACK
Department of Anatomy and Developmental Biology, Monash University,
Victoria, Australia
ABSTRACT
In most species including man, cardiomyocytes cease proliferating
soon after birth when they become terminally differentiated. A reduced
complement of cardiomyocytes in infancy may adversely impact on the
function and adaptive capabilities of the heart in later life. Low birthweight is associated with an increased risk of heart disease in adults, but
little is known about its effect on the number of cardiomyocytes. Using
naturally occurring differences in birthweight, our aim was to determine
the effect of birthweight on cardiomyocyte number in postnatal lambs. At
9 weeks after term birth, when the final number of cardiomyocytes is considered to be established, hearts were collected at necropsy from seven
singleton and seven twin lambs. Hearts were perfusion-fixed, and tissue
samples were systematically taken from the left ventricle plus intraventricular septum (LV1S) and the right ventricle (RV). The number of cardiomyocyte nuclei was estimated using an unbiased optical disector–fractionator stereological technique, and the total number of cardiomyocytes
was determined. Weights of the total heart, LV1S and RV were significantly related to both birthweight and necropsy weight. In the LV1S but
not the RV, cardiomyocyte number was significantly and directly related
to heart tissue weight, birthweight, and necropsy weight. We conclude
that the final number of cardiomyocytes in the LV1S is related to prenatal and early postnatal growth, and is proportionate to the weight of
heart tissue. A low cardiomyocyte number in the LV1S following restricted fetal growth may contribute to the increased incidence of heart
disease in adults born with low birthweight. Anat Rec, 292:29–37,
2009. Ó 2008 Wiley-Liss, Inc.
Key words: fetus; newborn; heart; cardiomyocytes; growth
Epidemiological studies have shown that low birthweight due to intrauterine growth restriction (IUGR) is
associated with an increased risk of cardiovascular disease in adult life (Barker et al., 1993; Zhao et al., 2002;
Lawlor et al., 2004). Previous experimental studies have
shown that IUGR alters the development and function
of major components of the cardiovascular system such
as the kidneys (Manalich et al., 2000), vasculature
(Goodfellow et al., 1998; Martin et al., 2000), and the activity of the sympathetic nervous system (Phillips and
Barker, 1997). However, the effects of IUGR on the development of the heart have not been well defined, with
Ó 2008 WILEY-LISS, INC.
Grant sponsor: National Health and Medical Research Council of Australia.
*Correspondence to: Richard Harding, Ph.D., D.Sc., Department of Anatomy and Developmental Biology, PO Box 13C,
Monash University, VIC 3800, Australia. Fax: 1613-9905-2330.
E-mail: richard.harding@med.monash.edu.au
Received 7 February 2008; Accepted 7 July 2008
DOI 10.1002/ar.20789
Published online 24 October 2008 in Wiley InterScience (www.
interscience.wiley.com).
30
STACY ET AL.
little being known about the effects of IUGR on the generation of cardiomyocytes.
The proliferation of cardiomyocytes is thought to cease
soon after birth (Oparil et al., 1984; Li et al., 1996), after
which postnatal growth of the heart muscle is predominantly due to cardiomyocyte hypertrophy and deposition
of extracellular matrix. If cardiomyocyte number is
reduced by IUGR, this could have important implications
for cardiovascular health later in life. A reduced number
of cardiomyocytes could contribute to cardiac dysfunction
and susceptibility to heart disease, especially after catchup growth in body size, which is common in individuals
subjected to restricted fetal growth (Eriksson and Forsen,
2002); catch-up in body growth (and even more so with
obesity) would result in a reduced number of cardiomyocytes per kilogram of adult body weight.
A recent study in our laboratory, using an experimental model of late gestational placental insufficiency to
induce IUGR, has shown that the hearts of IUGR
fetuses were smaller and structurally and functionally
less mature than in non-IUGR fetuses (Bubb et al.,
2007). Similar findings of cardiac muscle immaturity at
birth following IUGR induced by placental restriction in
sheep have recently been reported (Morrison et al.,
2007). However, in neither of these studies was cardiomyocyte number determined. A study of neonatal rat
pups born to mothers subjected to reduced protein
intake during pregnancy has shown that there were
fewer cardiomyocytes in the hearts of IUGR pups when
compared with normally grown pups (Corstius et al.,
2005). The growth-restricted pups also had significantly
lighter hearts, a finding made in another study of rat
pups, whose mothers were subjected to a restriction in
global food intake (Desai et al., 2005). To date, little is
known about the effects of differences in birthweight
within the normal range on heart development. In particular, it is unknown whether individuals who have low
birthweight due to mild, naturally occurring prenatal
growth restriction, such as that induced by twinning,
have altered development of heart muscle. In the present study, we have used sheep as, in this species, the
timing of cardiomyocyte differentiation during late gestation and early postnatal life is similar to that in
humans (Oparil et al., 1984; Burrell et al., 2003).
Our aim was to determine the effects of naturally
occurring differences in birthweight, as an index of fetal
nutrition and growth, on the number of cardiomyocytes
in the hearts of postnatal lambs; our hypothesis was that
low birthweight would be associated with a decreased
number of cardiomyocytes. In order to increase the range
of birthweights, we studied the hearts of singleton and
twin lambs. We analyzed the heart muscle at 9 weeks
after birth, as at this age, ovine cardiomyocytes are
thought to be terminally differentiated and binuclear
(Burrell et al., 2003). As postnatal nutrition, growth, and
arterial pressure could also influence cardiomyocyte differentiation, we have measured postnatal growth rates
and arterial pressure in these animals. We have also
compared data between males and females, as gender
differences in cardiac development may exist.
MATERIALS AND METHODS
All experimental procedures were approved by the
Monash University Animal Welfare Committee. In order
to measure cardiomyocyte numbers, we used 14 lambs
from 14 date-mated Border Leicester 3 Merino ewes;
seven carried singletons (three males, four females), and
seven carried twins. Only one lamb (randomly chosen)
from each twin pair was studied (two males, five
females). The ewes delivered their lambs vaginally at
term. After birth, lambs were raised with their mothers
and were weighed daily; at 5–6 weeks after birth, they
were separated from their mothers and provided
with lucerne chaff and free access to water. At 3 weeks
of age, the lambs underwent surgical implantation of
a catheter into the femoral artery for later measurement of arterial pressure and heart rate. At 58 6 1
days after birth, the lambs were placed prone in a
sling, while awake, for recording of arterial pressure
for 2 hours on 2 consecutive days.
Necropsy
At 64 6 1 days after birth, the lambs were humanely
killed by an overdose of sodium pentobarbital (325 mg/
mL i.v.), and body and organ weights recorded. Hearts
were removed intact and weighed. A catheter was placed
in the aortic arch and heparin sodium (to prevent clotting), papaverine hydrochloride (to maximally dilate the
vasculature), and potassium chloride (to relax the cardiomyocytes) were administered via the catheter. Hearts
were perfused via the aortic arch with saline to clear the
coronary vasculature of blood and then perfusion-fixed
with 4% formaldehyde. The fixed hearts were stored in
10% buffered formalin.
Sampling of Ventricular Muscle
The fixed hearts were trimmed of connective tissue
and weighed; the atria were removed and both ventricles
weighed. The ventricles were cut into a series of transverse ( 4 mm) slices. A central slice was used for the
measurement of ventricular wall thickness (see later). In
each slice, the right ventricle (RV) was separated from
the left ventricle plus interventricular septum (LV1S).
The LV1S were kept together and treated as a unit as
the septum resembles the LV both functionally and
structurally. Each slice of the LV1S was cut into four
pieces, and the pieces were laid out from largest to
smallest. Using a smooth-fractionator approach, five to
seven pieces were sampled; these were further sectioned
using a slicing device and again, using a smooth fractionator approach (Gundersen, 2002), eight to 10 pieces of
LV1S were sampled. The sampled pieces of LV1S were
placed in 70% ethanol in preparation for tissue processing. A similar sampling procedure was repeated for the
RV, resulting in the generation of eight to 10 pieces of
RV.
Measurement of Ventricular Wall Thickness
A transverse slice of the ventricles, as described earlier, from the mid region of the heart was used to measure ventricular wall thickness. A digital image was
taken of the slice and thicknesses of the LV wall, and
septum and RV wall were measured (Image ProPlus Version 6.0 for WindowsTM, Media Cybernetics, Silver
Spring).
FETAL GROWTH AND CARDIOMYOCYTE NUMBER
Tissue Processing
The sampled LV1S and RV tissue was embedded in
glycol-methacrylate (Technovit 7100 resin, Heraeus
Kulzer, Germany), and the blocks were exhaustively sectioned at 20 mm. Every thirtieth section for LV1S and
every twentieth section for RV (commencing at a randomly selected number between 1 and 10) were collected
and mounted onto a glass slide and stained with hematoxylin in a 1,000-Watt microwave oven (50% power for
2.5 min).
Estimation of the Number of Cardiomyoctye
Nuclei
An optical disector–fractionator approach was used to
estimate the total number of cardiomyocyte nuclei in the
LV1S and the RV (Corstius et al., 2005). Counting was
performed using a computer-assisted stereologic package
(CAST 2002; Olympus, Albertsland, Denmark). An
unbiased counting frame was superimposed over the sections using a 1003 objective lens. A systematic uniform
random sample of fields was obtained on a 2,000 3
2,000 mm grid for the LV1S and a 1,500 3 1,500 mm
grid for the RV; nuclei were counted in a ‘‘disector’’ (area
of 329.6 mm2) within a depth of 10 mm. To allow for
inconsistencies in cut surfaces and loss of material, the
upper and lower 5 mm of the section were used as a
‘‘guard" area. Nuclei were counted when they came into
clear focus within the volume of the disector; however, if
any part of a nucleus touched the ‘‘forbidden lines’’ of
the grid that nucleus was not counted. The cardiomyocyte nuclei were easily distinguishable from other cells
(e.g., endothelial cells and fibroblasts) by their elongated
oval shape, light purple staining, and visible chromatin
and prominent nucleoli.
The total number of cardiomyocytes in the individual
ventricles was determined by the following equation:
Ncardiomyocytes5 Q2 3 1/f1 3 1/f2 3 1/f3 3 1/f4 3 1/f5,
where Q2 is the number of cardiomyocyte nuclei
counted, f1 and f2 are the initial fixed tissue sampling
fractions, f3 is the third level of sampling where every
thirtieth section was chosen from a random start, f4 is
the fraction of the section area actually counted (LV:
329.6/(2,000 3 2,000); RV: 329.6/(1,500 3 1,500), and f5
is the fraction of the depth of the section (10/20)
counted. The total number of cardiomyocytes was subsequently determined by adjusting for the proportion of
mononucleates/binucleates (see later).
Proportion of Binuclearity
To determine the proportion of binucleated cells to
mononucleated cells, we used two additional singleton
lambs. At 9 weeks of age, these lambs were humanely
killed by an overdose of sodium pentobarbital (325 mg/
mL i.v). The freshly dissected hearts were attached to a
Langendorff apparatus via an intra-aortic cannula and
perfused with 0-Ca21 physiological saline at 358C for 8
min to clear remaining blood. To digest the ventricles,
saline-containing collagenase (Worthington CL-2, 200 U/
mL21) was infused for 25 min. To relax the cardiomyocytes, hearts were then perfused for 20 min with Hepesbuffered high K1 solution: 117 mM KCl, 36 mM NaCl, 1
mM MgSO4, 60 mM Hepes, 8 mM ATP-Na, 50 mM
31
EGTA; pH adjusted to 7.0 with KOH (Burrell et al.,
2003). The LVs and RVs were separated and placed in
Hepes-buffered high K1 solution and gently agitated for
5 min. The isolated cells were filtered through gauze,
and cardiomyocytes from the LV and RV were separately
fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 5 7.4) (Bubb et al., 2007).
Samples of fixed LV and RV cardiomyocytes were
smeared onto slides and stained with hematoxylin and
eosin. Cardiomyocytes were analyzed under a light
microscope (4003 magnification); all cells that fell
entirely within a field of view were analyzed. Six hundred cells were counted, and the proportions of mononucleated and binucleated cells determined (Bubb et al.,
2007).
Cardiomyocyte Proliferation
To confirm that cardiomyocytes had ceased proliferating at 9 weeks of age, we performed immunohistochemistry on paraffin-embedded tissue sections from the LVs
and RVs using an antibody against the Ki67 protein
(1:100, mouse anti-human monoclonal antibody; DakoCytomation, Glostrup, Denmark). The Ki67 protein is
expressed during all active phases of the cell cycle but is
absent in resting cells and is therefore used as a marker
of cell proliferation (Gerdes et al., 1984). Tissue sections
were boiled for 20 min in a microwave oven (on high
power) in an antigen retrieval solution (0.01 M sodium
citrate, pH 6.0) and an EnVision 1 Dual Link SystemHRP (DAB1) immunohistochemistry kit (DakoCytomation, CA) was used to visualize positively stained nuclei.
Specificity of immunostaining for Ki67 was confirmed by
omission of the primary antibody. A section from the
heart of a normal sheep fetus at 133 days of gestation
( 14 days before term) was included as a positive control, as many cardiomyocytes are not terminally differentiated at this age. Sections were counterstained with
hematoxylin, and the proportion of cardiomyocytes
undergoing cell proliferation was qualitatively analyzed
using a light microscope.
Analysis of Collagen Content of Cardiac Muscle
To assess the collagen content of the cardiac muscle,
we stained 5-mm sections with Masson’s Trichrome stain.
Using image analysis software (Image ProPlus; Version
6.0 for Windows), we analyzed eight sections per ventricle and nine fields per section. In each field, we measured the proportion of the area occupied by collagen
(stained blue) in the LVs and RVs.
Data Analysis
Data are presented as mean 6 SEM. Data were analyzed using a two-way ANOVA, using male vs. female
and singleton vs. twin as factors. Associations between
cardiomyocyte number and birthweight, necropsy
weight, and heart weight were tested using linear
regression analysis. Differences between data from the
LV1S and RV (cardiomyocyte number and density) were
analyzed by paired t test. Significance was taken at
P < 0.05.
32
STACY ET AL.
TABLE 1. Body and heart weights in 9-week-old lambs: data are presented comparing singletons and twins,
comparing males and females and for all animals combined
Single (n 5 7)
Birth weight (kg)
Autopsy weight (kg)
Heart weight (g)
Heart weight/autopsy weight (g/kg)
LV1S weight (g)
LV1S/autopsy weight (g/kg)
RV weight (g)
RV/autopsy weight (g/kg)
LV1S wall thickness (mm)
RV wall thickness (mm)
4.79
18.89
83.1
4.4
52.3
2.8
16.9
0.9
10.5
5.4
6
6
6
6
6
6
6
6
6
6
0.23
0.88
4.7
0.3
2.7
0.1
1.1
0.1
0.4
0.3
Twin (n 5 7)
Male (n 5 5)
3.67
13.58
57.0
4.2
35.6
2.6
11.3
0.8
8.7
4.1
4.06
16.80
71.8
4.3
44.9
2.7
14.3
0.8
9.2
4.7
6
6
6
6
6
6
6
6
6
6
0.31*
1.10*
4.4*
0.2
2.4*
0.1
0.9*
0.1
0.2*
0.3*
6
6
6
6
6
6
6
6
6
6
0.57
2.15
10.2
0.3
6.4
0.8
2.3
0.1
0.3
0.4
Female (n 5 9)
4.32
15.92
69.1
4.4
43.4
2.7
14.0
0.9
9.8
4.7
6
6
6
6
6
6
6
6
6
6
0.26
1.21
5.8
0.2
3.5
0.1
1.2
0.1
0.4
0.4
All (n 5 14)
4.23
16.24
70.0
4.3
43.9
2.7
14.1
0.9
9.6
4.7
6 0.24
6 1.00
6 4.8
6 0.1
6 2.9#
6 0.1#
6 1.0
6 0.03
6 0.3#
60.3
Abbreviations: LV1S, left ventricle plus septum; RV, right ventricle. Values shown are the means 6 SEM.
*Indicates twins significantly different from singles (P < 0.05).
Indicates significant difference between LV1S and RV (P < 0.05).
#
RESULTS
Fetal and Postnatal Growth
All lambs were born at term (146 6 0 days, range,
144–148 days). The mean birthweight was 4.23 6 0.24
kg (Table 1), with individual weights ranging from 2.8–
5.6 kg. As a group, twins were 20% lighter at birth than
singletons (3.67 6 0.31 kg vs. 4.79 6 0.23 kg; P < 0.01);
there was no difference between males and females.
Growth rates after birth are shown in Fig. 1. On average, body weights increased 3.8-fold between birth
and 9 weeks. Twins had a significantly lower absolute
growth rate (g/day) during the first 9 weeks after birth
compared with singletons (153.6 6 15.8 g/day vs. 227.4
6 8.1 g/day, P < 0.01); however, there was no significant
difference between the weekly percentage weight gain in
the two groups. There was no difference in postnatal
growth rates between males and females (Fig. 1). During
the experimental period of birth to 9 weeks of age, there
was no evidence of catch-up growth in the lower birthweight lambs.
The absolute weights of both the LV1S (twins, 35.6 6
2.4 g vs. singletons, 52.3 6 2.7 g; P < 0.001) and RV
(twins 11.3 6 0.9 g vs. singletons 16.9 6 1.1 g; P < 0.001)
were significantly lower in twins than singletons; when
these values were adjusted for body weight, there was
no difference between groups. There were no differences
in weights of the LV1S or RV between males and
females (Table 1).
Ventricular Wall Thickness
The walls of LV1S were approximately twice as thick
as those of the RV (Table 1). Compared with singletons,
twins had significantly thinner LV (twins, 8.7 6 0.2 mm
vs. singletons, 10.5 6 0.4 mm; P < 0.001) and RV (twins,
4.1 6 0.3 mm vs. singletons, 5.4 6 0.3mm; P < 0.001)
walls. Ventricular wall thicknesses (both LV1S and RV)
were significantly and positively correlated with birthweight in all lambs (LV1S, r2 5 0.337, P 5 0.037; RV,
r2 5 0.470, P 5 0.010). There were no differences in ventricular wall thicknesses between males and females
(Table 1).
Arterial Pressure
At 8 weeks, the mean arterial pressure in all lambs
averaged 75 6 4 mmHg, the systolic pressure was 96 6
4 mmHg, and the diastolic pressure was 65 6 5 mmHg.
There were no significant differences between twins and
singletons or between males and females in mean arterial pressure, systolic pressure, or diastolic pressure.
There were no significant correlations between arterial
pressure (mean, diastolic or systolic) and birthweight or
body weight at 8 weeks.
Heart Weights
At 9 weeks after birth, the mean heart weight in all
lambs was 70.0 6 4.8 g (4.3 6 0.1 g/kg body weight). In
twins, the heart weight was 32% lower than in singletons (57.0 6 4.4 g vs. 83.1 6 4.7 g; P < 0.001), but when
adjusted for body weight there was no significant difference between groups (Table 1). There was no difference
between males and females in either absolute or relative
heart weights. Among all animals, the weight of LV1S
was 62.7% of the total heart weight, and the RV was
20.1%. In relation to body weight (all animals combined),
the weight of the RV was significantly lower than that
of the LV1S (Table 1).
Number of Cardiomyocytes in the LV1S
In the LV1S, the mean number of cardiomyocytes in
the 14 lambs was 31.8 3 108 6 2.61 3 108; when
adjusted for tissue weight, this was 7.31 3 107 6 0.48 3
107 cardiomyocytes/g (Table 2). The absolute number of
cardiomyocytes in LV1S was significantly correlated
with birthweight (r2 5 0.578, P 5 0.002), body weight at
necropsy (r2 5 0.414, P 5 0.013), and heart weight (r2 5
0.511, P 5 0.004) (Fig. 2). There were significantly more
cardiomyocytes in the LV1S in singletons than in twins,
but their density was not different (Table 2). In the
LV1S, there was a trend for males to have fewer cardiomyocytes than females (males, 27.4 3 108 6 3.81 3 108
cells vs. females, 34.3 3 108 6 3.48 3 108 cells; P 5
0.086); however, their density was not different (Table 2).
Number of Cardiomyocytes in the RV
In the RV, the mean number of cardiomyocytes for all
animals was 8.13 3 108 6 0.50 3 108; the number of
cardiomyocytes per gram of RV tissue was 6.03 3 107 6
0.45 3 107 (Table 2). The number of cardiomyocytes in
the RV was not significantly correlated with birthweight
(r2 5 0.113, P 5 0.239) or necropsy weight (r2 5 0.058,
FETAL GROWTH AND CARDIOMYOCYTE NUMBER
33
Fig. 1. Postnatal growth rates between birth and necropsy at 9
weeks. The upper panels compare singleton lambs (filled circles, n 5
7) and twin lambs (open circles, n 5 7): (A) shows the increase in
body weight and (B) shows the mean percent weight gain per week.
The lower panels show the data for males (filled triangles, n 5 5) and
females (open triangles, n 5 9): (C) shows the increase in body weight
and (D) shows the mean percent weight gain per week. Data points
show the mean 6 SEM. *indicates that twins are significantly different
from singletons (P < 0.05).
P 5 0.406), but there was a trend for a correlation with
heart weight (r2 5 0.271, P 5 0.056). There was no difference in cardiomyocyte number between twins and singletons; however, there was a trend for a higher density
of cardiomyocytes in the twins compared with the singletons (P 5 0.055). In the RV, there was no difference for
either cardiomyocyte number or density between males
and females.
The total number of cardiomyocytes was significantly
lower in the RV than the LV1S (P < 0.001), and there
was a trend for a lower density of cells in the RV compared with the LV1S (P 5 0.062).
34
STACY ET AL.
TABLE 2. Cardiomyocyte number and density in 9-week-old lambs: data are presented comparing singletons
and twins, comparing males and females and for all animals combined
Single (n 5 7)
8
LV1S (10 )
LV1S/LV1S weight (107 #/g)
RV (108)
RV/RV weight (107 #/g)
LV1S 1 RV (108)
36.6
6.99
8.93
5.29
45.5
6
6
6
6
6
3.67
0.61
0.83
0.32
4.36
Twin (n 5 7)
27.0
7.63
7.33
6.80
34.4
6
6
6
6
6
3.11*
0.82
0.48
0.79
2.79*
Male (n 5 5)
27.4
6.23
7.69
5.74
35.1
6
6
6
6
6
3.81
0.54
0.78
0.87
4.54
Female (n 5 9)
34.3
7.91
8.38
6.20
42.7
6
6
6
6
6
3.48
0.63
0.69
0.58
3.73
All (n 5 14)
31.8
7.31
8.13
6.03
40.0
6
6
6
6
6
2.61#
0.48
0.50
0.45
2.88
Abbreviations: LV1S, left ventricle plus septum; RV, right ventricle. Values shown are the means 6 SEM.
*Indicates twins significantly different from singles (P < 0.05).
#
Indicates difference between LV1S and RV (P < 0.05).
Number of Cardiomyocytes in Both Ventricles
In the LV1S and RV combined, the average total
number of cardiomyocytes (from all hearts examined)
was 40.0 3 108 6 2.88 3 108. Twins had significantly
fewer ventricular cardiomyocytes than singletons (twins,
34.4 3 108 6 2.79 3 108 cells vs. singletons, 45.5 3 108
6 4.36 3 108 cells; P 5 0.031). There was a trend for
males to have fewer ventricular cardiomyocytes than
females (males, 35.1 3 108 6 4.54 3 108 cells vs. females,
42.7 3 108 6 3.73 3 108 cells; P 5 0.069) (Table 2).
When the data for all lambs were pooled, there were
positive, significant associations between total ventricular cardiomyocyte number and birthweight (r2 5 0.560,
P 5 0.002), necropsy weight (r2 5 0.391, P 5 0.017), and
heart weight (r2 5 0.546, P 5 0.003).
Proportion of Binucleated Cardiomyocytes
In the LV, 99% of the cells were binucleated in both
lambs studied, whereas in the RV 93% of the cells
were binucleated and the remaining 7% were mononucleated.
Cardiomyocyte Proliferation
There were no Ki67-positive cells in the LVs or RVs
from all lambs studied, confirming that cardiomyocytes
had ceased proliferating by 9 weeks of age. In contrast,
there was evidence of cell proliferation in fetal heart tissue at 133 days of gestation (Fig. 3).
Ventricular Collagen Content
The area of collagen in the RV (1.03% 6 0.12%) was
significantly greater than in the LV (0.70% 6 0.06%, P
< 0.05) (Fig. 4). There was no significant relation
between the amount of collagen in the ventricles and either birthweight or necropsy weight. Similarly, there
were no differences between singletons and twins, nor
between males and females.
DISCUSSION
The major finding of this study is that the number of
cardiomyocytes in the LV1S, at a time of development
when cardiomyocyte proliferation has ceased, is directly
related to body weight and heart tissue weight. This
was found to be true across a wide range of birthweights
within the normal range, including both singleton and
twin offspring of both genders. The same was not true
for the number of cardiomyocytes in the RV. Our findings have important implications for cardiac structure
and function in adult life, especially in low birthweight
(small for gestational age) individuals following catch-up
growth or exposure to cardiovascular challenges.
To examine the effects of a wide range of birthweights
on cardiomyocyte development, we included twin and
singleton lambs in our study, as well as male and female
lambs. Twins are normally 20%–30% lighter than singletons at birth, in both sheep (Ross et al., 2005; Bloomfield
et al., 2007; De Matteo et al., 2008) and humans (de
Geus et al., 2001), presumably because of a mild restriction of fetal growth imposed by a smaller placenta. We
found that twin lambs, as a group, were not different
from singleton lambs in that heart weight and cardiomyocyte number were directly related to body weight, and
cardiomyocyte data from singletons and twins showed
the same relation to body and heart weight (i.e., data
fell on the same line of identity). This suggests that
across the normal range of birthweights for singletons or
twins, the final number of cardiomyocytes is determined
primarily by the size of the heart, which in turn is
related to fetal and early postnatal growth. Hence,
larger individuals born at term will likely begin life with
a greater complement of cardiomyocytes than those who
are smaller at term birth and in early infancy. Conversely, individuals who are small for gestational age, as
a result of a chronic reduction in fetal nutrient or oxygen availability, are likely to have a reduced final complement of cardiomyocytes.
In the present study, we chose to examine the hearts
of lambs at 9 weeks after birth, as by this age, it was
expected that the cardiomyocytes would be terminally
differentiated and binuclear, and thus would have
ceased dividing (Smolich et al., 1989; Burrell et al.,
2003). Our use of Ki67 immunohistochemistry confirmed
the absence of cardiomyocyte proliferation at 9 weeks,
whereas marked proliferation was observed before birth.
Interestingly, when we examined enzymatically isolated
cardiomyocytes, there were negligible mononuclear cardiomyocytes (1%) in the LV1S, whereas in the RV, 7% of the cardiomyocytes were mononuclear, suggesting
that these cardiomyocytes were still capable of division.
Therefore, we can confidently conclude that the total
number of cardiomyocytes we counted in the LV1S (the
dominant chamber of the heart after birth) represents
the final complement of cardiomyocytes; in contrast, in
the RV the final number of cardiomyocytes may increase
further if the mononucleated cells continue to divide.
The difference in the proportion of mononucleated/
binucleated cells between the LV and RV may relate to
the hemodynamic transition at birth, whereby the LV
becomes the dominant pumping chamber; it is thus con-
FETAL GROWTH AND CARDIOMYOCYTE NUMBER
35
Fig. 3. High-power (bar 5 10 mm) images demonstrating Ki67-positive cardiomyocytes (brown staining), which are indicative of cells that
are proliferating. Note the absence of staining in heart tissue from 9week-old lambs (A), and the positive staining in heart tissue from a
sheep fetus at 133 days of gestation (C). Staining was absent in the
negative control sections in which the primary antibody was omitted
(B, 9-week-old heart; D, fetal heart).
Fig. 2. Relationship between cardiomyocyte number in the left
ventricle plus septum (LV1S) and (A) birthweight, (B) necropsy weight
at 9 weeks, and (C) heart weight. Data for singleton lambs (n 5 7) are
shown by filled circles; data for twin lambs (n 5 7) are shown by open
circles.
ceivable that cardiomyocytes in the LV will have accelerated maturation compared with cardiomyocytes in the
RV. The number of cardiomyocytes in the RV was considerably less than in the LV1S, and there was a trend for
reduced density in the RV as well. Although we have not
estimated cardiomyocyte number at birth, the differences between ventricles are again likely due to the considerable remodeling (including apoptosis) within the RV
soon after birth (Smolich et al., 1989; Kajstura et al.,
1995; Burrell et al., 2003), following the hemodynamic
transition at birth. In the fetus, both ventricles effectively operate in concert, because of the presence of the
ductus arteriosus that shunts blood from the pulmonary
artery to the aorta. After birth, when the ductus arteriosus closes and the right heart perfuses only the low resistance, low pressure pulmonary circulation, there is
marked apoptosis in the RV, resulting in the loss of cardiomyocytes (Kajstura et al., 1995; Burrell et al., 2003).
This could account for the greatly reduced number of
cardiomyocytes in the RV compared with the LV1S, and
the lack of a significant relationship between the number of RV cardiomyocytes, with both birthweight and
body weight at 9 weeks of age.
The number of cardiomyocytes that we measured in
the LV1S at 9 weeks (3.2 3 109) is similar to the number obtained in a previous study (Burrell et al., 2003) of
lambs at 4 and 6 weeks after birth ( 2.0 3 109). The
difference between the absolute values obtained between
studies is likely due to differences in postnatal ages (a
small proportion of cardiomyocytes may still be dividing
36
STACY ET AL.
Fig. 4. Representative images showing collagen staining (blue) in
the left ventricle (A) and right ventricle (B) of lambs at 9 weeks of age.
Bar 5 10 mm.
at 6 weeks) or alternatively, differences in methodologies
or breed of sheep. In the earlier study (Burrell et al.,
2003) cardiomyocyte number was derived indirectly from
mean cardiomyocyte volume and LV mass, whereas we
calculated the number using an unbiased optical disector–fractionator technique.
As we studied lambs at 9 weeks after birth, it is likely
that both prenatal and postnatal nutrition and growth
contributed to the differences in both heart weight and
cardiomyocyte number. Between birth and 9 weeks of
age, the animals grew in proportion to their body
weight, with no evidence of catch-up growth in animals
born with low birthweight; that is, for individual animals postnatal growth rates (% increase per day) were
likely to have been similar to fetal growth rates. Differences in the birthweights of our animals were not due to
differences in gestation length, as all were born at term,
and presumably were a result of differences in fetal
growth resulting from either genetic factors or differences in fetal nutrition and/or oxygenation. As we found
that arterial pressure was unrelated to body weight, differences in cardiomyocyte number between animals of
different body weights could not be attributed to arterial
pressure differences.
Heart sparing, as a result of preferential perfusion of
the myocardium, has often been described in growth-restricted human fetuses (al-Ghazali et al., 1989; Rizzo
and Arduini, 1991; Baschat et al., 1997). In the present
study, the growth of the heart was proportional to body
weight, with no evidence of disproportionately large
hearts in the smaller animals. Although redistribution
of cardiac output favoring the heart has previously been
described in the presence of acute fetal hypoxia in sheep
(Sheldon et al., 1979; Smolich et al., 1989), increases in
relative heart weight were not detected in IUGR lambs
following placental embolization (Bubb et al., 2007) or
carunclectomy (Morrison et al., 2007) in which hypoxia
would be chronic. Similarly, there were no effects on relative heart weight at 8 weeks of age (Manalich et al.,
2000) or at 2 years (Louey et al., 2000) following IUGR
induced by placental embolization. In the present study,
as the hearts were analyzed at 9 weeks of age, we cannot exclude the possibility that there may have been
accelerated cardiac growth in the smaller fetuses
in utero, which subsequently normalized after birth.
There have been a number of experimental studies
linking elevations in fetal cortisol levels with altered
cardiomyocyte growth; however, the findings are not consistent; studies have shown increased cardiomyocyte
proliferation (Giraud et al., 2006), cardiomyocyte hypertrophy (Martin et al., 2000), or no effect (Phillips and
Barker, 1997). In this regard, it is important to note
that we have previously shown that plasma cortisol levels are not elevated as a result of the spontaneous
growth restriction associated with twinning (De Matteo
et al., 2008), and so potentially confounding effects of
elevations in fetal cortisol levels do not apply in the
present study.
In animal models, an increase in fibrosis has often
been described in the RV when compared with the LV in
the adult heart (Weber and Brilla, 1991). Interestingly,
by 9 weeks of age in the present study, there was already an increased level of fibrosis in the RV compared
with the LV, which is likely a result of replacement fibrosis following cardiomyocyte apoptosis accompanying the
postnatal RV remodeling.
Implications for Adult Cardiovascular
Function and Disease
Although there is convincing epidemiological data linking low birthweight with an increased incidence of heart
disease later in life (Eriksson and Forsen, 2002), very
few studies have investigated the effects of IUGR and
low birthweight on the structure of the heart. In two
recent sheep studies (one from our laboratory), it has
been shown that IUGR, as a result of chronic placental
insufficiency induced by either placental embolization
(Bubb et al., 2007) or carunclectomy (Morrison et al.,
2007), leads to immaturity of the cardiac muscle in late
gestation. In both studies the proportion of undifferentiated, mononucleated cardiomyocytes was significantly
greater in the IUGR hearts compared with the hearts of
normally grown fetuses; this implies delayed differentiation and/or increased proliferation of cardiomyocytes in
these IUGR hearts. What effect the immaturity of the
myocardium has on the final number of cardiomyocytes
in these IUGR hearts is yet to be determined.
As cardiomyocytes, in general, cease proliferating soon
after birth, our present findings suggest that infants of
low birthweight may have a reduced capacity for physiological hypertrophy of the heart later in life, especially if
their adaptive capabilities are challenged; this could
occur, for example, as a result of catch-up growth, induction of obesity, or development of hypertensive LV hypertrophy. In support of this concept, maternal protein
restriction in rats leads to fewer cardiomyocytes in the
heart at birth (Corstius et al., 2005), and this is associated with an increased deposition of interstitial fibrosis
within the myocardium by early adulthood (Lim et al.,
2006). In addition, the hearts of IUGR rat offspring
exposed to hypoxia in utero are more vulnerable to ischemia and reperfusion injury in adulthood (Li et al., 2003).
CONCLUSIONS
We have shown that the number of cardiomyocytes in
the LV, at a time when cardiomyocyte differentiation has
essentially ceased, is related to birthweight and heart
weight. That is, individuals with a low birthweight,
FETAL GROWTH AND CARDIOMYOCYTE NUMBER
regardless of whether they are singleton or multiple
births, are likely to begin life with fewer LV cardiomyocytes than individuals born heavier. As cardiomyocytes
cease proliferating soon after birth, cardiomyocyte number is unlikely to be affected even if catch-up growth
occurs after infancy. A lower cardiomyocyte number following restricted fetal growth, and hence low birthweight and low neonatal weight may compromise the
postnatal adaptive capabilities and function of the heart,
especially if challenged in adulthood. It is possible that
a reduced number of cardiomyocytes in the LV of individuals born with lower birthweights may contribute to
the association between low birthweight and increased
risk of heart disease in adulthood, especially in individuals who catch up in body weight or who become obese
later in life.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the help of
N. Wreford, A. Satragno, N. Blasch, M. Tare, and S.
Connell.
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