Alcohol exposure during late gestation adversely

26 Jun Alcohol exposure during late gestation adversely

Question
Alcohol exposure during late gestation adversely affects
myocardial development with implications for postnatal
cardiac function

Joanna M. Goh, Jonathan G. Bensley, Kelly Kenna, Foula Sozo, Alan D. Bocking, James
Brien, David Walker, Richard Harding and M. Jane Black
Am J Physiol Heart Circ Physiol 300:H645-H651, 2011. First published 12 November 2010;
doi: 10.1152/ajpheart.00689.2010
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American Journal of Physiology – Heart and Circulatory Physiology publishes original investigations on the
physiology of the heart, blood vessels, and lymphatics, including experimental and theoretical studies of
cardiovascular function at all levels of organization ranging from the intact animal to the cellular, subcellular, and
molecular levels. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville
Pike, Bethesda MD 20814-3991. Copyright © 2011 the American Physiological Society. ISSN: 0363-6135, ESSN:
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Am J Physiol Heart Circ Physiol 300: H645H651, 2011.
First published November 12, 2010; doi:10.1152/ajpheart.00689.2010.

Alcohol exposure during late gestation adversely affects myocardial
development with implications for postnatal cardiac function
Joanna M. Goh,1 Jonathan G. Bensley,1 Kelly Kenna,1 Foula Sozo,1 Alan D. Bocking,2 James Brien,3
David Walker,4 Richard Harding,1* and M. Jane Black1*
1

Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia; 2Department of
Obstetrics and Gynaecology, University of Toronto, Ontario, Canada; 3Department of Pharmacology and Toxicology,
Queens University, Kingston, Ontario, Canada; and 4Monash Institute of Medical Research, Clayton, Victoria, Australia

Goh JM, Bensley JG, Kenna K, Sozo F, Bocking AD, Brien
J, Walker D, Harding R, Black MJ. Alcohol exposure during late
gestation adversely affects myocardial development with implications for postnatal cardiac function. Am J Physiol Heart Circ
Physiol 300: H645H651, 2011. First published November 12,
2010; doi:10.1152/ajpheart.00689.2010.Prenatal exposure to high
levels of ethanol is associated with cardiac malformations, but the
effects of lower levels of exposure on the heart are unclear. Our aim
was to investigate the effects of daily exposure to ethanol during late
gestation, when cardiomyocytes are undergoing maturation, on the
developing myocardium. Pregnant ewes were infused with either
ethanol (0.75 g/kg) or saline for 1 h each day from gestational days 95
to 133 (term 145 days); tissues were collected at 134 days. In sheep,
cardiomyocytes mature during late gestation as in humans. Within the
left ventricle (LV), cardiomyocyte number was determined using
unbiased stereology and cardiomyocyte size and nuclearity determined using confocal microscopy. Collagen deposition was quantied
using image analysis. Genes relating to cardiomyocyte proliferation
and apoptosis were examined using quantitative real-time PCR. Fetal
plasma ethanol concentration reached 0.11 g/dL after EtOH infusions.
Ethanol exposure induced signicant increases in relative heart
weight, relative LV wall volume, and cardiomyocyte cross-sectional
area. Ethanol exposure advanced LV maturation in that the proportion
of binucleated cardiomyocytes increased by 12%, and the number of
mononucleated cardiomyocytes was decreased by a similar amount.
Apoptotic gene expression increased in the ethanol-exposed hearts,
although there were no signicant differences between groups in total
cardiomyocyte number or interstitial collagen. Daily exposure to a
moderate dose of ethanol in late gestation accelerates the maturation
of cardiomyocytes and increases cardiomyocyte and LV tissue volume in the fetal heart. These effects on cardiomyocyte growth may
program for long-term cardiac vulnerability.
cardiomyocyte; heart; pregnancy; maturation

alcohol (ethanol, EtOH)
during pregnancy (8, 16). It is well established that exposure to
high levels of EtOH during pregnancy can lead to congenital
cardiac defects such as atrial and septal defects (22, 24, 37);
furthermore, cardiac function may be affected in the absence of
structural abnormalities (21). However, the effects of moderate
levels of EtOH exposure on cardiac muscle development during gestation are not well understood; they are difcult to
ascertain in the human infant due to the many confounding
factors including exposure at multiple time points and uncertainties regarding the level of exposure. To address this ques-

MANY WOMEN CONTINUE TO CONSUME

* R. Harding and M. J. Black are co-senior authors.
Address for reprint requests and other correspondence: M. J. Black, Dept. of
Anatomy and Developmental Biology, Monash Univ., Clayton Campus, Bldg.
76, Victoria 3800 Australia (e-mail: Jane.[email protected].edu).
http://www.ajpheart.org

tion it is appropriate to use carefully controlled animal studies,
in a species in which cardiomyocyte maturation resembles that
in the human.
Previous studies have reported that exposure to EtOH induces apoptosis of cardiomyocytes both in vivo and in vitro (7,
30); in addition, reductions in the in vivo circulating concentrations of the cardiomyocyte growth factor, insulin-like
growth factor (IGF)-1, have been reported following EtOH
exposure (14). We therefore hypothesized that fetal exposure
to EtOH during late gestation would adversely impact on the
growth and maturation of cardiomyocytes, as a result of an
increase in apoptotic activity and a decrease in IGF expression,
leading to a reduction in the complement of cardiomyocytes at
birth; we also expected increased extracellular matrix deposition in the myocardium because EtOH increases brosis in the
adult heart (29, 34).
In this study we have used an ovine model, because the
gestational timing of cardiomyocyte maturation in sheep
closely resembles that in the human (6). We have specically
targeted the developmental window late in gestation at a time
when cardiomyocytes are undergoing maturation (20). Our
aims were to determine the effects of prenatal exposure to a
moderate dose of EtOH during late gestation, equivalent to 3 to
4 standard drinks in 1 h, on cardiomyocyte growth parameters
and key genes associated with cardiomyocyte growth and on
the deposition of extracellular matrix.
METHODS

All experimental procedures were approved by the Monash University School of Biomedical Sciences Animal Ethics Committee and
were conducted in accordance with the Australian National Health
and Medical Research Council guidelines.
Animal groups. Pregnant crossbred ewes underwent aseptic surgery
at 91 days of gestational age (DGA; term, 147 days) for implantation of arterial and venous catheters (31). Between 95 and 124 DGA,
ewes were intravenously infused with either 0.75 g ethanol/kg body
weight or saline for 1 h each day. At 126 DGA, the ewes underwent
further aseptic surgery for the implantation of catheters into a fetal
brachial artery, for arterial pressure measurement and blood sampling,
and the amniotic sac for the measurement of intra-amniotic pressure.
After recovery from surgery the daily maternal infusion of EtOH or
saline continued from 127 DGA to 133 DGA. In the EtOH group,
plasma EtOH concentrations in the ewe and fetus reached maximal
values of 0.12 and 0.11 g/dL, respectively, at 1 h after the start of the
infusion; EtOH concentrations had returned to baseline (0 g/dL) by 8
h after the end of the infusion (31). Ethanol concentrations were
measured in maternal and fetal plasma using the Dade Behring
Dimension RxL Clinical Chemistry System with assay sensitivity
range of 0 65 mmol/l (12). Between 130 DGA and 132 DGA, we
measured fetal blood gas status and arterial pressure. Necropsy was

0363-6135/11 Copyright © 2011 the American Physiological Society

H645

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Submitted 12 July 2010; accepted in nal form 8 November 2010

H646

ALCOHOL EXPOSURE AFFECTS MYOCARDIAL DEVELOPMENT

performed at 134 DGA when some of the hearts were perfusion xed
(EtOH group, n
8; saline group, n
6), and in others the
myocardium was sampled and snap frozen (EtOH group, n
5;
saline group, n
6).
Perfusion xation of the heart. At necropsy, fetal hearts were
perfusion xed via the aorta with 4% formaldehyde in 0.1 M phosphate buffer. Before xation, the cardiac vasculature was cleared of
blood using saline and maximally dilated with papaverine hydrochloride (DBL Pharmaceuticals, Australia); the cardiomyocytes were
relaxed with potassium chloride. The xed hearts were stored in 10%
buffered formalin before tissue sampling.
Heart muscle preparation and sampling. Fat and connective tissue
were removed from the xed hearts, and the hearts weighed. The atria
were separated from the ventricles. The right ventricle (RV) was then
separated from the left ventricle plus septum (LV S). The ventricles
were cut into slices 3 mm thick, and the wall volumes of the RV and
LV
S were determined using the Cavalieri principle (25). Subsequent sampling of the LV
S for morphological and stereological
analyses was performed using a smooth fractionator approach (32);
the selected samples were then embedded in either glycolmethacrylate
or parafn.
Interstitial collagen quantication. Parafn-embedded samples of
LV S were sectioned at 5 m and stained with 0.001% picrosirius
red. The sections were uniformly, systematically sampled, and the
percentage of collagen within the tissue was quantied using image
analysis (Image-Pro Plus Version 6.0, Media Cybernetics) (2, 32).

Estimation of cardiomyocyte number. Glycolmethacrylate-embedded samples of LV S were serially sectioned at 20 m, and every
30th section was stained with Harriss Haematoxylin in a 1,000-watt
microwave oven set at 50% power. Sections were uniformly, systematically sampled, and the number of cardiomyocyte nuclei per unit
volume of tissue was determined using an optical disector stereological approach (2, 32). The total number of nuclei in the LV S wall
was calculated by multiplying the number of nuclei per unit volume of
tissue by the total LV
S tissue volume. Total cardiomyocyte
number in the LV S was then determined following correction for
binucleation (see below) (11).
Cardiomyocyte nuclearity. The nuclearity of cardiomyocytes
within the LV
S (i.e., the proportions of mononucleated and
binucleated cells) was examined using confocal microscopy in thick
parafn sections stained with wheat germ agglutinin-Alexa Fluor 488
conjugate (Invitrogen) to stain cell boundaries and 46-diamidino-2phenylindole, dihydrochloride (DAPI) to stain cell nuclei (Invitrogen)
(2). Sections were systematically sampled, and at least 200 cardiomyocytes per fetus were examined. Cardiomyocytes were recognized
by the appearance of striations and the appearance of cardiomyocyte
nuclei (long, round ended and dense nucleoli) (see Fig. 3).
Analyzing cell size. LV
S sections stained with wheat germ
agglutinin-Alexa Fluor 488 conjugate, and DAPI (see above) were
systematically sampled. Each eld of view with cardiomyocytes seen
in cross-section was analyzed for cardiomyocyte cross-sectional area
(Fig. 1). The boundaries of the cardiomyocytes were traced and

Table 1. Primer sequences
Primer Sequences
Gene of
Interest

18S rRNA
c-Myc
IGF-1
IGF-2
IGF-1R
BAX
Caspase 3

Reference

Accession
Number

15
15
15
13
13

X01117
NM_001009426
DQ152962
M89789
AY162434
AF163774
AF068837

Forward

GTC
CAT
TTG
GCT
AAG
TGT
CCA

TGT
ACA
GTG
TCT
AAC
CTG
ATG

GAT
TCC
GAT
TGC
CAT
AAG
GAC

GCC
TGT
GCT
CTT
GCC
CGC
CCG

CTT
CGG
CTC
CTT
TGC
ATT
TCG

Primer,
M

Reverse

AGA
TCC
CAG
GGC
AGA
GGA
ATC

TGT C
AA
TTC
CTT
AGG
GAT
T

AAG
CAA
AGC
TCG
GGA
AGG
GTC

CTT
CTG
AGC
GTT
TTC
GCC
TGC

ATG
TTC
ACT
TAT
TCA
TTG
CTC

ACC
TCG
CAT
GCG
GGT
AGC
AAC

CGC
CCT
CCA
GCT
TCT
ACC
TGG

ACT
CTT
CGA
GGA
GGC
AGT
TAT

TAC
CC
TTC
T
CAT T
TT
TTT CTG A

cDNA,
ng/ l

10
10
10
10
4
4
10

200
200
500
62.5
500
500
500

Sequences for each forward and reverse primer (5=-3=) were used to amplify each gene of interest. Primer sequences were designed based on the nucleotide
sequence that corresponds to the listed Genbank accession number. The starting primer and cDNA concentrations used for the amplication of each gene during
quantitative PCR are also shown. The annealing temperature used for all primers was 60°C. IGF, insulin-like growth factor.
AJP-Heart Circ Physiol VOL

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Fig. 1. Representative confocal image of left ventricle
plus septum (LV S; from a control lamb) stained with
wheat germ agglutinin-Alexa Fluor 488 conjugate (to
stain the cell boundaries; appears green) and TO-PRO-3
(Invitrogen; to stain the cell nuclei; appears blue). In
cardiomyocytes cut in cross-section and nuclei centrally located, the boundaries of the cardiomyocytes
were traced and cross-sectional area was determined.
Two cardiomyocytes are delineated in white. Scale
bar is 10 m.

H647

ALCOHOL EXPOSURE AFFECTS MYOCARDIAL DEVELOPMENT

Table 2. Body weight and heart dimensions in ethanol-exposed and control fetuses
Ethanol Exposed

8
3.84
34.1
9.0
6.7
1.7
12.8
3.4
7.58
1.96
14.15
3.71

6
0.26
2.6
0.7
0.6
0.1
0.9
0.1
0.68
0.06
0.95
0.11

4.02
28.8
7.2
6.7
1.7
12.2
3.0
7.49
1.86
13.57
3.37

0.16
1.7
0.5
0.3
0.1
0.6
0.1
0.49
0.09
0.80
0.10

Values are means
SE. For heart weight/body weight in ethanol-exposed group, P
0.049. For left ventricle
septum weight/body weight in
ethanol-exposed group, P 0.06. For left ventricle septum wall volume/body weight in ethanol-exposed group, P 0.04. Denotes signicantly different
from control.

cross-sectional area determined using NIS-Elements software (Nikon,
Japan). Only cardiomyocytes in which the nuclei could be seen in the
center were traced. The cross-sectional area of 200 cardiomyocytes
from each fetus was measured.
Immunohistochemical analysis of cell proliferation. Parafn LV
S sections (5 m; see above) were used to measure cell proliferation. Antigen retrieval was achieved using 0.01 M citrate buffer
(Ajax Finechem, Australia) with 0.1% Triton X-100 (SigmaAldrich) in a microwave oven for 20 min. The primary Ki-67
antibody (Ki-67; MIB-1 Clone, Dako, Australia) was diluted 1:100
before use, and the tissue was incubated with the primary antibody
overnight at 4°C in a humidied chamber. Detection was performed using the Dako EnVision Dual Link HRP/DAB immunohistochemistry kit (Dako). Positive controls were Zymed Laboratories Ki-67 Control Slides (mouse tonsil, a known Ki-67
positive tissue) (Invitrogen). Negative controls were sections that
were not incubated with the primary antibody. Staining was repeated using a secondary antibody conjugated to Alexa Fluor 594
(anti-mouse IgG Alexa Fluor 594; Invitrogen).
Gene expression analysis. Relative mRNA levels of a proliferation
marker (c-Myc), apoptotic markers (caspase 3 and BAX), and growth
promoting genes (IGF-1 and IGF-2) and the IGF-1 receptor (IGF-1R)
were measured in the myocardium using quantitative real-time PCR
(qPCR) as previously described (31). Total RNA was extracted and
DNase treated (Qiagen) and then reverse transcribed into cDNA
(M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant Kit;
Promega, WI). qPCR was performed using a SYBR green detection
method (Platinum SYBR Green qPCR SuperMix-UDG; Invitrogen
Life Technologies) and a Stratagene Mx3000P (Agilent Technologies) detection system. The thermal prole used to amplify the PCR
products included an initial 2-min hold step at 95°C, followed by 40
cycles of denaturation at 95°C for 3 s, annealing at 60°C for 20 s and
elongation at 72°C for 20 s. The uorescence readings were recorded
after each 72°C step. Each sample was measured in triplicate, and a
negative control sample (which contained no template cDNA) was

included in each run. A dissociation curve was performed following
each PCR run to ensure that a single PCR product was amplied per
primer set. The relative mRNA levels for each gene for each animal
were normalized to the 18S rRNA values for the same animal using
the
Ct method (where Ct is cycle threshold) and expressed relative
to the mean gene mRNA levels in control fetuses (31). The nucleotide
sequence for each primer is shown in Table 1.
Statistical analysis. All data were analyzed using an unpaired
Students t-test. The analyses and generation of graphs were performed using GraphPad Prism Version 5.0 (GraphPad Software). Data
are expressed as means SE, and differences were considered to be
statistically signicant at P 0.05.
RESULTS

Effects of ethanol exposure on fetal blood gas status, arterial
pressure, and heart rate. Between 130 DGA and 132 DGA,
there were no signicant differences between control and
EtOH fetuses in fetal PaO2 (23.7 1.6 mmHg vs. 23.1 0.8
mmHg), PaCO2 (49.3
1.4 mmHg vs. 49.1
2.0 mmHg),
arterial pH (7.364 0.006 vs. 7.355 0.003), SaO2 (66.2
1.8% vs. 63.0
2.5%), mean arterial pressure (39
0.5
mmHg vs. 40 0.4 mmHg), and heart rate (154 4 beats/min
vs. 164 5 beats/min).
Effects of EtOH exposure on body and heart growth, as
shown in Table 2 and Fig. 2. There were no signicant differences between the EtOH-exposed and control fetuses in body
weight and absolute heart weight, RV weight, or LV S weight;
similarly, the absolute volume of wall tissue in the ventricles did
not differ between groups. However, when heart weights and
combined ventricular weights were expressed in relation to body
weight, values in EtOH fetuses were signicantly greater than in
controls; a similar trend was seen for LV S weight (P 0.06).

Fig. 2. The weight (A) and volume (vol; B) of the LV
S, adjusted for body weight (body wt), of ethanol
exposed fetuses and controls. *P 0.05.

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n
Body weight, kg
Heart weight, g
Heart weight/body weight, g/kg
Right ventricle weight, g
Right ventricle weight/body weight, g/kg
Left ventricle septum weight, g
Left ventricle septum weight/body weight, g/kg
Right ventricle wall volume, 103 mm3
Right ventricle wall volume/body weight, mm3/kg
Left ventricle septum wall volume, 103 mm3
Left ventricle septum wall volume/body weight, mm3/kg

Controls

H648

ALCOHOL EXPOSURE AFFECTS MYOCARDIAL DEVELOPMENT

In accordance with differences in relative tissue weights, the
relative tissue volumes of the combined ventricles and LV S
were signicantly greater in EtOH fetuses than in controls.
Interstitial brosis. The levels of interstitial brosis were
low in the LV S of all fetal hearts. There was no signicant
difference between levels of brosis in the LV
S of EtOH
fetuses and controls (1.96 0.16% vs. 2.19 0.27%).
Cardiomyocyte nuclearity and maturation, as shown in Fig. 3.
The percentage of mononucleated cardiomyocytes in the LV
S of EtOH fetuses was signicantly lower than in controls
(23.6
1.0% vs. 36.0
1.5%; P
0.0001). Accordingly,
there was a signicantly higher percentage of binucleated
cardiomyocytes in EtOH-exposed hearts than in controls
(76.4
1.0% vs. 64.1
1.5%; P
0.0001).

Cardiomyocyte number and size, as shown in Fig. 4. The
total number of cardiomyocytes in the LV S was not signicantly different between EtOH fetuses and controls (4.08 109
3.0
108 vs. 4.38
109
2.6
108; P
0.5). The mean
cross-sectional area of LV S cardiomyocytes was signicantly
greater in EtOH fetuses compared with controls (48.7 2.1 m2
vs. 42.1 0.9 m2; P 0.02).
Myocardial cell proliferation. The rate of cell proliferation
as measured by Ki-67 immunohistochemistry (brighteld
and uorescence), in the control and ethanol-treated hearts,
was too low to quantify ( 1 per 5,000 nuclei positively
stained). The relative expression of the proliferation marker
c-Myc within the myocardium was not different between the
EtOH and control groups (1.07
0.10 vs. 1.00
0.13).

Fig. 4. The total number of cardiomyocytes (A) and
their cross-sectional area (B) in the LV S of ethanolexposed and control fetal hearts. *P 0.05.

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Fig. 3. Proportions of mononucleated (A) and
binucleated (B) cardiomyocytes within the LV S
of ethanol exposed and control fetal hearts. ***P
0.0001. Representative left ventricular section from
an ethanol-treated lamb. Cardiomyocytes following
staining with wheat germ agglutinin-Alexa Fluor
488 (appears green) and TO-PRO-3 (appears blue)
are shown. A mononucleate and binucleate cardiomyocyte (arrow) are delineated in yellow. Cardiomyocytes are recognized due to the appearance of
striations. Scale bar is 8 m.

ALCOHOL EXPOSURE AFFECTS MYOCARDIAL DEVELOPMENT

Growth factor gene expression, as shown in Fig. 5A. There
was signicantly greater relative expression of IGF-1 in the
ventricles of EtOH fetuses compared with controls (2.21
0.39 vs. 1.00
0.18; P
0.02). There were no signicant
differences in ventricular relative IGF-2 or IGF-1R mRNA
levels between EtOH and control fetuses.
Apoptotic marker gene expression, as shown in Fig. 5, B and
C. There was a signicant upregulation of relative caspase 3
mRNA levels in the ventricles of EtOH fetuses compared with
controls (1.91 0.12 vs. 1.00 0.22; P 0.007). Similarly,

AJP-Heart Circ Physiol VOL

there was a trend for relative BAX mRNA levels in the
ventricles of EtOH fetuses to be greater than in the controls
(2.00 0.36 vs. 1.00 0.29; P 0.057).
DISCUSSION

Our study clearly demonstrates that the fetal myocardium
is structurally altered as a result of exposure to a moderate
dose of EtOH during late gestation; in particular, the volume
of the LV wall was increased together with accelerated
maturation and an increase in cardiomyocyte size. Hence,
although it is well recognized that exposure to high doses of
EtOH during early pregnancy can lead to overt cardiac
malformations, the ndings of the present study indicate that
the heart remains highly vulnerable to EtOH exposure at
later time points in gestation, after the heart is fully formed.
The changes in cardiomyocyte maturation may persist into
postnatal life and may therefore have adverse programming
effects on cardiac function.
Of concern, the ndings of this study are highly relevant to
the substantial number of individuals who have been exposed
to moderate levels of EtOH during gestation. Until recently,
women were often advised that moderate consumption of
alcohol was not detrimental during pregnancy and many pregnant women continue to consume alcohol (8). In our animal
model the maximal blood alcohol concentration of the ewes is
approximately equivalent to that achieved by women 1 h after
the consumption of 3 to 4 standard drinks (26); similar blood
levels have been measured in young women who have been
drinking alcohol socially (28).
In general, exposure to high levels of EtOH during gestation
leads to reduced heart weight in parallel with reductions in
body weight and in the weights of other organs (18, 33).
However, in the present study we showed that, when the EtOH
exposure is more moderate and restricted to a window late in
gestation, there is induction of LV hypertrophy in the offspring. Whether this LV hypertrophy persists postnatally is yet
to be elucidated, but if so, it is likely to lead to elevated
cardiovascular risk (23). In support of our ndings, an apparent
increase in relative heart weight has also been described in fetal
sheep following acute EtOH exposure on days 116, 117, and
118 of gestation; however, in that study heart weight was
preserved in the presence of a signicant reduction in fetal
body weight (14).
Abnormal growth of the heart is often associated with
increased deposition of interstitial collagen (cardiac brosis)
(35, 36). Increased collagen deposition leads to stiffening of
the ventricular waAlcohol exposure during late gestation adversely affects
myocardial development with implications for postnatal
cardiac function

Joanna M. Goh, Jonathan G. Bensley, Kelly Kenna, Foula Sozo, Alan D. Bocking, James
Brien, David Walker, Richard Harding and M. Jane Black
Am J Physiol Heart Circ Physiol 300:H645-H651, 2011. First published 12 November 2010;
doi: 10.1152/ajpheart.00689.2010
You might find this additional info useful…

This article has been cited by 2 other HighWire-hosted articles:
http://ajpheart.physiology.org/content/300/2/H645#cited-by
Updated information and services including high resolution figures, can be found at:
http://ajpheart.physiology.org/content/300/2/H645.full
Additional material and information about American Journal of Physiology – Heart and Circulatory
Physiology can be found at:
http://www.the-aps.org/publications/ajpheart
This information is current as of August 28, 2012.

American Journal of Physiology – Heart and Circulatory Physiology publishes original investigations on the
physiology of the heart, blood vessels, and lymphatics, including experimental and theoretical studies of
cardiovascular function at all levels of organization ranging from the intact animal to the cellular, subcellular, and
molecular levels. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville
Pike, Bethesda MD 20814-3991. Copyright © 2011 the American Physiological Society. ISSN: 0363-6135, ESSN:
1522-1539. Visit our website at http://www.the-aps.org/.

Downloaded from http://ajpheart.physiology.org/ at Univ North Carolina Charlotte on August 28, 2012

This article cites 33 articles, 11 of which you can access for free at:
http://ajpheart.physiology.org/content/300/2/H645.full#ref-list-1

Am J Physiol Heart Circ Physiol 300: H645H651, 2011.
First published November 12, 2010; doi:10.1152/ajpheart.00689.2010.

Alcohol exposure during late gestation adversely affects myocardial
development with implications for postnatal cardiac function
Joanna M. Goh,1 Jonathan G. Bensley,1 Kelly Kenna,1 Foula Sozo,1 Alan D. Bocking,2 James Brien,3
David Walker,4 Richard Harding,1* and M. Jane Black1*
1

Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia; 2Department of
Obstetrics and Gynaecology, University of Toronto, Ontario, Canada; 3Department of Pharmacology and Toxicology,
Queens University, Kingston, Ontario, Canada; and 4Monash Institute of Medical Research, Clayton, Victoria, Australia

Goh JM, Bensley JG, Kenna K, Sozo F, Bocking AD, Brien
J, Walker D, Harding R, Black MJ. Alcohol exposure during late
gestation adversely affects myocardial development with implications for postnatal cardiac function. Am J Physiol Heart Circ
Physiol 300: H645H651, 2011. First published November 12,
2010; doi:10.1152/ajpheart.00689.2010.Prenatal exposure to high
levels of ethanol is associated with cardiac malformations, but the
effects of lower levels of exposure on the heart are unclear. Our aim
was to investigate the effects of daily exposure to ethanol during late
gestation, when cardiomyocytes are undergoing maturation, on the
developing myocardium. Pregnant ewes were infused with either
ethanol (0.75 g/kg) or saline for 1 h each day from gestational days 95
to 133 (term 145 days); tissues were collected at 134 days. In sheep,
cardiomyocytes mature during late gestation as in humans. Within the
left ventricle (LV), cardiomyocyte number was determined using
unbiased stereology and cardiomyocyte size and nuclearity determined using confocal microscopy. Collagen deposition was quantied
using image analysis. Genes relating to cardiomyocyte proliferation
and apoptosis were examined using quantitative real-time PCR. Fetal
plasma ethanol concentration reached 0.11 g/dL after EtOH infusions.
Ethanol exposure induced signicant increases in relative heart
weight, relative LV wall volume, and cardiomyocyte cross-sectional
area. Ethanol exposure advanced LV maturation in that the proportion
of binucleated cardiomyocytes increased by 12%, and the number of
mononucleated cardiomyocytes was decreased by a similar amount.
Apoptotic gene expression increased in the ethanol-exposed hearts,
although there were no signicant differences between groups in total
cardiomyocyte number or interstitial collagen. Daily exposure to a
moderate dose of ethanol in late gestation accelerates the maturation
of cardiomyocytes and increases cardiomyocyte and LV tissue volume in the fetal heart. These effects on cardiomyocyte growth may
program for long-term cardiac vulnerability.
cardiomyocyte; heart; pregnancy; maturation

alcohol (ethanol, EtOH)
during pregnancy (8, 16). It is well established that exposure to
high levels of EtOH during pregnancy can lead to congenital
cardiac defects such as atrial and septal defects (22, 24, 37);
furthermore, cardiac function may be affected in the absence of
structural abnormalities (21). However, the effects of moderate
levels of EtOH exposure on cardiac muscle development during gestation are not well understood; they are difcult to
ascertain in the human infant due to the many confounding
factors including exposure at multiple time points and uncertainties regarding the level of exposure. To address this ques-

MANY WOMEN CONTINUE TO CONSUME

* R. Harding and M. J. Black are co-senior authors.
Address for reprint requests and other correspondence: M. J. Black, Dept. of
Anatomy and Developmental Biology, Monash Univ., Clayton Campus, Bldg.
76, Victoria 3800 Australia (e-mail: Jane.[email protected].edu).
http://www.ajpheart.org

tion it is appropriate to use carefully controlled animal studies,
in a species in which cardiomyocyte maturation resembles that
in the human.
Previous studies have reported that exposure to EtOH induces apoptosis of cardiomyocytes both in vivo and in vitro (7,
30); in addition, reductions in the in vivo circulating concentrations of the cardiomyocyte growth factor, insulin-like
growth factor (IGF)-1, have been reported following EtOH
exposure (14). We therefore hypothesized that fetal exposure
to EtOH during late gestation would adversely impact on the
growth and maturation of cardiomyocytes, as a result of an
increase in apoptotic activity and a decrease in IGF expression,
leading to a reduction in the complement of cardiomyocytes at
birth; we also expected increased extracellular matrix deposition in the myocardium because EtOH increases brosis in the
adult heart (29, 34).
In this study we have used an ovine model, because the
gestational timing of cardiomyocyte maturation in sheep
closely resembles that in the human (6). We have specically
targeted the developmental window late in gestation at a time
when cardiomyocytes are undergoing maturation (20). Our
aims were to determine the effects of prenatal exposure to a
moderate dose of EtOH during late gestation, equivalent to 3 to
4 standard drinks in 1 h, on cardiomyocyte growth parameters
and key genes associated with cardiomyocyte growth and on
the deposition of extracellular matrix.
METHODS

All experimental procedures were approved by the Monash University School of Biomedical Sciences Animal Ethics Committee and
were conducted in accordance with the Australian National Health
and Medical Research Council guidelines.
Animal groups. Pregnant crossbred ewes underwent aseptic surgery
at 91 days of gestational age (DGA; term, 147 days) for implantation of arterial and venous catheters (31). Between 95 and 124 DGA,
ewes were intravenously infused with either 0.75 g ethanol/kg body
weight or saline for 1 h each day. At 126 DGA, the ewes underwent
further aseptic surgery for the implantation of catheters into a fetal
brachial artery, for arterial pressure measurement and blood sampling,
and the amniotic sac for the measurement of intra-amniotic pressure.
After recovery from surgery the daily maternal infusion of EtOH or
saline continued from 127 DGA to 133 DGA. In the EtOH group,
plasma EtOH concentrations in the ewe and fetus reached maximal
values of 0.12 and 0.11 g/dL, respectively, at 1 h after the start of the
infusion; EtOH concentrations had returned to baseline (0 g/dL) by 8
h after the end of the infusion (31). Ethanol concentrations were
measured in maternal and fetal plasma using the Dade Behring
Dimension RxL Clinical Chemistry System with assay sensitivity
range of 0 65 mmol/l (12). Between 130 DGA and 132 DGA, we
measured fetal blood gas status and arterial pressure. Necropsy was

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ALCOHOL EXPOSURE AFFECTS MYOCARDIAL DEVELOPMENT

performed at 134 DGA when some of the hearts were perfusion xed
(EtOH group, n
8; saline group, n
6), and in others the
myocardium was sampled and snap frozen (EtOH group, n
5;
saline group, n
6).
Perfusion xation of the heart. At necropsy, fetal hearts were
perfusion xed via the aorta with 4% formaldehyde in 0.1 M phosphate buffer. Before xation, the cardiac vasculature was cleared of
blood using saline and maximally dilated with papaverine hydrochloride (DBL Pharmaceuticals, Australia); the cardiomyocytes were
relaxed with potassium chloride. The xed hearts were stored in 10%
buffered formalin before tissue sampling.
Heart muscle preparation and sampling. Fat and connective tissue
were removed from the xed hearts, and the hearts weighed. The atria
were separated from the ventricles. The right ventricle (RV) was then
separated from the left ventricle plus septum (LV S). The ventricles
were cut into slices 3 mm thick, and the wall volumes of the RV and
LV
S were determined using the Cavalieri principle (25). Subsequent sampling of the LV
S for morphological and stereological
analyses was performed using a smooth fractionator approach (32);
the selected samples were then embedded in either glycolmethacrylate
or parafn.
Interstitial collagen quantication. Parafn-embedded samples of
LV S were sectioned at 5 m and stained with 0.001% picrosirius
red. The sections were uniformly, systematically sampled, and the
percentage of collagen within the tissue was quantied using image
analysis (Image-Pro Plus Version 6.0, Media Cybernetics) (2, 32).

Estimation of cardiomyocyte number. Glycolmethacrylate-embedded samples of LV S were serially sectioned at 20 m, and every
30th section was stained with Harriss Haematoxylin in a 1,000-watt
microwave oven set at 50% power. Sections were uniformly, systematically sampled, and the number of cardiomyocyte nuclei per unit
volume of tissue was determined using an optical disector stereological approach (2, 32). The total number of nuclei in the LV S wall
was calculated by multiplying the number of nuclei per unit volume of
tissue by the total LV
S tissue volume. Total cardiomyocyte
number in the LV S was then determined following correction for
binucleation (see below) (11).
Cardiomyocyte nuclearity. The nuclearity of cardiomyocytes
within the LV
S (i.e., the proportions of mononucleated and
binucleated cells) was examined using confocal microscopy in thick
parafn sections stained with wheat germ agglutinin-Alexa Fluor 488
conjugate (Invitrogen) to stain cell boundaries and 46-diamidino-2phenylindole, dihydrochloride (DAPI) to stain cell nuclei (Invitrogen)
(2). Sections were systematically sampled, and at least 200 cardiomyocytes per fetus were examined. Cardiomyocytes were recognized
by the appearance of striations and the appearance of cardiomyocyte
nuclei (long, round ended and dense nucleoli) (see Fig. 3).
Analyzing cell size. LV
S sections stained with wheat germ
agglutinin-Alexa Fluor 488 conjugate, and DAPI (see above) were
systematically sampled. Each eld of view with cardiomyocytes seen
in cross-section was analyzed for cardiomyocyte cross-sectional area
(Fig. 1). The boundaries of the cardiomyocytes were traced and

Table 1. Primer sequences
Primer Sequences
Gene of
Interest

18S rRNA
c-Myc
IGF-1
IGF-2
IGF-1R
BAX
Caspase 3

Reference

Accession
Number

15
15
15
13
13

X01117
NM_001009426
DQ152962
M89789
AY162434
AF163774
AF068837

Forward

GTC
CAT
TTG
GCT
AAG
TGT
CCA

TGT
ACA
GTG
TCT
AAC
CTG
ATG

GAT
TCC
GAT
TGC
CAT
AAG
GAC

GCC
TGT
GCT
CTT
GCC
CGC
CCG

CTT
CGG
CTC
CTT
TGC
ATT
TCG

Primer,
M

Reverse

AGA
TCC
CAG
GGC
AGA
GGA
ATC

TGT C
AA
TTC
CTT
AGG
GAT
T

AAG
CAA
AGC
TCG
GGA
AGG
GTC

CTT
CTG
AGC
GTT
TTC
GCC
TGC

ATG
TTC
ACT
TAT
TCA
TTG
CTC

ACC
TCG
CAT
GCG
GGT
AGC
AAC

CGC
CCT
CCA
GCT
TCT
ACC
TGG

ACT
CTT
CGA
GGA
GGC
AGT
TAT

TAC
CC
TTC
T
CAT T
TT
TTT CTG A

cDNA,
ng/ l

10
10
10
10
4
4
10

200
200
500
62.5
500
500
500

Sequences for each forward and reverse primer (5=-3=) were used to amplify each gene of interest. Primer sequences were designed based on the nucleotide
sequence that corresponds to the listed Genbank accession number. The starting primer and cDNA concentrations used for the amplication of each gene during
quantitative PCR are also shown. The annealing temperature used for all primers was 60°C. IGF, insulin-like growth factor.
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Fig. 1. Representative confocal image of left ventricle
plus septum (LV S; from a control lamb) stained with
wheat germ agglutinin-Alexa Fluor 488 conjugate (to
stain the cell boundaries; appears green) and TO-PRO-3
(Invitrogen; to stain the cell nuclei; appears blue). In
cardiomyocytes cut in cross-section and nuclei centrally located, the boundaries of the cardiomyocytes
were traced and cross-sectional area was determined.
Two cardiomyocytes are delineated in white. Scale
bar is 10 m.

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ALCOHOL EXPOSURE AFFECTS MYOCARDIAL DEVELOPMENT

Table 2. Body weight and heart dimensions in ethanol-exposed and control fetuses
Ethanol Exposed

8
3.84
34.1
9.0
6.7
1.7
12.8
3.4
7.58
1.96
14.15
3.71

6
0.26
2.6
0.7
0.6
0.1
0.9
0.1
0.68
0.06
0.95
0.11

4.02
28.8
7.2
6.7
1.7
12.2
3.0
7.49
1.86
13.57
3.37

0.16
1.7
0.5
0.3
0.1
0.6
0.1
0.49
0.09
0.80
0.10

Values are means
SE. For heart weight/body weight in ethanol-exposed group, P
0.049. For left ventricle
septum weight/body weight in
ethanol-exposed group, P 0.06. For left ventricle septum wall volume/body weight in ethanol-exposed group, P 0.04. Denotes signicantly different
from control.

cross-sectional area determined using NIS-Elements software (Nikon,
Japan). Only cardiomyocytes in which the nuclei could be seen in the
center were traced. The cross-sectional area of 200 cardiomyocytes
from each fetus was measured.
Immunohistochemical analysis of cell proliferation. Parafn LV
S sections (5 m; see above) were used to measure cell proliferation. Antigen retrieval was achieved using 0.01 M citrate buffer
(Ajax Finechem, Australia) with 0.1% Triton X-100 (SigmaAldrich) in a microwave oven for 20 min. The primary Ki-67
antibody (Ki-67; MIB-1 Clone, Dako, Australia) was diluted 1:100
before use, and the tissue was incubated with the primary antibody
overnight at 4°C in a humidied chamber. Detection was performed using the Dako EnVision Dual Link HRP/DAB immunohistochemistry kit (Dako). Positive controls were Zymed Laboratories Ki-67 Control Slides (mouse tonsil, a known Ki-67
positive tissue) (Invitrogen). Negative controls were sections that
were not incubated with the primary antibody. Staining was repeated using a secondary antibody conjugated to Alexa Fluor 594
(anti-mouse IgG Alexa Fluor 594; Invitrogen).
Gene expression analysis. Relative mRNA levels of a proliferation
marker (c-Myc), apoptotic markers (caspase 3 and BAX), and growth
promoting genes (IGF-1 and IGF-2) and the IGF-1 receptor (IGF-1R)
were measured in the myocardium using quantitative real-time PCR
(qPCR) as previously described (31). Total RNA was extracted and
DNase treated (Qiagen) and then reverse transcribed into cDNA
(M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant Kit;
Promega, WI). qPCR was performed using a SYBR green detection
method (Platinum SYBR Green qPCR SuperMix-UDG; Invitrogen
Life Technologies) and a Stratagene Mx3000P (Agilent Technologies) detection system. The thermal prole used to amplify the PCR
products included an initial 2-min hold step at 95°C, followed by 40
cycles of denaturation at 95°C for 3 s, annealing at 60°C for 20 s and
elongation at 72°C for 20 s. The uorescence readings were recorded
after each 72°C step. Each sample was measured in triplicate, and a
negative control sample (which contained no template cDNA) was

included in each run. A dissociation curve was performed following
each PCR run to ensure that a single PCR product was amplied per
primer set. The relative mRNA levels for each gene for each animal
were normalized to the 18S rRNA values for the same animal using
the
Ct method (where Ct is cycle threshold) and expressed relative
to the mean gene mRNA levels in control fetuses (31). The nucleotide
sequence for each primer is shown in Table 1.
Statistical analysis. All data were analyzed using an unpaired
Students t-test. The analyses and generation of graphs were performed using GraphPad Prism Version 5.0 (GraphPad Software). Data
are expressed as means SE, and differences were considered to be
statistically signicant at P 0.05.
RESULTS

Effects of ethanol exposure on fetal blood gas status, arterial
pressure, and heart rate. Between 130 DGA and 132 DGA,
there were no signicant differences between control and
EtOH fetuses in fetal PaO2 (23.7 1.6 mmHg vs. 23.1 0.8
mmHg), PaCO2 (49.3
1.4 mmHg vs. 49.1
2.0 mmHg),
arterial pH (7.364 0.006 vs. 7.355 0.003), SaO2 (66.2
1.8% vs. 63.0
2.5%), mean arterial pressure (39
0.5
mmHg vs. 40 0.4 mmHg), and heart rate (154 4 beats/min
vs. 164 5 beats/min).
Effects of EtOH exposure on body and heart growth, as
shown in Table 2 and Fig. 2. There were no signicant differences between the EtOH-exposed and control fetuses in body
weight and absolute heart weight, RV weight, or LV S weight;
similarly, the absolute volume of wall tissue in the ventricles did
not differ between groups. However, when heart weights and
combined ventricular weights were expressed in relation to body
weight, values in EtOH fetuses were signicantly greater than in
controls; a similar trend was seen for LV S weight (P 0.06).

Fig. 2. The weight (A) and volume (vol; B) of the LV
S, adjusted for body weight (body wt), of ethanol
exposed fetuses and controls. *P 0.05.

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n
Body weight, kg
Heart weight, g
Heart weight/body weight, g/kg
Right ventricle weight, g
Right ventricle weight/body weight, g/kg
Left ventricle septum weight, g
Left ventricle septum weight/body weight, g/kg
Right ventricle wall volume, 103 mm3
Right ventricle wall volume/body weight, mm3/kg
Left ventricle septum wall volume, 103 mm3
Left ventricle septum wall volume/body weight, mm3/kg

Controls

H648

ALCOHOL EXPOSURE AFFECTS MYOCARDIAL DEVELOPMENT

In accordance with differences in relative tissue weights, the
relative tissue volumes of the combined ventricles and LV S
were signicantly greater in EtOH fetuses than in controls.
Interstitial brosis. The levels of interstitial brosis were
low in the LV S of all fetal hearts. There was no signicant
difference between levels of brosis in the LV
S of EtOH
fetuses and controls (1.96 0.16% vs. 2.19 0.27%).
Cardiomyocyte nuclearity and maturation, as shown in Fig. 3.
The percentage of mononucleated cardiomyocytes in the LV
S of EtOH fetuses was signicantly lower than in controls
(23.6
1.0% vs. 36.0
1.5%; P
0.0001). Accordingly,
there was a signicantly higher percentage of binucleated
cardiomyocytes in EtOH-exposed hearts than in controls
(76.4
1.0% vs. 64.1
1.5%; P
0.0001).

Cardiomyocyte number and size, as shown in Fig. 4. The
total number of cardiomyocytes in the LV S was not signicantly different between EtOH fetuses and controls (4.08 109
3.0
108 vs. 4.38
109
2.6
108; P
0.5). The mean
cross-sectional area of LV S cardiomyocytes was signicantly
greater in EtOH fetuses compared with controls (48.7 2.1 m2
vs. 42.1 0.9 m2; P 0.02).
Myocardial cell proliferation. The rate of cell proliferation
as measured by Ki-67 immunohistochemistry (brighteld
and uorescence), in the control and ethanol-treated hearts,
was too low to quantify ( 1 per 5,000 nuclei positively
stained). The relative expression of the proliferation marker
c-Myc within the myocardium was not different between the
EtOH and control groups (1.07
0.10 vs. 1.00
0.13).

Fig. 4. The total number of cardiomyocytes (A) and
their cross-sectional area (B) in the LV S of ethanolexposed and control fetal hearts. *P 0.05.

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Fig. 3. Proportions of mononucleated (A) and
binucleated (B) cardiomyocytes within the LV S
of ethanol exposed and control fetal hearts. ***P
0.0001. Representative left ventricular section from
an ethanol-treated lamb. Cardiomyocytes following
staining with wheat germ agglutinin-Alexa Fluor
488 (appears green) and TO-PRO-3 (appears blue)
are shown. A mononucleate and binucleate cardiomyocyte (arrow) are delineated in yellow. Cardiomyocytes are recognized due to the appearance of
striations. Scale bar is 8 m.

ALCOHOL EXPOSURE AFFECTS MYOCARDIAL DEVELOPMENT

Growth factor gene expression, as shown in Fig. 5A. There
was signicantly greater relative expression of IGF-1 in the
ventricles of EtOH fetuses compared with controls (2.21
0.39 vs. 1.00
0.18; P
0.02). There were no signicant
differences in ventricular relative IGF-2 or IGF-1R mRNA
levels between EtOH and control fetuses.
Apoptotic marker gene expression, as shown in Fig. 5, B and
C. There was a signicant upregulation of relative caspase 3
mRNA levels in the ventricles of EtOH fetuses compared with
controls (1.91 0.12 vs. 1.00 0.22; P 0.007). Similarly,

AJP-Heart Circ Physiol VOL

there was a trend for relative BAX mRNA levels in the
ventricles of EtOH fetuses to be greater than in the controls
(2.00 0.36 vs. 1.00 0.29; P 0.057).
DISCUSSION

Our study clearly demonstrates that the fetal myocardium
is structurally altered as a result of exposure to a moderate
dose of EtOH during late gestation; in particular, the volume
of the LV wall was increased together with accelerated
maturation and an increase in cardiomyocyte size. Hence,
although it is well recognized that exposure to high doses of
EtOH during early pregnancy can lead to overt cardiac
malformations, the ndings of the present study indicate that
the heart remains highly vulnerable to EtOH exposure at
later time points in gestation, after the heart is fully formed.
The changes in cardiomyocyte maturation may persist into
postnatal life and may therefore have adverse programming
effects on cardiac function.
Of concern, the ndings of this study are highly relevant to
the substantial number of individuals who have been exposed
to moderate levels of EtOH during gestation. Until recently,
women were often advised that moderate consumption of
alcohol was not detrimental during pregnancy and many pregnant women continue to consume alcohol (8). In our animal
model the maximal blood alcohol concentration of the ewes is
approximately equivalent to that achieved by women 1 h after
the consumption of 3 to 4 standard drinks (26); similar blood
levels have been measured in young women who have been
drinking alcohol socially (28).
In general, exposure to high levels of EtOH during gestation
leads to reduced heart weight in parallel with reductions in
body weight and in the weights of other organs (18, 33).
However, in the present study we showed that, when the EtOH
exposure is more moderate and restricted to a window late in
gestation, there is induction of LV hypertrophy in the offspring. Whether this LV hypertrophy persists postnatally is yet
to be elucidated, but if so, it is likely to lead to elevated
cardiovascular risk (23). In support of our ndings, an apparent
increase in relative heart weight has also been described in fetal
sheep following acute EtOH exposure on days 116, 117, and
118 of gestation; however, in that study heart weight was
preserved in the presence of a signicant reduction in fetal
body weight (14).
Abnormal growth of the heart is often associated with
increased deposition of interstitial collagen (cardiac brosis)
(35, 36). Increased collagen deposition leads to stiffening of
the ventricular wall (35, 36) and impaired myocardial conductivity and contractility (3, 5). We therefore considered it
important to measure interstitial brosis in the fetal heart
exposed to EtOH, because in the adult, chronic alcohol consumption typically leads to brosis…ll (35, 36) and impaired myocardial conductivity and contractility (3, 5). We therefore considered it
important to measure interstitial brosis in the fetal heart
exposed to EtOH, because in the adult, chronic alcohol consumption typically leads to brosis…
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