Alcohol exposure during late gestation adversely
Write a 4-5 pg. paper proposal on the attached file. Include an argument for why this paper deserves to be evaluated and why it might be important, interesting, or groundbreaking in the field. Give at least 3 reference.
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
You might find this additional info useful…
This article has been cited by 2 other HighWire-hosted articles:
http://ajpheart.physiology.
Updated information and services including high resolution figures, can be found at:
http://ajpheart.physiology.
Additional material and information about American Journal of Physiology – Heart and Circulatory
Physiology can be found at:
http://www.the-aps.org/
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.
This article cites 33 articles, 11 of which you can access for free at:
http://ajpheart.physiology.
Am J Physiol Heart Circ Physiol 300: H645H651, 2011.
First published November 12, 2010; doi:10.1152/ajpheart.00689.
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.
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.Black@monash.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
Downloaded from http://ajpheart.physiology.
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
Our website has a team of professional writers who can help you write any of your homework. They will write your papers from scratch. We also have a team of editors just to make sure all papers are of HIGH QUALITY & PLAGIARISM FREE. To make an Order you only need to click Order Now and we will direct you to our Order Page at Litessays. Then fill Our Order Form with all your assignment instructions. Select your deadline and pay for your paper. You will get it few hours before your set deadline.
Fill in all the assignment paper details that are required in the order form with the standard information being the page count, deadline, academic level and type of paper. It is advisable to have this information at hand so that you can quickly fill in the necessary information needed in the form for the essay writer to be immediately assigned to your writing project. Make payment for the custom essay order to enable us to assign a suitable writer to your order. Payments are made through Paypal on a secured billing page. Finally, sit back and relax.