Proposal paper: 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: .physiology.org/content/300/2/H645#cited-by">http://ajpheart.physiology.org/content/300/2/H645#cited-by Updated information and services including high resolution figures, can be found at: .physiology.org/content/300/2/H645.full">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: .the-aps.org/publications/ajpheart">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 .the-aps.org/">http://www.the-aps.org/. Downloaded from .physiology.org/">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: .physiology.org/content/300/2/H645.full#ref-list-1">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: [email protected]">[email protected]). .ajpheart.org/">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 .physiology.org/">http://ajpheart.physiology.org/ at Univ North Carolina Charlotte on August 28, 2012 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
