Reprod Toxicol. 2016 Jun;61:82-96. doi: 10.1016/j.reprotox.2016.03.039.
Embryonic Exposures of Lithium and Homocysteine and Folate Protection Affect Lipid Metabolism during Mouse Cardiogenesis and Placentation
Han M, Evsikov AV, Zhang L, Lastra-Vicente R, Linask KK.
Department of Pediatrics, USF Morsani College of Medicine, Children’s Research Institute, St. Petersburg, FL 33701
Embryonic exposures can increase the risk of congenital cardiac birth defects and adult disease. The present study identifies the predominant pathways modulated by an acute embryonic mouse exposure during gastrulation to lithium or homocysteine that induces cardiac defects. High dose periconceptional folate supplementation normalized development. Microarray bioinformatic analysis of gene expression demonstrated that primarily lipid metabolism is altered after the acute exposures. The lipid-related modulation demonstrated a gender bias with male embryos showing greater number of lipid-related Gene Ontology biological processes altered than in female embryos. RT-PCR analysis demonstrated significant change of the fatty acid oxidation gene Acadm with homocysteine exposure primarily in male embryos than in female. The perturbations resulting from the exposures resulted in growth- restricted placentas with disorganized cellular lipid droplet distribution indicating lipids have a critical role in cardiac-placental abnormal development. High folate supplementation protected normal heart-placental function, gene expression and lipid localization.
The causes of congenital heart defects (CHDs) are generally unknown. CHD occur in approximately 1% of live born children but in a much higher percentage of those aborted spontaneously or are stillborn. The genetic risks of CHD have been difficult to define because 90 – 97% of subsequent human pregnancies, after an affected child, proceed without recurrence. A hypothesis that is gaining acceptance is that the maternal/placental microenvironment prior to, and within 5 – 8 weeks of conception influences the development of the fetal organs. Additionally, as based upon evidence from animal studies, neural and cardiac defects can be explained by abnormalities occurring early in pregnancy in the intrauterine microenvironment of the fetus. Little research has been done to analyze biomarkers of CHD at the time of human cardiac development between 2.5 to 8 weeks of gestation, because most women do not know they are pregnant until after the abnormal placental/cardiac microenvironment already has affected the early gestational period.
My laboratory began our studies on environmental influences, specifically the therapeutic drug lithium affecting heart development, by analyzing when is the earliest time point during mouse gestation that we can induce cardiac defects by just one-time, exposure of the pregnant mouse. We exposed the embryo by using injection of lithium intraperitoneally (i.p.). We began first by exposing the pregnant mouse on embryonic day (E) 9, the earliest gestational day that we found being used in the literature. Heart defects usually were not being analyzed, but only effects on the developing brain. We continued exposing at earlier times to define the earliest time-point at which cardiac defects were seen and where viability also was noted. The earliest time-point of a single exposure inducing cardiac defects at a relatively high percentage was E 6.75, that is when the i.p. injection was done at 17:30 hrs on E6. This timing would coincide with gastrulation when the heart cells are being specified (Figure 1). This timing of acute exposure was used in all of our subsequent studies.
The three environmental factors that we have analyzed were lithium, elevated homocysteine and alcohol, all of which are known to induce cardiac defects in human pregnancy at a higher incidence than is present in the normal population. To define whether embryonic cardiac defects were occurring, we used noninvasive Doppler ultrasound monitoring of embryonic mouse heart function in utero (Gui and others, 1996), similar to use in human pregnancy to determine whether heart defects in the developing fetus may be present.
Fig. 2A (left image). Pregnant mouse is shown sedated and undergoing Doppler ultrasound monitoring of embryonic heart function on E15.5 of gestation. Fig. 2B. On right are shown some typical abnormal ultrasound patterns that are obtained showing arrhythmia, atrioventricular valvle regurgitation, semilunar valve regurgitation or semilunar valve stenosis.
For the present published study we asked the question: What genes are being misexpressed in the heart by the acute environmental exposures and what biological processes are predominantly affected by lithium and by an elevation of homocysteine, with and without folate prevention. To answer this we used Affymetrix microarray analysis of microdissected cardiac tissue (right ventricle and outflow tract) that predominantly displayed abnormalities with either exposure, as compared to control normal tissues and those with folate supplementation. The methodology is shown in Figure 3 below.
Figure 3. After ultrasound monitoring of embryonic heart function, the embryonic heart was isolated, and the right ventricle and outflow tract were microdissected for microarray analyses.
Bioinformatic analyses of the more than 45,000 probesets on the chips indicated that with our various experimental manipulations and folate prevention described in this article, predominantly genes associated with lipid metabolism within Gene Ontology (GO) categories were being altered and this was happening in a gender-associated manner: With exposure, more genes relating to lipid metabolism were altered in the male embryonic tissues than in the female. When we carried out Oil Red O staining for neutral lipids, we found that neutral lipids in the heart and placenta were reduced and placental tissue was disorganized with exposure in comparison to normal, control tissues. In relation to effects on lipid metabolism, we recently published similar results with alcohol exposure during early pregnancy (Linask and Han, 2016)
These results have important clinical implications:
(1) A means of prevention of the cardiac birth defects induced by lipid changes appears to exist by dietary supplementation with a higher folate dose than currently formulated in prenatal vitamin preparations and that the supplementation be initiated early, that is peri-conceptionally (Huhta and Linask, 2015).
(2) Our results using the mouse model, when extrapolated to human pregnancy, demonstrate that in the first month of pregnancy, specifically in the second to third week post-conception, the human embryonic heart would be highly vulnerable to exposure to environmental factors encountered by the pregnant mother. This critical window that we targeted coincides with a period of cell specification and differentiation of cardiomyocytes and placental trophoblast cells. This is a time-period, however, that a woman usually is not aware of her pregnancy and is not taking precautionary measures to protect the embryo (Linask, 2013).
(3) An early intrauterine exposure before a woman recognizes her pregnancy may have long-lasting effects on tissue and organ function not only during pregnancy, but also postnatally as evidenced that certain adult disease, including cardiovascular, may have a developmental origin (Barker, 2007).
(4) The gender bias observed in lipid modulation, as evidenced by acyl-CoA-dehydrogenase-medium length chain, Acadm, gene expression, we suggest may relate to the increased severity of heart defects observed in male children, when compared to the female in the human population (Lindinger and others, 2010; Marelli and others, 2010; Zhao and others, 2013).
(5) As evidenced here with lithium and homocysteine exposure, and separately that we reported with alcohol (Linask and Han, 2016), all alter lipids in the fetal heart and placenta. Other disease states affecting lipid metabolism are diabetes and obesity, and when present during early pregnancy, can result in a higher incidence of CHDs in the offspring. We thus propose that fetoplacental dyslipidemia may be a common factor in the development of certain CHDs occurring during early gestation.
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Acknowledgements: The support from Suncoast Cardiovascular Research and Education Foundation founded by Helen Harper Brown (KKL) and the David and Janice Mason Foundation (KKL) of the USF Morsani College of Medicine for these studies is gratefully acknowledged.
Dr. Kersti K. Linask, MA., Ph.D., FAAA
Emeritus Professor of Translational Cardiology
Department of Pediatrics
USF Morsani College of Medicine
USF/All Children’s Hospital, Bldg 3
St. Petersburg, FL 33701