* College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131; Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131
1 To whom correspondence should be addressed at College of Pharmacy, University of New Mexico, 2502 Marble NE, Albuquerque, NM 87131. Fax: (505) 272-0704. E-mail: mkwalker{at}unm.edu.
Received July 21, 2005; accepted August 22, 2005
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ABSTRACT |
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Key Words: TCDD; aryl hydrocarbon receptor; cardiomyocyte proliferation; cardiac hypertrophy; ECG; bradycardia.
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INTRODUCTION |
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In addition to the alterations in vascular structure and permeability, changes in cardiac structure and function also have been described in piscine and avian embryos exposed to TCDD. In the piscine embryo, TCDD reduces heart size, and this is associated with a significant reduction in cardiomyocyte proliferation (Antkiewicz et al., 2005; Hornung et al., 1999
). Additionally, these structural defects are associated with functional deficits, including reduced cardiac output and, eventually, ventricular standstill (Antkiewicz et al., 2005
), suggesting that cardiac conduction may be disrupted. Similarly, in the chick embryo, TCDD induces ventricular cavity dilation associated with thinner ventricle walls and induces a significant reduction in cardiomyocyte proliferation (Ivnitski-Steele and Walker, 2003
; Walker et al., 1997
; Walker and Catron, 2000
), while functional deficits include cardiac arrhythmias and reduced responsiveness to ß-adrenergic stimulation (Fan et al., 2000
; Sommer et al., 2005
; Walker and Catron, 2000
). The importance of the timing of TCDD exposure to cardiac teratogenic end points has not been studied in detail; however, chick embryos exposed to TCDD after organogenesis exhibit less severe cardiac structural or functional deficits than when they are exposed shortly after fertilization (Ivnitski-Steele et al., 2005
; Sommer et al., 2005
).
In contrast to the responses of piscine and avian embryos to TCDD-induced cardiovascular teratogenicity, there is only limited evidence that the cardiovascular system also is a target of TCDD in the developing mammalian embryo. One common feature shared among these vertebrate species to TCDD during development is edema and hemorrhage. Subcutaneous edema and intestinal hemorrhages have been observed in rat and hamster fetuses after exposure to TCDD in utero (Olson et al., 1990), suggesting that TCDD-induced alterations in vascular structure and function may occur during mammalian development. It is noteworthy, however, that in both piscine and avian embryos exposed to TCDD, the presence of cardiac structural defects and functional deficits occurs prior to the appearance of edema (Antkiewicz et al., 2005
; Walker and Catron, 2000
).
TCDD-induced cardiac structural defects and/or functional deficits have not been reported in the mammalian fetus, and the reasons for these species differences are not known. It is possible that TCDD fails to induce cardiac teratogenicity during mammalian development or, alternatively, that the effects are subtle in nature and have not been studied in sufficient detail to identify their occurrence. Thus, in this study we tested the hypothesis that two of the most sensitive effects of TCDD on the developing piscine and avian heart, altered heart size and reduced cardiac proliferation, would also be observed after in utero exposure of the fetal mouse. We further hypothesized that these effects would not be associated with edema or mortality, but rather would be associated with altered cardiac development after birth. To test these hypotheses, first we exposed pregnant mice to graded doses of TCDD during a developmental window when cardiomyocyte proliferation peaks, and we assessed fetal heart weight and cardiac proliferative index. Second, we investigated the consequences of the combination of in utero and lactational TCDD exposure on postnatal cardiac development by assessing postnatal heart weight and cardiac conduction. Our results demonstrate that the fetal murine heart is a sensitive target of TCDD-induced teratogenicity and that subtle, but measurable, effects on the fetal heart are associated with altered cardiac development postnatally.
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MATERIALS AND METHODS |
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Immunohistochemistry.
Two immunohistochemical methods were used to assess proliferative index of the fetal murine heart: detection of proliferating cell nuclear antigen (PCNA) and incorporation of 5'-bromo-2'-deoxyuridine (BrdU, Sigma Chemical, St. Louis, MO). For PCNA analysis, fetal hearts were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 7.0 µm, stained with anti-PCNA antibody (BD Biosciences, San Diego, CA) followed by goat antimouse-IgG conjugated to horseradish peroxidase, and color developed using 3,3'diaminobenzidine as described elsewhere (Ivnitski et al., 2001). Sections were then stained with propidium iodide to visualize all nuclei (control, n = 4 litters; TCDD, n = 4; 24 hearts/litter). Identical photographs of 46 fields per heart were captured under brightfield or fluoresence illumination with a Texas Red filter, and PCNA positive nuclei and total nuclei, respectively, were counted in each field, using ImagePro Plus software. Results were expressed as percent PCNA-positive nuclei. For BrdU incorporation experiments, pregnant mice were injected intraperitoneally (ip) with 50 µg BrdU/kg in sterile saline 2 h prior to the GD 17.5 collection. Fetal hearts were fixed in 30% glacial acetic acid in ethanol, embedded in paraffin, and sectioned at 7.0 µm. Sections were stained with anti-BrdU antibody (Development Studies Hybridoma Bank, The University of Iowa, Iowa City, IA) followed by goat antimouse-IgG conjugated to horseradish peroxidase, and color developed using 3,3'diaminobenzidine as described elsewhere (Ivnitski et al., 2001
). Sections were then stained with propidium iodide, and 46 fields per heart were analyzed as described for PCNA (control, n = 4 litters; TCDD, n = 4; 24 hearts/litter). Neither of these methods distinguished the type of cell that was staining positive for the proliferation markers and thus, we report the cardiac proliferative index for all cardiac cells or cardiocytes on the section.
Real-Time PCR.
To quantify the expression of cardiac hypertrophy markers, total RNA was isolated from neonatal hearts on P 21 using Trizol reagent (Invitrogen, Carlsbad, CA). cDNA was generated using reverse transcriptase (Promega, Madison, WI) with oligo dT and an 18sRT primer to amplify the 18S control rRNA. The sequences of the primers used have been previously reported (Lund et al., 2003, 2005
). mRNA expression was then quantified using an I-Cycler (BioRad, Hercules, CA). The efficiency of each primer set was determined from a standard curve with known quantities of cDNA, and all sets used were >90% efficient. Real-time polymerase chain reactions (PCR) reactions were run in triplicate for the genes of interest and 18 s control simultaneously, and the difference between the CT values was determined. Values were then converted to mean relative expression using Q-Gene software (Simon, 2003
), and expressed as a percent of control values (control, n = 6 litters; TCDD, n = 6; one pup analyzed per litter).
Electrocardiograms.
The P 21 mice were anesthetized with 2.5% avertin (20 µl/g) and a three-lead surface electrocardiogram (ECG) was recorded for 5 min from subcutaneous 26-gauge needles using a PM-1000 high performance transducer amplifier, DI-720 data acquisition unit, and WinDaq waveform software (DATAQ Instruments, Akron, OH). Mice were then injected ip with 8.0 ng isoproterenol (ISO, in 100 µl sterile saline), ECGs were recorded for 5 min, the animals were injected with a second, 160-ng, dose of ISO, and ECGs were again recorded for 5 min (control, n = 5 litters; TCDD, n = 4; 35 pups randomly selected and analyzed per litter). The ECGs were analyzed using Advance Codas software (DATAQ Instruments, Akron, OH) to calculate RR, PQ, QRS, and QT intervals. Heart rate was determined from the RR interval, and QT corrected for heart rate (QTc) was calculated as described previously (Mitchell et al., 1998).
Statistics.
Student's t-test was used for individual comparisons, and one-way analysis of variance (ANOVA) was used for dose- and time-related comparisons with post hoc comparisons made using the Holm-Sidak test. Two-way repeated measures ANOVA was used to compared ISO-related changes in heart rate. A p < 0.05 was considered statistically significant in all cases.
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RESULTS |
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DISCUSSION |
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In this study, we found that a maternal dose as low as 3.0 µg and 6.0 µg TCDD/kg on GD 14.5 significantly reduces fetal heart-to-body weight ratio and cardiac proliferation, respectively, on GD 17.5. These doses are comparable to those that induce some of the most sensitive teratogenic effects of TCDD reported in the developing murine fetus. For example, a single maternal dose of 3.0 µg TCDD/kg on GD 14 induces a 54% incidence of hydronephrosis, while doses of 12 and 24 µg/kg increase the incidence to 76% and 64%, respectively (Couture et al., 1990). Similarly, the severity of hydronephrosis is increased significantly at a 3.0-µg/kg maternal dose and increased further at 12 and 24 µg/kg. And, while TCDD doses of 324 µg/kg fail to induce cleft palate on GD 14, doses of 12 and 24 µg/kg induce 76% and 100% incidence of cleft palate, respectively, when administered on GD 12 (Couture et al., 1990
). Thus, the fetal heart appears to be among the more sensitive organs to TCDD-induced teratogenesis on GD 14 in the mouse.
In this study, TCDD treatment at GD 14.5 likely avoided the potential for major morphological cardiac defects, because cardiac morphogenesis in the mouse is complete by this stage of development (Vuillemin and Pexieder, 1989); however, we specifically targeted this developmental window because it coincides with a period of peak cardiomyocyte proliferation (Soonpaa et al., 1996
). TCDD has been shown to decrease cardiomyocyte proliferation during development in both the chick embryo and the zebrafish embryo (Antkiewicz et al., 2005
; Ivnitski et al., 2001
), and our data demonstrate for the first time that in utero TCDD exposure also significantly reduces cardiocyte proliferation in the murine fetus. Although we did not distinguish specific cell types, cardiomyocytes likely account for a significant proportion of the cells affected by TCDD, as they account for the majority of cells in the developing heart. Although the mechanism by which TCDD induces fetal cardiocyte growth arrest is not known, the reduction in proliferation is associated with a reduction in the mRNA expression of cyclins A2, E1, and E2 as reported in our companion article (Thackaberry et al., 2005
). The only cyclin to be downregulated at the mRNA level during cardiogenesis is cyclin A2, and this downregulation is coincident with withdrawal of cardiomyocytes from the cell cycle (Chaudhry et al., 2004
). Additionally, bone morphogenetic protein 10 expression (BMP10) is required for normal proliferation of fetal cardiomyocytes (Chen et al., 2004
), and we also found that BMP10 mRNA expression was reduced in the GD 17.5 fetal heart as reported in our companion article (Thackaberry et al., 2005
). Thus, it is plausible that one mechanism by which TCDD reduces cardiocyte proliferation is by disrupting cell cycle regulation.
To determine if the reduced cardiac size at GD 17.5 was transient or persisted into postnatal development, we also determined heart weight at 7 and 21 days after birth in pups born to dams treated with control or 6.0 µg TCDD/kg. The mean heart/body weight ratio of TCDD-exposed pups on P 7 was reduced by 14%, compared to control pups; however, the relatively small sample size and lack of statistical difference at p < 0.05 precludes concluding that there is a difference in heart weights 1 week after birth. In contrast, the heart/body weight ratio of TCDD-exposed pups was significantly increased at P 21 as compared to control offspring. These results parallel those in a previous report which shows that offspring of dams exposed to a single 5.0 µg TCDD/kg dose on GD 13 exhibit increased heart weight postnatally (Lin et al., 2001). Cardiomyocytes exit the cell cycle prior to birth, and all DNA synthesis in cardiomyocytes after birth contributes solely to binucleation (Soonpaa et al., 1996
). Thus, all cardiac growth that occurs postnatally in mice is due to hypertrophy. The enlargement of hearts in P 21 pups from TCDD litters suggests that inappropriate cardiac hypertrophy is occurring. This explanation is supported by the fact that P 21 pups also exhibit increased expression of the cardiac hypertrophy marker gene, ANF. ANF, and ß-MHC are normally expressed in the heart during fetal development and subsequently downregulate after birth (Mercadier et al., 1989
; Morkin, 2000
). However, their expression upregulates under conditions of pathological cardiac hypertrophy. Induction of ANF mRNA, in particular, is an early response of cardiac myocytes to stretch that occurs during load-induced hypertrophy (Torsoni et al., 2003
). Furthermore, induction of cardiac ANF mRNA occurs in neonatal mice as cardiac hypertrophy develops, even in the absence of changes in ßMHC expression (Yoshimine et al., 1997
). Thus, one explanation may be that the TCDD-induced reduction in cardiocyte proliferation prior to birth results in a smaller heart, which then undergoes abnormal hypertrophy postnatally as a mechanism to maintain appropriate cardiac output as body weight increases. Future histological studies will be needed to determine whether structural hypertrophy occurs postnatally.
To determine if the early signs of cardiac hypertrophy in P 21 pups were associated with altered cardiac conduction or arrhythmias, we conducted ECG analysis. Our most significant finding from these studies was that pups exposed in utero and via lactation to TCDD exhibit bradycardia, reflected by 12% reduction in heart rate compared to control pups. It seems unlikely that the bradycardia results from an effect of TCDD on the developing sinoatrial node, because these pacemaker cells and the entire murine cardiac conduction system are fully differentiated and functional by GD 13 (Rentschler et al., 2001
), 1.5 days prior to TCDD exposure. Alternatively, the TCDD-induced bradycardia may be a result of the increased ANF expression. During early events in cardiac hypertrophy, ANF enhances vagal activity and potentiates reflex bradycardia (Woods, 2004
).
In addition, our results, which show that TCDD reduces basal heart rate but does not alter the responsiveness to ß-adrenergic stimulation of heart rate, differ from those reported in other TCDD studies. For example, overtly toxic doses of TCDD in the adult rat reduce both basal and ß-adrenergicstimulated heart rate (Hermansky et al., 1988; Kelling et al., 1987
), whereas TCDD exposure of the developing chick embryo does not alter basal heart rate but does reduce the chronotropic response to ß-adrenergic stimulation (Sommer et al., 2005
; Walker and Catron, 2000
). The reasons for these differences are unclear; however, they may result from differences in the timing and dose of TCDD used and suggest that multiple mechanisms may be involved.
Finally, the degree to which the effects of TCDD on postnatal heart development result from in utero versus lactational exposure is unknown. Based on toxicokinetic models, a single maternal dose of 6.0 µg TCDD/kg would result in a fetal TCDD concentration of approximately 84 ng TCDD/kg, whereas this same in utero dose would lead to body burdens as high as 2.4 µg TCDD/kg in pups after lactational transfer (Gasiewicz et al., 1983; Nau et al., 1986
; Weber and Birnbaum, 1985
). Thus, it is possible that the effects of TCDD on heart size and cardiac chronotropy at P 21 result, in part, from the considerably higher dose of TCDD received during lactation. Future cross-fostering studies will need to be conducted to determine the importance of lactational TCDD exposure to the cardiovascular effects observed postnatally.
In conclusion, our results demonstrate that the fetal murine heart is a sensitive target of TCDD-induced teratogenicity, resembling many of the TCDD-induced effects observed in fish and avian embryos, including reduced cardiocyte proliferation and altered fetal heart size. In addition, these results confirm that in utero and lactational TCDD exposure induces postnatal cardiac hypertrophy and demonstrate that this exposure also induces postnatal bradycardia. More detailed assessment of the effects of TCDD on postnatal cardiovascular physiology and function is needed to determine the potential to which early TCDD exposure may represent a previously unidentified risk factor for cardiovascular disease in adulthood.
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ACKNOWLEDGMENTS |
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