![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Departments of Human Genetics, Pediatrics, and Biology, McGill University-Montreal Children's Hospital Research Institute, Montreal, PQ, Canada
2 To whom correspondence should be addressed. E-mail: rima.rozen{at}mcgill.ca.
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
KEY WORDS: • methylenetetrahydrofolate reductase • folate • proliferation • apoptosis • myocardium
Embryonic heart development is a complex and delicate process. Proliferation and apoptosis play important roles during critical periods of heart development (1,2). Inadequate proliferation or excess apoptosis can directly or indirectly result in pathological consequences such as congenital heart defects (CHD)3 and myocardial hypoplasia (1,2). Both genetic and environmental factors can dramatically disturb these processes and lead to abnormal development. One important environmental determinant, dietary folate, was reported to have great influence on proliferation and apoptosis in murine brain (3,4). This finding is part of the considerable body of evidence demonstrating that folate deficiency during pregnancy is a risk factor for neural tube defects. Interestingly, folate deficiency in humans and mice was also shown to affect heart development, leading to CHD and myocardial hypoplasia (5–8). It remains to be determined, however, whether folate deficiency alters proliferation or apoptosis in the heart, as reported for neural tissues.
With the recognition of the important role of folate in brain and heart development, genes involved in folate metabolism are being studied increasingly in mice (8–10). Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate and homocysteine metabolism because it synthesizes the primary circulatory form of folate, 5-methyltetrahydrofolate, which serves as a major methyl donor for homocysteine conversion to methionine. A common MTHFR polymorphism (677C
T), present in the homozygous state in 10–15% of many populations, results in a mild enzymatic deficiency and hyperhomocysteinemia (11). Some clinical studies showed an association of this polymorphism with CHD (12–14) and we recently reported that Mthfr +/– female mice, with a single null allele of Mthfr, give rise to offspring with CHD (mainly ventricular septal defects) and myocardial hypoplasia on gestational day (GD) 14.5 (8). Our group also showed recently that there is decreased proliferation and increased apoptosis in neural cells in mice with a complete knockout of the Mthfr gene (Mthfr –/– mice) (15); these findings may account for the abnormal cerebellar development in these mice (16). Whether similar disturbances occur in early heart development in Mthfr +/– mice remains to be determined.
To test the hypothesis that a disturbance in folate metabolism leads to an alteration in proliferation or apoptosis in the heart, and ultimately to heart defects, we used the Mthfr heterozygous knockout mouse model and a folic acid–deficient diet. We measured the levels of proliferation and apoptosis in embryonic hearts on GD 12.5, an earlier time point than that of our previous study (GD 14.5), to examine the interventricular septum when it is beginning to close, and the myocardium in a period of rapid growth (17).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Animal experimentation was approved by the Montreal Children's Hospital Animal Care Committee, according to the guidelines of the Canadian Council on Animal Care. Mthfr knockout mice were generated as reported (16) and backcrossed for at least 10 generations onto a BALB/c background. Mice were maintained in plastic-bottomed cages embedded with shavings in a temperature-controlled room on a 12-h light:dark cycle. After weaning, female mice (either Mthfr +/+ or +/–) were fed a control diet (CD, TD 01369, Harlan Teklad, Indianapolis), or a folic acid–deficient diet (FADD, TD 01546). These amino acid–defined diets, also utilized in our previous study (8), were composed according to the recommendation of the AIN (18,19). The CD contained the recommended amount of folic acid for rodents (2 mg/kg diet); the FADD contained 0.3 mg/kg diet, 
14% of the recommended amount. Both diets contained 1% succinyl sulfathiazole, an antibiotic, to prevent generation of folate by intestinal bacteria (20). At weaning, female mice were fed their allotted diets for 6 wk, followed by overnight mating with Mthfr +/– males (80–100 d of age). Males were fed a standard rodent diet 5001 (Agribrands Purina, Saint-Hubert, Canada) before mating. The following morning, females were checked for the presence of vaginal plugs. If a plug was present, that morning was designated as GD 0.5. Females were fed their designated diets during the breeding period and throughout pregnancy, creating 4 groups according to maternal diets and genotypes: CD +/+, CD +/–, FADD +/+, FADD +/–. On GD 12.5, 5–7 pregnant mice were selected randomly from each group and injected i.p. with 5-bromo-2'-deoxyuridine (BrdU; Roche). They were injected 3–4 h before killing at a dose of 30 mg/kg body weight. Pregnant mice were killed by CO2 asphyxiation, and blood was collected by cardiac puncture. All visible embryos and resorption sites were recorded. Resorption rates were calculated as the ratio of resorption sites/total number of implantation sites per litter. All visible embryos were dissected and examined for gross malformations, and crown-rump lengths and individual weights were measured. Embryos from the BrdU-injected dams were then fixed in 4% paraformaldehyde overnight and transferred to 70% ethanol for storage. Embryos from dams not injected with BrdU (5–7 litters/group) were used for embryonic heart collection. On GD 12.5, embryonic hearts were quickly removed, placed in PBS, immediately frozen in liquid nitrogen, and stored at –70°C. The yolk sacs from all embryos were dissected and washed in PBS for subsequent embryonic Mthfr genotyping.
Mthfr genotyping
Genomic DNA from maternal toes or livers and embryonic yolk sacs was isolated by phenol/chloroform extraction. PCR and genotyping was performed according to previously described procedures (16).
Plasma total folate concentrations
At the time of killing, blood was collected into tubes containing EDTA. Plasma was immediately separated from blood by centrifugation at 4000 x g for 7 min at 4°C. Plasma was then frozen on dry ice and stored at –70°C until analysis. Total folate was measured by microbial assay using Lactobacillus casei and 96-well microtiter plates (21) with some modifications (22).
Detection of BrdU labeling
Fixed embryos were dehydrated through an ethanol series, embedded in paraffin, and sectioned transversely at 6-µm intervals. One embryo from each litter (5–7 embryos/group) was randomly selected for BrdU staining. Every 33rd or 34th section (counting rostrally from the 1st section, which shows the outflow tract and pulmonary valves) was chosen for BrdU detection. Slides were heated in 10 mmol/L citrate buffer (pH 6.0) for 20 min for BrdU epitope retrieval and then stained with a BrdU labeling and detection kit, Alkaline Phosphatase (Roche) according to the protocol recommended by the manufacturer, and counterstained with hematoxylin. Cells that incorporated BrdU displayed dark blue precipitates in their nuclei.
Detection of apoptosis
TdT-mediated dUTP nick end labeling (TUNEL) staining. The slides adjacent to the slides used for BrdU staining were used for TUNEL staining. TUNEL staining was conducted with the In Situ Cell Death Detection Kit, POD (Roche) according to the manufacturer's instructions. Hematoxylin was used for counterstaining. TUNEL-stained cells displayed dark brown precipitates in their nuclei.
Caspase-3/7 activity assay. All hearts from a litter (5–7 litters/group) were pooled, ground into powder in liquid nitrogen, and lysed in buffer (50 mmol/L potassium phosphate, 0.3 mmol/L EDTA, pH 8.0). Protein concentration was determined with the Bio-Rad Protein Assay solution using bovine serum albumin as a standard. Caspase-3/7 activities were measured using the Caspase-Glo® 3/7 Assay kit (Promega). Protein (2 µg) was used for each reaction and each sample was assayed in duplicate. The entire assay was repeated once. The mean of 4 replicates was used for each sample.
Image analysis and quantitation
BrdU- and TUNEL-stained sections were photographed at 100X by a digital photography system with visualization software (Spot Advanced version 4.1) and quantified with Northern Eclipse software (version 6.0). The number of BrdU-positive cells and TUNEL-positive cells was counted per section. The thickness of the left and right ventricle compact walls was quantified on the same slides that had been used for BrdU staining. For each slide, we recorded the shortest cross distance along the compact wall to represent the thickness of the wall. Two individuals, unaware of the genotype and dietary information, performed the counting and measuring independently; the reported values represent the mean score.
Statistical analysis
Each litter or dam was considered as a statistical unit and all data are presented as mean ± SEM. Nonparametric data (resorption rate) was transformed into ranked data before statistical analysis. Two-factor ANOVA was conducted to determine the effects of maternal Mthfr genotype and dietary treatment. To analyze the distribution of Mthfr genotypes among embryos, the 
2 test was used. All statistical analyses were performed with SPSS for WINDOWS (version 11.0; SPSS). P-values < 0.05 were considered significant.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
|
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Proliferation and apoptosis play important roles in heart development; an imbalance in these processes can result in a variety of cardiac malformations (2). For example, ventricular septal defects, a common type of CHD, often result from insufficient proliferation and/or excess apoptosis of the septum or neighboring tissue. Myocardial hypoplasia can result from altered proliferation or apoptosis of the myocardium (25). The effects of folate on proliferation and apoptosis in cardiac tissue were not examined previously, although some recent studies showed that folate deficiency or knockouts of genes in folate metabolism, e.g., Mthfr or Folbp1, resulted in increased apoptosis and poor proliferation in neural tissues (3,4,15,26). In this study, we examined the potential influences of maternal dietary folate deficiency and Mthfr deficiency on proliferation and apoptosis in the myocardium of the offspring. Our results suggest that maternal folate deficiency significantly affects myocardial proliferation, but does not affect apoptosis, whereas maternal Mthfr deficiency does not significantly affect proliferation or apoptosis in the myocardium. We also examined 2–3 embryos per group at a later time point, GD 14.5, and obtained similar results (data not shown). Some of the embryos examined were Mthfr –/– embryos, because we had randomly selected embryos for measurements of proliferation and apoptosis. These embryos did not differ from the wild-type embryos (data not shown). This finding is consistent with our earlier report suggesting that maternal folate status and maternal Mthfr genotype, not embryonic genotype, are associated with the risk of CHD. We also observed the expected distribution of embryonic genotypes in this study, indicating that Mthfr–/– embryos are not preferentially resorbed in utero. The increase in resorptions due to low dietary folate must also depend on maternal folate rather than embryonic genotypes, as suggested earlier (8).
There are several possible mechanisms by which folate can affect proliferation. Folate derivatives are involved in a variety of critical cellular reactions, including nucleotide synthesis, amino acid synthesis, and methylation reactions. During embryonic development, myocardial cells undergo rapid mitosis or differentiation. Thus, it is not surprising that folate deficiency can affect proliferation. Another metabolic consequence of folate deficiency is an elevation of homocysteine, which was shown to inhibit proliferation in vitro (27,28), and a decrease in methionine, which is required for the synthesis of S-adenosylmethionine for methylation reactions. Folate deficiency could therefore affect the expression of genes involved in cell proliferation, through an effect on DNA methylation. The effect of folate deficiency on proliferation can account in part for the observed phenotypes (ventricular septal defects and myocardial hypoplasia) that we reported earlier (8). On GD 12.5, the compact layers of ventricles were thinner in embryos from FADD-treated dams than those from CD-treated dams, although this effect was not as dramatic as that observed on GD 14.5.
Although maternal dietary folate affected levels of proliferation in this study at GD 12.5, we did not find significant effects of maternal Mthfr genotype. Dietary folate was clearly associated with a marked incidence of CHD at GD 14.5 in our earlier report (mean of 17% of litters), whereas Mthfr genotype was associated with a more modest rate (7% of litters). It is possible that the more modest effects of Mthfr might not be manifest at this earlier time point. In the 2 d between GD 12.5 and GD 14.5, the embryonic weight increases 3-fold; an effect of genotype might become manifest during this rapid growth phase. On the other hand, the Mthfr +/– genotype may contribute to CHD risk by influencing molecular events other than proliferation or apoptosis, e.g., methylation.
On GD 12.5, maternal folate deficiency caused a high rate of embryonic loss (50%). This value is very similar to the resorption rate that we observed on GD 14.5, suggesting that events contributing to fetal demise must have occurred at an earlier embryonic stage. Of the 4 groups of embryos, the lowest mean body lengths and weights of viable embryos occurred in dams fed FADD that were also Mthfr +/–, although the genotype effect was not significant (P = 0.09); this pattern was similar to that seen on GD 14.5 (8). Again, the influence of diet or genotype on embryonic growth would presumably have begun at a time point before GD 12.5.
In conclusion, we found that maternal folate deficiency in mice increased the rate of resorption, decreased embryonic length and weight, and inhibited myocardial proliferation. Folic acid supplementation during pregnancy, in addition to the well-documented role in prevention of neural tube defects, may also reduce pregnancy loss and cardiac malformations.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: BrdU, 5-bromo-2'-deoxyuridine; CD, control diet; CHD, congenital heart defects; FADD, folic acid–deficient diet; GD, gestational day; MTHFR, methylenetetrahydrofolate reductase; TUNEL, TdT-mediated dUTP nick end labeling.
Manuscript received 21 March 2006. Initial review completed 6 April 2006. Revision accepted 20 April 2006.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Fisher SA, Langille BL, Srivastava D. Apoptosis during cardiovascular development. Circ Res. 2000;87:856–64.
2. Gittenberger-de Groot AC, Bartelings MM, Deruiter MC, Poelmann RE. Basics of cardiac development for the understanding of congenital heart malformations. Pediatr Res. 2005;57:169–76.[Medline]
3. Kruman II, Mouton PR, Emokpae R Jr, Cutler RG, Mattson MP. Folate deficiency inhibits proliferation of adult hippocampal progenitors. Neuroreport. 2005;16:1055–9.[Medline]
4. Craciunescu CN, Brown EC, Mar MH, Albright CD, Nadeau MR, Zeisel SH. Folic acid deficiency during late gestation decreases progenitor cell proliferation and increases apoptosis in fetal mouse brain. J Nutr. 2004;134:162–6.
5. Kapusta L, Haagmans ML, Steegers EA, Cuypers MH, Blom HJ, Eskes TK. Congenital heart defects and maternal derangement of homocysteine metabolism. J Pediatr. 1999;135:773–4.[Medline]
6. Botto LD, Mulinare J, Erickson JD. Occurrence of congenital heart defects in relation to maternal multivitamin use. Am J Epidemiol. 2000;151:878–84.
7. Burgoon JM, Selhub J, Nadeau M, Sadler TW. Investigation of the effects of folate deficiency on embryonic development through the establishment of a folate deficient mouse model. Teratology. 2002;65:219–27.[Medline]
8. Li D, Pickell L, Liu Y, Wu Q, Cohn JS, Rozen R. Maternal methylenetetrahydrofolate reductase deficiency and low dietary folate lead to adverse reproductive outcomes and congenital heart defects in mice. Am J Clin Nutr. 2005;82:188–95.
9. Maddox DM, Manlapat A, Roon P, Prasad P, Ganapathy V, Smith SB. Reduced-folate carrier (RFC) is expressed in placenta and yolk sac, as well as in cells of the developing forebrain, hindbrain, neural tube, craniofacial region, eye, limb buds and heart. BMC Dev Biol. 2003; July 29. doi:10.1186/1471-213X-3-6.
10. Tang LS, Wlodarczyk BJ, Santillano DR, Miranda RC, Finnell RH. Developmental consequences of abnormal folate transport during murine heart morphogenesis. Birth Defects Res A Clin Mol Teratol. 2004;70:449–58.[Medline]
11. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ, den Heijer M, Kluijtmans LA, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet. 1995;10:111–3.[Medline]
12. Wenstrom KD, Johanning GL, Johnston KE, DuBard M. Association of the C677T methylenetetrahydrofolate reductase mutation and elevated homocysteine levels with congenital cardiac malformations. Am J Obstet Gynecol. 2001;184:806–12.[Medline]
13. Junker R, Kotthoff S, Vielhaber H, Halimeh S, Kosch A, Koch HG, Kassenbohmer R, Heineking B, Nowak-Gottl U. Infant methylenetetrahydrofolate reductase 677TT genotype is a risk factor for congenital heart disease. Cardiovasc Res. 2001;51:251–4.[Medline]
14. Storti S, Vittorini S, Lascone MR, Sacchelli M, Collavoli A, Ripoli A, Cocchi G, Biagini A, Clerico A. Association between 5,10-methylenetetrahydrofolate reductase C677T and A1298C polymorphisms and conotroncal heart defects. Clin Chem Lab Med. 2003;41:276–80.[Medline]
15. Chen Z, Schwahn BC, Wu Q, He X, Rozen R. Postnatal cerebellar defects in mice deficient in methylenetetrahydrofolate reductase. Int J Dev Neurosci. 2005;23:465–74.[Medline]
16. Chen Z, Karaplis AC, Ackerman SL, Pogribny IP, Melnyk S, Lussier-Cacan S, Chen MF, Pai A, John SW, et al. Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition. Hum Mol Genet. 2001;10:433–43.
17. Kaufman MH. The atlas of mouse development 1st edition. London (UK): Academic Press; 1994.
18. Reeves PG, Nielsen FH, Fahey GC Jr. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr. 1993;123:1939–51.
19. Reeves PG. Components of the AIN-93 diets as improvements in the AIN-76A diet. J Nutr. 1997;127:838S–41.[Medline]
20. Sepehr E, Peace RW, Storey KB, Jee P, Lampi BJ, Brooks SP. Folate derived from cecal bacterial fermentation does not increase liver folate stores in 28-d folate-depleted male Sprague-Dawley rats. J Nutr. 2003;133:1347–54.
21. Horne DW, Patterson D. Lactobacillus casei microbiological assay of folic acid derivatives in 96-well microtiter plates. Clin Chem. 1988;34:2357–9.
22. Tamura T, Freeberg LE, Cornwell PE. Inhibition of EDTA of growth of Lactobacillus casei in the folate microbiological assay and its reversal by added manganese or iron. Clin Chem. 1990;36:1993.[Medline]
23. Doseff AI. Apoptosis: the sculptor of development. Stem Cells Dev. 2004;13:473–83.[Medline]
24. Hoffman JI, Kaplan S. The incidence of congenital heart disease. J Am Coll Cardiol. 2002;39:1890–900.
25. Oh H, Schneider MD. The emerging role of telomerase in cardiac muscle cell growth and survival. J Mol Cell Cardiol. 2002;34:717–24.[Medline]
26. Tang LS, Santillano DR, Wlodarczyk BJ, Miranda RC, Finnell RH. Role of Folbp1 in the regional regulation of apoptosis and cell proliferation in the developing neural tube and craniofacies. Am J Med Genet C Semin Med Genet. 2005;135:48–58.[Medline]
27. Chen JZ, Zhu JH, Wang XX, Zhu JH, Xie XD, Sun J, Shang YP, Guo XG, Dai HM, Hu SJ. Effects of homocysteine on number and activity of endothelial progenitor cells from peripheral blood. J Mol Cell Cardiol. 2004;36:233–9.[Medline]
28. Buemi M, Marino D, Di Pasquale G, Floccari F, Ruello A, Aloisi C, Corica F, Senatore M, Romeo A, Frisina N. Effects of homocysteine on proliferation, necrosis, and apoptosis of vascular smooth muscle cells in culture and influence of folic acid. Thromb Res. 2001;104:207–13.[Medline]
This article has been cited by other articles:
|
|
 |
|
  E. Knock, L. Deng, Q. Wu, A. K. Lawrance, X.-l. Wang, and R. Rozen Strain Differences in Mice Highlight the Role of DNA Damage in Neoplasia Induced by Low Dietary Folate J. Nutr., April 1, 2008; 138(4): 653 - 658. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Knock, L. Deng, Q. Wu, D. Leclerc, X.-l. Wang, and R. Rozen Low Dietary Folate Initiates Intestinal Tumors in Mice, with Altered Expression of G2-M Checkpoint Regulators Polo-Like Kinase 1 and Cell Division Cycle 25c Cancer Res., November 1, 2006; 66(21): 10349 - 10356. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
