OBJECTIVETo construct the folic acid deficient model in zebrafish and observe the abnormal cardiac phenotypes, to find the optimal period for supplementing folic acid that can most effectively prevent the heart malformation induced by folic acid deficiency, and to investigate the possible mechanisms by which folic acid deficiency induces malformations of heart.

METHODThe folic acid deficient zebrafish model was constructed by using both the folic acid antagonist methotrexate (MTX) and knocking-down dhfr (dihydrofolate reductase gene). Exogenous tetrahydrofolic acid rescue experiment was performed. Folic acid was given to folic acid deficient groups in different periods. The percent of cardiac malformation, the cardiac phenotypes, the heart rate and the ventricular shortening fraction (VSF) were recorded. The out flow tract (OFT) was observed by using fluorescein micro-angiography. Whole-mount in situ hybridization and real-time PCR were performed to detect vmhc, amhc, tbx5 and nppa expressions.

RESULTAbout (78.00 ± 3.74)% embryos in MTX treated group and (68.00 ± 6.32)% embryos in dhfr knocking-down group had heart malformations, including the abnormal cardiac shapes, the hypogenesis of OFT and the reduced heart rate and VSF. Giving exogenous tetrahydrofolic acid rescued the above abnormalities. Given the folic acid on 8 - 12 hours post-fertilization (hpf), both the MTX treated group (20.20% ± 3.77%) and dhfr knocking-down group (43.40% ± 4.51%) showed the most significantly reduced percent of cardiac malformation and the most obviously improved cardiac development. In folic acid deficient group, the expressions of tbx5 and nppa were reduced while the expressions of vmhc and amhc appeared normal. After being given folic acid to MTX treated group and dhfr knocking-down group, the expressions of tbx5 and nppa were increased.

CONCLUSIONSThe synthesis of tetrahydrofolic acid was decreased in our folic acid deficient model. Giving folic acid in the middle period, which is the early developmental stage, can best prevent the abnormal developments of hearts induced by folic acid deficiency. Folic acid deficiency did not disrupt the differentiations of myosins in ventricle and atrium. The cardiac malformations caused by folic acid deficiency were related with the reduced expressions of tbx5 and nppa.