Investigating the effects of zinc on rat neural crest cells using an in vitro model

Abstract

Maternal zinc (Zn) deficiency increases the incidence of heart malformation in rat fetuses, particularly in regions and structures derived from the outflow tract (OFT). To test the hypothesis that Zn deficiency kills cardiac neural crest cells (NCC) and result in heart malformations, we fed gestation day (GD) 1.5 pregnant rat dams a Zn‐adequate (ZnA) or a Zn‐deficient (ZnD) diet. On GD 10.5, we isolated the portion of the neural tube containing the NCC and cultured these explants in control (CT)media, in media containing a Zn chelator (TPEN, 3 uM), or in TPEN‐treated media with ZnCl2 (TZNC, 5 uM) for 4, 6 and 8h. Our results show that the NCC population isolated from dams fed a ZnD diet was smaller compared to NCC from dams fed the ZnA or restricted fed. Regarding the effects of TPEN, cell viability assessed by ATP production was reduced at 4h compared to that of CT or TZNC media; these differences grew more noticeable at 6 and 8h. We observed increased expression of active caspase‐3 and cytochrome c, two markers of apoptotic cell death, and decreased fluozin expression, an intracellular Zn probe, in 6h TPEN‐treated NCC, compared to NCC cultured in CT or TZNC media. Data from this in vitro model demonstrate the vulnerability of cardiac NCC to zinc deficiency, and suggest that Zn deficiency‐induced cell death may contribute to reduce the population of NCC leading to abnormal heart development. (Supported by NIH 2 R01 HD01743).