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  • Title: Hypoglycemia induced changes in cell death and cell proliferation in the organogenesis stage embryonic mouse heart.
    Author: Ghatnekar GS, Barnes JA, Dow JL, Smoak IW.
    Journal: Birth Defects Res A Clin Mol Teratol; 2004 Mar; 70(3):121-31. PubMed ID: 15039926.
    Abstract:
    BACKGROUND: Hypoglycemia is a side effect of diabetes therapy and causes abnormal heart development. Embryonic heart cells are largely resistant to teratogen-induced apoptosis. METHODS: Hypoglycemia was tested for effects on cell death and cell proliferation in embryonic heart cells by exposing mouse embryos on embryonic day (E) 9.5 (plug = E0.5) to hypoglycemia (30-50 mg/dl glucose) in vivo or in vitro for 24 hr. Long-term effects of in vivo exposure on conceptus viability were evaluated at E18.5. Cell death was evaluated on E10.5 by: 1) two TUNEL assays in sectioned embryos to demonstrate DNA fragmentation; 2) confocal microscopy in whole embryos stained with Lysotracker; 3) flow cytometry in dispersed heart cells stained for TUNEL and myosin heavy chain (MHC) to quantify and characterize cell type susceptibility; and 4) immunohistochemistry (IHC) and Western analysis in sectioned embryos to evaluate potential involvement of caspase-3 active subunit and p53. Effects on cell proliferation were evaluated by IHC and Western analysis of proliferating cell nuclear antigen (PCNA). RESULTS: In vivo hypoglycemic exposure on E9.5 reduced viability in conceptuses examined on E18.5. Hearts examined on E10.5 demonstrated increased TUNEL and Lysotracker staining. In hearts of embryos exposed to hypoglycemia, flow cytometry demonstrated increased TUNEL-positive cells and cells dual-labeled for TUNEL and MHC. Protein expression of caspase-3 active subunit and p53 was increased and PCNA was markedly reduced in hearts of embryos exposed to hypoglycemia. CONCLUSIONS: Hypoglycemia reduces embryonic viability, induces significant cell death, and reduces cell proliferation in the E9.5 mouse heart, and these processes may involve active caspase-3 and p53.
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