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Hypoglycemia induced changes in cell death and cell proliferation in the organogenesis stage embryonic mouse heart
Gautam S Ghatnekar1, Jill A Barnes, Janet L Dow
1Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA. gsghatne@unity.ncsu.edu
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.
Insights
Hypoglycemia during early pregnancy harms embryonic development, causing heart cell death and reduced proliferation in mouse models. This may involve caspase-3 and p53 pathways, impacting fetal viability.
Area of Science:
- Developmental Biology
- Cardiovascular Research
- Teratology
Background:
- Hypoglycemia, a common side effect of diabetes therapy, is linked to abnormal embryonic heart development.
- Embryonic heart cells exhibit resistance to teratogen-induced apoptosis.
Purpose of the Study:
- To investigate the effects of hypoglycemia on embryonic heart cell death and proliferation.
- To assess the impact of hypoglycemia on conceptus viability in a mouse model.
Main Methods:
- Mouse embryos at embryonic day (E) 9.5 were exposed to hypoglycemia (30-50 mg/dl glucose) in vivo or in vitro for 24 hours.
- Cell death was assessed using TUNEL assays, Lysotracker staining, and flow cytometry for TUNEL and myosin heavy chain (MHC) positive cells.
- Protein expression of active caspase-3, p53, and proliferating cell nuclear antigen (PCNA) was evaluated via immunohistochemistry and Western analysis.
Main Results:
- In vivo hypoglycemic exposure reduced conceptus viability at E18.5.
- Increased cell death markers (TUNEL, Lysotracker) and caspase-3/p53 expression were observed in embryonic hearts.
- Proliferating cell nuclear antigen (PCNA) levels were markedly reduced, indicating decreased cell proliferation.
Conclusions:
- Hypoglycemia significantly reduces embryonic viability and induces cell death and proliferation defects in the developing mouse heart.
- Active caspase-3 and p53 may play a role in mediating the detrimental effects of hypoglycemia on embryonic heart development.
