Programmed cell death in the developing heart: Regulation by BMP4 and FGF2

Inhibition of cell proliferation in the embryonic myocardium by A1 adenosine receptor activation

“…For in vitro explant studies, dissected ventricular tissues (500 ϫ 500 m) were placed on Millipore filters (Millipore) and cultured in Dulbecco's modified Eagle medium (DMEM; GIBCO) containing 10% fetal bovine serum (GIBCO) in modified Trowel's organ culture system (Vainio et al, 1993;Zhao and Rivkees, 2000). Cultures were incubated at 37°C in 5% CO 2 and 95% room air.…”

“…Explants were collected after 4, 8, or 24 hr of treatment. One hour before explants were harvested, BrdU (30 g/ml final concentration) was added to the media (Zhao and Rivkees, 2000). Specimens were then embedded and frozen in OCT medium.…”

“…To assess apoptosis, the TUNEL assay was performed as described by Zhao and Rivkees (2000). Briefly, sections were fixed in 4% PFA in PBS for 20 min, permeated with 0.1% Triton X-100 (10 min), and incubated with 10 g/ml proteinase K (10 -15 min) at room temperature.…”

“…BrdU incorporation was used to assess cell proliferation as described by Zhao and Rivkees (2000). Briefly, sections or whole mount specimens were fixed with 4% PFA, incubated with 4 M HCl for 30 min, and neutralized with 100 mM Tris-HCl (pH 8.5).…”

“…Formation of the heart involves differentiation of the primary heart tube into the four major cardiac chambers, the right and left ventricles and atria (Pexieder, 1975b). This process involves complex temporal and spatial patterns of gene expression and dynamic changes in cell proliferation and programmed cell death (Olson and Srivastava, 1996;Pexieder, 1975a;Zhao and Rivkees, 2000). Whereas the influence of growth factors and transcription factors on cardiac development has received considerable attention (Olson and Srivastava, 1996), little is known about the influence of hormonal agents, including neurotransmitters, on cardiac structural development.…”