Hypoxia induces cardiac malformations via A1 adenosine receptor activation in chicken embryos

BACKGROUND: The current understanding of the effects of hypoxia on early embryogenesis is limited. Potential mediators of hypoxic effects include adenosine, which increases dramatically during hypoxic conditions and activates A₁ adenosine receptors (A₁ARs). METHODS: To examine the influences of hypoxia and adenosine signaling on cardiac development, chicken embryos were studied. Real time RT-PCR assay was used to examine the A ₁AR gene expression during embryogenesis and after siRNA- mediated knock down. Cell proliferation was determined by counting cell nuclei and PhosphoHistone H3 positive cells. Apoptosis was determined by TUNEL assay. RESULTS: A₁ARs were found to be expressed in chicken embryos during early embryogenesis. Treatment of Hamburger and Hamilton stage 4 embryos with the A₁AR agonist N⁶-cyclopentyladenosine caused cardiac bifida and looping defects in 55% of embryos. Hamburger and Hamilton stage 4 embryos exposed to 10% oxygen for 6, 12, 18, and 24 h followed by recovery in room air until stage 11, exhibited cardia bifida and looping defects in 34, 45, 60, and 86% of embryos respectively. Hypoxia-induced abnormalities were reduced when A₁AR signaling was inhibited by the A₁AR antagonist 1,3 dipropyl-8-cyclopentylxanthine or by siRNA-targeting A₁ARs. Hypoxia treatment did not increase apoptosis, but decreased embryonic cell proliferation. CONCLUSIONS: These data indicate that hypoxia adversely influences cardiac malformations during development, in part by A₁AR signaling.