Overexpression of A3 adenosine receptors in smooth, cardiac, and skeletal muscle is lethal to embryos.

The profile of expression of the A3 adenosine receptor (A3AR) and its importance during embryo development were explored. To this end, different ages of mouse embryos (8.5 days and older) were subjected to in situ hybridization with an A3AR riboprobe. No expression was found in any embryonic tissue except for the aorta and heart of 15.5-day embryos. To investigate further the role of the A3AR gene in development, we overexpressed this gene in A3AR knockout and wild-type mice by using the SM22 alpha promoter. This promoter is active in smooth, cardiac, and skeletal muscle lineages during early embryogenesis (at 8.5 days or earlier), becoming restricted to vascular and visceral smooth muscle cells in late fetal development and adult mice. We observed that moderate copy number incorporation (four copies) of the A3AR gene driven by the SM22 alpha promoter is sufficient to induce lethality at an early stage of embryo development. Remains of 8.5-day transgenic embryos were collected, including fragmented DNA. Hence, we speculate that A3AR homeostasis is critical for embryo viability and proper development. This finding is intriguing in view of the reported effects of sustained activation of the A3AR on induction of DNA fragmentation and apoptosis in cultured myocytes and other cell types.