Adenosine kinase regulation of cardiomyocyte hypertrophy

There is evidence that extracellular adenosine can attenuate cardiac hypertrophy, but the mechanism by which this occurs is not clear. Here we investigated the role of adenosine receptors and adenosine metabolism in attenuation of cardiomyocyte hypertrophy. Phenylephrine (PE) caused hypertrophy of neonatal rat cardiomyocytes with increases of cell surface area, protein synthesis, and atrial natriuretic peptide (ANP) expression. These responses were attenuated by 5 μM 2-chloroadenosine (CADO; adenosine deaminase resistant adenosine analog) or 10 μM adenosine. While antagonism of adenosine receptors partially blocked the reduction of ANP expression produced by CADO, it did not restore cell size or protein synthesis. In support of a role for intracellular adenosine metabolism in regulating hypertrophy, the adenosine kinase (AK) inhibitors iodotubercidin and ABT-702 completely reversed the attenuation of cell size, protein synthesis, and expression of ANP by CADO or ADO. Examination of PEinduced phosphosignaling pathways revealed that CADO treatment did not reduce AKT^Ser473 phosphorylation but did attenuate sustained phosphorylation of Raf^Ser338 (24-48 h), mTOR^Ser2448 (24-48 h), p70S6k^Thr389 (2.5-48 h), and ERK^{Thr202/Tyr204} (48 h). Inhibition of AK restored activation of these enzymes in the presence of CADO. Using dominant negative and constitutively active Raf adenoviruses, we found that Raf activation is necessary and sufficient for PEinduced mTORC1 signaling and cardiomyocyte hypertrophy. CADO treatment still blocked p70S6k^Thr389 phosphorylation and hypertrophy downstream of constitutively active Raf, however, despite a high level phosphorylation of ERK^{Thr202/Tyr204} and AKT^Ser473. Reduction of Raf-induced p70S6k^Thr389 phosphorylation and hypertrophy by CADO was reversed by inhibiting AK. Together, these results identify AK as an important mediator of adenosine attenuation of cardiomyocyte hypertrophy, which acts, at least in part, through inhibition of Raf signaling to mTOR/p70S6k.