Differential AMPK phosphorylation sites associated with phenylephrine vs. antihypertrophic effects of adenosine agonists in neonatal rat ventricular myocytes

Stimulation of cardiac AMP-activated protein kinase (AMPK) has been demonstrated in both prohypertrophic and antihypertrophic settings, although the reasons for such discrepant results are not well understood. We determined how AMPK is regulated in response to phenylephrine-induced cardiomyocyte hypertrophy and assessed whether AMPK activity may be a factor underlying the antihypertrophic effect of adenosine receptor agonists. The role of AMPK in hypertrophic responses was determined by assessing the effect of the AMPK activator 5-aminoimidazole-4-carboxyamide ribonucleoside on three hypertrophic indexes, including protein synthesis, cell surface area, and fetal gene expression. The changes in phosphorylation of the catalytic α-subunit of AMPK at two different sites, Thr 172 and Ser 485/491 , in response to phenylephrine and adenosine receptor agonists were also examined. 5-Aminoimidazole-4-carboxyamide ribonucleoside completely abolished phenylephrine-induced increases in protein synthesis, cell surface area, and fetal gene expression. AMPK phosphorylation time course studies revealed that phenylephrine induced a time-dependent activation at site Ser 485/491 , in contrast to adenosine receptor agonists, which demonstrated rapid AMPK phosphorylation at Thr 172 . Furthermore, the phosphorylation at Ser 485/491 by phenylephrine was not affected by the addition of adenosine receptor agonists, although, conversely, phosphorylation of AMPK at Thr 172 by adenosine receptor agonists was abrogated by the addition of phenylephrine. We propose from these results that cardiomyocyte hypertrophic and antihypertrophic responses, at least with respect to inhibition of phenylephrine-induced hypertrophy by adenosine receptor agonists, are mediated by multisite AMPK regulation. The latter are reflected by increased phosphorylation at Ser 485/491 and at Thr 172 , associated with prohypertrophic and antihypertrophic responses, respectively.