PDE4 and mAKAPb are nodal organizers of b2-ARs nuclear PKA signalling in cardiac myocytes

Abstract

Aims b1- and b2-adrenergic receptors (b-ARs) produce different acute contractile effects on the heart partly because they impact on different cytosolic pools of cAMP-dependent protein kinase (PKA). They also exert different effects on gene expression but the underlying mechanisms remain unknown. The aim of this study was to understand the mechanisms by which b1- and b2-ARs regulate nuclear PKA activity in cardiomyocytes. Methods We used cytoplasmic and nuclear targeted biosensors to examine cAMP signals and PKA activity in adult rat ven- and results tricular myocytes upon selective b1- or b2-ARs stimulation. Both b1- and b2-AR stimulation increased cAMP and activated PKA in the cytoplasm. Although the two receptors also increased cAMP in the nucleus, only b1-ARs increased nuclear PKA activity and up-regulated the PKA target gene and pro-apoptotic factor, inducible cAMP early repressor (ICER). Inhibition of phosphodiesterase (PDE)4, but not Gi, PDE3, GRK2 nor caveolae disruption disclosed nuclear PKA activation and ICER induction by b2-ARs. Both nuclear and cytoplasmic PKI prevented nuclear PKA activation and ICER induction by b1-ARs, indicating that PKA activation outside the nucleus is required for subsequent nuclear PKA activation and ICER mRNA expression. Cytoplasmic PKI also blocked ICER induction by b2-AR stimulation (with concomitant PDE4 inhibition). However, in this case nuclear PKI decreased ICER up-regulation by only 30%, indicating that other mechanisms are involved. Down-regulation of mAKAPb partially inhibited nuclear PKA activation upon b1-AR stimulation, and drastically decreased nuclear PKA activation upon b2-AR stimulation in the presence of PDE4 inhibition. Conclusions b1- and b2-ARs differentially regulate nuclear PKA activity and ICER expression in cardiomyocytes. PDE4 insulates a mAKAPb-targeted PKA pool at the nuclear envelope that prevents nuclear PKA activation upon b2-AR stimulation.