Differential Regulation of Cytoplasmic and Nuclear PKA Activities by β1- and β2-Adrenoceptors in Adult Cardiac Myocytes

Abstract

Functional differences between β1-adrenoceptors (β1-ARs) and β2-ARs in the regulation of cardiac excitation-contraction coupling have been widely studied. Much less is known regarding differences between β1- and β2-ARs in the regulation of gene expression and the mechanisms involved. Here, we studied the mechanisms regulating nuclear versus bulk cytoplasmic PKA activation by β1- and β2-ARs and the functional consequences on gene expression.PKA activity was measured using targeted FRET-based A-kinase activity reporters. For specific β1- or β2-AR stimulation, isoprenaline (Iso) was used in combination with the β2-AR antagonist ICI118551 or with the β1-AR antagonist CGP20712A, respectively. At all Iso concentrations tested, β1-AR stimulation was more efficient than β2-AR stimulation to increase cytoplasmic and nuclear PKA activities. For a similar activation of cytoplasmic PKA, nuclear PKA activity was 3-fold higher with β1-AR than with β2-AR stimulation. Inhibition of Gi protein, caveolae disruption, and inhibition of GRK2-mediated desensitization potentiated β2-AR-induced cytoplasmic but not nuclear PKA activity. PDE4 inhibition strongly potentiated cytoplasmic and nuclear PKA responses to both β1- and β2-AR stimulation. In contrast, PDE3 inhibition had no significant effect on β1-AR induced PKA activation in both compartments, while it increased cytoplasmic but not nuclear PKA activity upon β2-AR stimulation. Downregulation of mAKAP, a nuclear envelope associated scaffold protein, decreased β1-AR stimulation of nuclear PKA activity. Consistently, β1-ARs but not β2-ARs were able to induce the expression of the PKA-regulated pro-apoptotic gene, ICER. These results show that i) β1- and β2-ARs differentially regulate cytoplasmic versus nuclear PKA activities, ii) nuclear PKA activation can be dissociated from bulk cytoplasmic PKA activity upon β2-AR stimulation; and iii) PDE4 and mAKAP are critical components of nuclear PKA signalling.