PKA catalytic subunit compartmentation regulates contractile and hypertrophic responses to β-adrenergic signaling

Abstract

β-Adrenergic signaling is spatiotemporally heterogeneous in the cardiac myocyte, conferring exquisite control to sympathetic stimulation. Such heterogeneity drives the formation of protein kinase A (PKA) signaling microdomains, which regulate Ca(2+) handling and contractility. Here, we test the hypothesis that the nucleus independently comprises a PKA signaling microdomain regulating myocyte hypertrophy. Spatially-targeted FRET reporters for PKA activity identified slower PKA activation and lower isoproterenol sensitivity in the nucleus (t50=10.6±0.7 min; EC50=89.0 nmol/L) than in the cytosol (t50=3.71±0.25 min; EC50=1.22 nmol/L). These differences were not explained by cAMP or AKAP-based compartmentation. A computational model of cytosolic and nuclear PKA activity was developed and predicted that differences in nuclear PKA dynamics and magnitude are regulated by slow PKA catalytic subunit diffusion, while differences in isoproterenol sensitivity are regulated by nuclear expression of protein kinase inhibitor (PKI). These were validated by FRET and immunofluorescence. The model also predicted differential phosphorylation of PKA substrates regulating cell contractility and hypertrophy. Ca(2+) and cell hypertrophy measurements validated these predictions and identified higher isoproterenol sensitivity for contractile enhancements (EC50=1.84 nmol/L) over cell hypertrophy (EC50=85.9 nmol/L). Over-expression of spatially targeted PKA catalytic subunit to the cytosol or nucleus enhanced contractile and hypertrophic responses, respectively. We conclude that restricted PKA catalytic subunit diffusion is an important PKA compartmentation mechanism and the nucleus comprises a novel PKA signaling microdomain, insulating hypertrophic from contractile β-adrenergic signaling responses.

Keywords: 3-isobytl-1-methylxanthine; A-kinase anchoring protein; AKAP; CREB; Compartmentation; FSK; Hypertrophy; IBMX; ISO; Modeling; NES; NLS; Nuclear; PDE; PKA; PKI; PP2A; WGA; cAMP response element binding protein; forskolin; isoproterenol; nuclear export sequence; nuclear localization sequence; phosphodiesterase; protein kinase A; protein kinase inhibitor; protein phosphatase 2A; wheat germ agglutinin; β-AR; β-Adrenergic; β-adrenergic receptor.