Abstract
In the heart cyclic AMP‐dependent Protein Kinase A (PKA) mediates the catecholaminergic control over the force and frequency of cardiac contraction via phosphorylation of proteins that are involved in excitation‐contraction coupling (ECC). As part of the ECC machinery, catecholamine dependent activation of PKA leads to phosphorylation of the L‐type Ca2+ channels (LTCC) and phospholamban (PLB), resulting in increased systolic Ca2+. PKA also phosphorylates phospholemman (PLM), a regulator of the cardiac Na+/K+ ATPase (NKA) which, by setting the Na2+ gradient for Na2+/Ca2+ exchanger, promotes Ca2+ extrusion from the cytosol. How cAMP/PKA signaling coordinates these apparently opposing effects on intracellular Ca2+ remains unclear.The aim of this study was to investigate cAMP/PKA signaling at the A Kinase Anchoring Protein 79(AKAP79)/150 which binds PKA that phosphorylates LTCC and at the PLM/NKA complex and determine whether the two sites are under distinct cAMP pools which could explain the resulting opposing effects on Ca2+ ion flux.Fluorescence Resonance Energy Transfer (FRET)‐based sensors for cAMP (CUTie) and PKA‐dependent phosphorylation (AKAR4) targeted to PLM/NKA and AKAP79/LTCC nanodomains at the plasmalemma were used for real time monitoring of cAMP levels and PKA activity. Experiments were conducted in rat ventricular myocytes.A heterogeneous cAMP response was observed at the two sites on β‐AR stimulation with Isoproterenol (Iso), with the cAMP increase at PLM/NKA being significantly lower than that at AKAP79/LTCC. We demonstrated that this differential local regulation of cAMP response is due to phosphodiesterases (PDEs) which degrade cAMP and contain its increase at the PLM/NKA site. We found that PDE2 and PDE8 play a major role in selectively shielding the PLM/NKA complex from cAMP generated on activation of the b‐adrenergic receptor. However, despite the difference in the amplitude of the cAMP response to catecholamines at the AKAP79/LTCC and PLM/NKA complexes, we found that the PKA‐dependent phosphorylation of local targets is similar at the two sites. This apparent discrepancy appears to depend on a more robust phosphatase activity at AKAP79 compared to PLM. Our findings show profound differences in local handling of cAMP levels and phosphatase activity at these two plasmalemma sites and reveal that adrenergic regulation of Ca2+ flux across the plasmalemma relies on submicroscopic compartmentalization of cAMP/PKA signals.Support or Funding InformationThis research was funded by the British Heart Foundation, (PG/10/75/28537 and RG/17/6/32944), the BHF Centre of Research Excellence, Oxford (RE/13/1/30181), the Graduate School of Science and Engineering (GSSE), and Groningen Research Institute of Pharmacy (GRIP)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Published Version
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