Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

Simon Diering,Konstantina Stathopoulou,Mara Goetz,Laura Rathjens,Sönke Harder,Angelika Piasecki,Janice Raabe,Steven Schulz,Mona Brandt,Julia Pflaumenbaum,Ulrike Fuchs,Sonia Donzelli,Sakthivel Sadayappan,Viacheslav O Nikolaev,Frederik Flenner,Elisabeth Ehler,Friederike Cuello

doi:10.1074/jbc.ra120.014467

Abstract

The contraction and relaxation of the heart is controlled by stimulation of the β1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation. Phosphorylation is counteracted by the main cardiac protein phosphatases, PP2A and PP1. Both kinase and phosphatases are sensitive to intramolecular disulfide formation in their catalytic subunits that inhibits their activity. Additionally, intermolecular disulfide formation between PKA type I regulatory subunits (PKA-RI) has been described to enhance PKA's affinity for protein kinase A anchoring proteins, which alters its subcellular distribution. Nitroxyl donors have been shown to affect contractility and relaxation, but the mechanistic basis for this effect is unclear. The present study investigates the impact of several nitroxyl donors and the thiol-oxidizing agent diamide on cardiac myocyte protein phosphorylation and oxidation. Although all tested compounds equally induced intermolecular disulfide formation in PKA-RI, only 1-nitrosocyclohexalycetate (NCA) and diamide induced reproducible protein phosphorylation. Phosphorylation occurred independently of β1-AR activation, but was abolished after pharmacological PKA inhibition and thus potentially attributable to increased PKA activity. NCA treatment of cardiac myocytes induced translocation of PKA and phosphatases to the myofilament compartment as shown by fractionation, immunofluorescence, and proximity ligation assays. Assessment of kinase and phosphatase activity within the myofilament fraction of cardiac myocytes after exposure to NCA revealed activation of PKA and inhibition of phosphatase activity thus explaining the increase in phosphorylation. The data suggest that the NCA-mediated effect on cardiac myocyte protein phosphorylation orchestrates alterations in the kinase/phosphatase balance.

Highlights

The contraction and relaxation of the heart is controlled by stimulation of the b1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation
The NCAinduced significant prolongation of the time to peak was visible after 1 min of treatment and this effect was maintained during the new plateau state (Fig. 1D)
This suggests that the contribution of HNO-mediated partial restoration of sarcomeric protein phosphorylation by an orchestrated modulation of receptor-independent kinase translocation and activation combined with phosphatase inhibition would assume greater significance under heart failure conditions with the were treated with vehicle, NCA (100 mmol/liter, n = 32), or DIA (500 mmol/liter, n = 7) and sensor activation was recorded by fluorescence detection

Summary

Introduction

The contraction and relaxation of the heart is controlled by stimulation of the b1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation. Comparable signals were detected in response to the PKA-activating b1-AR agonist ISO, suggesting an impact of oxidant-mediated phosphorylation of PKA substrate proteins.

Results

Conclusion