Background —cAMP-dependent protein kinase (PKA) regulates a broad range of cellular responses in the cardiac myocyte. Downstream regulation of the PKA pathway is mediated by a class of scaffolding proteins called A-kinase anchoring proteins (AKAPs), which sequester PKA to specific subcellular locations through binding to its regulatory subunit (R). However, the effect of RII autophosphorylation on AKAP binding and the degree of RII autophosphorylation in failing and nonfailing human hearts remains unknown. Methods and Results —We investigated AKAP-RII binding by overlay analysis and surface plasmon resonance spectroscopy and measured RII autophosphorylation in human hearts by backphosphorylation. Binding of Ht31 peptide (representing the RII-binding region of AKAPs) to cardiac RII was increased ≈145% ( P <0.01) for autophosphorylated RII relative to unphosphorylated control. By surface plasmon resonance, RII autophosphorylation significantly increased binding affinity to Ht31 by ≈200% ( P <0.01). Baseline PKA-dependent phosphorylation of RII was significantly decreased ≈30% ( P <0.05) in human hearts with dilated cardiomyopathy compared with nonfailing controls. Conclusions —These results suggest that AKAP binding of PKA in the heart is regulated by RII autophosphorylation. Therefore AKAP targeting of PKA may be reduced in patients with end-stage heart failure. This mechanism may be responsible for the decreased cAMP-dependent phosphorylation of proteins in dilated cardiomyopathy that we and others have previously observed.
Downstream regulation of the cAMP-dependent protein kinase (PKA) pathway is mediated by anchoring proteins (AKAPs) that sequester PKA to specific subcellular locations through binding to PKA regulatory subunits (RI or RII). The RII-binding domain of all AKAPs forms an amphipathic α-helix with similar secondary structure. However, the importance of sequence differences in the RII-binding domains of different AKAPs is unknown, and mechanisms that regulate AKAP-PKA affinity are not clearly defined. Using surface plasmon resonance (SPR) spectroscopy, we measured real-time kinetics of RII interaction with various AKAPs. Base-line equilibrium binding constants (Kd) for RII binding to Ht31, mAKAP, and AKAP15/18 were 10 nm, 119 nm, and 6.6 μm, respectively. PKA stimulation of intact Chinese hamster ovary cells increased RIIα binding to AKAP100/mAKAP and AKAP15/18 by ∼7- and 82-fold, respectively. These results suggest that differences in primary sequence of the RII-binding domain may be responsible for the selective affinity of RII for different AKAPs. Furthermore, RII autophosphorylation may provide additional localized regulation of kinase anchoring. In cardiac myocytes, disruption of RII-AKAP interaction decreased PKA phosphorylation of the PKA substrate, myosin-binding protein C. Thus, these mechanisms may be involved in adding additional specificity in intracellular signaling in diverse cell types and under conditions of cAMP/PKA activation.
Stress-induced cardiomyopathy (SIC) is characterized by reversible left ventricular (LV) systolic dysfunction, which appears to be triggered by an intense, stressful event in the absence of significant coronary artery disease. It manifests typically with transient left ventricular wall motion abnormalities (WMA) involving the apical and/or mid-ventricular myocardial segments, associated with minimal troponin rise (<5 ng/ml), and typical EGG changes. Described are 3 cases of stress-induced cardiomyopathy with atypical distribution of wall motion abnormalities. Possible contributing mechanisms to the pathogenesis and the variability in WMA are discussed.
Background —Most studies indicate that failing human hearts have greater baseline myofibrillar Ca 2+ sensitivity of tension development than nonfailing hearts. Phosphorylation of cardiac troponin I (TnI) by cAMP-dependent protein kinase (PKA) decreases the affinity of the troponin complex for Ca 2+ , thus altering the Ca 2+ sensitivity of force production. We tested the hypothesis that PKA-dependent TnI phosphorylation is altered in the failing human heart and investigated changes in PKA regulatory subunits as a potential mechanism. Methods and Results —Using in vitro back-phosphorylation with [γ- 32 P]ATP, we demonstrated a significant ( P <0.05) ≈25% reduction in baseline PKA-dependent TnI phosphorylation in human hearts with dilated cardiomyopathy (DCM) compared with nonfailing (NF) human hearts. There was no significant difference in cAMP content or maximal PKA activity between DCM and NF hearts, but expression of the regulatory subunits of PKA-I (RI) and PKA-II (RII) was significantly decreased in DCM versus NF hearts (RI by ≈40%, P <0.05; RII by ≈30%, P <0.01). Conclusions —PKA activity is regulated at the substrate level through interactions of PKA regulatory subunits with A-kinase anchoring proteins. The reduced baseline PKA-dependent phosphorylation of TnI in DCM may be due to decreased expression of RI and RII and consequently reduced anchoring of PKA holoenzyme. These findings provide new evidence of deficiencies in downstream regulation of the β-adrenergic pathway in the failing human heart and may account for increased baseline myofibrillar Ca 2+ sensitivity.
