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Phosphodiesterase type 3A regulates basal myocardial contractility through interacting with sarcoplasmic reticulum calcium ATPase type 2a signaling complexes in mouse heart.

ABSTRACT: RATIONALE:cAMP is an important regulator of myocardial function, and regulation of cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is a critical determinant of the amplitude, duration, and compartmentation of cAMP-mediated signaling. The role of different PDE isozymes, particularly PDE3A vs PDE3B, in the regulation of heart function remains unclear. OBJECTIVE:To determine the relative contribution of PDE3A vs PDE3B isozymes in the regulation of heart function and to dissect the molecular basis for this regulation. METHODS AND RESULTS:Compared with wild-type littermates, cardiac contractility and relaxation were enhanced in isolated hearts from PDE3A(-/-), but not PDE3B(-/-), mice. Furthermore, PDE3 inhibition had no effect on PDE3A(-/-) hearts but increased contractility in wild-type (as expected) and PDE3B(-/-) hearts to levels indistinguishable from PDE3A(-/-). The enhanced contractility in PDE3A(-/-) hearts was associated with cAMP-dependent elevations in Ca(2+) transient amplitudes and increased sarcoplasmic reticulum (SR) Ca(2+) content, without changes in L-type Ca(2+) currents of cardiomyocytes, as well as with increased SR Ca(2+)-ATPase type 2a activity, SR Ca(2+) uptake rates, and phospholamban phosphorylation in SR fractions. Consistent with these observations, PDE3 activity was reduced ?8-fold in SR fractions from PDE3A(-/-) hearts. Coimmunoprecipitation experiments further revealed that PDE3A associates with both SR calcium ATPase type 2a and phospholamban in a complex that also contains A-kinase anchoring protein-18, protein kinase type A-RII, and protein phosphatase type 2A. CONCLUSIONS:Our data support the conclusion that PDE3A is the primary PDE3 isozyme modulating basal contractility and SR Ca(2+) content by regulating cAMP in microdomains containing macromolecular complexes of SR calcium ATPase type 2a-phospholamban-PDE3A.

SUBMITTER: Beca S

PROVIDER: S-EPMC3579621 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Phosphodiesterase type 3A regulates basal myocardial contractility through interacting with sarcoplasmic reticulum calcium ATPase type 2a signaling complexes in mouse heart.

Beca Sanja S Ahmad Faiyaz F Shen Weixing W Liu Jie J Makary Samy S Polidovitch Nazari N Sun Junhui J Hockman Steven S Chung Youn Wook YW Movsesian Matthew M Murphy Elizabeth E Manganiello Vincent V Backx Peter H PH

Circulation research 20121119 2

<h4>Rationale</h4>cAMP is an important regulator of myocardial function, and regulation of cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is a critical determinant of the amplitude, duration, and compartmentation of cAMP-mediated signaling. The role of different PDE isozymes, particularly PDE3A vs PDE3B, in the regulation of heart function remains unclear.<h4>Objective</h4>To determine the relative contribution of PDE3A vs PDE3B isozymes in the regulation of heart function and to ...[more]

PMID: 23168336

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Project description:Cyclic nucleotide phosphodiesterase 3A (PDE3) regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects and that murine PDE3A1 associates with sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2), phospholamban (PLB), and AKAP18 in a multiprotein signalosome in human sarcoplasmic reticulum (SR). Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB, and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct high molecular weight (HMW) and low molecular weight (LMW) peaks. HMW peaks contained PDE3A1 and PDE3A2, whereas LMW peaks contained PDE3A1, PDE3A2, and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immunoprecipitation of SERCA2, cav3, PKA regulatory subunit (PKARII), PP2A, and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of recombinant human PDE3A isoforms by recombinant PKA catalytic subunit increased co-immunoprecipitation with rSERCA2 and rat rAKAP18 (recombinant AKAP18). Deletion of the recombinant human PDE3A1/PDE3A2 N terminus blocked interactions with recombinant SERCA2. Serine-to-alanine substitutions identified Ser-292/Ser-293, a site unique to human PDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of human PDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation, and SERCA2 activity.

Dataset Information

Phosphodiesterase type 3A regulates basal myocardial contractility through interacting with sarcoplasmic reticulum calcium ATPase type 2a signaling complexes in mouse heart.

Publications

Phosphodiesterase type 3A regulates basal myocardial contractility through interacting with sarcoplasmic reticulum calcium ATPase type 2a signaling complexes in mouse heart.

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