Dataset Information

Effect of troponin I Ser23/24 phosphorylation on Ca2+-sensitivity in human myocardium depends on the phosphorylation background.

ABSTRACT: Protein kinase A (PKA)-mediated phosphorylation of Ser23/24 of cardiac troponin I (cTnI) causes a reduction in Ca(2+)-sensitivity of force development. This study aimed to determine whether the PKA-induced modulation of the Ca(2+)-sensitivity is solely due to cTnI phosphorylation or depends on the phosphorylation status of other sarcomeric proteins. Endogenous troponin (cTn) complex in donor cardiomyocytes was partially exchanged (up to 66+/-1%) with recombinant unphosphorylated human cTn and in failing cells similar exchange was achieved using PKA-(bis)phosphorylated cTn complex. Cardiomyocytes immersed in exchange solution without complex added served as controls. Partial exchange of unphosphorylated cTn complex in donor tissue significantly increased Ca(2+)-sensitivity (pCa(50)) to 5.50+/-0.02 relative to the donor control value (pCa(50)=5.43+/-0.04). Exchange in failing tissue with PKA-phosphorylated cTn complex did not change Ca(2+)-sensitivity relative to the failing control (pCa(50)=5.60+/-0.02). Subsequent treatment of the cardiomyocytes with the catalytic subunit of PKA significantly decreased Ca(2+)-sensitivity in donor and failing tissue. Analysis of phosphorylated cTnI species revealed the same distribution of un-, mono- and bis-phosphorylated cTnI in donor control and in failing tissue exchanged with PKA-phosphorylated cTn complex. Phosphorylation of myosin-binding protein-C in failing tissue was significantly lower compared to donor tissue. These differences in Ca(2+)-sensitivity in donor and failing cells, despite similar distribution of cTnI species, could be abolished by subsequent PKA-treatment and indicate that other targets of PKA are involved the reduction of Ca(2+)-sensitivity. Our findings suggest that the sarcomeric phosphorylation background, which is altered in cardiac disease, influences the impact of cTnI Ser23/24 phosphorylation by PKA on Ca(2+)-sensitivity.

SUBMITTER: Kooij V

PROVIDER: S-EPMC2854313 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Effect of troponin I Ser23/24 phosphorylation on Ca2+-sensitivity in human myocardium depends on the phosphorylation background.

Kooij Viola V Saes Martina M Jaquet Kornelia K Zaremba Ruud R Foster D Brian DB Murphy Anne M AM Dos Remedios Cris C van der Velden Jolanda J Stienen Ger J M GJ

Journal of molecular and cellular cardiology 20100115 5

Protein kinase A (PKA)-mediated phosphorylation of Ser23/24 of cardiac troponin I (cTnI) causes a reduction in Ca(2+)-sensitivity of force development. This study aimed to determine whether the PKA-induced modulation of the Ca(2+)-sensitivity is solely due to cTnI phosphorylation or depends on the phosphorylation status of other sarcomeric proteins. Endogenous troponin (cTn) complex in donor cardiomyocytes was partially exchanged (up to 66+/-1%) with recombinant unphosphorylated human cTn and in ...[more]

PMID: 20079747

Similar Datasets

Project description:AimsProtein kinase C? (PKC?) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKC? is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKC? phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.Methods and resultsEndogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (?69%) with PKC?-treated recombinant human cTn (cTn (DD+PKC?)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKC?. Isometric force was measured at various [Ca(2+)] after exchange. The maximal force (Fmax) in the cTn (DD+PKC?) group (17.1±1.9 kN/m(2)) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m(2)). Exchange of endogenous cTn with cTn (DD+PKC?) increased Ca(2+)-sensitivity of force (pCa50?=?5.59±0.02) compared to cTn (DD) (pCa50?=?5.51±0.02). In contrast, subsequent PKC? treatment of the cells exchanged with cTn (DD+PKC?) reduced pCa50 to 5.45±0.02. Two PKC?-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKC? and showed the highest phosphorylation increase on Thr143.ConclusionPKC?-mediated phosphorylation of the cTn complex decreases Fmax and increases myofilament Ca(2+)-sensitivity, while subsequent treatment with PKC? in situ decreased myofilament Ca(2+)-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKC? are phosphorylated in human cTn complex treated with PKC? with a high degree of specificity for Thr143.

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

Effect of troponin I Ser23/24 phosphorylation on Ca2+-sensitivity in human myocardium depends on the phosphorylation background.

Publications

Effect of troponin I Ser23/24 phosphorylation on Ca2+-sensitivity in human myocardium depends on the phosphorylation background.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets