Dataset Information

Characterization of the cardiac myosin binding protein-C phosphoproteome in healthy and failing human hearts.

ABSTRACT: INTRODUCTION:Cardiac myosin binding protein-C (cMyBP-C) becomes dephosphorylated in the failing heart and reduced phosphorylation-dependent regulation of cMyBP-C has been implicated in contractile dysfunction. To date, several phosphorylation sites have been identified for human cMyBP-C; however, a comprehensive characterization of the cMyBP-C phosphoproteome is lacking. This study aimed to characterize the cMyBP-C phosphoproteome using two different proteomic-based methods in explanted donor and end-stage failing hearts. METHODS:The first approach used to characterize the cMyBP-C phosphoproteome employed a strong-cation exchange chromatography (SCX) fractionation method (10 pooled samples, technical replicates=4) and the second employed a sodium dodecylsulfate polyacrylamide gel electrophoresis method (n=10; technical replicates=2). Each subsequently underwent titanium dioxide (TiO2) affinity chromatography to enrich for the tryptic phosphopeptides, which were analyzed using an LTQ-Orbitrap mass spectrometer. Moreover, recombinant C0-C2 fragment of mouse cMyBP-C incubated with PKA, PKC, CaMKII and CK2 was analyzed to identify the kinases involved with phosphorylation of cMyBP-C. RESULTS:Seventeen phosphorylation sites on cMyBP-C were identified in vivo, with the majority localized in the N-terminal domains C0-C2. The three most abundant phosphorylated sites, Ser284, Ser286 and Thr290, are located in the regulatory M-domain of cMyBP-C. Ser284 showed a significant reduction in phosphorylation in HF. CONCLUSION:This study demonstrates that cMyBP-C harbors more phosphorylation sites than previously known, with a total of 17 (9 novel) identified phosphorylation sites in vivo. Most sites were primarily located within the N-terminal side of the protein. The most highly phosphorylated site on cMyBP-C was Ser284 and this site showed decreased phosphorylation in the failing heart, which implicates importance for fine-tuning contractility. To date, the functional importance of Ser286 and Thr290 is unknown. In addition, 16 sites were identified after in vitro kinase incubation. The data have been deposited to the ProteomeXchange with identifier PXD000158.

SUBMITTER: Kooij V

PROVIDER: S-EPMC3710717 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Characterization of the cardiac myosin binding protein-C phosphoproteome in healthy and failing human hearts.

Kooij Viola V Holewinski Ronald J RJ Murphy Anne M AM Van Eyk Jennifer E JE

Journal of molecular and cellular cardiology 20130422

<h4>Introduction</h4>Cardiac myosin binding protein-C (cMyBP-C) becomes dephosphorylated in the failing heart and reduced phosphorylation-dependent regulation of cMyBP-C has been implicated in contractile dysfunction. To date, several phosphorylation sites have been identified for human cMyBP-C; however, a comprehensive characterization of the cMyBP-C phosphoproteome is lacking. This study aimed to characterize the cMyBP-C phosphoproteome using two different proteomic-based methods in explanted ...[more]

PMID: 23619294

Similar Datasets

Project description:Introduction: Cardiac myosin binding protein-C (cMyBP-C) becomes dephosphorylated in the failing heart and reduced phosphorylation-dependent regulation of cMyBP-C has been implicated in contractile dysfunction. To date, several phosphorylation sites have been identified for human cMyBP-C; however, a comprehensive characterization of the cMyBP-C phosphoproteome is lacking. This study aimed to characterize the cMyBP-C phosphoproteome using two different proteomic-based methods in explanted control and end-stage failing hearts. Methods: The first approach used to characterize the cMyBP-C phosphoproteome employed a strong-cation exchange chromatography (SCX)-based fractionation method (10 pooled samples, technical replicates = 4) and the second employed a sodium dodecylsulfate polyacrylamide gel electrophoresis method (n = 10; technical replicates = 2). Each subsequently underwent titanium dioxide (TiO2) affinity chromatography to enrich for the tryptic phosphopeptides, which were analyzed using an LTQ-Orbitrap mass spectrometer. Moreover, recombinant C0-C2 fragment of mouse cMyBP-C incubated with PKA, PKC, CamKII and CK2 was analyzed to identify the kinases involved with phosphorylation of cMyBP-C. Results: Seventeen phosphorylation sites on cMyBP-C were identified, with the majority localized in the N-terminal domains C0-C2. The three most abundant phosphorylated sites, Ser284, Ser286 and Thr290, are located in the regulatory M-domain of cMyBP-C. Ser284 showed a significant reduction in phosphorylation in HF, while Ser286 and Thr290 showed a trend towards a reduced phosphorylation. Conclusion: This study demonstrates that cMyBP-C is more extensively phosphorylated than previously known, with 10 novel sites identified. Most sites were primarily located within the N-terminal side of the protein. The three most highly phosphorylated sites on cMyBP-C were Ser284, Ser286 and Thr290 and these three sites showed decreased phosphorylation in the failing heart, which implicate their importance for fine-tuning contractility. To date, the functional importance of Ser286 and Thr290 is unknown. In addition, 16 sites were identified after in vitro kinase incubation. Bioinformatics pipeline: All RAW files were searched using the Sorcerer 2 Sequest algorithm. Human samples were searched against the IPI human database version 3.79 and mouse samples were searched against the IPI mouse database version 3.80. All searches were carried out with static modification of +57 on C and differential modifications of +16 on M and +80 on S,T,Y. Parent mass tolerance was set to 50 ppm and fragment mass tolerance was set to 1 Da. Post-search analysis was performed using Scaffold version 3.2.0 and site localization was determined using Scaffold PTM version 1.1.2 (Proteome Software, Inc., Portland, OR, USA).

Project description:A common denominator for patients with heart failure is the correlation between elevated serum levels of proinflammatory cytokines and adverse clinical outcomes. Furthermore, lipoxygenase-induced inflammation is reportedly involved in the pathology of heart failure. Cardiac fibroblasts, which are abundant in cardiac tissue, are known to be activated by inflammation. We previously showed high expression of the lipoxygenase arachidonate 15 lipoxygenase (ALOX15), which catalyzes the conversion of arachidonic acid to 15-hydroxy eicosatetraenoic acid (15-HETE), in ischemic cardiac tissue. The exact roles of ALOX15 and 15-HETE in the pathogenesis of heart failure are however unknown. Biopsies were collected from all chambers of explanted failing human hearts from heart transplantation patients, as well as from the left ventricles from organ donors not suffering from chronic heart failure. Biopsies from the left ventricles underwent quantitative immunohistochemical analysis for ALOX15/B. Gene expression of ALOX enzymes, as well as 15-HETE levels, were examined in cardiac fibroblasts which had been cultured in either hypoxic or normoxic conditions after isolation from failing hearts. After the addition of fibroblast supernatants to human induced pluripotent stem cell-derived cardiomyocytes, intracellular calcium concentrations were measured to examine the effect of paracrine signaling on cardiomyocyte beating frequency. While ALOX15 and ALOX15B were expressed throughout failing hearts as well as in hearts from organ donors, ALOX15 was expressed at significantly higher levels in donor hearts. Hypoxia resulted in a significant increase in gene and protein expression of ALOX15 and ALOX15B in fibroblasts isolated from the different chambers of failing hearts. Finally, preconditioned medium from hypoxic fibroblasts decreased the beating frequency of human cardiomyocytes derived from induced pluripotent stem cells in an ALOX15-dependent manner. In summary, our results demonstrate that ALOX15/B signaling by hypoxic cardiac fibroblasts may play an important role in ischemic cardiomyopathy, by decreasing cardiomyocyte beating frequency.

Dataset Information

Characterization of the cardiac myosin binding protein-C phosphoproteome in healthy and failing human hearts.

Publications

Characterization of the cardiac myosin binding protein-C phosphoproteome in healthy and failing human hearts.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets