PKCα-Specific Phosphorylation of the Troponin Complex in Human Myocardium: A Functional and Proteomics Analysis

Viola Kooij; Pingbo Zhang; Sander R. Piersma; Vasco Sequeira; Nicky M. Boontje; Paul J.M. Wijnker; Connie R. Jiménez; Kornelia E. Jaquet; Cris dos Remedios; Anne M. Murphy; Jennifer E. Van Eyk; Jolanda van der Velden; Ger JM Stienen

doi:10.1371/journal.pone.0074847

PKCα-Specific Phosphorylation of the Troponin Complex in Human Myocardium: A Functional and Proteomics Analysis

Viola Kooij, Pingbo Zhang, Sander R. Piersma, Vasco Sequeira, Nicky M. Boontje, Paul J.M. Wijnker, Connie R. Jiménez, Kornelia E. Jaquet, Cris dos Remedios, Anne M. Murphy, Jennifer E. Van Eyk, Jolanda van der Velden, Ger JM Stienen

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Abstract

Aims:Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα 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 PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.Methods and Results:Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca²⁺] after exchange. The maximal force (F_max) in the cTn (DD+PKCα) group (17.1±1.9 kN/m²) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m²). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca²⁺-sensitivity of force (pCa₅₀ = 5.59±0.02) compared to cTn (DD) (pCa₅₀ = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa₅₀ to 5.45±0.02. Two PKCα-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 PKCα and showed the highest phosphorylation increase on Thr143.Conclusion:PKCα-mediated phosphorylation of the cTn complex decreases F_max and increases myofilament Ca²⁺-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca²⁺-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.

Original language	English (US)
Article number	e74847
Journal	PloS one
Volume	8
Issue number	10
DOIs	https://doi.org/10.1371/journal.pone.0074847
State	Published - Oct 7 2013
Externally published	Yes

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Kooij, V., Zhang, P., Piersma, S. R., Sequeira, V., Boontje, N. M., Wijnker, P. J. M., Jiménez, C. R., Jaquet, K. E., dos Remedios, C., Murphy, A. M., Van Eyk, J. E., van der Velden, J., & Stienen, G. JM. (2013). PKCα-Specific Phosphorylation of the Troponin Complex in Human Myocardium: A Functional and Proteomics Analysis. PloS one, 8(10), Article e74847. https://doi.org/10.1371/journal.pone.0074847

Kooij, V, Zhang, P, Piersma, SR, Sequeira, V, Boontje, NM, Wijnker, PJM, Jiménez, CR, Jaquet, KE, dos Remedios, C, Murphy, AM, Van Eyk, JE, van der Velden, J & Stienen, GJM 2013, 'PKCα-Specific Phosphorylation of the Troponin Complex in Human Myocardium: A Functional and Proteomics Analysis', PloS one, vol. 8, no. 10, e74847. https://doi.org/10.1371/journal.pone.0074847

@article{ce2bc8928f6d42d1a13ef76b03572a60,

title = "PKCα-Specific Phosphorylation of the Troponin Complex in Human Myocardium: A Functional and Proteomics Analysis",

abstract = "Aims:Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα 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 PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.Methods and Results:Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca2+] after exchange. The maximal force (Fmax) in the cTn (DD+PKCα) group (17.1±1.9 kN/m2) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m2). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca2+-sensitivity of force (pCa50 = 5.59±0.02) compared to cTn (DD) (pCa50 = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa50 to 5.45±0.02. Two PKCα-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 PKCα and showed the highest phosphorylation increase on Thr143.Conclusion:PKCα-mediated phosphorylation of the cTn complex decreases Fmax and increases myofilament Ca2+-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca2+-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.",

author = "Viola Kooij and Pingbo Zhang and Piersma, {Sander R.} and Vasco Sequeira and Boontje, {Nicky M.} and Wijnker, {Paul J.M.} and Jim{\'e}nez, {Connie R.} and Jaquet, {Kornelia E.} and {dos Remedios}, Cris and Murphy, {Anne M.} and {Van Eyk}, {Jennifer E.} and {van der Velden}, Jolanda and Stienen, {Ger JM}",

year = "2013",

month = oct,

day = "7",

doi = "10.1371/journal.pone.0074847",

language = "English (US)",

volume = "8",

journal = "PloS one",

issn = "1932-6203",

publisher = "Public Library of Science",

number = "10",

}

TY - JOUR

T1 - PKCα-Specific Phosphorylation of the Troponin Complex in Human Myocardium

T2 - A Functional and Proteomics Analysis

AU - Kooij, Viola

AU - Zhang, Pingbo

AU - Piersma, Sander R.

AU - Sequeira, Vasco

AU - Boontje, Nicky M.

AU - Wijnker, Paul J.M.

AU - Jiménez, Connie R.

AU - Jaquet, Kornelia E.

AU - dos Remedios, Cris

AU - Murphy, Anne M.

AU - Van Eyk, Jennifer E.

AU - van der Velden, Jolanda

AU - Stienen, Ger JM

PY - 2013/10/7

Y1 - 2013/10/7

N2 - Aims:Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα 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 PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.Methods and Results:Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca2+] after exchange. The maximal force (Fmax) in the cTn (DD+PKCα) group (17.1±1.9 kN/m2) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m2). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca2+-sensitivity of force (pCa50 = 5.59±0.02) compared to cTn (DD) (pCa50 = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa50 to 5.45±0.02. Two PKCα-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 PKCα and showed the highest phosphorylation increase on Thr143.Conclusion:PKCα-mediated phosphorylation of the cTn complex decreases Fmax and increases myofilament Ca2+-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca2+-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.

AB - Aims:Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα 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 PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.Methods and Results:Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca2+] after exchange. The maximal force (Fmax) in the cTn (DD+PKCα) group (17.1±1.9 kN/m2) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m2). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca2+-sensitivity of force (pCa50 = 5.59±0.02) compared to cTn (DD) (pCa50 = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa50 to 5.45±0.02. Two PKCα-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 PKCα and showed the highest phosphorylation increase on Thr143.Conclusion:PKCα-mediated phosphorylation of the cTn complex decreases Fmax and increases myofilament Ca2+-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca2+-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.

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DO - 10.1371/journal.pone.0074847

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VL - 8

JO - PloS one

JF - PloS one

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PKCα-Specific Phosphorylation of the Troponin Complex in Human Myocardium: A Functional and Proteomics Analysis

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