A novel phosphorylation site, Serine 199, in the C-terminus of cardiac troponin I regulates calcium sensitivity and susceptibility to calpain-induced proteolysis

P.J.M. Wijnker, Y. Li, P. Zhang, D.B. Foster, C. dos Remedios, J.E. van Eyk, G.J.M. Stienen, A.M. Murphy, J. van der Velden

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium. The functional consequences of cTnI-Ser199 phosphotylation in the heart are unknown. Therefore, we investigated the impact of phosphorylation of cTnI-Ser199 on myofilament function in human cardiac tissue and the susceptibility of cTnI to proteolysis. cTnI-Serl 99 was replaced by aspartic acid (1990) or alanine (199A) to mimic phosphorylation and dephosphorylation, respectively, with recombinant wild-type (Wt) cTn as a negative control. Force development was measured at various [Ca2+] and at sarcomere lengths of 1.8 and 2.2 mu m in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca2+-sensitivity (pCa(50)) at 2.2 pm was significantly higher in 199D (pCa(50) = 5.79 +/- 0.01) compared to 199A (pCa(50) = 5.65 +/- 0.01) and Wt (pCa(50) = 5.66 +/- 0.02) at -63% cTn exchange. Myofilament Ca2+-sensitivity was significantly higher even with only 5.9 +/- 2.5% 199D exchange compared to 199A, and saturated at 123 +/- 2.6%199D exchange. Ser199 pseudo-phosphorylation decreased cTnI binding to both actin and actin-tropomyosin. Moreover, altered susceptibility of cTnI to proteolysis by calpain I was found when Ser199 was pseudo-phosphorylated. Our data demonstrate that low levels of cTnI-Ser199 pseudo-phosphorylation (similar to 6%) increase myofilament Ca2+-sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudophosphorylation or mutation regulates calpain I mediated proteolysis of cTnI. (C) 2015 Elsevier Ltd. All rights reserved.
Original languageEnglish
Pages (from-to)93-103
JournalJournal of Molecular and Cellular Cardiology
Volume82
DOIs
Publication statusPublished - 2015

Cite this

@article{c2adab854fc945f8ba06bf15b53fdc50,
title = "A novel phosphorylation site, Serine 199, in the C-terminus of cardiac troponin I regulates calcium sensitivity and susceptibility to calpain-induced proteolysis",
abstract = "Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium. The functional consequences of cTnI-Ser199 phosphotylation in the heart are unknown. Therefore, we investigated the impact of phosphorylation of cTnI-Ser199 on myofilament function in human cardiac tissue and the susceptibility of cTnI to proteolysis. cTnI-Serl 99 was replaced by aspartic acid (1990) or alanine (199A) to mimic phosphorylation and dephosphorylation, respectively, with recombinant wild-type (Wt) cTn as a negative control. Force development was measured at various [Ca2+] and at sarcomere lengths of 1.8 and 2.2 mu m in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca2+-sensitivity (pCa(50)) at 2.2 pm was significantly higher in 199D (pCa(50) = 5.79 +/- 0.01) compared to 199A (pCa(50) = 5.65 +/- 0.01) and Wt (pCa(50) = 5.66 +/- 0.02) at -63{\%} cTn exchange. Myofilament Ca2+-sensitivity was significantly higher even with only 5.9 +/- 2.5{\%} 199D exchange compared to 199A, and saturated at 123 +/- 2.6{\%}199D exchange. Ser199 pseudo-phosphorylation decreased cTnI binding to both actin and actin-tropomyosin. Moreover, altered susceptibility of cTnI to proteolysis by calpain I was found when Ser199 was pseudo-phosphorylated. Our data demonstrate that low levels of cTnI-Ser199 pseudo-phosphorylation (similar to 6{\%}) increase myofilament Ca2+-sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudophosphorylation or mutation regulates calpain I mediated proteolysis of cTnI. (C) 2015 Elsevier Ltd. All rights reserved.",
author = "P.J.M. Wijnker and Y. Li and P. Zhang and D.B. Foster and {dos Remedios}, C. and {van Eyk}, J.E. and G.J.M. Stienen and A.M. Murphy and {van der Velden}, J.",
year = "2015",
doi = "10.1016/j.yjmcc.2015.03.006",
language = "English",
volume = "82",
pages = "93--103",
journal = "Journal of Molecular and Cellular Cardiology",
issn = "0022-2828",
publisher = "Academic Press Inc.",

}

A novel phosphorylation site, Serine 199, in the C-terminus of cardiac troponin I regulates calcium sensitivity and susceptibility to calpain-induced proteolysis. / Wijnker, P.J.M.; Li, Y.; Zhang, P.; Foster, D.B.; dos Remedios, C.; van Eyk, J.E.; Stienen, G.J.M.; Murphy, A.M.; van der Velden, J.

In: Journal of Molecular and Cellular Cardiology, Vol. 82, 2015, p. 93-103.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - A novel phosphorylation site, Serine 199, in the C-terminus of cardiac troponin I regulates calcium sensitivity and susceptibility to calpain-induced proteolysis

AU - Wijnker, P.J.M.

AU - Li, Y.

AU - Zhang, P.

AU - Foster, D.B.

AU - dos Remedios, C.

AU - van Eyk, J.E.

AU - Stienen, G.J.M.

AU - Murphy, A.M.

AU - van der Velden, J.

PY - 2015

Y1 - 2015

N2 - Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium. The functional consequences of cTnI-Ser199 phosphotylation in the heart are unknown. Therefore, we investigated the impact of phosphorylation of cTnI-Ser199 on myofilament function in human cardiac tissue and the susceptibility of cTnI to proteolysis. cTnI-Serl 99 was replaced by aspartic acid (1990) or alanine (199A) to mimic phosphorylation and dephosphorylation, respectively, with recombinant wild-type (Wt) cTn as a negative control. Force development was measured at various [Ca2+] and at sarcomere lengths of 1.8 and 2.2 mu m in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca2+-sensitivity (pCa(50)) at 2.2 pm was significantly higher in 199D (pCa(50) = 5.79 +/- 0.01) compared to 199A (pCa(50) = 5.65 +/- 0.01) and Wt (pCa(50) = 5.66 +/- 0.02) at -63% cTn exchange. Myofilament Ca2+-sensitivity was significantly higher even with only 5.9 +/- 2.5% 199D exchange compared to 199A, and saturated at 123 +/- 2.6%199D exchange. Ser199 pseudo-phosphorylation decreased cTnI binding to both actin and actin-tropomyosin. Moreover, altered susceptibility of cTnI to proteolysis by calpain I was found when Ser199 was pseudo-phosphorylated. Our data demonstrate that low levels of cTnI-Ser199 pseudo-phosphorylation (similar to 6%) increase myofilament Ca2+-sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudophosphorylation or mutation regulates calpain I mediated proteolysis of cTnI. (C) 2015 Elsevier Ltd. All rights reserved.

AB - Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium. The functional consequences of cTnI-Ser199 phosphotylation in the heart are unknown. Therefore, we investigated the impact of phosphorylation of cTnI-Ser199 on myofilament function in human cardiac tissue and the susceptibility of cTnI to proteolysis. cTnI-Serl 99 was replaced by aspartic acid (1990) or alanine (199A) to mimic phosphorylation and dephosphorylation, respectively, with recombinant wild-type (Wt) cTn as a negative control. Force development was measured at various [Ca2+] and at sarcomere lengths of 1.8 and 2.2 mu m in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca2+-sensitivity (pCa(50)) at 2.2 pm was significantly higher in 199D (pCa(50) = 5.79 +/- 0.01) compared to 199A (pCa(50) = 5.65 +/- 0.01) and Wt (pCa(50) = 5.66 +/- 0.02) at -63% cTn exchange. Myofilament Ca2+-sensitivity was significantly higher even with only 5.9 +/- 2.5% 199D exchange compared to 199A, and saturated at 123 +/- 2.6%199D exchange. Ser199 pseudo-phosphorylation decreased cTnI binding to both actin and actin-tropomyosin. Moreover, altered susceptibility of cTnI to proteolysis by calpain I was found when Ser199 was pseudo-phosphorylated. Our data demonstrate that low levels of cTnI-Ser199 pseudo-phosphorylation (similar to 6%) increase myofilament Ca2+-sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudophosphorylation or mutation regulates calpain I mediated proteolysis of cTnI. (C) 2015 Elsevier Ltd. All rights reserved.

U2 - 10.1016/j.yjmcc.2015.03.006

DO - 10.1016/j.yjmcc.2015.03.006

M3 - Article

VL - 82

SP - 93

EP - 103

JO - Journal of Molecular and Cellular Cardiology

JF - Journal of Molecular and Cellular Cardiology

SN - 0022-2828

ER -