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J Mol Cell Cardiol. 2015 May;82:93-103. doi: 10.1016/j.yjmcc.2015.03.006. Epub 2015 Mar 11.

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

Author information

1
Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands; Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: p.wijnker@vumc.nl.
2
Department of Pediatrics, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, USA.
3
Muscle Research Unit, Bosch Institute, The University of Sydney, Sydney, Australia.
4
The Advanced Clinical Biosystems Research Institute, The Heart Institute, Department of Medicine, Cedars Sinai Medical Center, Los Angeles, CA, USA.
5
Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands; Department of Physics and Astronomy, VU University, Amsterdam, The Netherlands.
6
Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands; ICIN-Netherlands Heart Institute, Utrecht, The Netherlands.

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 phosphorylation 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-Ser199 was replaced by aspartic acid (199D) 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 [Ca(2+)] and at sarcomere lengths of 1.8 and 2.2 μm in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca(2+)-sensitivity (pCa50) at 2.2 μm was significantly higher in 199D (pCa50 = 5.79 ± 0.01) compared to 199A (pCa50 = 5.65 ± 0.01) and Wt (pCa50 = 5.66 ± 0.02) at ~63% cTn exchange. Myofilament Ca(2+)-sensitivity was significantly higher even with only 5.9 ± 2.5% 199D exchange compared to 199A, and saturated at 12.3 ± 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 (~6%) increase myofilament Ca(2+)-sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudo-phosphorylation or mutation regulates calpain I mediated proteolysis of cTnI.

KEYWORDS:

Cardiac troponin I; Cardiomyocyte; Heart failure; Myofilament function; Protein phosphorylation; Proteolysis

PMID:
25771144
PMCID:
PMC4405491
DOI:
10.1016/j.yjmcc.2015.03.006
[Indexed for MEDLINE]
Free PMC Article

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