The multifunctional Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) phosphorylates cardiac titin's spring elements

Carlos G. Hidalgo, Charles S. Chung, Chandra Saripalli, Mei Methawasin, Kirk R. Hutchinson, George Tsaprailis, Siegfried Labeit, Alicia Mattiazzi, Hendrikus "Henk" Granzier

Research output: Contribution to journalArticle

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Abstract

Titin-based passive stiffness is post-translationally regulated by several kinases that phosphorylate specific spring elements located within titin's elastic I-band region. Whether titin is phosphorylated by calcium/calmodulin dependent protein kinase II (CaMKII), an important regulator of cardiac function and disease, has not been addressed. The aim of this work was to determine whether CaMKIIδ, the predominant CaMKII isoform in the heart, phosphorylates titin, and to use phosphorylation assays and mass spectrometry to study which of titin's spring elements might be targeted by CaMKIIδ. It was found that CaMKIIδ phosphorylates titin in mouse LV skinned fibers, that the CaMKIIδ sites can be dephosphorylated by protein phosphatase 1 (PP1), and that under baseline conditions, in both intact isolated hearts and skinned myocardium, about half of the CaMKIIδ sites are phosphorylated. Mass spectrometry revealed that both the N2B and PEVK segments are targeted by CaMKIIδ at several conserved serine residues. Whether phosphorylation of titin by CaMKIIδ occurs in vivo, was tested in several conditions using back phosphorylation assays and phospho-specific antibodies to CaMKIIδ sites. Reperfusion following global ischemia increased the phosphorylation level of CaMKIIδ sites on titin and this effect was abolished by the CaMKII inhibitor KN-93. No changes in the phosphorylation level of the PEVK element were found suggesting that the increased phosphorylation level of titin in IR (ischemia reperfusion) might be due to phosphorylation of the N2B element. The findings of these studies show for the first time that titin can be phosphoryalated by CaMKIIδ, both in vitro and in vivo, and that titin's molecular spring region that determines diastolic stiffness is a target of CaMKIIδ.

Original languageEnglish (US)
Pages (from-to)90-97
Number of pages8
JournalJournal of Molecular and Cellular Cardiology
Volume54
Issue number1
DOIs
StatePublished - Jan 2013

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Connectin
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Phosphorylation
Reperfusion
Mass Spectrometry
Ischemia
Phospho-Specific Antibodies
Protein Phosphatase 1
Calcium-Calmodulin-Dependent Protein Kinases

Keywords

  • CaMKII
  • Diastolic function
  • Myofilament
  • Passive stiffness
  • Regulation
  • Titin

ASJC Scopus subject areas

  • Molecular Biology
  • Cardiology and Cardiovascular Medicine

Cite this

The multifunctional Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) phosphorylates cardiac titin's spring elements. / Hidalgo, Carlos G.; Chung, Charles S.; Saripalli, Chandra; Methawasin, Mei; Hutchinson, Kirk R.; Tsaprailis, George; Labeit, Siegfried; Mattiazzi, Alicia; Granzier, Hendrikus "Henk".

In: Journal of Molecular and Cellular Cardiology, Vol. 54, No. 1, 01.2013, p. 90-97.

Research output: Contribution to journalArticle

Hidalgo, Carlos G. ; Chung, Charles S. ; Saripalli, Chandra ; Methawasin, Mei ; Hutchinson, Kirk R. ; Tsaprailis, George ; Labeit, Siegfried ; Mattiazzi, Alicia ; Granzier, Hendrikus "Henk". / The multifunctional Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) phosphorylates cardiac titin's spring elements. In: Journal of Molecular and Cellular Cardiology. 2013 ; Vol. 54, No. 1. pp. 90-97.
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AU - Hutchinson, Kirk R.

AU - Tsaprailis, George

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