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Circ Res. 2013 Feb 15;112(4):664-74. doi: 10.1161/CIRCRESAHA.111.300105. Epub 2013 Jan 2.

Crucial role for Ca2(+)/calmodulin-dependent protein kinase-II in regulating diastolic stress of normal and failing hearts via titin phosphorylation.

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  • 1Department of Cardiovascular Physiology, Ruhr University Bochum, Bochum, Germany.

Abstract

RATIONALE:

Myocardial diastolic stiffness and cardiomyocyte passive force (F(passive)) depend in part on titin isoform composition and phosphorylation. Ca(2+)/calmodulin-dependent protein kinase-II (CaMKII) phosphorylates ion channels, Ca(2+)-handling proteins, and chromatin-modifying enzymes in the heart, but has not been known to target titin.

OBJECTIVE:

To elucidate whether CaMKII phosphorylates titin and modulates F(passive) in normal and failing myocardium.

METHODS AND RESULTS:

Titin phosphorylation was assessed in CaMKIIδ/γ double-knockout (DKO) mouse, transgenic CaMKIIδC-overexpressing mouse, and human hearts, by Pro-Q-Diamond/Sypro-Ruby staining, autoradiography, and immunoblotting using phosphoserine-specific titin-antibodies. CaMKII-dependent site-specific titin phosphorylation was quantified in vivo by mass spectrometry using stable isotope labeling by amino acids in cell culture mouse heart mixed with wild-type (WT) or DKO heart. F(passive) of single permeabilized cardiomyocytes was recorded before and after CaMKII-administration. All-titin phosphorylation was reduced by >50% in DKO but increased by up to ≈100% in transgenic versus WT hearts. Conserved CaMKII-dependent phosphosites were identified within the PEVK-domain of titin by quantitative mass spectrometry and confirmed in recombinant human PEVK-fragments. CaMKII also phosphorylated the cardiac titin N2B-unique sequence. Phosphorylation at specific PEVK/titin N2B-unique sequence sites was decreased in DKO and amplified in transgenic versus WT hearts. F(passive) was elevated in DKO and reduced in transgenic compared with WT cardiomyocytes. CaMKII-administration lowered F(passive) of WT and DKO cardiomyocytes, an effect blunted by titin antibody pretreatment. Human end-stage failing hearts revealed higher CaMKII expression/activity and phosphorylation at PEVK/titin N2B-unique sequence sites than nonfailing donor hearts.

CONCLUSIONS:

CaMKII phosphorylates the titin springs at conserved serines/threonines, thereby lowering F(passive). Deranged CaMKII-dependent titin phosphorylation occurs in heart failure and contributes to altered diastolic stress.

PMID:
23283722
[PubMed - indexed for MEDLINE]
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