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

Nazha Hamdani, Judith Krysiak, Michael M. Kreusser, Stefan Neef, Cristobal G. Dos Remedios, Lars S. Maier, Markus Krüger, Johannes Backs, Wolfgang A. Linke

Research output: Contribution to journalArticlepeer-review

154 Scopus citations

Abstract

Rationale: Myocardial diastolic stiffness and cardiomyocyte passive force (Fpassive) depend in part on titin isoform composition and phosphorylation. Ca/calmodulin-dependent protein kinase-II (CaMKII) phosphorylates ion channels, Ca-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 Fpassive 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 s 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 Fpassive 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.(Table is included in full-text article.) Conclusions: CaMKII phosphorylates the titin springs at conserved serines/threonines, thereby lowering Fpassive. Deranged CaMKII-dependent titin phosphorylation occurs in heart failure and contributes to altered diastolic stress.

Original languageEnglish
Pages (from-to)664-674
Number of pages11
JournalCirculation Research
Volume112
Issue number4
DOIs
StatePublished - 15 Feb 2013
Externally publishedYes

Keywords

  • Ca/calmodulin-dependent protein kinase-II
  • diastolic function
  • passive stiffness
  • phosphoproteomics
  • titin

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