Crucial Role for Ca2+/Calmodulin-Dependent Protein Kinase-II in Regulating Diastolic Stress of Normal and Failing Hearts via Titin Phosphorylation
Rationale: Myocardial diastolic stiffness and cardiomyocyte passive force (Fpassive) depend in part on titin isoform composition and phosphorylation. Ca2+/calmodulin-dependent protein kinase-II (CaMKII) phosphorylates ion channels, Ca22+-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 (TG) 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 SILAC mouse heart mixed with wildtype (WT) or DKO heart. Fpassive 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 TG versus WT hearts. Conserved CaMKII-dependent phosphosites were identified within titin's PEVK-domain by quantitative mass spectrometry and confirmed in recombinant human PEVK-fragments. CaMKII also phosphorylated the cardiac titin N2B-unique sequence (N2Bus). Phosphorylation at specific PEVK/N2Bus sites was decreased in DKO and amplified in TG versus WT hearts. Fpassive was elevated in DKO and reduced in TG compared to 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/N2Bus sites than non-failing donor hearts.
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.
- Received September 17, 2012.
- Revision received December 23, 2012.
- Accepted December 31, 2012.
- Copyright © 2013, Circulation Research