Rationale:

Myocardial diastolic stiffness and cardiomyocyte passive force (F_passive) depend in part on titin isoform composition and phosphorylation. Ca²⁺/calmodulin-dependent protein kinase-II (CaMKII) phosphorylates ion channels, Ca²⁺-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 CaMKIId/g double-knockout (DKO) mouse, transgenic CaMKIIdC-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. 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 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. F_passive was elevated in DKO and reduced in TG compared to 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/N2Bus 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.