Abstract 18454: Calcium Calmodulin Dependent Protein Kinase II delta Modulates Cardiomyocytes Passive Stiffness by Phosphorylating Titin Spring Elements

Abstract

Background: High diastolic stiffness of the failing heart can result from altered cardiomyocyte mechanical properties. Cardiomyocyte passive force (F passive ) is attributed mainly to the mechanical properties of titin, particularly isoform composition and phosphorylation. Titin can be phosphorylated by protein kinase(PK)A or PKG at the N2-Bus, which decreases F passive . However, PKCα phosphorylates titin's PEVK-region, which increases F passive . Whether other kinases relevant for cardiomyocyte function phosphorylate the titin springs has been unknown. Aim: To investigate whether Ca 2+ /calmodulin dependent protein kinase IIδ (CaMKIIδ) phosphorylates cardiac titin and affects cardiomyocyte F passive . Methods: Cardiac titin phosphorylation was assessed in CaMKIIδ/γ double knockout (DKO; n=8) and transgenic CaMKIIδC overexpressing mice (TG; n=6). A SILAC-DKO mouse was generated for quantitative proteomics in vivo . Cardiac titin separated by titin gel electrophoresis was also studied for phosphorylation sites using MudPIT mass spectrometry. Recombinant wildtype (WT) and mutant fragments of human cardiac titin were expressed and phosphorylated in vitro by CaMKIIδ. F passive of permeabilized isolated WT, DKO and TG mouse cardiomyocytes was recorded between 1.8 and 2.4 µm sarcomere length before and after CaMKIIδ administration. Results: Total titin phosphorylation was reduced in DKO and elevated in TG compared to WT hearts. CaMKIIδ treatment of cardiac tissue ex vivo increased titin phosphorylation. A CaMKII-dependent phosphosite was identified within the N2-Bus (S4062) by MudPIT and two phosphosites within the PEVK-region ( T12007 and S12022 ) by SILAC. Western blotting using phosphospecific antibodies against these sites in cardiac tissues or recombinant WT/mutant titin constructs confirmed that CaMKIIδ indeed phosphorylates these 3 sites. F passive was elevated in DKO and lowered in TG, compared to WT cardiomyocytes. Administering CaMKIIδ ex vivo lowered F passive in DKO but did not affect TG cardiomyocytes. Conclusion: CaMKIIδ phosphorylates the titin springs , thereby lowering F passive . Reducing titin-based stiffness through CaMKIIδ-dependent phosphorylation of titin N2-Bus and PEVK may benefit diastolic function.