Titin-based passive stiffness is post-translationally regulated by several
kinases that phosphorylate specific spring elements located within
titin's elastic I-band region. Whether
titin is phosphorylated by
calcium/calmodulin dependent protein kinase II (
CaMKII), an important regulator of cardiac function and disease, has not been addressed. The aim of this work was to determine whether CaMKIIδ, the predominant
CaMKII isoform in the heart, phosphorylates
titin, and to use phosphorylation assays and mass spectrometry to study which of
titin's spring elements might be targeted by CaMKIIδ. It was found that CaMKIIδ phosphorylates
titin in mouse LV skinned fibers, that the CaMKIIδ sites can be dephosphorylated by
protein phosphatase 1 (PP1), and that under baseline conditions, in both intact isolated hearts and skinned myocardium, about half of the CaMKIIδ sites are phosphorylated. Mass spectrometry revealed that both the N2B and PEVK segments are targeted by CaMKIIδ at several conserved
serine residues. Whether phosphorylation of
titin by CaMKIIδ occurs in vivo, was tested in several conditions using back phosphorylation assays and
phospho-specific antibodies to CaMKIIδ sites. Reperfusion following global
ischemia increased the phosphorylation level of CaMKIIδ sites on
titin and this effect was abolished by the
CaMKII inhibitor
KN-93. No changes in the phosphorylation level of the PEVK
element were found suggesting that the increased phosphorylation level of
titin in IR (
ischemia reperfusion) might be due to phosphorylation of the N2B
element. The findings of these studies show for the first time that
titin can be phosphoryalated by CaMKIIδ, both in vitro and in vivo, and that
titin's molecular spring region that determines diastolic stiffness is a target of CaMKIIδ.