Crucial Role for Ca2+/Calmodulin-Dependent Protein Kinase-II Delta in Regulating Diastolic Stress of Normal and Failing Hearts via Titin Phosphorylation
Copyright policy )Crucial Role for Ca2+/Calmodulin-Dependent Protein Kinase-II Delta in Regulating Diastolic Stress of Normal and Failing Hearts via Titin Phosphorylation
Rationale: Myocardial diastolic stiffness and cardiomyocyte passive force (F passive ) depend in part on titin isoform composition and phosphorylation. Ca 2+ /calmodulin-dependent protein kinase-II (CaMKII) phosphorylates ion channels, Ca 2+ -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 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 table 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 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/titin N2B-unique sequence 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.
- Max Planck Society Germany
- Heidelberg University Germany
- Amsterdam UMC Netherlands
- University of Sydney Australia
- Ruhr University Bochum Germany
Heart Failure, Mice, Knockout, Molecular Sequence Data, Biophysics, Muscle Proteins, Mice, Transgenic, Biomechanical Phenomena, Mice, Phosphoserine, Phosphothreonine, Diastole, Animals, Humans, Connectin, Myocytes, Cardiac, Amino Acid Sequence, Phosphorylation, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Protein Kinases, Cells, Cultured, Compliance
Heart Failure, Mice, Knockout, Molecular Sequence Data, Biophysics, Muscle Proteins, Mice, Transgenic, Biomechanical Phenomena, Mice, Phosphoserine, Phosphothreonine, Diastole, Animals, Humans, Connectin, Myocytes, Cardiac, Amino Acid Sequence, Phosphorylation, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Protein Kinases, Cells, Cultured, Compliance
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