Crucial Role for Ca2+/Calmodulin-Dependent Protein Kinase-II in Regulating Diastolic Stress of Normal and Failing Hearts via Titin Phosphorylation

• Published in: Circulation research Vol. 112; no. 4; pp. 664 - 674
• Main Authors: Hamdani, Nazha, Krysiak, Judith, Kreusser, Michael M, Neef, Stefan, dos Remedios, Cristobal G, Maier, Lars S, Krüger, Markus, Backs, Johannes, Linke, Wolfgang A
• Format: Journal Article
• Language: English
• Published: United States American Heart Association, Inc 15.02.2013
• Subjects: Amino Acid Sequence; Animals; Biomechanical Phenomena; Calcium-Calmodulin-Dependent Protein Kinase Type 2 - deficiency; Calcium-Calmodulin-Dependent Protein Kinase Type 2 - genetics; Calcium-Calmodulin-Dependent Protein Kinase Type 2 - physiology; Cells, Cultured - drug effects; Cells, Cultured - metabolism; Compliance; Connectin; Diastole - physiology; Heart Failure - enzymology; Heart Failure - physiopathology; Humans; Mice; Mice, Knockout; Mice, Transgenic; Molecular Sequence Data; Muscle Proteins - metabolism; Myocytes, Cardiac - enzymology; Myocytes, Cardiac - physiology; Phosphorylation; Phosphoserine - metabolism; Phosphothreonine - metabolism; Protein Kinases - metabolism; Protein Processing, Post-Translational; Protein Structure, Tertiary; Rats; Recombinant Fusion Proteins - physiology
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/CIRCRESAHA.111.300105

RATIONALE:Myocardial diastolic stiffness and cardiomyocyte passive force (Fpassive) depend in part on titin isoform composition and phosphorylation. Ca/calmodulin-dependent protein kinase-II (CaMKII) phosphorylates ion channels, Ca-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 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 isotope labeling by amino acids in cell culture mouse heart mixed with wild-type (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 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. Fpassive was elevated in DKO and reduced in transgenic compared with 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/titin N2B-unique sequence sites than nonfailing donor hearts.(Table is included in full-text article.) 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.