Crucial Role for Ca ²⁺ /Calmodulin-Dependent Protein Kinase-II in Regulating Diastolic Stress of Normal and Failing Hearts via Titin Phosphorylation

Abstract

A lterations in diastolic myocardial mechanics, such as slowed left-ventricular relaxation and elevated left-ventricular stiffness, cause diastolic dysfunction and can lead to heart failure (HF).1 Diastolic left-ventricular stiffness is determined, among others, by the extracellular matrix and the cardiomyocytes, 2 and both components can be stiffer than normal in HF, particularly in HF with preserved ejection fraction (HFpEF). 3,4 In cardiomyocytes, passive stiffness (F passive ) is attributable largely to the giant elastic protein titin.2 The mechanical properties of titin are altered in HF, including human HF with reduced ejection fraction (HFrEF) and HFpEF. 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 sitespecific titin phosphorylation was quantified in vivo by mass spectrometry using stable 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:

The Sydney Heart Bank: improving translational research while eliminating or reducing the use of animal models of human heart disease

“…For example, Wolfgang Linke and his group work on the giant protein titin which is very sensitive to even very low levels of proteases, so they reasonably assumed that only biopsy samples were likely to be free of degradation. Thus, several years passed before it was discovered (Krüger et al 2009;Hamdani et al 2013;Koetter et al 2013) that the titin in our donors were indeed free of proteolysis. RNAs are also highly sensitive to hydrolysis, but several colleagues have used LV samples from failing and donors with good results including Kuster et al (2013), Kong et al (2010) and Huang et al (2016).…”

Renin overexpression leads to increased titin-based stiffness contributing to diastolic dysfunction in hypertensive mRen2 rats

“…We found increased phosphorylation at Ser-12742, without changes in the phosphorylation level of Ser-12884 in mRen2 hearts. PEVK Ser-12742 hyperphosphorylation was previously demonstrated in mice with transverse aortic constriction (22), in an old HTN dog model (18), as well as in human HF (17,26). Moreover, a recent translational work implicated the hyperphosphorylation of the very same site (Ser-12742) in human HFpEF (45).…”

“…*P Ͻ 0.05, significant differences between the groups (mRen2 vs. SD; unpaired comparison, nonparametric Mann-Whitney test. phosphorylated by CaMKII␦ (21), proposed to be a contributor to diastolic stiffness of the failing myocardium (17).…”

Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

“…Site-directed mutagenesis was performed using a two-step PCR reaction (Hamdani et al, 2013), as described in Table S1. Titin fragments were ligated into the pGEX4T2 expression vector and transformed into competent E. coli XL1-blue (Agilent Technologies) or BL21De3 pLys cells (New England Biolabs, Inc.).…”

“…Cardiomyocyte force measurement Demembranated single human cardiomyocytes were prepared from donor heart tissue as described previously (Hamdani et al, 2013). In brief, samples were thawed in relaxing buffer (1 mmol/l of free Mg, 100 mmol/l KCl, 2 mmol/l EGTA, 4 mmol/l Mg-ATP, and 10 mmol/l imidazole, pH 7.4), mechanically disrupted, and incubated for 5 min in relaxing solution supplemented with 0.5% Triton X-100.…”