Department of Cardiovascular Physiology, Ruhr University Bochum, Bochum, Germany.
Circ Res. 2013 Feb 15;112(4):664-74. doi: 10.1161/CIRCRESAHA.111.300105. Epub 2013 Jan 2.
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.
To elucidate whether CaMKII phosphorylates titin and modulates F(passive) in normal and failing myocardium.
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 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.
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.
心肌舒张僵硬和心肌细胞被动力（F(passive)）部分依赖于肌联蛋白异构体组成和磷酸化。钙/钙调蛋白依赖性蛋白激酶-II（CaMKII）在心脏中磷酸化离子通道、钙处理蛋白和染色质修饰酶，但尚未发现其靶向肌联蛋白。
阐明 CaMKII 是否磷酸化肌联蛋白并调节正常和衰竭心肌中的 F(passive)。
通过 Pro-Q-Diamond/Sypro-Ruby 染色、放射自显影和磷酸化肌联蛋白抗体的免疫印迹，在 CaMKIIδ/γ 双敲除（DKO）小鼠、转基因 CaMKIIδC 过表达小鼠和人类心脏中评估肌联蛋白磷酸化。通过使用稳定同位素标记的细胞培养物中的氨基酸混合野生型（WT）或 DKO 心脏的体内质谱法，定量测量 CaMKII 依赖性肌联蛋白特定磷酸化位点。在 CaMKII 给药前后记录单个通透的心肌细胞的 F(passive)。与 WT 心脏相比，DKO 中的全肌联蛋白磷酸化减少了 >50%，而转基因中的肌联蛋白磷酸化增加了约 ≈100%。通过定量质谱法在肌联蛋白的 PEVK 结构域内鉴定出保守的 CaMKII 依赖性磷酸化位点，并在重组人 PEVK 片段中得到证实。CaMKII 还磷酸化了心脏肌联蛋白的 N2B 独特序列。在 DKO 中，特定的 PEVK/肌联蛋白 N2B 独特序列位点的磷酸化减少，而在转基因中则增加。与 WT 心肌细胞相比，DKO 和转基因心肌细胞的 F(passive)升高。与 WT 相比，CaMKII 给药增加了 DKO 和转基因心肌细胞的 F(passive)，而肌联蛋白抗体预处理则减弱了这种作用。与非衰竭供体心脏相比，人类终末期衰竭心脏的肌联蛋白的 CaMKII 表达/活性和 PEVK/肌联蛋白 N2B 独特序列位点的磷酸化更高。
CaMKII 磷酸化肌联蛋白的弹性能量储存区（PEVK）和 N2B 独特序列上的保守丝氨酸/苏氨酸，从而降低 F(passive)。心脏衰竭时出现异常的 CaMKII 依赖性肌联蛋白磷酸化，并导致舒张压力改变。
