Crucial Role for Ca2+/Calmodulin-Dependent Protein Kinase-II in Regulating Diastolic Stress of Normal and Failing Hearts via Titin Phosphorylation
Crucial Role for Ca2+/Calmodulin-Dependent Protein Kinase-II in Regulating Diastolic Stress of Normal and Failing Hearts via Titin Phosphorylation
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DOI：
10.1161/circresaha.111.300105
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发表时间：
2013-02-15
影响因子：
20.1
通讯作者：
Linke, Wolfgang A.
中科院分区：
文献类型：
--
作者：
Hamdani, Nazha;Krysiak, Judith;Linke, Wolfgang A.
原理：心肌舒张僵硬度和心肌细胞被动力（F-被动）部分取决于肌联蛋白亚型组成和磷酸化。Ca 2 +/钙调蛋白依赖性蛋白激酶-II（CaMKII）磷酸化心脏中的离子通道、Ca 2+处理蛋白和染色质修饰酶，但尚未知道靶向titin.Objective：阐明CaMKII是否磷酸化titin并调节正常和衰竭心肌中的F-被动。通过Pro-Q-Diamond/Sypro-Ruby染色，放射自显影，和使用磷酸丝氨酸特异性肌联蛋白抗体的免疫印迹。在与野生型（WT）或DKO心脏混合的细胞培养小鼠心脏中，通过质谱法使用氨基酸稳定同位素标记，在体内定量CaMKII依赖性位点特异性肌联蛋白磷酸化。在CaMK II给药之前和之后记录单个透化心肌细胞的F-被动。DKO中的全肌联蛋白磷酸化减少了>50%，但与WT心脏相比，转基因心脏中的全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结构域内鉴定了保守的CaMKII依赖性磷酸化位点，并在重组人PEVK片段中得到证实。CaMKII也磷酸化心肌肌联蛋白N2 B独特序列。特异性PEVK/肌联蛋白N2 B独特序列位点的磷酸化在DKO中降低，并在转基因心脏与WT心脏中扩增。与WT心肌细胞相比，DKO中F-被动升高，转基因中F-被动降低。CaMKII给药降低WT和DKO心肌细胞的F-被动反应，这种作用被肌联蛋白抗体预处理减弱。人终末期衰竭心脏显示更高的CaMKII的表达/活性和磷酸化PEVK/肌联蛋白N2 B独特的序列sites than nonfailing donor hearts.Conclusions：CaMKII磷酸化肌联蛋白弹簧在保守的丝氨酸/苏氨酸，从而降低F-被动。CaMKII依赖性肌联蛋白磷酸化紊乱发生在心力衰竭中，并导致舒张应力改变。(Circ Res. 2013; 112：664-674）。
Rationale: Myocardial diastolic stiffness and cardiomyocyte passive force (F-passive) depend in part on titin isoform composition and phosphorylation. Ca2+/calmodulin-dependent protein kinase-II (CaMKII) phosphorylates ion channels, Ca2+-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 CaMKII delta/gamma double-knockout (DKO) mouse, transgenic CaMKII delta C-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 approximate 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. (Circ Res. 2013; 112:664-674.)