Circ Res：发现在心脏衰竭中过分活跃的酶

肌联蛋白磷酸化决定着肌肉细胞的机械张力

肌联蛋白（Titin）是人体内最大的蛋白，它像弹簧一样控制着肌肉细胞的收缩与放松。磷酸基团与肌联蛋白的特定位点结合（就是所谓的磷酸化），使肌肉细胞处于放松状态。已经有研究表明，CaM kinase II可以使心脏细胞的多种蛋白磷酸化，但是它是否同样能使肌联蛋白仍不清楚，这正是本研究所要探索的。

CaM kinase II可以使肌联蛋白磷酸化

在本研究中，研究人员选取“正常”老鼠的心脏细胞进行研究。这些老鼠有些是体内不产生CaM kinase II的，有些则产生的CaM kinase II的量要比正常水平的高。在没有CaM kinase II的细胞中，肌联蛋白磷酸化程度下降50%；而在含有较高水平的CaM kinase II的细胞中，肌联蛋白磷酸化程度是正常细胞的2倍。因此，CaM kinase II对肌联蛋白磷酸化具有重要的作用。

Linke的研究小组鉴定到了肌联蛋白分子中2个区域可以被磷酸化，这2个区域分别是PEVK和N2Bus区域。这些位点包含了丝氨酸和苏氨酸，而这些类型的氨基酸在进化过程中并没有发生很大的变化。

CaM kinase II决定着细胞刚度

在进一步的研究中，研究小组发现，CaM kinase II的不足或过多可以影响到细胞的刚度（stiffness）。缺少CaM  kinase II的细胞变得比较硬，而CaM kinase II过多的细胞会变得较为松弛。如果向不能产生CaM kinase II的细胞中添加CaM kinase II，细胞会变得松弛。研究小组发现，在衰竭的人类心脏中，CaM kinase II的活性要比正常的心脏要高，因此，PEVK和N2Bus区域会出现过度磷酸化。“这似乎改变了人类心肌的机械性能。”Wolfgang Linke说。

doi: 10.1161/​CIRCRESAHA.111.300105
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Crucial Role for Ca2+/Calmodulin-Dependent Protein Kinase-II in Regulating Diastolic Stress of Normal and Failing Hearts via Titin Phosphorylation

Nazha Hamdani1, Judith Krysiak2, Michael M. Kreusser3, Stefan Neef4, Cristobal G. dos Remedios5, Lars S. Maier6, Markus Krüger7, Johannes Backs8 and Wolfgang A. Linke9*

Rationale: Myocardial diastolic stiffness and cardiomyocyte passive force (Fpassive) depend in part on titin isoform composition and phosphorylation. Ca2+/calmodulin-dependent protein kinase-II (CaMKII) phosphorylates ion channels, Ca22+-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 (TG) 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 SILAC mouse heart mixed with wildtype (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 TG versus WT hearts. Conserved CaMKII-dependent phosphosites were identified within titin's PEVK-domain by quantitative mass spectrometry and confirmed in recombinant human PEVK-fragments. CaMKII also phosphorylated the cardiac titin N2B-unique sequence (N2Bus). Phosphorylation at specific PEVK/N2Bus sites was decreased in DKO and amplified in TG versus WT hearts. Fpassive was elevated in DKO and reduced in TG compared to 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/N2Bus sites than non-failing donor hearts. 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.