Modulation of Titin-Based Stiffness by Disulfide Bonding in the Cardiac Titin N2-B Unique Sequence
Modulation of Titin-Based Stiffness by Disulfide Bonding in the Cardiac Titin N2-B Unique Sequence
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DOI：
10.1016/j.bpj.2009.05.037
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发表时间：
2009-08-05
影响因子：
3.4
通讯作者：
Linke, Wolfgang A.
中科院分区：
文献类型：
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作者：
Gruetzner, Anika;Garcia-Manyes, Sergi;Linke, Wolfgang A.
巨蛋白肌联蛋白负责非活化肌肉肌节的弹性。肌联蛋白-亚型转换和蛋白激酶（PK）A-或PKG-依赖性肌联蛋白磷酸化调节心肌肌联蛋白被动僵硬。对肌联蛋白硬度的其他调节作用可能来自氧化应激下的二硫键，因为肌联蛋白弹簧区中的许多免疫球蛋白样（IG-）结构域具有S-S形成的潜力。使用单分子原子力显微镜（AFM）力延伸测量重组Ig结构域多蛋白构建体，我们表明，肌联蛋白Ig模块不含稳定的二硫键，与以前的信念相反。然而，我们证明，人类N2-B-独特的序列（N2-B-US），心脏特异性的，生理上可扩展的肌联蛋白片段，包含572个氨基酸残基，在氧化条件下含有多达三个二硫键。AFM力光谱对重组N2-B-US分子表现出更短的轮廓长度在没有还原剂比在其存在下，与分子内的S-S键合。在离体人心脏肌原纤维的拉伸实验中，还原剂硫氧还蛋白降低了肌联蛋白的硬度，其程度可以通过仅改变N2-B-us的可伸展性来解释（使用熵弹性理论）。我们的结论是，增加氧化应激可以提高肌联蛋白为基础的刚度的心肌细胞，这可能有助于全球心肌硬化常见的老化或衰竭的心脏。
The giant protein titin is responsible for the elasticity of nonactivated muscle sarcomeres. Titin-based passive stiffness in myocardium is modulated by titin-isoform switching and protein-kinase (PK)A- or PKG-dependent titin phosphorylation. Additional modulatory effects on titin stiffness may arise from disulfide bonding under oxidant stress, as many immunoglobulin-like (Ig-)domains in titin's spring region have a potential for S-S formation. Using single-molecule atomic force microscopy (AFM) force-extension measurements on recombinant Ig-domain polyprotein constructs, we show that titin Ig-modules contain no stabilizing disulfide bridge, contrary to previous belief. However, we demonstrate that the human N2-B-unique sequence (N2-B-us), a cardiac-specific, physiologically extensible titin segment comprising 572 amino-acid residues, contains up to three disulfide bridges under oxidizing conditions. AFM force spectroscopy on recombinant N2-B-us molecules demonstrated a much shorter contour length in the absence of a reducing agent than in its presence, consistent with intramolecular S-S bonding. In stretch experiments on isolated human heart myofibrils, the reducing agent thioredoxin lowered titin-based stiffness to a degree that could be explained (using entropic elasticity theory) by altered extensibility solely of the N2-B-us. We conclude that increased oxidant stress can elevate titin-based stiffness of cardiomyocytes, which may contribute to the global myocardial stiffening frequently seen in the aging or failing heart.