Department of Medicine, Division of Cardiology, Johns Hopkins University, Baltimore, MD, USA.
Circulation. 2012 Oct 9;126(15):1828-37. doi: 10.1161/CIRCULATIONAHA.112.096388. Epub 2012 Sep 12.
Human cardiac troponin I is known to be phosphorylated at multiple amino acid residues by several kinases. Advances in mass spectrometry allow sensitive detection of known and novel phosphorylation sites and measurement of the level of phosphorylation simultaneously at each site in myocardial samples.
On the basis of in silico prediction and liquid chromatography/mass spectrometry data, 14 phosphorylation sites on cardiac troponin I, including 6 novel residues (S4, S5, Y25, T50, T180, S198), were assessed in explanted hearts from end-stage heart failure transplantation patients with ischemic heart disease or idiopathic dilated cardiomyopathy and compared with samples obtained from nonfailing donor hearts (n=10 per group). Thirty mass spectrometry-based multiple reaction monitoring quantitative tryptic peptide assays were developed for each phosphorylatable and corresponding nonphosphorylated site. The results show that in heart failure there is a decrease in the extent of phosphorylation of the known protein kinase A sites (S22, S23) and other newly discovered phosphorylation sites located in the N-terminal extension of cardiac troponin I (S4, S5, Y25), an increase in phosphorylation of the protein kinase C sites (S41, S43, T142), and an increase in phosphorylation of the IT-arm domain residues (S76, T77) and C-terminal domain novel phosphorylation sites of cardiac troponin I (S165, T180, S198). In a canine dyssynchronous heart failure model, enhanced phosphorylation at 3 novel sites was found to decline toward control after resynchronization therapy.
Selective, functionally significant phosphorylation alterations occurred on individual residues of cardiac troponin I in heart failure, likely reflecting an imbalance in kinase/phosphatase activity. Such changes can be reversed by cardiac resynchronization.
已知多种激酶可使人心肌钙蛋白 I 的多个氨基酸残基发生磷酸化。质谱技术的进步允许同时灵敏地检测已知和新的磷酸化位点，并测量心肌样本中每个位点的磷酸化水平。
基于计算机预测和液相色谱/质谱数据，在患有缺血性心脏病或特发性扩张型心肌病的终末期心力衰竭移植患者的心脏样本中检测到心肌钙蛋白 I 上的 14 个磷酸化位点，包括 6 个新残基（S4、S5、Y25、T50、T180、S198），并与来自非衰竭供体心脏的样本进行比较（每组 10 个样本）。针对每个可磷酸化和相应的非磷酸化位点开发了 30 种基于质谱的多重反应监测定量胰酶肽测定法。结果表明，在心力衰竭中，已知蛋白激酶 A 位点（S22、S23）和位于心肌钙蛋白 I N 端延伸部分的其他新发现磷酸化位点（S4、S5、Y25）的磷酸化程度降低，蛋白激酶 C 位点（S41、S43、T142）的磷酸化增加，以及 IT 臂结构域残基（S76、T77）和心肌钙蛋白 I C 端结构域新磷酸化位点（S165、T180、S198）的磷酸化增加。在犬非同步性心力衰竭模型中，发现 3 个新位点的增强磷酸化在再同步治疗后趋于对照水平。
在心力衰竭中，心肌钙蛋白 I 的单个残基上发生了选择性的、具有功能意义的磷酸化改变，这可能反映了激酶/磷酸酶活性的失衡。这些变化可以通过心脏再同步来逆转。
