Multiple Reaction Monitoring to Identify Site-Specific Troponin I Phosphorylated Residues in the Failing Human Heart
Background—Human cardiac troponin I (cTnI) is known to be phosphorylated at multiple amino acid residues by several kinases. Advances in mass spectrometry (MS) allow sensitive detection of known and novel phosphorylation sites and measurement of the level of phosphorylation simultaneously at each site in myocardial samples.
Methods and Results—Based on in silico prediction and LC/MS/MS data, 14 phosphorylation sites on cTnI, including 6 novel residues (S4, S5, Y25, T50, T180, S198), were assessed in explanted hearts from end-stage heart failure transplant patients with ischemic heart disease or idiopathic dilated cardiomyopathy and compared to samples obtained from non-failing donor hearts (n = 10 per group). Thirty MS-based multiple reaction monitoring quantitative tryptic peptide assays were developed for each phosphorylatable and corresponding non-phosphorylated site. The results show in heart failure there is i) a decrease in the extent of phosphorylation of the known PKA sites (S22, S23) as well as other newly discovered phosphorylation sites located in the N-terminal extension of cTnI (S4, S5, Y25); ii) increase in phosphorylation of the PKC sites (S41, S43, T142); and iii) increase in phosphorylation of the IT-arm domain residues (S76, T77) and C-terminal domain novel phosphorylation sites of cTnI (S165, T180, S198). In a canine dyssynchronous heart failure model, enhanced phosphorylation at three of the novel sites was found to decline towards control following resynchronization therapy.
Conclusions—Selective, functionally significant phosphorylation alterations occurred on individual residues of cTnI in heart failure, likely reflecting an imbalance in kinase/phosphatase activity. Such changes can be reversed by cardiac resynchronization.
- Received January 27, 2012.
- Accepted August 10, 2012.
- Copyright © 2012, American Heart Association, Inc. All rights reserved. Unauthorized use prohibited