Abstract 17152: Identification and Site-Specific Quantification of Novel Phosphorylated Residues in Cardiac Troponin I: Implications for Heart Failure
Cardiac Troponin I (cTnI), a key regulatory and inhibitory myofilament protein, is known to be phosphorylated at six residues (Ser23/24, Ser42/44, Thr143, Ser150) by PKA, PKC, PKD, PKG and PAK etc. Although it is known that phosphorylation levels are altered in heart failure (HF) the exact sites involved are yet to be determined. Using the state-of-the-art targeted proteomic technique-multiple reaction monitoring (MRM) assay, we aimed to develop multiplex quantification in failing and non-failing human LV samples for all sites. Myofibril cTnIs were purified and identified from the banked end-stage HF with Ischemic Heart Disease (ISHD) and Idiopathic Cardiomyopathy (IDCM) patients and compared with non-failing hearts (n=30). Overall, we identified 12 phosphorylation sites including 6 novel sites: Ser4/5, Try26, Ser77, Ser166, Thr181 and Ser199. To quantify phosphorylation status of each site, total 30 assays and 145 transitions were designed for each mono or di-phosphorylated and the corresponding non-phosphorylated sequence and analyzed by 4000 Qtrap nano-LC-MS/MS in triplicate. Status on sites Ser4/5, Try26and Ser24 was significantly reduced in ISHD (↓42.2±0.7%; ↓67.2±0.6%, p<0.005) and IDCM (↓49.4±0.5%, p<0.001; ↓88.3±0.2%, p<0.005) patients compared with non-failing donors. However, phosphorylation in HF was higher on novel sites Ser77(ISHD ↑48.9±1.9% and IDCM ↑38.4±1.5, p<0.005); Ser166(ISHD ↑34.4±0.3% and IDCM ↑206.3±0.5%, p<0.01); Thr181(ISHD↑35.0±0.3% and IDCM ↑32.9±0.2%, p<0.001), Ser199 (ISHD ↑167.7±0.1%, p<0.005 and IDCM ↑133.3±0.1%, p<0.001) and the known Ser42 (ISHD ↑40.8±0.8% and IDCM ↑49.1±0.7%, p<0.005) and Thr143 (ISHD ↑10.1±0.8% and IDCM ↑11.7±0.7%, p<0.001) as compared to donor heart. In summary, this study provides the first identification and quantification of 6 novel phosphorylated sites. With HF there is i) a shift in the phosphorylation from PKA to PKC sites; ii) importantly shift with decreased in the N-terminal residues Ser4/5, 24 and Try26 and increased with the middle Ser77, Ser166, Thr181 and C-terminal Ser199. Thus, dynamic and selective alterations occurred to individual residues of cTnI most likely reflecting changes in the upstream signaling cascades eg. imbalance in kinase/phosphatase activity.
- © 2010 by American Heart Association, Inc.