Abstract 16409: Identification and Quantification of Novel Phosphorylation of Cardiac Troponin I in Human Heart Failure
Phosphorylation of human cardiac troponin I (cTnI), a key regulatory and inhibitory myofilament protein can alter cardaic muscle contractile. However, the site-specific phosphorylation and quantification have not been conclusively identified or quantified in heart failure in vivo.
Methods: 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). Initial mass spectrometry analysis was carried out to identify all phosphorylation sites on cTnI. Verification and quantification of each known and novel sites was carried out using the state-of-the-art targeted proteomic technique-multiple reaction monitoring (MRM) assay. A total 30 MRM assays comprising 145 transitions were developed for each mono or di-phosphorylated and the corresponding non-phosphorylated tryptic peptide and analyzed by 4000 Qtrap nano-LC-MS/MS in triplicate.
Results: 12 phosphorylation sites including 6 novel sites: S5S6, Y26, S77, T78, S166, T181 and S199 were identified. Status on sites S5/6, Y26 and S24 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 S77 (ISHD ↑48.9±1.9% and IDCM ↑38.4±1.5, p<0.005); S166 (ISHD ↑34.4±0.3% and IDCM ↑206.3±0.5%, p<0.01); T181 (ISHD↑35.0±0.3% and IDCM ↑32.9±0.2%, p<0.001), S199 (ISHD ↑167.7±0.1%, p<0.005 and IDCM ↑133.3±0.1%, p<0.001) and the known S42 (ISHD ↑40.8±0.8% and IDCM ↑49.1±0.7%, p<0.005) and T143 (ISHD ↑10.1±0.8% and IDCM ↑11.7±0.7%, p<0.001) as compared to donor heart.
Conclusions: This is the first identification and quantification of 6 novel phosphorylated sites on cTnI. 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 S5,S6,S5/6,S23,S24, S23/24 and Y26 and increased in the middle S77, T78, S166, T181 and C-terminal S199. 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.
- © 2011 by American Heart Association, Inc.