Cardiac troponins may be irreversibly modified by glycation: novel potential mechanisms of cardiac performance modulation

Figures

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  Figure 1. Novel AGE modification sites on human cardiac troponin-I (TnI). (A) MS/MS spectrum of trypsin-digested TnI unglycated peptide 193–204 (m/z 645.828). (B) MS/MS spectrum of trypsin-digested TnI peptide 193–204 with CML modification (+29 m/z = +58 Da) of Lys193 (m/z 674.830), confirmed by 4 peptides. (C) MS/MS spectrum of trypsin-digested TnI unglycated peptide 118–131 (m/z 787.444). (D) MS/MS spectrum of trypsin-digested TnI peptide 118–131 with CML modification (+29 m/z = +58 Da) of Lys120 (m/z 816.446), confirmed by 23 peptides. (E) MS/MS spectrum of AspN-digested TnI unglycated peptide 113–126 (m/z 772.931). (F) MS/MS spectrum of AspN-digested TnI peptide 113–126 with CML modification (+29 m/z = +58 Da) of Lys120 (m/z 801.934), confirmed by 7 peptides. (G) MS/MS spectrum of trypsin-digested rat TnI unglycated peptide 119–132 (m/z 787.443) corresponding to human TnI unglycated peptide 118–131. (H) MS/MS spectrum of trypsin-digested rat TnI peptide 119–132 with CML modification (+29 m/z = +58 Da) of Lys121 (m/z 816.449) corresponding to human TnI Lys120. The b-ions denote N-terminal ions and y-ions denote C-terminal ions. CML, N ε-carboxymethyl-lysine.

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  Figure 2. Novel AGE modification sites on human cardiac troponin-T (TnT). (A) MS/MS spectrum of trypsin-digested TnT unglycated peptide 227–240 (m/z 832.461). (B) MS/MS spectrum of trypsin-digested TnT peptide 227–240 with CML modification (+29 m/z = +58 Da) of Lys227 (m/z 861.464), confirmed by 4 peptides. (C) MS/MS spectrum of trypsin-digested TnT unglycated peptide 124–134 (m/z 666.374). (D) MS/MS spectrum of trypsin-digested TnT peptide 124–134 with CML modification (+29 m/z = +58 Da) of Lys125 (m/z 695.377), confirmed by 3 peptides. The b-ions denote N-terminal ions and y-ions denote C-terminal ions. CML, N ε-carboxymethyl-lysine.

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  Figure 3. Novel phosphorylation sites on human cardiac TnI. (A) MS/MS spectrum of trypsin-digested TnI unphosphorylated peptide 118–131 (m/z 787.444). (B) MS/MS spectrum of trypsin-digested TnI peptide 118–131 with phosphorylation (+40 m/z = +80 Da) of Thr119 (m/z 827.422), confirmed by 3 peptides. (C) MS/MS spectrum of trypsin-digested TnI unphosphorylated peptide 121–136 (m/z 945.521). (D) MS/MS spectrum of trypsin-digested TnI peptide 121–136 with phosphorylation (+40 m/z = +80 Da) of Thr123 (m/z 985.500). The b-ions denote N-terminal ions and y-ions denote C-terminal ions. Phos, phosphorylation.

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  Figure 4. Amino acid residue location of identified AGE and phosphorylation modification sites on human cardiac troponin complex proteins. (A,B) 3-dimensional structure of cardiac troponin-C (TnC, green), troponin-I (TnI, aqua) and troponin-T (TnT, mauve). Modification sites are shown in red. Location of Ca2+ binding pockets indicated by dark blue circles. TnI R204MG-H, TnI S23/24Phos, TnI S199Phos, TnT K107CML and TnT K125CML are not shown as the 3-dimensional structure is not resolved for the C-terminal domain of TnI and N-terminal of TnT. CML, N ε-carboxymethyl-lysine; CEL, N ε-carboxyethyl-lysine; MG-H, methylglyoxal-derived hydroimidazolone; Phos, Phosphorylation.

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  Figure 5. Domain structure of human troponin complex proteins indicating identified AGE and phosphorylation modification sites detected by LC-MS/MS. (A) Domain structure of human cardiac troponin-C. Ca2+ binding sites are depicted in green: dark green, high-affinity Ca2+ binding site; light green, low affinity Ca2+ binding sites. (B) Domain structure of human cardiac troponin-I. ID, inhibitory domain; RD, regulatory domain. Detected phosphorylation sites in blue, known but not detected site in grey. (C) Domain structure of human cardiac troponin-T. CML, N ε-carboxymethyl-lysine; CEL, N ε-carboxyethyl-lysine; MG-H, methylglyoxal-derived hydroimidazolone; P, phosphorylation.

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  Figure 6. In vitro exposure to glucose and fructose increases AGE-modification of human cardiac troponin-C (TnC). (A–C) Percentage of technical replicates with detection of a CML modification at lysine residues of purified human cardiac TnC using LC-MS/MS (total 5–7 technical replicates per condition). Dark grey: modified, detection above identity threshold; light grey: modified, detection below identity threshold. CML, carboxymethyllysine. (D–F) Number of TnC MS/MS peptide spectra with hexose, CML, or oxidation modifications as a percentage of the total number of TnC MS/MS spectra detected for a given peptide (n = 5 replicates/group). Criteria for identifying a modification: above homology threshold and expect value < 0.5. Data presented as mean ± SEM. One-way ANOVA, Bonferroni post-hoc tests, *p < 0.05.