Troponin I degradation and covalent complex formation accompanies myocardial ischemia/reperfusion injury

Selective troponin I(TnI) modification has been demonstrated to be in part responsible for the contractile dysfunction observed with myocardial ischem ia/reperfusion injury. We have isolated and characterized modified TnI prod ucts in isolated rat hearts after 0, 15, or 60 minutes of ischemia followed by 45 minutes of reperfusion using affinity chromatography with cardiac tr oponin C (TnC) and an anti-TnI antibody, immunological mapping, reversed-ph ase high-performance liquid chromatography, and mass spectrometry. Rat card iac TnI becomes progressively degraded from 210 amino acid residues to resi dues 1-193, 63-193, and 73-193 with increased severity of injury. Degradati on is accompanied by formation of covalent complexes between TnI 1-193 and, respectively, TnC residues 1-94 and troponin T (TnT) residues 191-298. The covalent complexes are likely a result of isopeptide bond formation betwee n lysine 193 of TnI and glutamine 191 of TnT by the cross linking enzyme tr ansglutaminase. With severe ischemia, cellular necrosis results in specific release of TnI 1-193 into the reperfusion effluent and TnT degradation in the myocardium (25-, 27-, and 33-kDa products). Two-dimensional electrophor esis demonstrated that phosphorylation of TnI prevents ischemia-induced deg radation. This study characterized the modified TnI products in isolated ra t hearts reperfused after a brief or severe period of ischemia, revealing t he progressive nature of TnI degradation, changes in phosphorylation, and c ovalent complexes with ischemia/reperfusion injury. Finally, we propose a m odel for ischemia/reperfusion injury in which the extent of proteolytic and transglutaminase activities ultimately determines whether apoptosis or nec rosis is achieved.