CARDIOMYOCYTE TROPONIN-T IMMUNOREACTIVITY IS MODIFIED BY CROSS-LINKING RESULTING FROM INTRACELLULAR CALCIUM OVERLOAD

Background During myocardial ischemia, the increase in cytosolic Ca2promotes the activation of neutral proteases such as calpains. Since t he troponin T subunit is a substrate for calpains, we investigated the effects of irreversible myocyte damage on troponin T immunoreactivity . Methods and Results Hearts from adult guinea pigs (n=32) were perfus ed under conditions of normoxia, ischemia, postischemic reperfusion, o r Ca2+ paradox. Hearts were frozen and processed for immunohistochemis try and Western blot with three anti-troponin T monoclonal antibodies. Two of these antibodies are unreactive on cryosections of freshly iso lated and normoxic hearts and of hearts exposed to 30 minutes of no-fl ow ischemia. In contrast, reactivity is detected in rare myocytes afte r 60 minutes of ischemia, in a large population of myocytes after 60 m inutes of ischemia followed by 30 minutes of reperfusion, and in every myocyte exposed to Ca2+ paradox. In Western blots, samples from ische mia-reperfusion and Ca2+-overloaded hearts show reactive polypeptides of about 240 to 260 kD and 65 to 66 kD in addition to troponin T. A si milar pattern of immunoreactivity is observed with an anti-troponin I antibody. Histochemical troponin T immunoreactivity and reactivity on high-molecular-weight polypeptides are detectable in normal heart samp les after preincubation with 10 mmol/L Ca2+ or with transglutaminase, whereas they are not if either transglutaminase or calpain is inhibite d. Conclusions The evolution of the ischemic injury is accompanied by changes in troponin T immunoreactivity as a consequence of the calcium -dependent activation of both calpain proteolysis and transglutaminase cross-linking.