BACKGROUND: During myocardial ischemia, the increase in cytosolic Ca2+ promotes the activation of neutral proteases such as calpains. Since the 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 perfused under conditions of normoxia, ischemia, postischemic reperfusion, or Ca2+ paradox. Hearts were frozen and processed for immunohistochemistry and Western blot with three anti-troponin T monoclonal antibodies. Two of these antibodies are unreactive on cryosections of freshly isolated and normoxic hearts and of hearts exposed to 30 minutes of no-flow ischemia. In contrast, reactivity is detected in rare myocytes after 60 minutes of ischemia, in a large population of myocytes after 60 minutes of ischemia followed by 30 minutes of reperfusion, and in every myocyte exposed to Ca2+ paradox. In Western blots, samples from ischemia-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 similar 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 samples after preincubation with 10 mmol/L Ca2+ or with transglutaminase, whereas they are not if either transglutaminase or calpain is inhibited. 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.

Cardiomyocyte troponin T immunoreactivity is modified by crosslinking resulting from intracellular calcium overload

BERGAMINI, Carlo;
1996

Abstract

BACKGROUND: During myocardial ischemia, the increase in cytosolic Ca2+ promotes the activation of neutral proteases such as calpains. Since the 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 perfused under conditions of normoxia, ischemia, postischemic reperfusion, or Ca2+ paradox. Hearts were frozen and processed for immunohistochemistry and Western blot with three anti-troponin T monoclonal antibodies. Two of these antibodies are unreactive on cryosections of freshly isolated and normoxic hearts and of hearts exposed to 30 minutes of no-flow ischemia. In contrast, reactivity is detected in rare myocytes after 60 minutes of ischemia, in a large population of myocytes after 60 minutes of ischemia followed by 30 minutes of reperfusion, and in every myocyte exposed to Ca2+ paradox. In Western blots, samples from ischemia-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 similar 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 samples after preincubation with 10 mmol/L Ca2+ or with transglutaminase, whereas they are not if either transglutaminase or calpain is inhibited. 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.
Gorza, L; Menabò, R; Vitadello, M; Bergamini, Carlo; Dilisa, F.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/1197335
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