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(Circulation. 1996;93:1896-1904.)
© 1996 American Heart Association, Inc.

Articles

Cardiomyocyte Troponin T Immunoreactivity Is Modified by Cross-linking Resulting From Intracellular Calcium Overload

Luisa Gorza, MD; Roberta Menabò, PhD; Maurizio Vitadello, MD; Carlo M. Bergamini, MD; Fabio Di Lisa, MD

From the Department of Biomedical Sciences, CNR-Unit for Muscle Biology and Physiopathology (L.G., M.V.), and Department of Biological Chemistry (R.M., F.D.L.), University of Padova, and the Department of Biochemistry and Molecular Biology, University of Ferrara (C.M.B.), Italy.

Correspondence to Dr Luisa Gorza, Department of Biomedical Sciences, CNR-Unit for Muscle Biology and Physiopathology, via Trieste 75, 35121 Padova, Italy.

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.

Key Words: calcium • reperfusion • troponin T • calpain • transglutaminase

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