Cardiomyocyte troponin T immunoreactivity is modified by cross-linking resulting from intracellular calcium overload.
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.


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
L
Department of Biomedical Sciences, University of Padova, Italy.
Menabò
R
R
Vitadello
M
M
Bergamini
C M
CM
Di Lisa
F
F
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000502:physiology; D006168:Guinea Pigs; D007150:Immunohistochemistry; D008297:Male; D017202:Myocardial Ischemia / Q000378:metabolism*; D015425:Myocardial Reperfusion; D009206:Myocardium / Q000737:chemistry*; D009994:Osmolar Concentration; D011503:Transglutaminases / Q000502:physiology; D014336:Troponin / Q000032:analysis* / Q000276:immunology; D020107:Troponin T
D014336:Troponin; D020107:Troponin T; D011503:Transglutaminases; D002154:Calpain; D002118:Calcium