Assessing experimental models in myocardial injury: Lack of activation of the proteases TACE and calpain in brief ischaemia and reperfusion

Abstract

Background: Calpain inhibitors are reportedly cardioprotective. Furthermore, oxidative stress may acutely activate the sheddase tumour necrosis factor (TNF)-α-cleaving enzyme (TACE). The aim of this study was to examine whether myocardial reperfusion leads to activation of the proteases μ- and m-calpain, and to evaluate which cardiac cells act as a source of TNF-α. Methods: Isolated hearts (guinea pig) were subjected to global ischaemia (15 min) and reperfused. Calpain activity was determined by zymography. Calpastatin (inhibitor) and troponin I (substrate) were quantified by western blotting. Immunohistology of hearts and a human mast cell line (HMC-1) was used to localise expression of TNF-α and TACE. Shedding of TNF-α was assessed in Mono Mach, Jurkat-T, HMC-1 and peripheral blood leucocytes with and without oxidative stress. Results: Neither of the ubiquitous calpains (μ- and m-calpain) was significantly activated by brief ischaemia/reperfusion, nor were calpastatin and troponin degraded more than in extracts of control hearts. Cardiac TNF-α immunoreactivity was localised to mast cells. None of the tested cell lines shed TNF-α in response to non-toxic amounts of oxidants. However, HMC-1 cells showed poor expression of proTNF-α, while TACE was abundant. Conclusions: Although the severity of ischaemia in the current model may have been insufficient, activation of calpain by ischaemia/reperfusion cannot be demonstrated simply in the Langendorff-mode perfused isolated heart. Mast cells are the prime source of myocardial TNF-α. A suitable whole-cell model remains to be found to demonstrate acute oxidative activation of TACE.