Activation of calpain in myocardial infarction: An immunohistochemical study using a calpain antibody raised against active site histidine-containingpeptide

Tissue damage resulting from ischemia due to myocardial infarction is thoug ht to be intensified by the proteolytic action of endogenous enzymes. Calpa in (calcium dependent cysteine protease) is considered to be a highly likel y candidate, since it is activated by calcium ion which increases in concen tration under conditions of ischemia. We prepared a mono-specific antibody against the active site histidine stretch, Lys-Leu-Val-Lys-Gly-His-Ala-Tyr- Ser-Val, in the calpain 80 kDa large subunit. The specificity of the antibo dy was verified by its inhibitory effect on the caseinolytic activity of bo th mu- and m-calpains, western blotting analysis, and by absorption with th e antigen peptide. The antibody was used to localize the intracellular dist ribution of activated calpains in infarcted regions of the human heart. The results showed that myocardial cells affected by ischemia were stained by the antibody, allowing damaged cells to be distinguished from cells of unaf fected regions and that the irnmunostained regions were essentially the sam e regions as those identified by dense eosinophilic staining with hematoxyl in and eosin. However, the staining pattern obtained with the antibody, was characteristic in denser staining at the cell periphery, whereas the damag ed cells were stained homogeneously by hematoxylin and eosin. By the former method, results of staining indicated that the activation site of the calp ain proenzyme was in the peri-plasma membrane, whereas by the latter method , diffusely distributed plasma proteins such as albumin and immunoglobulins were visualized as demonstrated in earlier reports. (C) 1998 by Elsevier S cience inc.