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

Tissue damage resulting from ischemia due to myocardial infarction is thought to be intensified by the proteolytic action of endogenous enzymes. Calpain (calcium dependent cysteine protease) is considered to be a highly likely candidate, since it is activated by calcium ion which increases in concentration 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 antibody was verified by its inhibitory effect on the caseinolytic activity of both μ- and m-calpains, western blotting analysis, and by absorption with the antigen peptide. The antibody was used to localize the intracellular distribution 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 unaffected regions and that the immunostained regions were essentially the same regions as those identified by dense eosinophilic staining with hematoxylin and eosin. However, the staining pattern obtained with the antibody, was characteristic in denser staining at the cell periphery, whereas the damaged cells were stained homogeneously by hematoxylin and eosin. By the former method, results of staining indicated that the activation site of the calpain 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.