[Role of calpain on hypoxic myocyte injury]

The calcium-activated neutral protease, calpain, was tested for its proteolytic effects to degrade the cell membrane spectrin-like protein, fodrin, during hypoxia. Cardiac myocytes, isolated from neonatal rat hearts, were incubated under hypoxic conditions for 6 hours. The cell death during hypoxia rose to 80% after 6 hours. Extracellular protease activity was much more elevated during hypoxia, than in aerobic states at 6 hours. Intracellular protease activity was also elevated in hypoxia. These protease activities were markedly inhibited by the cysteine protease inhibitor E-64 and the calpain specific inhibitor, calpastatin. Hypoxic cell death was also suppressed by E-64. Cell membrane proteins prepared from hypoxic myocytes were examined with electroblots stained for fodrin by the peroxidase method. A 125 kilodalton immunoreactive degradation product of fodrin was found under hypoxic conditions. Treatment with E-64 inhibited both the appearance of this degradation band and the decrease in the content of fodrin. These observations indicate that calpain is activated during hypoxia and that it is related to cell membrane protein degradation, especially in fodrin. The data also suggest that protease inhibitor E-64 treatment may be beneficial in protection against hypoxic myocyte injury.