Effects of Thiol Protease Inhibitors on Fodrin Degradation During Hypoxia in Cultured Myocytes

The effects of thiol protease inhibitors on fodrin (a cell membrane nonerythroid spectrum-like protein) degradation were studied during hypoxia in cultured myocytes. Cardiac myocytes, isolated from neonatal rat hearts, were incubated under hypoxic conditions for 6 h. Cell membrane proteins, including fodrin, prepared from hypoxic myocytes were examined with electroblots stained for fodrin by the peroxidase method. Cell death during hypoxia rose to 80% after 6 h. Intracellular protease activity was also elevated in hypoxia. This intracellular protease activity was markedly inhibited by the cysteine protease inhibitor E-64 and calpain inhibitor 1. Hypoxic cell death was also suppressed by E-64 and calpain inhibitor 1. A 125 kDa immunoreactive degradation product of fodrin was found in hypoxic conditions. Treatment with E-64 and calpain inhibitor 1 decreased both the appearance of this band and the degradation of fodrin. These observations indicate that intracellular thiol protease, including calpains, are activated during hypoxia and that these are related to cell membrane protein degradation, especially of fodrin. The data also suggest that protease inhibitor E-64 treatment may be beneficial in protection against hypoxic myocyte injury.