Calpain activation and cell death in HL-5 cardiacmyocytes

Calcium overload is considered one of the major causes of irreversibility in cell injury caused by ischemia. Among the several intracellular enzymes activated by Ca2 +, a crucial role has been attributed to calpains, a group of cysteine proteinases, which are activated by Ca2+ at neutral pH. In HL-5 cardiomyocytes prolonged calcium overload, caused by addition of A23187, led to calpain activation as inferred from: (a) proteolysis of desmin, an optimal substrate, and consequent modification of its immunoreactivity; (b) hydrolysis of a permeable synthetic substrate (suc-LLVY-AMC); (c) inhibition exerted on the proteolysis of above markers by calpeptin, a membrane permeable calpain inhibitor. The results show that calpain activation was always concomitant and proportional to the occurrence of cell death, detected following LDH release or propidium iodide staining. In conclusion, in this cellular model, calpain activation was detectable only after a sustained calcium overload, a condition hardly compatible with cellular viability.