Calpain activation and death of isolated cardiomyocytes exposed to intracellular calcium overload

The causal relationships linking cell death with calpain activation are still elusive. To this aim calpain activation was investigated in Hl-5 cardiomyocyte added with 1 μM A23187, a calcium ionophore. To obtain a persistent intracellular Ca2+ overload, as detected by fluo-3 fluorescence, NaCl of the incubation buffer was replaced with KCl and 1 mM vanadate was added to inhibit Ca2+ ATPases. Fluo3 fluorescence dropped immediately to background levels when EGTA was added. In this way it was possible to expose HL-5 cells to various durations of calcium overload. Calpain activation was investigated by means of immunoblot analyses of desmin degradation and fluorescence increases reflecting the hydrolysis of the synthetic peptide Suc-LLVY-AMC, a calpain substrate. Cell death was assessed as lactic dehydrogenase (LDH) release. Calpain activation became detectable after 20 min of calcium overload and was followed by the increase in LDH release, which approached to plateau after 40 min. The addition of EGTA after 30 min was no longer able to block the progression in cell death and calpain activation. More importantly, calpain inhibition by 10 μM PD150606 or 100 μM calpeptin reduced significantly LDH release although at a lesser extent than calpain mediated proteolysis. In conclusion, the present findings suggest that (i) calpain activation precedes the onset of cell death; (ii) intracellular calcium overload hampers cell viability in a process that eventually becomes independent of Ca2+; (iii) calpain activation is causally related to cell death, although the severity of the present protocol limits the protective efficacy of calpain inhibition.