Calpain and mitochondrial dysfunction in Ca2+ overloaded cardiomyocytes

Intracellular Ca2+ overload and mitochondrial dysfunction are considered major causes of ischemia-induced cell death. An increase in intracellular Ca2+ ([Ca2+]i) can also cause the activation of calpains. Although the increased activity of these proteinases is suggested to play a relevant role in different forms of cell death, the relationship between calpain activation and mitochondrial dysfunction is not elucidated yet. To this aim the fluorescent potentiometric probe JC-1 was used to assess mitochondrial membrane potential (MMP) in HL-1 cardiomyocytes. A persistent elevation in [Ca2+]i as detected by Fluo-4 FF fluorescence was obtained by adding HL-1 cells with the calcium ionophore A23187 (1 uM) in the presence of 1 mM vanadate to inhibit P-type ATPases and replacing NaCl with KCl in the incubation buffer. The rise in [Ca2+]i was followed by MMP fall which occurred in 50% of the cells 30 min after A23187 addition. Mitochondrial deenergization was concomitant with calpain activation as detected by the proteolysis of a synthetic peptide (Suc-LLVY-AMC) and the occurrence of cell necrosis as detected by LDH release. Interestingly the extent of enzyme release correlated with the number of cells displaying MMP fall. Cell death was likely due only to necrosis since cleavage of PARP-1 and caspase-3 was not detected. Calpains inhibitors, like PD150606 (30 uM) and calpeptin (100 uM), reduced by 60% both LDH release and mitochondrial deenergization. Therefore the increase in [Ca2+]i does not appear to cause mitochondrial deenergization per se. Interestingly, HL-1 staining with anti calpain antibodies demonstrated that calpains colocalize with mitochondria. In conclusion, the present data suggest that calpain activation is a causative factor of mitochondrial dysfunction under conditions of intracellular Ca2+ overload.