The Blockade of Transmembrane Cl⁻ Flux Mitigates I/R-Induced Heart Injury via the Inhibition of Calpain Activity.

AIMS The aim of this study was to determine whether calpain is involved in Cl(-)-induced myocardial ischemia/reperfusion (I/R) injury. METHODS Isolated rat hearts were subjected to either 45 min of global no-flow ischemia followed by reperfusion or successive perfusion with Ca(2+)-free KH solution for 3 min and normal KH solution for 30 min, also known as Ca(2+) paradox. RESULTS The hearts in the I/R group exhibited increases in myocardial injury area, LDH release, caspase 3 activity and apoptotic indices and a marked decline in cardiac performance. As was the case regarding the effects of MDL 28170, an inhibitor of calpain, treatment with 5 µM NPPB, 5 µM DIDS and low Cl(-) significantly attenuated cardiac injury. Moreover, each of the treatments significantly protected against Ca(2+) overload-induced injury in the setting of Ca(2+) paradox. The Western blot and immunofluorescence data revealed that there was an increase in the percentages of calpain membrane-positive cells and the numbers of fragments resulting from the calpain-mediated proteolysis of α-fodrin in both the I/R and the Ca(2+) paradox, indicating that the activation of calpain occurred. More importantly, these effects were mitigated by the blockade of transmembrane Cl(-) flux, as was accomplished via MDL 28170. CONCLUSION Our results provide evidence that the blockade of transmembrane Cl(-) flux mitigates I/R-induced cardiac injury via the inhibition of calpain activity. They also indicate that intracellular Ca(2+) overload regulates calpain activation in the setting of Cl(-)-induced injury.