Calpain translocation and activation as pharmacological targets during myocardial ischemia/reperfusion

Abstract 

Calpains contribute to reperfusion-induced myocardial cell death. However, it remains controversial whether its activation occurs during ischemia or reperfusion. We investigated the regulation and time-course of calpain activation secondary to transient ischemia and the efficacy of its inhibition at reperfusion as a therapeutic strategy to limit infarct size. In isolated rat hearts (Sprague–Dawley), ischemia induced a time-dependent translocation of m-calpain to the membrane that was not associated with calpain activation as assessed by proteolysis of its substrate α-fodrin. Translocation of calpain was dependent on Ca²⁺ entry through reverse mode Na⁺/Ca²⁺-exchange and was independent of acidosis. Calpain activation occurred during reperfusion, but only after intracellular pH (pHi) normalization, and was not prevented by inhibiting its translocation during ischemia with methyl-β-cyclodextrin. The intravenous infusion of MDL-28170 in an in vivo rat model with transient coronary occlusion during the first minutes of reperfusion resulted in a reduction of infarct size (43.9±3.9% vs. 60.2±4.7, P=0.046, n=18) and α-fodrin degradation. These results suggest that (1) Ca²⁺-induced calpain translocation to the membrane during ischemia is independent of its activation, (2) intracellular acidosis inhibits calpain activation during ischemia and pHi normalization allows activation upon reperfusion, and (3) calpain inhibition at the time of reperfusion appears as a potentially useful strategy to limit infarct size.

Abbreviations: BDM, 2,3-butanedione monoxime, DAPI, 4′,6′-diamidino-2-phenylindole, KHB, Krebs-Henseleit bicarbonate, LAD, left anterior descendent coronary artery, LVEDP, left ventricular end diastolic pressure, LVdevP, left ventricular developed pressure, LDH, lactate dehydrogenase, MβCX, methyl-β-cyclodextrine, SDS, sodium dodecyl sulfate

Keywords: Calpains, Ischemia, Reperfusion injury, Infarct, Calcium, pH