Ubiquitous calpains (calpain I & II) are generally recognized as cytosolic proteins. Recently, mitochondrial localized calpain I (μ-calpain) has been identified. Activation of mito-u-calpain cleaves apoptosis inducing factor (AIF), a flavoprotein located within the mitochondrial intermembrane space, in liver mitochondria, but not in brain mitochondria. We first tested if activation of mito-u-calpain cleaves AIF in isolated heart mitochondria. A decrease in AIF content within mitochondria increases cardiac injury during ischemia-reperfusion by augmenting oxidative stress. We hypothesize that the activation of mito-u-calpain by calcium overload during ischemia-reperfusion results in decreased AIF content within mitochondria by cleaving AIF. The u-calpain was present within mouse heart mitochondria, mostly in the intermembrane space. Exogenous calcium treatment induced a calpain-dependent decrease of mitochondrial AIF content in isolated mouse heart mitochondria. This process was blocked by a calpain inhibitor (MDL-28170). The Mitochondrial u-calpain activity was increased by 160% ± 15% during ischemia-reperfusion compared to time control. In contrast, the mitochondrial AIF content was decreased by 52% ± 7% during reperfusion vs. time control in the buffer perfused mouse heart. Inhibition of mito-u-calpain using MDL-28170 decreased cardiac injury by preserving AIF content within mitochondria during ischemia-reperfusion. Thus, activation of mito-u-calpain is required to release AIF from cardiac mitochondria. Inhibition of calpains using MDL-28170 decreases cardiac injury by inhibiting both cytosolic calpains and mito-u-calpain during ischemia-reperfusion.

A reversible inhibition of mitochondrial respiration by complex I inhibition at the onset of reperfusion decreases injury in buffer-perfused hearts. Administration of acidic reperfusate for a brief period at reperfusion decreases cardiac injury. We asked if acidification treatment decreased cardiac injury during reperfusion by inhibiting complex I. Exposure of isolated mouse heart mitochondria to acidic buffer decreased the complex I substrate-stimulated respiration, whereas respiration with complex II substrates was unaltered. Evidence of the rapid and reversible inhibition of complex I by an acidic environment was obtained at the level of isolated complex, intact mitochondria and in situ mitochondria in digitonin-permeabilized cardiac myocytes. Moreover, ischemia-damaged complex I was also reversibly inhibited by an acidic environment. In the buffer-perfused mouse heart, reperfusion with pH 6.6 buffer for the initial 5 min decreased infarction. Compared with untreated hearts, acidification treatment markedly decreased the mitochondrial generation of reactive oxygen species and improved mitochondrial calcium retention capacity and inner mitochondrial membrane integrity. The decrease in infarct size achieved by acidic reperfusion approximates the reduction obtained by a reversible, partial blockade of complex I at reperfusion. Extracellular acidification decreases cardiac injury during reperfusion in part via the transient and reversible inhibition of complex I, leading to a reduction of oxyradical generation accompanied by a decreased susceptibility to mitochondrial permeability transition during early reperfusion.