Ubiquitous calpains (calpain I and II) are generally recognized as cytosolic proteins. Recently, mitochondrial localized calpain I (mu-calpain) has been identified. Activation of mito-mu-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-mu-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-mu-calpain by calcium overload during ischemia-reperfusion results in decreased AIF content within mitochondria by cleaving AIF. The mu-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 mu-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-mu-calpain using MDL-28170 decreased cardiac injury by preserving AIF content within mitochondria during ischemia-reperfusion. Thus, activation of mito-mu-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-mu-calpain during ischemia-reperfusion. Published by Elsevier Inc.