Abstract 4142203: Reducing reperfusion injury in circulatory death hearts with calpain inhibitor

Background: Donation after Circulatory Death (DCD) donor hearts are rarely utilized for transplantation following more than 30 minutes of ischemia. Calpains 1 and 2 (CPN1/2) are ubiquitous proteases known to exist in cytosol and mitochondria that significantly contribute to reperfusion injury by degrading the ND5 (NADH dehydrogenase subunit 5) and releasing apoptosis inducing factor (AIF). We elected to study the role of CPN1/2 activation in the mitochondria and their inhibition on DCD heart reperfusion injury. Hypothesis: In the DCD hearts mitochondrial CPN1/2 are activated and administration of MDL-28170 (inhibitor of CPN1/2) during reperfusion will decrease cardiac injury. Methods: Male Sprague-Dawley rats were divided in three groups: control beating-heart donors (CBD n=6) without ischemia, DCD with 35 minutes of global ischemia (n=6), and DCD with 35 minutes of ischemia and 10 minutes of reperfusion (n=7). Hearts were procured and mitochondria isolated. Immunoblotting was used to measure CPN1/2 activation. In a separate group of DCD hearts, to assess the protective effect of MDL, hearts were reperfused for 90 minutes on Langendorff setup with MDL (10 μM n=8) while the control DCD hearts were reperfused without MDL (n=7). At the end hearts were collected to measure infarct size using triphenyl tetrazolium chloride staining. Results: There were no significant differences in either AIF or ND5 contents between CBD and DCD ischemia alone hearts (Panel A and B). However, a marked decrease in the contents of AIF and ND5 occurred in DCD hearts mitochondria following reperfusion (Panel A&B) indicating that CLP1/2 were mainly activated in DCD hearts during reperfusion. Although MDL treatment did not markedly improve cardiac function during reperfusion (Panel C), it did decrease infarct size in DCD hearts (Panel D), indicating that cardiac injury can be decreased by inhibiting CPN1/2 in DCD hearts even following 35 minutes of ischemia. Conclusion: Timely administration of selective CPN1/2 inhibitor is a practical approach to decrease cardiac injury in DCD hearts subjected to prolonged period of ischemia.