DEGRADATION OF MITOCHONDRIAL INNER MEMBRANE PROTEIN DISRUPTS THE STRUCTURAL INTERACTION BETWEEN MITOFILIN AND CYCLOPHILIN D AND DETERMINES THE EXTENT OF ISCHEMIA/REPERFUSION INJURY

Disorders characterized by ischemia/reperfusion (I/R), such as myocardial infarction, stroke, andperipheral vascular disease, continue to be among the most frequent causes of devastating disease and maincauses of death in the US. Reperfusion of ischemic tissue triggers many pro-death signaling pathways whichconverge on to the mitochondria. Indeed, reoxygenation of cardiomyocytes leads to mitochondrial Ca2+overload and an increase in reactive oxygen species (ROS) generation that triggers the opening of themitochondrial permeability transition pore (mPTP). Although several proteins have been proposed ascontributing to mPTP formation and function, its exact molecular identity and mechanism still need to beelucidated. Therefore, the current proposal seeks to establish the impact of mitofilin, which controlsmitochondrial cristae morphology, regulation in mPTP formation that is responsible for triggering mitochondrialpermeability transition is of fundamental importance for advancing our basic understanding of the mechanismsof I/R injury and represents a particularly exciting approach that will open new possibilities for therapeuticinterventions against various diseases including I/R injury. Using 2D-DIGE and mass spectrometry, weidentified mitofilin as a protein whose expression is significantly reduced after I/R versus sham. We found thatversus WT, mitofilin-/- mice subjected to I/R exhibit an increase in myocardial infarct size, a reduction in cardiacfunctional recovery and Ca2+ retention capacity required to induce the mPTP opening, as well as an increase inmitochondrial Parkin expression and mitofilin ubiquitination. We further found that knockdown of mitofilin inH9c2 myoblasts with siRNA led to an increase in apoptosis via the AIF-PARP1 pathway that is associated withS phase arrest of the cell cycle, an increase in mitochondrial cristae disorganization, ROS production andCalpain activity, as well as a decrease in intracellular ATP production and mitochondrial membrane potentialversus scramble siRNA. Interestingly, we also revealed that mitofilin structurally binds to Cyclophilin D and thisinteraction is abridged after mPTP opening triggered by Ca2+ overload. Our central hypothesis is thatdegradation mitofilin during I/R disrupts the CypD-mitofilin interaction resulting in pore formation that triggersmitochondrial permeability transition, thus activating necrotic signaling cascades. We will: 1) Define whetherprotection of mitofilin from degradation induces protective effects against I/R injury and anti-inflammatoryeffects in vivo, as well as establish the mechanism by which mitofilin down-regulation promotes apoptosis intransfected H9c2 myoblasts; 2. Define the impact of the MEK/ERK/GSK-3? pathway in I/R-induced mitofilindegradation, and reveal the mechanisms by which I/R stress induces mitofilin loss by increasing mitofilinubiquitination, promoting excessive mitophagy, and increasing Calpain activity; 3. Determine the impact of themitofilin-Cyclophilin D interaction in mPTP formation. In this proposal, we will test a panel of novel therapeuticapproaches that could be ultimately used to improve the survival and outcomes of I/R injury.