Degradation of Mitochondrial Inner Membrane Protein Disrupts the Structural Interaction between Mitofilin and Cyclophilin D and Determines the Extent of Ischemia/reperfusion injury

点击翻译按钮获取中文摘要

Disorders characterized by ischemia/reperfusion (I/R), such as myocardial infarction, stroke, and peripheral vascular disease, continue to be among the most frequent causes of devastating disease and main causes of death in the US. Reperfusion of ischemic tissue triggers many pro-death signaling pathways which converge 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 the mitochondrial permeability transition pore (mPTP). Although several proteins have been proposed as contributing to mPTP formation and function, its exact molecular identity and mechanism still need to be elucidated. Therefore, the current proposal seeks to establish the impact of mitofilin, which controls mitochondrial cristae morphology, regulation in mPTP formation that is responsible for triggering mitochondrial permeability transition is of fundamental importance for advancing our basic understanding of the mechanisms of I/R injury and represents a particularly exciting approach that will open new possibilities for therapeutic interventions against various diseases including I/R injury. Using 2D-DIGE and mass spectrometry, we identified mitofilin as a protein whose expression is significantly reduced after I/R versus sham. We found that versus WT, mitofilin-/- mice subjected to I/R exhibit an increase in myocardial infarct size, a reduction in cardiac functional recovery and Ca2+ retention capacity required to induce the mPTP opening, as well as an increase in mitochondrial Parkin expression and mitofilin ubiquitination. We further found that knockdown of mitofilin in H9c2 myoblasts with siRNA led to an increase in apoptosis via the AIF-PARP1 pathway that is associated with S phase arrest of the cell cycle, an increase in mitochondrial cristae disorganization, ROS production and Calpain activity, as well as a decrease in intracellular ATP production and mitochondrial membrane potential versus scramble siRNA. Interestingly, we also revealed that mitofilin structurally binds to Cyclophilin D and this interaction is abridged after mPTP opening triggered by Ca2+ overload. Our central hypothesis is that degradation mitofilin during I/R disrupts the CypD-mitofilin interaction resulting in pore formation that triggers mitochondrial permeability transition, thus activating necrotic signaling cascades. We will: 1) Define whether protection of mitofilin from degradation induces protective effects against I/R injury and anti-inflammatory effects in vivo, as well as establish the mechanism by which mitofilin down-regulation promotes apoptosis in transfected H9c2 myoblasts; 2. Define the impact of the MEK/ERK/GSK-3 pathway in I/R-induced mitofilin degradation, and reveal the mechanisms by which I/R stress induces mitofilin loss by increasing mitofilin ubiquitination, promoting excessive mitophagy, and increasing Calpain activity; 3. Determine the impact of the mitofilin-Cyclophilin D interaction in mPTP formation. In this proposal, we will test a panel of novel therapeutic approaches that could be ultimately used to improve the survival and outcomes of I/R injury.