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.

Public Health Relevance

PROJECT NARATIVE In this proposal, we will use a combination of intact animals, isolated working hearts, isolated cardiomyocytes and mitochondria to determine the impact of the interaction between mitofilin and cyclophilin D in the pore formation in that inner membrane of mitochondria that result in the initiation of ischemia/reperfusion injury. This will be accomplished by taking a multidisciplinary approach that will advance our basic understanding of pathology related to ischemia/reperfusion injury including myocardial infarction, stroke, organs transplant as well as Alzheimer?s disease, and ultimately provide novel therapeutic opportunities to develop new compounds to better treat complications of ischemia/reperfusion injury in both women and men.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL138093-04
Application #
9933987
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Wong, Renee P
Project Start
2017-07-15
Project End
2022-06-30
Budget Start
2020-07-01
Budget End
2021-06-30
Support Year
4
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Texas Health Science Center
Department
Physiology
Type
Schools of Medicine
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
Madungwe, Ngonidzashe B; Feng, Yansheng; Lie, Mihaela et al. (2018) Mitochondrial inner membrane protein (mitofilin) knockdown induces cell death by apoptosis via an AIF-PARP-dependent mechanism and cell cycle arrest. Am J Physiol Cell Physiol 315:C28-C43