Abstract 420: RIP3 Interacts With Mitofilin in the Inner Membrane of Mitochondria to Induce Cardiomyocytes Necrosis After Ischemia/reperfusion

Abstract

Introduction: Receptor-Interacting Protein Kinase 3 (RIP3/RIPK3), a kinase promoting necroptotic cell death, has been shown to mediate adverse remodeling after myocardial infarction. We recently showed that knockdown of the inner membrane of mitochondria protein, mitofilin induces cell death by increasing mitochondria dysfunction via the AIF-PARP axis in rat H9c2 myoblasts. As we found that mitofilin interacts with the mitochondrial permeability transition pore (mPTP) regulator, cyclophilin D (CypD), we investigated whether RIP3 promotes cardiomyocyte death after I/R by increasing mitofilin loss, resulting in mitochondrial cristae structural damage and mPTP opening. Methods: C57BL/6J and RIP3 knockout mice hearts were subjected to 35 min ischemia followed by 180 min reperfusion. Myocardial infarct size was assessed by TTC staining. Mitochondrial function was assessed by mitochondrial ΔΨm, ROS production, and calcium retention capacity (CRC) required to induce mPTP opening. Mitochondrial integrity was measured by analyzing cristae morphology. RIP3 translocation was assessed by measuring the levels in the cytosol and mitochondrial fractions using Western blot and immunofluorescence labeling. The interaction between RIP3, CypD, and mitofilin was measured by co-immunoprecipitation. Results: We found that RIP3 KO mice developed less myocardial infarction as compared to WT and that RIP3 is translocated into mitochondria after I/R. In mitochondria, RIP3 interacts with mitofilin and CypD in the inner mitochondrial membrane (IMM). These interactions were associated with increased ROS production and decreased mitochondrial ΔΨm and CRC. In addition, we also found that RIP3 affects the expression of CypD and mitofilin. Conclusion: After I/R, RIP3 translocates into mitochondria to interact with CypD and mitofilin in the IMM resulting in increased mitochondrial ROS production and a decrease in ΔΨm, both of which promote the opening of the mPTP and destruction of mitochondrial cristae, leading to cardiomyocyte death. This finding points to the key role of RIP3 in cardiomyocyte death.