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

以缺血/再灌注（I/R）为特征的疾病，如心肌梗死、中风和 外周血管疾病，仍然是最常见的原因，破坏性疾病和主要 美国的死亡原因。缺血组织的再灌注触发许多促死亡信号通路， 聚集到线粒体上事实上，心肌细胞的复氧导致线粒体Ca 2 + 超负荷和活性氧（ROS）产生的增加，触发开放的 线粒体通透性转换孔（mPTP）。尽管有几种蛋白质被认为是 尽管mPTP的形成和功能，其确切的分子身份和机制仍需要进一步研究。 阐明。因此，目前的建议旨在确定丝裂霉素的影响，它控制 线粒体嵴形态，负责触发线粒体mPTP形成的调节 渗透性转变对于推进我们对机制的基本理解具有根本重要性 I/R损伤，并代表了一个特别令人兴奋的方法，将开辟新的可能性， 包括I/R损伤在内的各种疾病的干预。使用2D-DIGE和质谱，我们 将丝裂素鉴定为在I/R后与假手术相比表达显著降低的蛋白质。我们发现 与WT相比，I/R的mitofilin-/-小鼠表现出心肌梗死面积的增加，心肌梗死面积的减少，心肌梗死面积的增加，心肌梗死面积的减少，心肌梗死面积的减少，心肌梗死面积的增加，心肌梗死面积的减少，心肌梗死面积的减少。 诱导mPTP开放所需的功能恢复和Ca 2+保留能力，以及 线粒体Parkin表达和丝裂素泛素化。我们进一步发现，在细胞中， siRNA诱导的H9 c2成肌细胞通过AIF-PARP 1途径导致凋亡增加， 细胞周期的S期阻滞，线粒体嵴紊乱，ROS产生和 钙蛋白酶活性，以及细胞内ATP产生和线粒体膜电位的降低 对比scramble siRNA。有趣的是，我们还发现丝裂素在结构上与亲环素D结合， Ca 2+超载触发mPTP开放后，相互作用被缩短。我们的核心假设是 I/R期间丝裂素降解破坏CypD-丝裂素相互作用，导致孔形成， 线粒体通透性转换，从而激活坏死信号级联。我们将：1）定义是否 保护丝裂素免于降解诱导了抗I/R损伤和抗炎的保护作用 在体内的影响，以及建立机制，其中丝裂素下调促进细胞凋亡， 转染的H9 c2成肌细胞; 2.确定MEK/ERK/GSK-3 β通路在I/R诱导的丝裂素中的影响 降解，并揭示了I/R应激通过增加丝裂素损失诱导丝裂素损失的机制。 泛素化，促进过度线粒体自噬，并增加钙蛋白酶活性; 3.确定 mPTP形成中的丝裂素-亲环素D相互作用。在这项提案中，我们将测试一组新的治疗方法， 这些方法最终可用于改善I/R损伤的生存率和结局。

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.