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，仍然是最常见的破坏性疾病的原因之一和主要 美国的死因。缺血组织再灌注触发许多促死亡信号通路，其中 汇聚到线粒体上。的确，心肌细胞的复氧导致线粒体钙离子的产生。 过载和活性氧物种(ROS)生成的增加，触发了 线粒体通透性转换孔(MPTP)。尽管有几种蛋白质被提出为 MPTP的形成和功能，其确切的分子同一性和作用机制尚需进一步研究。 已澄清。因此，目前的提案寻求确定米托菲林的影响，它控制 线粒体嵴形态，MPTP形成中的调节，负责触发线粒体 渗透性转变对于提高我们对其机理的基本认识具有重要意义。 这是一种特别令人兴奋的方法，将为治疗 对包括I/R损伤在内的各种疾病的干预。利用2D-DGE和质谱学，我们 结果表明，与假手术相比，I/R后丝裂霉素的表达显著降低。我们发现 与WT相比，接受I/R的MITOFILIN-/-小鼠表现出心肌梗死面积的增加，心脏的减少 诱导MPTP开放所需的功能恢复和钙保持能力，以及 线粒体Parkin表达与丝裂霉素泛素化。我们进一步发现米托菲林的基因敲除 携带siRNA的H9c2成肌细胞通过AIF-PARP1途径促进细胞凋亡 S期细胞周期停滞，线粒体脊解体增多，ROS生成增多 钙蛋白酶活性，以及细胞内ATP产量和线粒体膜电位的下降 而不是杂乱的siRNA。有趣的是，我们还揭示了丝裂霉素在结构上与亲环素D结合，这 钙超载触发MPTP开放后，相互作用减弱。我们的中心假设是 I/R过程中降解的丝裂霉素破坏CypD-Mmitofin的相互作用，导致孔道形成，从而触发 线粒体通透性转变，从而激活坏死信号级联。我们将：1)定义是否 米托菲林抗I/R损伤和抗炎作用的实验研究 在体内的作用，以及建立丝裂霉素下调促进细胞凋亡的机制 2.明确MEK/ERK/GSK-3通路在I/R诱导的丝裂原活化中的作用 降解，揭示了I/R应激通过增加丝裂霉素而导致丝裂霉素丢失的机制 泛素化，促进过度的丝裂原吞噬，并增加钙蛋白酶活性；3.确定 MPTP形成中的丝裂霉素-亲环素D相互作用。在这项提案中，我们将测试一组新颖的治疗方法 最终可用于改善I/R损伤的存活率和预后的方法。

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.