Mitochondrial Fission, Calcium, ROS in Right Ventricular Fibrosis

右心室纤维化中的线粒体裂变、钙、ROS

• 批准号: 10734675
• 负责人: Bong Sook Jhun
• 金额: $ 38.75万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10734675
• 关键词: Antioxidants; Apoptosis; Apoptotic; Attenuated; Bax protein; Calcium; Cardiac; Cardiac Myocytes; Cause of Death; Cell Membrane Permeability; Cell Nucleus; Chronic; Clinical; Collagen; Cyclic AMP-Dependent Protein Kinases; Data; Development; Dominant-Negative Mutation; Dynamin; Enzymes; Extracellular Matrix; Failure; Fibroblasts; Fibrosis; Functional disorder; Goals; Injury; Left; Link; Mediating; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Myofibroblast; Names; Nuclear; Nuclear Proteins; Outcome; Outer Mitochondrial Membrane; Pathologic; Patients; Phenotype; Phosphorylation; Physiologic intraventricular pressure; Process; Prognosis; Proliferating; Protein Inhibition; Proteins; Pulmonary Hypertension; Rattus; Reactive Oxygen Species; Reporting; Research; Resistance; Right Ventricular Function; Right Ventricular Hypertrophy; Role; Signal Transduction; Stress; Testing; Ventricular; conventional therapy; coronary fibrosis; design; improved; in vivo; interstitial; mitochondrial permeability transition pore; mortality; novel; novel strategies; novel therapeutic intervention; novel therapeutics; pre-clinical; pressure; pro-apoptotic protein; protein kinase D; pulmonary arterial hypertension; response; response to injury; right ventricular failure; therapeutic evaluation; uptake

Project Summary: Growing evidence suggests the link between right ventricular (RV) fibrosis, poor function of the pressure-overloaded RV, and mortality in pulmonary arterial hypertension (PAH). PAH patients with decompensated RV failure (RVF) have persistent RV fibrosis even when treated with the conventional therapies for PAH. RVF is the main cause of death in PAH and maintaining RV function in PAH is associated with improved patient survival. However, there are currently no available therapies that specifically target RV fibrosis. Therefore, identifying the molecular mechanisms underlying RV fibrosis in PAH is urgently needed to develop novel therapeutic approaches targeting RVF in PAH. We recently reported the significant roles of o xidative stress-sensitive protein kinase D (PKD) at outer mitochondrial membrane (OMM) and its substrate dynamin-related protein 1 (DRP1), a mitochondrial fission protein, in dysregulating CM functions. We also showed that DRP1-mediated mitochondrial fission limits the size of the matrix cavity, thus causing elevated and sustained mitochondrial Ca2+ (mtCa2+) transient in response to cytosolic Ca2+ elevation. Using a preclinical rat PAH model with RV hypertrophy, failure, and fibrosis that significant , we found PKD activation and DRP1 phosphorylation occurs specifically in cardiac fibroblasts (CFs) in the RV (RV-CFs), but not in CMs under PAH, which subsequently causes an PKD-dependent increase in mitochondrial fission, mitochondrial reactive oxygen species (mROS), and CF proliferation. Moreover, we found that PKD activation is associated with increased phosphorylation of a pro-apoptotic protein Bax, which inhibits apoptotic pore formation in the OMM and potentially contributes to the anti-apoptotic phenotype of RV-CFs in PAH. Lastly, we also found that mtCa2+ uptake via mtCa2+ uniporter (MCU) is required for mROS elevation and subsequent activation of proliferative signaling in CFs. Based on these findings, we hypothesize that 1) PKD-dependent Bax phosphorylation allows RV-CFs to be resistant to apoptosis under PAH; 2) PKD-dependent mitochondrial fission limits mtCa2+ and antioxidant capacity by decreasing the size of the matrix cavity and causing increased mtCa2+ and mROS levels, thus acting as a molecular “switch” for proliferative signaling for RV-CFs in PAH; and 3) CF-specific inhibition of PKD at the OMM in vivo can be leveraged as a novel therapy to attenuate cardiac fibrosis in response to stress/injury such as PAH. In Aim 1, we will establish Bax as a novel PKD substrate in the mitochondria and assess the impact of PKD-dependent Bax phosphorylation on OMM permeability. To specifically inhibit PKD activity only at the OMM, we will use an OMM-targeted dominant-negative PKD1 (mt-PKD- DN) that we have newly validated. In Aim 2, we will test whether PKD-dependent enhancement of mitochondrial fission facilitates RV-CF proliferation via increased mtCa2+ and mROS levels. In Aim 3, we will test the therapeutic potential of mitochondrial PKD inhibition by mt-PKD-DN in the quiescent CFs before they transform into myofibroblasts by CF- specifically expressing mt-PKD-DN in a preclinical rat PAH model. The proposed project is designed to determine the role of mitochondrial fission, Ca2+, and mROS in RV-CF hyperproliferation and RV fibrosis in PAH, which will lead to develop a novel strategy (i.e., PKD inhibition) for the management of RV fibrosis and failure in the setting of PAH.

项目摘要： 越来越多的证据表明，右心室（RV）纤维化，压力超负荷的功能差， RV和肺动脉高压（PAH）的死亡率。患有失代偿性RV衰竭（RVF）的PAH患者 持续性RV纤维化，即使接受常规PAH治疗。裂谷热是非洲主要的死亡原因， PAH和维持PAH患者的RV功能与改善患者生存率相关。然而，目前没有 现有的专门针对RV纤维化的治疗方法。因此，确定RV的分子机制 因此，迫切需要开发针对PAH中RVF的新治疗方法。 我们最近报道了O的重要作用 氧化应激敏感蛋白激酶 D（PKD）外部 线粒体膜（OMM）及其底物动力蛋白相关蛋白1（DRP 1），一种线粒体分裂蛋白， CM功能失调。我们还发现，DRP 1介导的线粒体分裂限制了基质的大小， 腔，从而引起升高和持续的线粒体Ca 2+（mtCa 2+）瞬态响应胞质Ca 2+升高。 使用具有RV肥大、衰竭和纤维化的临床前大鼠PAH模型， ，我们发现 PKD激活 DRP 1磷酸化特异性地 在RV（RV-CF）中的心脏成纤维细胞（CF）中，但在 PAH，随后导致PKD依赖性线粒体分裂增加，线粒体活性氧 种（mROS）和CF增殖。此外，我们发现PKD激活与增加的 促凋亡蛋白Bax的磷酸化，其抑制OMM中的凋亡孔形成，并可能 有助于PAH中RV-CF的抗凋亡表型。最后，我们还发现，通过mtCa 2+摄取mtCa 2 + 单转运体（MCU）是CF中mROS升高和随后增殖信号传导激活所需的。基于 根据这些发现，我们推测：1）PKD依赖的Bax磷酸化使RV-CF对凋亡具有抵抗性 2）PKD依赖的线粒体分裂通过减少线粒体的大小来限制线粒体Ca 2+和抗氧化能力。 基质腔，并引起增加的mtCa 2+和mROS水平，从而作为一个分子“开关”的增殖 PAH中RV-CF的信号传导;和3）在体内OMM处的PKD的CF特异性抑制可以作为新的免疫抑制剂来利用。 缓解对压力/损伤（如PAH）作出反应的心脏纤维化的治疗。在目标1中，我们将把Bax确立为一部小说， 线粒体中的PKD底物，并评估PKD依赖性Bax磷酸化对OMM渗透性的影响。 为了仅在OMM特异性抑制PKD活性，我们将使用OMM靶向的显性阴性PKD 1（mt-PKD-1）。 我们最近确认的。在目标2中，我们将测试PKD依赖的线粒体分裂增强是否 通过增加mtCa 2+和mROS水平促进RV-CF增殖。在目标3中，我们将测试 线粒体PKD抑制mt-PKD-DN在静止的CF中，然后通过CF转化为肌成纤维细胞， 在临床前大鼠PAH模型中特异性表达mt-PKD-DN。 拟议项目旨在确定 线粒体分裂、Ca 2+和mROS在PAH中RV-CF过度增殖和RV纤维化中的作用，这将导致 开发新的策略（即，PKD抑制）治疗PAH背景下的RV纤维化和失败。