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.

项目概要： 越来越多的证据表明右心室（RV）纤维化与压力超载功能不良之间存在联系 RV 和肺动脉高压 (PAH) 死亡率。患有失代偿性右心室衰竭 (RVF) 的 PAH 患者 即使使用 PAH 的常规疗法进行治疗，仍会出现持续性 RV 纤维化。裂谷热是导致死亡的主要原因 PAH 和维持 PAH 中的 RV 功能与提高患者生存率相关。不过，目前还没有 专门针对 RV 纤维化的可用疗法。因此，确定 RV 的分子机制 迫切需要开发针对 PAH 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) 位于外侧 线粒体膜 (OMM) 及其底物动力相关蛋白 1 (DRP1)（一种线粒体裂变蛋白） CM 功能失调。我们还表明 DRP1 介导的线粒体裂变限制了基质的大小 腔，从而导致线粒体 Ca2+ (mtCa2+) 短暂升高并持续，以响应胞质 Ca2+ 升高。 使用具有 RV 肥大、衰竭和纤维化的临床前大鼠 PAH 模型，显着 ，我们发现 公钥簿激活 DRP1 磷酸化特异发生 存在于右心室 (RV-CF) 中的心脏成纤维细胞 (CF) 中，但不存在于 CM 中 PAH，随后导致线粒体裂变、线粒体活性氧的 PKD 依赖性增加 物种（mROS）和CF增殖。此外，我们发现 PKD 激活与增加 促凋亡蛋白 Bax 的磷酸化，可抑制 OMM 中凋亡孔的形成，并可能 有助于 PAH 中 RV-CF 的抗凋亡表型。最后，我们还发现 mtCa2+ 通过 mtCa2+ 摄取 mROS 升高和随后 CF 中增殖信号的激活需要单向转运蛋白 (MCU)。基于 根据这些发现，我们假设 1) PKD 依赖性 Bax 磷酸化使 RV-CF 能够抵抗细胞凋亡 根据多环芳烃； 2) PKD 依赖性线粒体裂变通过减小线粒体的大小来限制 mtCa2+ 和抗氧化能力 基质空腔并导致 mtCa2+ 和 mROS 水平增加，从而充当增殖的分子“开关” PAH 中 RV-CF 的信号传导； 3) 体内 OMM 处的 CF 特异性抑制 PKD 可作为一种新的方法 减轻因压力/损伤（如肺动脉高压）而导致的心脏纤维化的治疗。在目标 1 中，我们将把 Bax 打造为一部小说 线粒体中的 PKD 底物并评估 PKD 依赖性 Bax 磷酸化对 OMM 通透性的影响。 为了仅在 OMM 处特异性抑制 PKD 活性，我们将使用 OMM 靶向显性失活 PKD1 (mt-PKD- DN）是我们新近验证的。在目标 2 中，我们将测试线粒体裂变是否依赖于 PKD 增强 通过增加 mtCa2+ 和 mROS 水平促进 RV-CF 增殖。在目标 3 中，我们将测试以下药物的治疗潜力： 在静息 CF 转化为肌成纤维细胞之前，mt-PKD-DN 抑制线粒体 PKD 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 线粒体裂变、Ca2+和 mROS 在 PAH 中 RV-CF 过度增殖和 RV 纤维化中的作用，这将导致 开发一种新策略（即 PKD 抑制）来管理 PAH 情况下的 RV 纤维化和衰竭。