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.

项目总结： 越来越多的证据表明，右室(RV)纤维化与压力超负荷功能低下之间存在联系 RV，以及肺动脉高压(PAH)的死亡率。伴有失代偿性RV衰竭(RVF)的PAH患者 持续的右室纤维化，即使用传统疗法治疗PAH也是如此。裂谷热是#年的主要死亡原因。 PAH和维持PAH中的RV功能与改善患者的存活率有关。然而，目前还没有 针对RV纤维化的可用治疗方法。因此，确定RV的分子机制 PAH中的纤维化迫切需要开发针对PAH中RVF的新的治疗方法。 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)在外部 线粒体膜(OMM)及其底物动力蛋白相关蛋白1(Drp1)是一种线粒体分裂蛋白 CM功能失调。我们还发现，drp1介导的线粒体分裂限制了基质的大小。 空洞，从而引起线粒体钙离子(mtCa~(2+))的升高和持续的一过性响应胞浆钙离子升高。 使用临床前大鼠右室肥大、衰竭和纤维化的模型 ，我们发现 PKD激活 而Drp1的磷酸化是专门发生的 在RV中的心脏成纤维细胞(CFs)中(RV-CFs)，而在CMS中不 PAH，随后导致依赖于PKD的线粒体分裂、线粒体活性氧增加 物种(MRO)和CFs增殖。此外，我们还发现，PKD的激活与 促凋亡蛋白Bax的磷酸化，它抑制OMM中的凋亡孔形成，并可能 有助于RV-CFs在PAH中的抗凋亡表型。最后，我们还发现线粒体通过线粒体摄取钙。 在CFs中，单转运蛋白(MCU)是MRO升高和随后的增殖信号激活所必需的。基于 这些发现，我们假设1)依赖PKD的Bax磷酸化使RV-CFs具有抗细胞凋亡的能力 在PAH下；2)依赖PKD的线粒体分裂通过减小线粒体的大小来限制mtCa+和抗氧化能力 基质空洞，并导致线粒体钙离子和MROS水平增加，从而充当增殖的分子“开关” RV-CFs在PAH中的信号转导；以及3)在活体OMM处CFs对PKD的特异性抑制可以作为一种新的 减轻因应激/损伤(如PAH)而导致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 并评估依赖PKD的Bax磷酸化对OMM通透性的影响。 为了仅在OMM处特异性地抑制PKD活性，我们将使用OMM靶向的显性负性PKD 1(mt-PKD- Dn)，这是我们最新验证的。在目标2中，我们将测试依赖于PKD的线粒体分裂增强 通过增加线粒体钙离子和MROS水平来促进RV-CF的增殖。在目标3中，我们将测试 线粒体PKD-dN对CFs转化为肌成纤维细胞前线粒体PKD的抑制作用 在临床前期大鼠PAH模型中特异性表达mt-PKD-dN。 拟议的项目旨在确定 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 开发一种新的策略(即，抑制PKD)来管理PAH中的RV纤维化和失败。