Targeting Mineralocorticoid Receptor Condensates to Optimize Donor Heart Preservation

靶向盐皮质激素受体浓缩物以优化供体心脏保护

• 批准号: 10555801
• 负责人: Paul Tang
• 金额: $ 68.29万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-15 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10555801
• 关键词: Acetylation; Biology; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cell Death; Cells; Cessation of life; Chromatin; Chronic; Clinical; Clinical Trials; Cloning; Combined Modality Therapy; Coupled; Crowding; Cryopreservation; DNA Binding Domain; Data; Dependovirus; Disease; Enhancers; Environment; Evaluation; Event; Evolution; Family suidae; Fibrosis; Future; Genetic Transcription; Geography; Goals; HDAC4 gene; Heart; Heart Hypertrophy; Heart Transplantation; Heart failure; Histidine; Histone Deacetylase; Histone Deacetylase Inhibitor; Hour; Human; Impairment; In Vitro; Inflammation; Inflammatory; Kidney; Kidney Failure; Knowledge; Length; Ligand Binding Domain; Ligands; Liver; Maintenance; Malignant Neoplasms; Mechanics; Mediating; Mediator; Medical; Mineralocorticoid Receptor; Modeling; Molecular; Mus; Myocardial Infarction; Myocardial Ischemia; Myocardial dysfunction; Neonatal; Nuclear; Nuclear Receptors; Organ; Outcome; Output; Oxidative Stress; Pathway interactions; Patients; Perfusion; Phase; Physical condensation; Play; Population; Process; Proteins; Receptor Inhibition; Receptor Signaling; Refractory; Reperfusion Therapy; Research; Resources; Response Elements; Risk; Role; Signal Transduction; Solid; Stroke; Structure; Temperature; Time; Translations; Transplantation; Treatment Failure; Tryptophan; Valproic Acid; Waiting Lists; antagonist; cold temperature; efficacy evaluation; graft dysfunction; heart function; heart preservation; improved; in vivo; interest; liver injury; member; mouse model; mutant; neuropathology; novel; organizational structure; pharmacologic; pre-clinical; preservation; promoter; protein expression; receptor; receptor expression; reconstitution; response; single-cell RNA sequencing; three dimensional structure; transcription factor; transplant model

Project Summary/Abstract Heart transplantation is recognized as the gold standard therapy for end-stage heart failure. However, demand for donor hearts currently far outstrips supply due to multiple factors. An important limitation is primary graft dysfunction (PDG) in 10-20% of transplants and is an important contributor to adverse clinical outcomes and increased resource utilization. PGD occurs when donor heart function is inadequate for end organ perfusion and the risk increases once cold preservation time is >4 hours. Mineralocorticoid receptor (MR) signaling has a key role in many cardiovascular diseases including heart failure, and cardiac hypertrophy. It mediates harmful processes such as oxidative stress, inflammation, and fibrosis. Using murine models with MR deletions in cardiomyocytes as well as clinically utilized mineralocorticoid receptor antagonists (e.g. canrenone), we show that MR antagonism can greatly improve donor heart function following preservation. We also demonstrate that cardiac preservation is associated with increased MR protein expression coupled with organization of MR into molecular condensates with organizational structures that are known to augment protein transcription and translation. Importantly we find this occurs both in murine models as well as human hearts suggesting conserved events during evolution. We also confirm that MR contains intrinsic disordered regions which mediate phase separation and condensate formation. Our plan is to: (1) Identify the MR domains that can mediate condensate formation at differing temperatures. We will also determine if MR ligands, MR response elements and histone deacetylases (HDAC) can facilitate MR condensate formation. This will be performed by in-vitro evaluation of condensate formation by cloning truncated MR constructs missing specific domains and then adding different mediators of interest (e.g. HDAC) in the presence of a crowding agent. (2) We will examine the effects of MR condensate formation by first deleting MR in cardiomyocytes and then use adeno- associated virus to reconstitute cardiomyocytes with a MR mutant that has an impaired ability to form condensates because it is missing the intrinsic disordered region. We will then compare the inflammatory, oxidative stress and cell death responses associated with full length MR and the MR mutant. This will be evaluated in-vivo in a transplant model which incorporates recipient responses, ex-vivo in a perfusion model consisting only of the native cardiac cells and in an in-vitro neonatal cardiomyocyte preservation-reperfusion culture model. (3) We will determine the efficacy of canrenone (a MR antagonist) with or without valproic acid (a HDAC inhibitor) for improving donor heart function in pigs and humans. Single-cell RNA sequencing will be performed in human hearts to determine the cell population in which MR signaling is important for preservation. Our findings will contribute towards decreasing PGD occurrence, increasing donor heart utilization, and improving transplant outcomes. This is expected to have broad clinical implications such as transplantation of other solid organs (e.g. liver, kidneys) and for warm cardiac ischemia settings (e.g. myocardial infarction).

项目摘要/摘要 心脏移植被公认为治疗终末期心力衰竭的金标准。然而，需求 由于多种因素，目前供者心脏远远供不应求。一个重要的限制是一次移植物。 10%-20%的移植患者出现功能障碍(PDG)，是导致不良临床结果的重要因素 提高了资源利用率。当供体心脏功能不足以维持终末器官灌流时，就会发生PGD 当冷藏时间为4小时时，风险增加。盐皮质激素受体(MR)信号转导途径 在许多心血管疾病中起关键作用，包括心力衰竭和心肌肥厚。它调停有害的 氧化应激、炎症和纤维化等过程。使用MR缺失的小鼠模型 心肌细胞以及临床上使用的盐皮质激素受体拮抗剂(例如坎利酮)，我们显示 MR拮抗可显著改善供心保存后的功能。我们还证明了 心脏保存与MR蛋白表达增加和MR组织成 分子缩合物具有已知的组织结构，可增强蛋白质转录和 翻译。重要的是，我们发现这种情况在小鼠模型和人类心脏中都存在，这表明 进化过程中的保守事件。我们还证实了MR包含本征无序区域， 中间相分离和凝析油形成。我们的计划是：(1)确定能够 调节不同温度下凝析油的形成。我们还将确定莱兰兹先生是否会做出回应 元素和组蛋白脱乙酰酶(HDAC)可以促进MR凝聚体的形成。这将由以下人员执行 克隆缺失特定结构域的截短型MR结构物体外评价凝集液的形成 然后在拥堵剂存在的情况下加入不同的感兴趣的介体(例如HDAC)。(2)我们会 通过首先删除心肌细胞中的MR，然后使用腺病毒来检查MR凝集形成的影响。 相关病毒用MR突变体重建心肌细胞，该突变体形成能力受损 凝聚是因为它缺少固有的无序区。然后我们将比较炎症性的， 与全长MR和MR突变体相关的氧化应激和细胞死亡反应。这将是 在包含受体反应的移植模型中进行体内评估，在体外灌流模型中进行评估 仅由天然心肌细胞组成的体外培养新生心肌细胞保存-再灌流 文化模式。(3)我们将确定坎利酮(MR拮抗剂)与丙戊酸一起或不与丙戊酸一起使用的疗效 (一种HDAC抑制剂)用于改善猪和人类的供体心脏功能。单细胞RNA测序将是 在人的心脏中进行，以确定在其中MR信号对保存重要的细胞群。 我们的发现将有助于减少PGD的发生，提高供体心脏的利用率，以及 改善移植结果。预计这将具有广泛的临床意义，例如移植 其他实体器官(如肝脏、肾脏)和热性心脏缺血(如心肌梗塞)。