Circadian Rhythm as a Therapeutic Target for Perioperative Cardioprotection

昼夜节律作为围手术期心脏保护的治疗目标

• 批准号: 10659089
• 负责人: Holger K. Eltzschig
• 金额: $ 66万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-11 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10659089
• 关键词: 3-Dimensional; AREG gene; ARNTL gene; Address; Amino Acids; Amphiregulin; Anticoagulation; Applications Grants; Area; Binding; Biochemical; Biopsy; Cardiac Surgery procedures; Circadian Rhythms; Clinical; Co-Immunoprecipitations; Complex; Critical Care; Cryoelectron Microscopy; DNA; Data; Exposure to; Genes; Genetic Transcription; Genetic study; Goals; Hemorrhage; Heterodimerization; Hour; Hypoxia; In Situ; In Vitro; Infarction; Injury; Intervention; Left; Left ventricular structure; Link; Maps; Mass Spectrum Analysis; Mediating; Medicine; Modeling; Morbidity - disease rate; Mus; Muscle Cells; Myocardial Ischemia; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardium; Myosin ATPase; Operative Surgical Procedures; Outcome; Pathway interactions; Patient-Focused Outcomes; Patients; Pattern; Periodicity; Perioperative; Pharmacology Study; Platelet Inhibitors; Prevention; Prospective Studies; Proteins; Regulation; Research Proposals; Resistance; Resolution; Response Elements; Retrospective Studies; Risk; Role; Severities; Signal Transduction; Site; Structure; Therapeutic Intervention; Time; Transgenic Mice; Variant; Ventricular; aortic valve replacement; bHLH-PAS factor HLF; biophysical analysis; cardioprotection; circadian; differential expression; electron density; in vivo; mortality; mouse model; myocardial injury; novel; novel strategies; novel therapeutic intervention; organ injury; percutaneous coronary intervention; pharmacologic; prevent; promoter; protective pathway; randomized, clinical trials; targeted treatment; therapeutic target; transcription factor; transcriptome sequencing; treatment strategy

PROJECT SUMMARY The main goal of this grant application is to target circadian rhythm for perioperative cardioprotection. Perioperative myocardial ischemia-reperfusion injury (IRI) and infarction continue to be major causes of morbidity and mortality in surgical patients. However, percutaneous coronary intervention in combination with anticoagulation and platelet inhibitors may be unsuitable in the perioperative setting due to the risk of bleeding from the surgical site. Finding novel pharmacologic or interventional strategies to render the myocardium more resistant to the deleterious effects of myocardial IRI would be highly significant for perioperative and critical care medicine. Previous studies indicate that the occurrence of myocardial IRI follows a circadian pattern which could be linked to an interaction between circadian rhythm and hypoxia signaling. Thus, the current application is focused on identifying circadian mechanisms that could be targeted therapeutically to dampen myocardial IRI. To study the circadian control of myocardial IRI, we established a murine model of in situ myocardial IRI and found the smallest infarct sizes at zeitgeber time (ZT) 8 (3 pm), and largest infarct sizes at ZT 20 (3 am). Subsequent unbiased RNA sequencing of the area at risk from the left ventricle of mice exposed to myocardial IRI at ZT8 versus ZT20 pointed us toward the core circadian transcription factor - Bmal1. Similarly, RNA-seq data from left ventricular biopsies of patients undergoing cardiac surgery in the morning versus afternoon groups confirmed BMAL1 as the most differentially expressed gene. In functional studies, using transgenic mice with myocyte-specific deletion of Bmal1 (Bmal1loxp/loxp Myosin Cre+ mice), we observed complete blunting of the cardioprotective effect at ZT8. Co-immunoprecipitation followed by mass spectrometry identified hypoxia- inducible factor 2-alpha (HIF2A) as a binding partner for BMAL1. Similar to Bmal1loxp/loxp Myosin Cre+ mice, we observed abolished cardioprotection at ZT8 in Hif2aloxp/loxp Myosin Cre+ mice. Subsequent biochemical and biophysical studies revealed a direct interaction between BMAL1 and HIF2A showing a 4.5 Å electron density map of the BMAL1/HIF2A heterodimer in complex with hypoxia-response element (HRE) DNA. Finally, we identified amphiregulin (AREG) as a critical transcriptional co-target for BMAL1/HIF2A heterodimer in mediating circadian-dependent cardioprotection. Thus, we hypothesize that BMAL1 and HIF2A form a transcriptionally active complex critical in mediating circadian-dependent cardioprotection via daytime-dependent regulation of AREG. We propose three Specific Aims to address this hypothesis: (1) Characterize the interaction between BMAL1 and HIF2A and their functional roles during hypoxia in vitro or myocardial IRI in vivo. (2) Study the co-target gene of BMAL1/HIF2A – AREG in mediating circadian-dependent cardioprotection. (3) Target BMAL1/HIF2A for cardioprotection and proof-of-principle studies during perioperative myocardial IRI.

项目摘要 这项拨款申请的主要目标是针对围手术期心脏保护的昼夜节律。 围手术期心肌缺血再灌注损伤（IRI）和梗死仍然是发病的主要原因 和手术患者的死亡率。然而，经皮冠状动脉介入治疗结合 由于出血风险，抗凝和血小板抑制剂可能不适用于围手术期 从手术部位。寻找新的药理学或介入策略，使心肌更多 抵抗心肌IRI的有害影响对于围手术期和重症监护具有重要意义 药先前的研究表明，心肌IRI的发生遵循昼夜节律模式， 与昼夜节律和缺氧信号之间的相互作用有关。因此，当前的应用是 重点是确定可以作为治疗靶点以抑制心肌IRI的昼夜节律机制。 为了研究心肌IRI的昼夜节律控制，我们建立了小鼠原位心肌IRI模型， 发现在Zeitgeber时间（ZT）8（3 pm）时梗死面积最小，在ZT 20（3am）时梗死面积最大。 暴露于心肌细胞的小鼠左心室风险区域的后续无偏RNA测序 ZT 8与ZT 20的IRI将我们指向核心昼夜节律转录因子Bmal 1。类似地，RNA-seq 上午与下午组接受心脏手术的患者的左心室活检数据 证实BMAL 1是最差异表达的基因。在功能研究中，使用转基因小鼠， 肌细胞特异性缺失Bmal 1（Bmal 1 loxp/loxp肌球蛋白Cre+小鼠），我们观察到完全钝化的 ZT 8的心脏保护作用。免疫共沉淀和质谱鉴定出缺氧- 诱导因子2-α（HIF 2A）作为BMAL 1的结合伴侣。与Bmal 1 loxp/loxp肌球蛋白Cre+小鼠相似，我们 观察到在ZT 8时在Hif 2aloxp/loxp肌球蛋白Cre+小鼠中消除了心脏保护。随后的生化和 生物物理学研究揭示了BMAL 1和HIF 2A之间的直接相互作用，显示出4.5电子密度 与缺氧反应元件（HRE）DNA复合的BMAL 1/HIF 2A异二聚体图谱。最后我们 鉴定了双调蛋白（AREG）作为BMAL 1/HIF 2A异二聚体介导 昼夜节律依赖的心脏保护作用。因此，我们假设BMAL 1和HIF 2A在转录水平上形成了一个转录调控因子。 活性复合物在介导昼夜节律依赖性心脏保护中的关键作用 AREG的规则。我们提出了三个具体的目标来解决这一假设：（1）表征的相互作用 BMAL 1和HIF 2A之间的关系及其在体外缺氧或体内心肌IRI中的功能作用。(2)研究 BMAL 1/HIF 2A-AREG介导昼夜节律依赖性心脏保护的共同靶基因。(3)目标 BMAL 1/HIF 2A用于围手术期心肌IRI期间的心脏保护和原理验证研究。