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; 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 该赠款申请的主要目的是针对定期心脏保护的昼夜节律。 围手术期心肌缺血 - 再灌注损伤（IRI）和梗塞继续是发病率的主要原因 手术患者的死亡率。但是，经皮冠状动脉干预与 由于出血的风险 来自手术部位。寻找新颖的药理学或介入策略，以使心肌更多 对心肌IRI的有害作用的抵抗力对于周期性和重症监护非常重要 药品。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传导之间的相互作用联系在一起。那是当前的应用程序 专注于识别昼夜节律机制，这些机制可以针对治疗方法来抑制心肌IRI。 为了研究心肌IRI的昼夜节律控制，我们建立了一种原位心肌IRI和 在Zeitgeber时间（ZT）8（3 pm）时发现了最小的梗塞大小，而ZT 20（上午3点）的梗塞大小最大。 随后从暴露于心肌的小鼠左心室风险的区域的无偏RNA测序 ZT8与ZT20的IRI将我们指向核心昼夜节律转录因子-BMAL1。同样，RNA-Seq 早晨与下午组的患者进行心脏手术的患者的左心室活检的数据 确认为BMAL1为最不同的基因。在功能研究中，使用具有转基因小鼠 BMAL1（BMAL1LOXP/LOXP肌球蛋白CRE+小鼠）的心肌细胞特异性缺失，我们观察到完全钝的 ZT8处的心脏保护作用。共免疫沉淀随后质谱法鉴定出缺氧 - 诱导因子2-α（HIF2A）作为BMAL1的结合伴侣。类似于bmal1loxp/loxp肌球蛋白Cre+小鼠，我们 观察到HIF2Aloxp/Loxp肌球蛋白CRE+小鼠在ZT8处取消的心脏保护作用。随后的生化和 生物物理研究揭示了BMAL1和HIF2A之间的直接相互作用，显示了4.5Å电子密度 BMAL1/HIF2A异二聚体的图与缺氧反应元件（HRE）DNA复合体。最后，我们 鉴定出介导的BMAL1/HIF2A异二聚体的鉴定为临界转录共同目标 依赖昼夜节律的心脏保护。那就是我们假设BMAL1和HIF2A形成了转录 活跃的复合物在通过白天依赖性介导昼夜节律的心脏保护方面至关重要 AREG的调节。我们提出了三个特定的目的，以解决这一假设：（1）表征相互作用 BMAL1和HIF2A及其在体外缺氧或心肌IRI中的功能作用之间。 （2）研究 BMAL1/HIF2A - AREG的共靶基因介导昼夜节律依赖性心脏保护作用。 （3）目标 BMAL1/HIF2A用于定期心肌IRI期间的心脏保护和原理研究证明。