Circadian Rhythm as a Therapeutic Target for Perioperative Cardioprotection

项目总结 这项拨款申请的主要目标是针对围术期心脏保护的昼夜节律。 围术期心肌缺血再灌注损伤(IRI)和心肌梗死仍然是发病率的主要原因 以及外科病人的死亡率。然而，经皮冠状动脉介入治疗联合 由于出血的风险，抗凝和血小板抑制药可能不适合围手术期治疗。 从手术现场找到的。寻找新的药理或干预策略以使心肌 抵抗心肌IRI的有害影响对围手术期和危重护理具有重要意义。 医药。先前的研究表明，心肌IRI的发生遵循昼夜节律模式，这可能 与昼夜节律和缺氧信号之间的相互作用有关。因此，当前的应用程序是 重点是确定可以在治疗上靶向抑制心肌IRI的昼夜节律机制。 为了研究心肌缺血再灌注损伤的昼夜节律控制，我们建立了小鼠原位心肌缺血再灌注损伤模型。 在ZT时间(ZT)8点(下午3点)发现最小的梗塞面积，在ZT 20时(凌晨3点)发现最大的梗塞面积。 心肌损伤小鼠左心室危险区域的后续无偏RNA测序 ZT8的IRI与ZT20的IRI指向了核心的昼夜节律转录因子-BMal1。类似地，RNA-seq 上午组和下午组心脏手术患者的左心室活检数据 证实BMAL1是差异表达最多的基因。在功能研究中，使用带有 肌细胞特异性缺失BMal1(BMal1loxp/loxP myosin CRE+小鼠)，我们观察到完全钝化 ZT8的心脏保护作用。免疫共沉淀和质谱学鉴定缺氧- 诱导因子2-α(HIF2A)作为BMAL1的结合伙伴。类似于BMal1loxp/loxP肌球蛋白Cre+小鼠，我们 观察到ZT8对Hif2aloxp/loxP肌球蛋白Cre+小鼠的心脏保护作用消失。随后的生化和 生物物理研究显示BMAL1和HIF2A之间的直接相互作用显示出4.5埃的电子密度 BMAL1/HIF2A异源二聚体与缺氧反应元件(HRE)DNA的复合体图谱。最后，我们 双调节蛋白(AREG)是BMAL1/HIF2A异源二聚体的关键转录共靶点 昼夜节律依赖的心脏保护。因此，我们假设BMAL1和HIF2A在转录上形成了一个 活性复合体在昼夜依赖性心肌保护中的关键作用 对AREG的监管。我们提出了三个具体目标来解决这一假设：(1)描述相互作用 BMAL1和HIF2A之间的关系及其在体外缺氧和体内心肌IRI中的功能作用。(2)研究 BMAL1/HIF2A-AREG在介导昼夜节律依赖性心脏保护中的共同靶基因(3)目标 BMal1/HIF2A用于围术期心肌缺血再灌注期间的心脏保护和原则验证研究。

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.