Circadian Rhythm as a Therapeutic Target for Perioperative Cardioprotection

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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.