Detailed information on experimental materials, reagents and mouse lines, including sources and identifiers, is provided in the key resources table in Supplementary Table 12.
Human participant research
Study design and participants
We examined samples from a prospective study of myocardial injury in humans undergoing cardiac surgery (ClinicalTrials.gov: NCT00281164). The study population consisted of consecutive patients (aged ≥20 years) with aortic stenosis referred to our cardiovascular surgery department at Brigham and Women’s Hospital for aortic valve replacement (with or without coronary artery bypass graft) between 1 January 2009 and 31 December 2014. Patients enrolled in a concurrent drug or device trial were excluded. This ongoing study involved 56 patients in the morning (samples collected between 08:00 and 12:00; median time, 10:32) and 17 patients who underwent the same procedure in the afternoon (samples collected between 15:00 and 21:00; median time, 17:15). Patients whose surgery fell outside of these time periods were excluded from the analysis. The ethics committee for the Protection of Human Subjects from Research Risks of Brigham and Women’s Hospital approved the protocol, and written informed consent was obtained from all of the patients.
Procedures
Patients underwent aortic valve replacement in the morning or afternoon by the same senior surgeon. Anaesthesia, cardiopulmonary bypass, cardioplegia and surgical procedures were done according to standard guidelines. Anaesthesia was induced with intravenous fentanyl or sufentanil and propofol (0.5–1.5 mg per kg) and maintained with isoflurane. Surgery was done using normothermic cardiopulmonary bypass and repeated antegrade and retrograde cold blood cardioplegia. LV biopsy samples were obtained from both the morning and afternoon patient cohorts before and after approximately 80 min of aortic cross-clamping—a procedure that temporarily halts blood flow to the heart. The samples were taken from the site of the routinely placed LV vent.
Animal care and use
Animal care was performed according to the guide for the care and use of laboratory animals of the National Institutes of Health. All experimental procedures were approved by the UTHealth Institutional Animal Care and Use Committee. The sample size was estimated based on published literature on previous murine models of myocardial IRI27. All of the mice were housed under a standard 12 h–12 h light–dark photoperiod at 22 °C with ad libitum access to a standard chow diet, and maintained at a humidity level of 40–60%.
Generation of transgenic mouse models
To generate cardiomyocyte-specific deletion, myosin-Cre+ (A1cfTg[Myh6-cre/Esr1*]1Jmk/J, The Jackson Laboratory, 005650), Hif1aloxP/loxP (B6.129-Hif1atm3Rsjo/J, The Jackson Laboratory, 007561), Hif2aloxP/loxP (Epas1tm1Mcs/J, The Jackson Laboratory, 008407), LSL-HIF2dPA (Gt(ROSA)26Sortm4(HIF2A*)Kael/J, Jackson Laboratory, 009674), and Bmal1loxP/loxP (B6.129S4(Cg)-Bmal1tm1Weit/J, The Jackson Laboratory, 007668) mice, aged 8 weeks, were purchased from The Jackson Laboratory and crossbred as previously described16,27. To address sex as a biological variable, both male and female mice aged 8 to 16 weeks underwent myocardial ischaemia and reperfusion surgery. Our sex-specific analysis, which included assessments of infarct size, troponin levels and cardiac function, revealed no significant differences between sexes29,50. To induce Cre-recombinase activity, mice received an i.p. injection of tamoxifen at a dosage of 1 mg per day for five consecutive days, as previously described29,30,50. A recovery period of 7 days was allowed after the final tamoxifen dose before proceeding with further experimental procedures. In these experiments, myosin-Cre+ mice were used as controls. Moreover, Areg−/− mice (B6;129-Aregtm1Dle/Mmnc, MMRRC, MMRRC_011533-UNC)51 aged 8 weeks were purchased from Mutant Mouse Resources & Research Centers. To circumvent lactation difficulties observed in younger female mice, Areg−/− mice were bred using a heterozygous (Areg+/−) strategy16,41. Control mice for these experiments were C57BL/6J (The Jackson Laboratory, 000664) mice aged 8 weeks, chosen for their same genetic background as the Areg−/− mice. Genotyping for all mouse strains was conducted by GeneTyper (NY).
Mouse model of myocardial IRI
The mouse model used in the study was subjected to a period of entrainment for at least 2 weeks within circadian cabinets (Actimetrics) to establish a stable circadian rhythm42. After this acclimatization period, the mice underwent an in situ procedure for myocardial IRI at different times of day (ZT2, ZT8, ZT14 or ZT20), as described in previous publications27,29,30,50,52. In brief, the mice were anaesthetized using 1–3% isoflurane delivered by a compact small-animal anaesthesia device (RWD), followed by endotracheal intubation and ventilation using a VentStar Small Animal Ventilator (RWD). Once anaesthetized, the mice were placed into a supine position and received pre-incisional analgesia with sustained-release buprenorphine (0.1 mg per kg, subcutaneous; ZooPharm). Throughout the procedure, their body temperature was consistently maintained at 37 °C with a ThermoStar Homeothermic Blanket equipped with rectal feedback control (RWD). The surgical procedure was meticulously performed using a research stereomicroscope system, SZX10 (Olympus). We temporarily ligated the proximal left anterior descending coronary artery approximately 2 mm from its emergence beneath the left atrium, using Surgipro II 7–0 monofilament polypropylene sutures (Covidien). The success of this ligation was confirmed by observing blanching or a pale discolouration of the anterior wall of the heart, a reduction in wall movement and the presence of ST-segment elevation on an electrocardiogram (ECG). After 45 min of induced ischaemia, reperfusion was initiated to restore the blood flow. The effectiveness of reperfusion was verified both by the resolution of ST-segment elevation on the ECG and through direct visual inspection, where a return of colour and movement in the previously affected area of the heart was noted. After surgery, the mice were carefully transferred back to the circadian cabinets for recovery and continued observation. Any mice that did not survive within the first 48 h after MI were considered to have experienced technical complications and were consequently excluded from further analysis. Blinding was implemented for the individuals analysing the data, including those calculating infarct size and measuring serum troponin I levels, to ensure unbiased data interpretation. However, blinding the surgeons performing the surgeries was logistically challenging due to the nature of the procedures. Despite this, the surgeries were carried out by experienced surgeons, ensuring consistency. To confirm the similarity of conditions, we calculated the AAR/LV, which showed comparable values across ZT and treatment/vehicle groups. This suggests that any differences observed in the study are largely due to experimental conditions rather than variations in the surgical procedure.
Cell line
All cell lines and primary cells used in this study, including HEK293 cells (ATCC, CRL-1573) and human primary cardiomyocytes (ScienCell Research Laboratories, 6200), were authenticated by short-tandem-repeat profiling, as performed by the manufacturers. Moreover, the cell lines were tested for mycoplasma contamination by the manufacturers and were subsequently tested monthly for potential contamination throughout the study. All mycoplasma contamination tests yielded negative results. Furthermore, none of the cell lines used in this study are listed in the database of commonly misidentified cell lines maintained by the International Cell Line Authentication Committee (ICLAC) and NCBI Biosample.
Timed administration of AREG and NOB in mouse models
Timed administration of AREG
To simulate the clinical scenario in which treatment is initiated after the onset of MI, mouse recombinant AREG protein (R&D Systems) was prepared in 0.9% NaCl solution, and a dosage of 10 µg AREG was administered at the start of the reperfusion phase. Furthermore, to assess the long-term effects of timed AREG treatment on cardiac function, a post-injury treatment regimen was implemented. In this protocol, AREG (10 µg) was administered through i.p. injection at either ZT8 or ZT20 daily for the first 3 days after myocardial IRI. For control purposes, an equivalent volume of 0.9% NaCl solution was administered as a vehicle.
Administration of NOB
In the NOB treatment experiments, Mice were administered either DMSO (as a vehicle control) or NOB at a dose of 200 mg per kg body weight. The administration was carried out through i.p. injection and repeated on an every-other-day basis. This treatment protocol was followed for 2 weeks before the surgical procedure and was specifically timed within the ZT14–ZT20 time window. To evaluate the long-term effects of NOB on cardiac function and remodelling, the same dosing regimen was continued after IRI. The chosen dosing regimen was based on several considerations: first, the dosage range was consistent with those used in previous studies (100–200 mg per kg per day)42,53, and it was aligned with the active phase of the mice. Second, daily dosing was intentionally avoided to prevent the entrainment of the experimental mice to the dosing schedule as an artificial zeitgeber. Third, previous single-dose pharmacokinetic assays demonstrated a favourable pharmacokinetic profile for NOB, with significant exposure detected in the serum, brain and liver42. Considering that NOB levels were typically undetectable within 8–24 h after administration42,54, we opted for an every-other-day dosing strategy to prevent incomplete clearance over the 4-week experimental period.
Mouse RNA-seq and bioinformatic analysis
The AAR of the heart was collected after 2 h of reperfusion from the C57BL/6J mice subjected to myocardial IRI at either ZT8 or ZT20. Total RNA was extracted using the RNeasy Mini kit (Qiagen) and was used to construct RNA-seq libraries, which were then sequenced using the Illumina 1.9 platform (75 bp paired end). Alignment of RNA sequencing tags was restricted to those mapping to the same DNA strand as annotated in the GRCm38 reference genome, using STAR (v.2.7.10a). Quantification of transcripts was conducted by calculating the fragments per kilobase of transcript per million mapped reads (FPKM) values, along with transcript counts. In the preprocessing step of mRNA analysis, any gene identified as non-expressed, defined as having an FPKM expression level less than 1 in more than 80% of samples, was excluded. The normalized expression profiles of these mRNAs were then subjected to PCA for quality control and to evaluate sample similarity. Differential expression analysis was performed using the DESeq255 pipeline, with DEGs being identified based on a threshold of 1.5-fold change and an adjusted P < 0.05, as determined using the Benjamini–Hochberg method56.
The functional enrichment analysis of the identified DEGs was conducted using the GO and KEGG pathway databases with the WebGestalt tool57 (http://www.webgestalt.org/; v.2019). For rigorous validation, only pathways and GO terms with an adjusted P < 0.05 were included. In constructing the gene regulation network, we used known transcription factor (TF)–mRNA interactions from the TRRUST_v258 and Chipbase_v259 databases. Moreover, the mouse protein–protein interaction (PPI) network was integrated from the STRING database (v.11.5)60, with a focus on interactions having a combined score above 900. Considering the limited sample size at each timepoint, we merged six samples from both conditions to calculate the Spearman’s correlation coefficient (SCC), ensuring robust and meaningful analysis. In finalizing the gene regulation network, we merged the TF–mRNA regulation network with the PPI data, specifically targeting interactions where both genes were differentially expressed. We also selectively incorporated edges with high SCC into our network, tailored to align with the specific conditions of our experimental design.
Human RNA-seq and bioinformatic analysis
For human RNA-seq and subsequent bioinformatic analysis, we began by excluding outlier samples that failed to cluster appropriately, resulting in a total of 73 samples for analysis, including 56 morning and 17 afternoon samples derived from various sequencing protocols. Quality control of raw sequencing reads identified adaptor sequences from the Illumina Nextera platform in some samples, which were subsequently trimmed using Cutadapt (v.4.1)61. kallisto (v.0.46.1)62 was used to quantify transcript-level expression by mapping to a transcript index built from GENCODE human transcript (v.44)63. We next used tximport to convert these transcript-level quantifications to gene read counts64. Differential expression analysis was performed with DESeq2 (v.1.34.0)55, with particular attention paid to sequencing batch to control for batch effects. Genes displaying a log2-transformed fold change of greater than 0.5 and P < 0.01 were deemed to be significant. For enrichment analysis, we used the GO annotation (v.1.1) for humans, using all genes annotated by at least one GO term65 as the background in Fisher’s exact test to identify over-represented biological processes. Moreover, the R package KEGGREST (v.1.36.0) was used to extract KEGG pathway annotations, and Cytoscape (v.3.10.0) was used to construct network plots for the identified KEGG pathways.
Microarray data reanalysis
The microarray assay for gene expression transcript levels of post-ischaemic myocardium from myosin-Cre+ or Hif2aloxP/loxP myosin-Cre+ mice was reanalysed using data obtained from the GEO (GSE67308)16. Differential expression analysis was performed using GEO2R, with limma precision weights applied and the remaining options set to default66.
Assessment of infarct size in the acute phase
The assessment of myocardial infarct size was conducted by determining the percentage of infarcted myocardium within the AAR using a previously established method16,27,29,30,50. After 2 h or 1 day of reperfusion, the hearts were flushed with PBS and then subjected to permanent occlusion of the left coronary artery. 1 ml of 1% Evans blue dye (Sigma-Aldrich, E2129) was then infused through the carotid artery catheter. After infusion, the hearts were excised and sectioned into 1 mm slices using a microtome (Roboz, SA-4130). These heart sections were subsequently double-stained with 1% TTC (Sigma-Aldrich, T8877) for 10 min at 37 °C and then fixed in 10% formalin overnight. The double-stained heart slices were photographed, and ImageJ software (NIH, Fiji v.2.1.0) was used to calculate the infarct size.
Cardiac troponin I enzyme-linked immunosorbent assay
Serum samples were collected from mice subjected to myocardial IRI at various ZTs through the inferior vena cava after 2 h of reperfusion. The measurement of serum troponin I, a highly sensitive biomarker for cardiac injury, was conducted using the mouse cardiac troponin-I SPARCL kit (Life Diagnostics, CTNI-SP-1), as we have done previously16,27,29,30,50.
STE analysis of mouse cardiac function
Ultrasound imaging
Cardiac function and structure were assessed by transthoracic echocardiography using the Vevo3100 Ultrasound system (VisualSonics). Mice were lightly sedated with 0.5–1.0% isoflurane and positioned on a heated platform equipped with ECG monitoring. Validation criteria included consistent, uninterrupted tracking of the endocardium throughout the cardiac cycles, maintaining a heart rate between 450 and 550 beats per minute, and a frame rate exceeding 250 fps. Two-dimensional grey-scale echocardiographic images were obtained from parasternal long-axis and short-axis views. Longitudinal strain represented myocardial shortening at the endocardium, while radial strain indicated shortening at the mesocardium. All image acquisitions and subsequent offline measurements were conducted by a single investigator who was blinded to the grouping of the animals.
Speckle-based deformation mapping and analysis
For strain analysis using Vevo LAB (FUJIFILM VisualSonics, v.5.7.1), we carefully selected suitable B-mode loops, ensuring a clear view of the endocardial border and no image artefacts. Three consecutive cardiac cycles with distinct ECG recordings were chosen for in-depth analysis. Semi-automated tracing of the endocardial and epicardial borders was performed, with adjustments made as necessary to maintain optimal tracking quality throughout each cine loop. The tracked images were then processed frame by frame for strain measurements34,67,68. The resulting strain values were averaged across these cardiac cycles, providing comprehensive data on the LV systolic function (EF, FS and GLS), LV size (end-diastolic volume and end-systolic volume) and LV mass (end-diastolic LV mass and end-systolic LV mass).
In analysing regional and global cardiac dynamics, such as contractility and synchronicity, the LV endocardium was delineated using 48 sampling points34,67. This method divided the chamber into six segments for detailed examination in the long-axis view: basal anterior, mid anterior, apical anterior, basal posterior, mid posterior and apical posterior segments67. In myocardial IRI models, the mid anterior, apical anterior and apical posterior wall segments were designated as the infarct region, and the remaining segments were categorized as the non-infarct region34. Peak systolic strain, indicating the percentage change in length during myocardial contraction and relaxation, was calculated using the formula: ε = (L1 − L0)/L0, where L0 is the initial length and L1 is the final length69. This calculation was applied to each segment to evaluate the peak systolic strain (%) and the time-to-peak systolic strain (ms). Abnormal ventricular contractility patterns were evaluated on the basis of the magnitude of systolic strain (segment peak strain) and its timing (peak of shortening). Dyssynchrony was specifically identified by a pattern of reduced systolic strain magnitude, early opposing deflection and a delayed time-to-peak systole. Akinesis was defined as minimal or no contractility, with peak systolic strain values between −5% and 5%. Dyskinesis was described as systolic motion of the ventricle occurring in the opposite direction of normal contraction. Moreover, intraventricular disparity was measured by assessing the intraventricular delay in the time-to-peak strain and the s.d. of the time-to-peak strain across segments34,70.
Sequential hypoxia treatment after synchronization in vitro
HCMs were obtained from ScienCell Research Laboratories (ScienCell, 6200) and isolated from human hearts71. The cells were synchronized using 200 nM dexamethasone, which was replaced with complete media (ScienCell, 6101) after 1 h. After synchronization, we initiated a series of hypoxia treatments, exposing different batches of cells to 1% oxygen for 4 h at systematic 4-h intervals to capture cellular responses at various circadian phases. The first hypoxia session started immediately after synchronization (CT0) and lasted for 4 h (referred to as the CT4 timepoint). Subsequent treatments were applied every 4 h: from CT4 to CT8 (CT8 timepoint), and so on, continuing up to CT40. Immediately after each hypoxia period, cells were collected to evaluate transcript and protein levels.
To ensure valid results, we carefully selected timepoints for post-synchronization analysis. After synchronization, cells require a stabilization period to re-establish their circadian rhythms, and early evaluations may capture this adjustment phase rather than true circadian behaviour72. Moreover, dexamethasone induces immediate stress responses and activates signalling pathways unrelated to circadian changes73,74. Administering hypoxia treatments too soon after synchronization could overlap these stress responses, confounding the circadian-related changes we aim to measure. For this reason, early timepoints like CT8 were excluded, and we focused on later timepoints to minimize these effects and ensure that the data reflect circadian-driven changes.
ChIP–qPCR
HCMs were synchronized by dexamethasone (200 nM) for 1 h and then exposed to normoxia or hypoxia (1% oxygen) for 4 h at CT20 and CT32 in Fig. 3q. HEK293 cells were transfected with pcDNA3-mBmal1 (a gift from A. Sancar, Addgene, 31367)75 and treated with normoxia or hypoxia (1% oxygen) for 4 h in Fig. 3s. ChIP–qPCR was conducted using the SimpleChIP enzymatic chromatin IP kit (Cell Signaling Technology, 9002). In brief, cells were fixed with 1% formaldehyde, quenched with 125 mM glycine, washed and sonicated to digest the DNA to an average length of 150–500 bp. Lysates were then incubated overnight at 4 °C with 2 µg of ChIP grade anti-HIF2A antibody (Novus Biologicals, NB100-122) and anti-BMAL1 antibody (Cell Signaling Technology, 14020), anti-HIF1B antibody (Cell Signaling Technology, 5537) or IgG as a negative control. After washing, the antibody–protein–DNA complexes were eluted from the beads. The cross-links were then reversed through overnight incubation at 65 °C, followed by DNA purification. For the qPCR analysis, specific primer pairs were designed to amplify the predicted common binding sequence (CAGGTG) on the human AREG promoter region or E-box regions on the human HIF2A promoter region. The enrichment was quantitatively compared against input controls and IgG negative controls to confirm the specificity of the ChIP-qPCR assay.
Co-IP analysis
HEK293 cell lines (Fig. 2b and Extended Data Fig. 2b) overexpressing the pcDNA3-mBmal1 (a gift from A. Sancar, Addgene, 31367)75 were exposed to either normoxia or hypoxia (1% oxygen) for 4 h. In Fig. 3r, HCMs were initially synchronized using dexamethasone (200 nM) for 1 h and then exposed to hypoxia (1% oxygen) for 4 h at CT20 and CT32. After exposure, the cytoplasmic and nuclear fractions were separately extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, 78835), according to the manufacturer’s instructions. The cell lysates were centrifuged and pre-cleared with protein A/G beads (Thermo Fisher Scientific, 53133) for 1 h with rotation. For the IP, the pre-cleared lysates were incubated overnight at 4 °C with specific antibodies: mouse anti-Flag (Sigma-Aldrich, F1804) or rabbit anti-HIF2A (Novus Biologicals, NB100-122) antibodies. Mouse IgG (Cell Signaling Technology, 5415) and rabbit IgG (Abcam, ab172730) were used as isotype controls for the respective antibodies. After the overnight incubation, 40 μl of protein A/G beads were added to each sample, and the incubation was continued for an additional 4 h at 4 °C. The proteins were then eluted using an SDS sample buffer and subjected to heating at 95 °C for 5 min with vigorous shaking. The eluted proteins were subsequently stored at −80 °C for further analysis. In Fig. 2d, HCMs were subjected to hypoxic conditions (1% oxygen) for various durations: 0, 0.5, 1, 2, 4 or 8 h. After each exposure period, HCMs from a single 10-cm dish were lysed using 330 µl of HEPES extraction buffer containing 20 mM HEPES pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 5% glycerol, and a cocktail of protease, phosphatase and RNase inhibitors. Co-IP was then performed according to the previously described protocol. In Extended Data Fig. 7w,x, HEK293 cells were transfected with UB–HA, HIF2A–MYC or BMAL1–Flag expression constructs. To assess the ubiquitination of HIF2A, cells were treated with 10 µM MG132 for 16 h. After treatment, the cells were collected and lysed using HEPES extraction buffer. Immunoprecipitation was then performed using rabbit anti-HIF2A antibodies (Novus, NB100-122) as previously described.
CHX chase assay
HCMs were transduced with Bmal1-AAV (105 GC per cell) or shBMAL1-AAV (105 GC per cell) and exposed to 1% O2 for 4 h to stabilize HIF1A and HIF2A. After the return to normoxic conditions, CHX (20 μg ml−1, Sigma-Aldrich, C4859) was added to inhibit de novo protein synthesis. Cells were collected at 15, 30, 60, 90 and 120 min after CHX treatment, washed with cold PBS, and lysed in RIPA buffer containing protease and phosphatase inhibitors. After centrifugation, protein concentrations were determined using a BCA protein assay kit. Equal amounts of protein (10–20 μg) were separated by SDS–PAGE and probed with primary antibodies against HIF2A (Novus Biologicals, NB100-122, 1:1,000), HIF1A (Novus Biologicals, NB100-479, 1:1,000), BMAL1 (Cell Signaling Technology, 14020, 1:1,000) and β-actin (Cell Signaling Technology, 4967, 1:1,000). The relative protein levels at each timepoint were quantified using ImageJ (NIH, v.2.1.0) and normalized to β-actin as a loading control. For each experimental group, normalized protein levels at 15, 30, 60, 90 and 120 min were expressed as the percentage of the 0-min timepoint (set as 100%). The degradation rates of the proteins were then plotted as the percentage of remaining protein over time.
PLA
Confluent HCMs were subjected to hypoxic conditions (1% oxygen) for various durations (0, 1, 4 or 8 h). After exposure, the cells were fixed using 4% paraformaldehyde (Millipore Sigma, P6148), permeabilized with 0.1% Triton X-100 (Amresco, M143), and subjected to a blocking step to reduce non-specific binding. Rabbit anti-BMAL1 (Abcam, ab3350, 1:100), mouse anti-HIF2A (Novus Biologicals, NB100-132, 1:100) and mouse anti-HIF1A (Novus Biologicals, NB100-105, 1:100) were then applied. For PLA, pairs of primary antibodies raised in different species were used as mentioned. After primary antibody incubation, cells were treated with Duolink In Situ PLA Probes (Millipore Sigma, DUO92002 and DUO92004) conjugated to these antibodies. These probes, when in close proximity of less than 40 nm, facilitated the ligation of adjacent oligonucleotides attached to them. Subsequently, the ligated oligonucleotides were amplified and detected using fluorescently labelled probes. The resulting fluorescence signals, which are indicative of PPIs, were visualized using a Nikon Eclipse Ti2 confocal microscope (Nikon) and analysed with NIS Element AR software (Nikon, v6.10.01). To confirm the specificity of the observed interactions, control experiments were performed using only the BMAL1 antibody.
Plasmid construction and site-directed mutagenesis
To overexpress proteins in Escherichia coli, the DNA sequences encoding the N-terminal portions of mouse HIF2A (residues 3–361) and mouse HIF1A (residues 13–357), with a 6×His tag at their N terminus, were inserted into the pET15b vector. The DNA encoding the N-terminal region of mouse BMAL1 protein (residues 68–488), with a C-terminal Flag tag, was ligated into the pET24b vector.
For GST pull-down, the DNA encoding residues 3–361 of mouse HIF2A was inserted into pGEX-6P-1 to generate GST–HIF2A for expression. Six BMAL1 single-site mutants (L95E, L115E, L150E, F248D, R343A and F423D) and three double-site mutants (L95E/L115E, L150E/F248D and R343A/F423D) were generated using site-directed mutagenesis. The DNA sequences of all of the mutants were verified by DNA sequencing.
Protein expression and purification
The recombinant plasmids were individually transformed into E. coli BL21 Rosetta (DE3) or co-transformed together to express either individual proteins or the BMAL1–HIF2A complex. Expression of the proteins or BMAL1–HIF2A complex was carried out at 18 °C by induction with 0.1 mM isopropyl-β-d-thiogalactoside for 16 h. The BMAL1–HIF2A heterodimer (including bHLH, PAS-A and PAS-B domains) was first purified through a Ni-NTA affinity column (Thermo Fisher Scientific, 88222) followed by heparin chromatography. The heterodimer eluted from the heparin column was further analysed by size-exclusion chromatography using the Superdex 200 Increase 10/300 GL column. The peak fractions were assessed using SDS–PAGE and Coomassie blue staining. Subsequently, the samples were combined and concentrated. The concentrated protein was then used for cryo-EM sample preparation or stored at −80 °C for further use. His–HIF2A and His–HIF1A were purified individually using Ni-NTA affinity columns, followed by dialysis to eliminate imidazole. The proteins were concentrated and stored at −80 °C for further use.
Pull-down assay
To confirm the interaction between BMAL1 and HIF2A or HIF1A in Fig. 2h, BMAL1-expressing cells were lysed using binding buffer (20 mM HEPES (pH 7.4), 10% glycerol, 0.01% Triton X-100, 300 mM NaCl, 5 mM MgCl2, 1 mM DTT, and protease inhibitors). After centrifugation, the supernatants were incubated with anti-Flag M2 beads (Millipore Sigma, A2220) for 40 min at 4 °C. Subsequently, the beads were washed three times using the binding buffer, followed by the addition of purified His–HIF2A or His–HIF1A proteins. After incubation for 40 min at 4 °C, the beads were washed five times using the binding buffer. The proteins bound to beads were then eluted using Flag peptide (0.3 mg ml−1) and further analysed by SDS–PAGE and western blotting using anti-Flag (Sigma-Aldrich, F1804, 1:1,000) and anti-His (Thermo Fisher Scientific, MA1-21315, 1:1,000) antibodies.
To compare the interactions between HIF2A and WT BMAL1 or BMAL1 mutants in Fig. 5e, the cell pellets of GST–HIF2A and GST (as a control) were lysed by sonication in binding buffer (20 mM HEPES (pH 7.4), 10% glycerol, 0.05% Triton X-100, 300 mM NaCl, 5 mM MgCl2, 1 mM DTT and protease inhibitors). After centrifugation, the supernatants were incubated with Glutathione magnetic agarose beads (GE Healthcare, 17-0756-01) for 30 min at 4 °C. The beads were subsequently washed three times using the binding buffer, followed by the addition of Flag-tagged WT or mutated BMAL1. After 40 min incubation at 4 °C, the beads were washed five times using the binding buffer. The proteins bound to beads were eluted by heating with SDS–PAGE loading dye at 95 °C. The eluted samples were further analysed by SDS–PAGE and western blotting using anti-GST (Genscript, A00865, 1:1,000) and anti-Flag (Sigma-Aldrich, F1804, 1:1,000) antibodies.
Electrophoretic mobility shift assay
Five fmol of 22-bp biotin-labelled dsDNA fragment containing an HRE consensus sequence was incubated with 1, 2, 3, 4 and 6 pmol of purified BMAL1–HIF2A heterodimer in a total volume of 10 µl in 10 mM MES (pH 6.0), 50 mM KCl, 1 mM DTT, 2.5% glycerol, 0.05% NP-40 and 5 mM MgCl2. After incubation at room temperature for 20 min, the binding reactions were directly loaded onto a native 4–20% polyacrylamide gel and electrophoresed in 0.5× TBE buffer. DNA on the gel was transferred onto a positively charged nylon membrane and detected using an HRP-conjugated streptavidin with chemiluminescent substrates.
Surface plasma resonance
The interaction analysis of the BMAL1–HIF2A heterodimer with 22-bp DNA duplexes (HRE or E-box) was performed using OpenSPR (Nicoya Life Sciences). A 5′-end biotin-labelled dsDNA fragment (2 μM) containing either an HRE or E-box consensus sequence was immobilized in channel 2 of the biotin–streptavidin sensor, while channel 1 remained unmodified and served as a control. The BMAL1–HIF2A heterodimer at various concentrations (from 5 to 50 nM) was injected and flowed slowly at a rate of 20 μl min−1 over the sensor chip for 5 min in a running buffer (20 mM MES pH 5.5, 300 mM NaCl, 0.05% Tween-20, 0.02% BSA). After the injection, a 10-min dissociation phase was collected. The resulting data were analysed using Trace Drawer Kinetic Data Analysis software (v.1.9.2), using a one-to-one model, which assumes one monovalent ligand binding to one target.
Transactivation assay
To assess the transcriptional activation activity of HIF2A and BMAL1 on the promoter of human AREG, the 3 × 300 bp sequences (−300/0) from the human AREG promoter harbouring the common binding sequence (CAGGTG) were PCR-amplified and cloned into the pEZX-PG04-promoter luciferase vector to generate GLuc-hAREG. HEK293 cells were co-transfected with several constructs: pcDNA3-mHif2a-P405A/P530V/N851A (oxygen-regulation insensitive, a gift from C. Simon, Addgene, 44027)76, pcDNA3-mBmal1 (a gift from A. Sancar, Addgene, 31367)75, hAREG-gluc and SEAP vectors (GeneCopoeia, LF031). Transfection was performed using Lipofectamine 3000 reagent (Invitrogen, L3000015). After 48 h, the culture medium was collected, and the transcriptional activity was quantified using the Secrete-pair Dual Luminescence Assay Kit (GeneCopoeia, LF031) on the Cytation5 (Agilent Technologies) device with Gen5 software (v.3.12, Agilent Technologies). To investigate the transcriptional activity of HIF2A and BMAL1 on a general HIF target gene, HEK293 cells were transfected with the hPGK1-luc reporter (a gift from C. V. Dang, Addgene plasmid, 128095) along with HIF2A or BMAL1 expression constructs. After 48 h of transfection, firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, E1690). The luciferase activity was detected on the Cytation5 (Agilent Technologies) system and analysed using Gen5 software (v.3.12, Agilent Technologies).
Immunofluorescence staining and TUNEL assay
On day 1 after MI, collected heart tissues were fixed in 10% neutral-buffered formalin and then dehydrated through a graded alcohol series. After dehydration, the tissues were cleared in xylene and embedded in paraffin blocks. Sections of 5–6-μm thickness were prepared and underwent antigen retrieval. These sections were then blocked with diluted donkey serum, followed by overnight incubation with primary antibodies at 4 °C. In the immunofluorescence staining process, a range of primary antibodies was used to specifically target and identify various proteins within the tissue sections. These primary antibodies included: AREG (Santa Cruz, sc-74501, 1:50), HIF2A (Novus Biologicals, NB100-122, 1:200; Novus Biologicals, NB100-132, 1:200), BMAL1 (Abcam, ab3350, 1:200), α-sarcomeric (Abcam, ab137346, 1:200), vimentin (Cell Signaling Technology, 5741, 1:200), and α-smooth muscle actin (Cell Signaling Technology, 19245, 1:200) antibodies. For antigen visualization, the sections were then incubated with Alexa fluorescence-conjugated secondary antibodies (Invitrogen). Moreover, TUNEL staining was conducted using in situ Click-iT Plus TUNEL assay kits (Thermo Fisher Scientific, C10617) according to the manufacturer’s protocol to detect apoptotic cells as previously described77. The samples were counterstained with DAPI (1 μg ml−1, Invitrogen, D3571) and mounted using SlowFade Gold Antifade reagent (Invitrogen, S36936). The stained sections were then imaged with Nikon Eclipse Ti2 confocal microscopy (Nikon) and analysed using ImageJ software (NIH, Fiji v.2.1.0).
RT–qPCR
Total RNA was extracted from both tissue samples and isolated cells using the RNeasy Mini Kit (Qiagen, 74106) according to the manufacturer’s guidelines. This procedure is consistent with our established protocols as described in previous publications16,29,50. After the extraction, cDNA was synthesized from the extracted RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4368814). qPCR was then performed using SYBR Green PCR Master Mix (Qiagen, 204145). The reactions were performed on the Bio-Rad CFX384 Touch Real-Time PCR Detection System. For the analysis of gene expression levels, we applied the comparative Ct (ΔΔCt) method. The final data were presented as mean expression ratios relative to β-actin, allowing for the comparison of gene expression across different samples.
Western blotting
Mouse tissues and isolated cells were prepared for western blot analysis by lysing in RIPA lysis buffer (Thermo Fisher Scientific, 89900), supplemented with both protease (Thermo Fisher Scientific, 78425) and phosphatase inhibitor cocktails (Thermo Fisher Scientific, 78420). Protein concentrations were quantified, and 10–20 μg of total protein was separated on 4–12% SDS-PAGE gels (Bio-Rad Laboratories). After electrophoresis, the proteins were transferred to membranes for immunoblotting. The membranes were probed with the following primary antibodies: anti-HIF1A (Novus, NB100-105, 1:1,000), anti-HIF2A (Novus, NB100-122, 1:1,000), anti-HIF2A (Bethyl Laboratories, A700-003, 1:1,000), anti-HIF1A (Bethyl Laboratories, A700-001, 1:1,000), anti-HIF1B (Cell Signaling Technology, 5537. 1:2,000), anti-BMAL1 (Cell Signaling Technology, 14020, 1:2,000), anti-CLOCK (Cell Signaling Technology, 5157, 1:1,000), anti-RORα (Abcam, ab60134, 1:1,000), anti-AREG (Santa Cruz, sc-74501, 1:1,000), anti-caspase-3 (Cell Signaling Technology, 9662, 1:1,000), anti-cleaved caspase-3 (Cell Signaling Technology, 9661, 1:1,000), anti-Bax (Cell Signaling Technology, 2772, 1:1,000), anti-Flag (Sigma-Aldrich, F1804, 1:1,000), anti-ubiquitin (Santa Cruz, sc-8017, 1:1,000), anti-lamin B1 (Cell Signaling Technology, 12586, 1:2,000), anti-lamin A/C (Cell Signaling Technology, 4777, 1:2,000), anti-TBP (Cell Signaling Technology, 8515, 1:1,000), anti-α-tubulin (Cell Signaling Technology, 2144, 1:2,000) and anti-β-actin (Santa Cruz, 47778, 1:2,000). After incubation with secondary antibodies, the membranes were developed using appropriate substrates. The signal intensity was detected and quantified using ImageJ software (NIH, Fiji v.2.1.0). The results were normalized to the appropriate internal control, and data were expressed as the relative fold changes in protein levels.
Cryo-EM sample preparation
The purified BMAL1–HIF2A heterodimer was mixed with a twofold molar excess of the HRE dsDNA, followed by incubation on ice for 20 min. The mixture was then applied onto the Superdex 200 Increase 10/300 GL column. The eluate fractions from the column were analysed by SDS–PAGE and stained with Coomassie blue. The peak fractions corresponding to the BMAL1–HIF2A–DNA complex were combined and concentrated to 0.5 mg ml−1 for preparing cryo-EM specimens. In brief, 3 μl of the purified complex was applied onto freshly glow-discharged R 2/1 holey carbon 300 mesh copper grids (C-flat). The grids were then vitrified in liquid ethane using an FEI Vitrobot (Mark IV).
Cryo-EM data collection and processing
Single-particle cryo-EM images were collected using the FEI Titan Krios (300 KV) electron microscope with a Gatan GIF Quantum K2 direct electron detector. The images were automatically acquired using EPU (Thermo Fisher Scientific, v.2.10) at underfocus values ranging from 1.0 to 2.5 μm with a pixel size of 0.85 Å px−1. Each micrograph was exposed for 9 s with a total dose of about 75 e− Å−2, which was fractionated into 45 frames. The movie frames were aligned using MotionCor2 (v.1.4.0)78, resulting in a total of 18,039 micrographs. Gctf (v.1.18)79 was used to estimate the parameters of contrast transfer function (CTF) for each micrograph. crYOLO (v.1.10)80 was used for reference-free automatic particle picking, yielding a total of approximately 710,000 particles. The particles were extracted with a box size of 352. Multiple rounds of Cryosparc (v.-2.5)81 2D classification were initially performed to remove junk particles. Next, the remaining particles were subjected to an additional round of 2D classification using RELION (v.3.1)82, yielding a stack of 420,000 particles. An ab initio model, showing secondary structural features bound to DNA, was generated using CryoSPARC and then used as a reference for subsequent 3D analysis. Multiple rounds of 3D classification were performed in RELION, resulting in 141,218 particles. The particles were rescaled to a pixel size of 1.02 Å px−1 with a box size of 328 pixels and subsequently subjected to 3D refinement. The reference map for 3D refinement was filtered to 25 Å, and a low-pass filtered mask at 25 Å was generated using a threshold that included the protein and DNA regions, followed by a 4-pixel dilation and 5-pixel soft padding. 3D refinement in RELION produced a 4.7 Å-resolution map of the BMAL1–HIF2A–DNA complex. Next, these particles were further processed by CTF refinement and Bayesian polishing in RELION, resulting in 141,218 shiny particles. These particles were then subjected to 3D refinement, producing a map with a resolution of 4.3 Å. Multiple rounds of 3D classification, with and without alignment, were then conducted. A final set of 43,098 shiny particles was used for 3D refinement, resulting in a map with an average resolution of 3.6 Å. The resolution was calculated using a low-pass filtered mask at 15 Å, created with a threshold of 0.008, a 3-pixel dilation and 3-pixel soft padding. The b-factor for the RELION map was −61. The final map was sharpened using DeepEMhancer (v.0.14) with the wideTarget training mode83. The local resolutions were calculated using CryoSPARC. The resolutions of the 3D maps were estimated using gold-standard Fourier shell correlation (FSC) curves with a 0.143 cut-off criteria84. The directional resolution anisotropy was quantified by the 3D FSC algorithm (v.3.0)85. The final set of 43,098 particles had an Eod value of 0.74 with the best and worst PSF resolutions of 2.8 and 5.3 Å, respectively, as calculated by cryoEF (v.1.1.0)86, indicating minor anisotropy. The cryo-EM data collection and image analysis procedures are shown in Supplementary Table 11 and Extended Data Fig. 9, respectively.
Model building and refinement
To build an atomic model of the BMAL1–HIF2A–DNA complex, the crystal structure of HIF2A (Protein Data Bank (PDB): 4ZP4) and a 22-bp B-form DNA duplex were used as templates and fitted into the deepEMhancer map of BMAL1–HIF2A–DNA by rigid-body fitting in Chimera (v.1.15)87. Owing to the structural arrangement, the bHLH, PAS-A and PAS-B domains of the BMAL1 crystal structure (PDB: 4F3L) were individually fitted into the map. The model was then manually built and adjusted in Coot (v.1.1)88, followed by real-space refinement in Phenix (v.1.21)89 (Supplementary Table 11). In the final BMAL1–HIF2A–DNA atomic model, amino acids for HIF2A (3–12, 76–88, 151–163, 174–185, 201–219 and 356–361) and BMAL1 (68–74, 102–110, 126–144, 164–167, 210–242, 254–279, 291–311, 321–337, 345–349, 407–412 and 441–488) as well as a four DNA bases near the terminal ends were not built because of missing or poor densities. The refined model statics are shown in Supplementary Table 11. All molecular graphic figures were generated by Chimera, ChimeraX (v.1.7)90 and PyMOL (v.2.5.5)91.
Quantification and statistical analysis
Unless otherwise specified, all results are presented as the mean ± s.e.m., with the precise number of biological replicates (n) described in the figure legends. All data were plotted from independent biological replicates. The Shapiro–Wilk normality test was used to assess normal distribution. The equality of variances for unpaired t-tests was determined using the F-test. Statistical analysis was performed using GraphPad Prism 10.0, using unpaired t-tests (two-tailed), Welch’s t-tests (for unequal variances), Mann–Whitney U-tests (when normality assumptions were not met) or one-way ANOVA with Bonferroni’s multiple-comparison analysis, depending on the dataset. Outliers were identified using the ROUT method (Q = 1%) in GraphPad Prism. For ChIP–qPCR data analysis in Fig. 3s, values identified as outliers were excluded from the statistical analysis. Cardiac function assessment in Bmal1loxP/loxP myosin-Cre+, Hif2aloxP/loxP myosin-Cre+ and Hif1aloxP/loxP myosin-Cre+ mice was compared to a shared myosin-Cre+ control group. Cardiac function data from Bmal1loxP/loxP myosin-Cre+ mice were used as the control for comparing NOB-treated Bmal1loxP/loxP myosin-Cre+ mice. Similarly, cardiac function data from Areg−/− mice were used as the control for comparing NOB-treated Areg−/− mice. Cardiac function data from C57BL/6J mice were partially used as the control group for Areg−/− mice. This strategy minimized animal use and ensured a consistent baseline, optimizing research efficiency while enabling reliable comparative analysis.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
