In a groundbreaking study published in Nature, researchers have unveiled a critical molecular mechanism underlying the circadian regulation of myocardial injury, shedding new light on the intricate interplay between the body’s internal clock and cardiac response to ischemic stress. The study focuses on the BMAL1–HIF2A heterodimer, a protein complex that orchestrates rhythmic gene expression in heart tissue, dramatically influencing how myocardial damage fluctuates over the course of the day.
Myocardial ischemia-reperfusion injury (IRI) remains a major clinical challenge, with variations in tissue damage observed depending on the time of day when the injury occurs. The mystery behind this temporal variability has persisted despite advances in cardiovascular research. Here, the authors present compelling evidence implicating the BMAL1–HIF2A complex as a pivotal driver of diurnal changes in myocardial injury, providing a molecular explanation for these daily fluctuations.
Central to their discoveries is the gene Areg, which encodes Amphiregulin (AREG), a member of the epidermal growth factor family. The study reveals that among several potential HIF2A target genes upregulated during myocardial IRI, Areg displays the most pronounced diurnal fold change. Specifically, AREG levels peak in the affected heart tissue at Zeitgeber Time 8 (ZT8), resembling mid-light phase, whereas levels decline by ZT20, corresponding to the early dark phase. This rhythmic expression pattern of Areg mirrors the presence and activity of the BMAL1–HIF2A heterodimer within the cardiomyocytes of the ischemic border zone.
Detailed immunofluorescence analysis corroborated these findings, localizing elevated AREG expression precisely to cardiomyocytes adjacent to the infarcted region at ZT8. Remarkably, other cardiac cell types such as fibroblasts and smooth muscle cells exhibited negligible AREG induction, underscoring a cell-type-specific regulatory mechanism. This spatial restriction coincides with nuclear co-localization of BMAL1 and HIF2A in the same region and circadian time frame, supporting the hypothesis that the heterodimer directly regulates Areg transcription in response to ischemic stress.
Moving beyond in vivo studies, the team employed synchronized human cardiomyocytes (HCMs) cultivated in vitro under controlled circadian conditions. Upon exposure to hypoxia (1% oxygen for 4 hours), these cells exhibited a starkly time-dependent induction of AREG mRNA. Intriguingly, robust transcript induction occurred at circadian time 32 (CT32), equivalent to ZT8 in vivo, while expression remained comparatively low at other times including CT20. This suggests that the molecular clock tunes cardiomyocyte responsiveness to hypoxic stress in a circadian fashion, modulating protective gene programs like Areg expression.
Mechanistic investigations employing siRNA-mediated knockdown identified both BMAL1 and HIF2A as indispensable for hypoxia-triggered AREG induction, whereas silencing of closely related factors such as CLOCK or HIF1A failed to affect expression levels. These results elucidate a highly specific transcriptional regulatory axis, highlighting BMAL1–HIF2A as the critical heterodimeric complex governing this circadian response.
Extending these observations to genetically modified mice, myocardial IRI-induced Areg expression was significantly attenuated in cardiomyocytes deficient for either Bmal1 or Hif2a. This in vivo genetic evidence confirms the central role of the BMAL1–HIF2A pathway in orchestrating cardiomyocyte adaptive responses to ischemia-reperfusion injury on a time-of-day basis.
Biophysical analyses further elucidated the interaction dynamics within this transcriptional network. Surface plasmon resonance studies demonstrated direct binding of the BMAL1–HIF2A complex to the AREG promoter at conserved DNA motifs, including hypoxia response elements (HRE) and E-box sequences. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) validated the occupancy of these factors on the AREG promoter in both human embryonic kidney cells and cardiomyocytes, with significant circadian modulation observed.
Protein-protein interaction studies using co-immunoprecipitation confirmed the stable formation of the BMAL1–HIF2A heterodimer at specific circadian times in cardiomyocytes, correlating tightly with periods of high AREG expression. In luciferase reporter assays, the AREG promoter was potently activated by the BMAL1–HIF2A complex under hypoxic conditions, further underscoring the functional relevance of this heterodimeric interaction.
Collectively, these findings establish the BMAL1–HIF2A heterodimer as a master regulator of circadian-dependent gene expression programs in the ischemic heart, orchestrating protective mechanisms through rhythmic induction of factors like AREG. This work not only deepens our understanding of molecular cardiology but also points toward novel therapeutic avenues that leverage circadian biology to minimize myocardial injury.
The implications of these discoveries are profound. Circadian modulation of myocardial injury suggests that timing of interventions such as reperfusion therapies or pharmacological treatments could be optimized according to the patient’s internal biological clock. Targeting the BMAL1–HIF2A axis may enable development of drugs that enhance cardioprotection specifically during vulnerable time windows, mitigating damage and improving outcomes.
Moreover, the study elegantly integrates hypoxic signaling with circadian transcriptional control, illuminating how stress responses are temporally gated by intrinsic cellular clocks. This intersection of oxygen-sensing pathways and molecular chronobiology catalyzes a paradigm shift in how cardiovascular diseases are conceptualized, highlighting the importance of chronotherapy in clinical practice.
Future research building on these insights may explore the broader landscape of circadian-regulated genes involved in cardiac repair and remodeling, uncovering comprehensive temporal maps of gene networks activated during ischemic episodes. Additionally, the potential interplay between BMAL1–HIF2A and other circadian regulators or hypoxia-inducible factors warrants detailed dissection.
This landmark investigation sets the stage for translational applications aiming to harness circadian biology for cardioprotection. Through meticulous experimental design combining animal models, cellular assays, molecular biology techniques, and biophysical analyses, the authors have provided a compelling narrative linking molecular clock components to temporal dynamics of myocardial injury. Their work exemplifies the power of integrative science in addressing complex physiological phenomena.
As the global burden of ischemic heart disease continues to rise, understanding the temporal dimension of cardiac injury and repair emerges as a critical research frontier. The BMAL1–HIF2A heterodimer stands at this frontier as a promising molecular target, heralding new possibilities for personalized medicine tailored to the rhythms of human biology.
Subject of Research: Circadian regulation of myocardial injury and gene expression via the BMAL1–HIF2A heterodimer.
Article Title: BMAL1–HIF2A heterodimer modulates circadian variations of myocardial injury.
Article References:
Ruan, W., Li, T., Bang, I.H. et al. BMAL1–HIF2A heterodimer modulates circadian variations of myocardial injury. Nature (2025). https://doi.org/10.1038/s41586-025-08898-z
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