Cold exposure aggravates myocardial ischemia-reperfusion injury via m6A-mediated circRNA-mRNA regulatory networks

Abstract

Objective: Myocardial ischemia-reperfusion (I/R) injury remains a major contributor to cardiac morbidity and mortality, and accumulating evidence suggests that epitranscriptomic regulation may critically influence cardiac stress responses. N6-methyladenosine (m6A) modification and circular RNAs (circRNAs) have emerged as important regulators of cardiovascular pathology; however, their integrated roles in myocardial I/R injury, particularly under chronic cold stress, remain poorly defined. Methods: A mouse model of myocardial I/R injury was established under room-temperature or chronic cold exposure conditions. Cardiac function, infarct size, histopathology, and serum injury markers were assessed. Global m6A levels were quantified, and m6A-modified circRNA profiles were analyzed using epitranscriptomic microarrays and bioinformatics approaches. Differentially expressed circRNAs were validated in vivoand in hypoxia-reoxygenation-treated neonatal cardiomyocytes. Circular structures and stability were confirmed by Sanger sequencing, divergent/convergent PCR, and actinomycin D assays. Competing endogenous RNA (ceRNA) networks were constructed to identify downstream regulatory pathways. Results: Myocardial I/R injury resulted in significant cardiac dysfunction, increased infarct size, histological damage, and elevated serum CK-MB and LDH levels, accompanied by a marked increase in global m6A methylation. Epitranscriptomic profiling identified 391 circRNAs with altered m6A modification following I/R injury, involving pathways related to molecular binding, cellular processes, and kinase signaling. Multiple circRNAs exhibited consistent dysregulation in both in vivo and in vitroI/R models and displayed high structural stability. Importantly, chronic cold exposure significantly exacerbated I/R-induced cardiac dysfunction and infarct severity while further modulating the expression of specific m6A-modified circRNAs. ceRNA network analysis revealed that cold-responsive circRNAs potentially regulate myocardial injury through miRNA-mediated signaling pathways. Conclusion: This study identifies m6A-modified circRNAs as key epitranscriptomic regulators of myocardial I/R injury and demonstrates that chronic cold stress amplifies circRNA-mediated regulatory networks. These findings provide novel mechanistic insight into temperature-dependent epigenetic regulation in ischemic heart disease and highlight m6A-circRNAs as potential therapeutic targets.

Keywords

circRNAs / m6A / myocardial I/R injury / cold stress

Acknowledgements

Not applicable.

Research ethics

All animal experiments were conducted in accordance with the guidelines of the American Association for the Accreditation of Laboratory Animal Care International and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). All experimental procedures were approved by the Institutional Animal Care and Use Committee of Harbin Medical University (ethical approval number: IRB3017723).

Informed consent

Not applicable.

Author contributions

Yang B F and Bian Y designed the experiments and supervised the project. Wu H and Jiang WT performed the molecular biology experiments and drafted the manuscript. Zhang X Y, Yao F T, and Pang P conducted the design and implementation of the animal experiments. Pan T F and Lutokhina Y critically revised the manuscript. All authors reviewed and approved the final version of the manuscript.

Use of large language models, AI and machine learning tools

No large language models, AI or machine learning tool was used for any part of the present study.

Conflict of interests

Yang B F is the Editor-in-Chief of Frigid Zone Medicine. The manuscript was handled according to the journal’s standard editorial procedures, with peer review conducted independently of the Editor-in-Chief and his research groups.

Research funding

This work was supported by the National Natural Science Foundation of China (Nos. 82330011); Key Research and Development Program of Heilongjiang Province (Grant No. 2025ZX05A01); the Basic Research Support Program for Excellent Young Teachers in Heilongjiang Province (YQJH2025126).

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

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