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Cold exposure aggravates myocardial ischemia-reperfusion injury via m6A-mediated circRNA-mRNA regulatory networks
doi: 10.1515/fzm-2026-0001
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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 vivo and 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 vitro I/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. -
Key words:
- circRNAs /
- m6A /
- myocardial I/R injury /
- cold stress
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Figure 1. Establishment of a mouse myocardial ischemia-reperfusion (I/R) model
(A) Representative echocardiographic images showing cardiac function. Ejection fraction (EF%). (C) Fractional shortening (FS%). **P < 0.01 versus Sham, N = 5. (D-E) Representative images and quantitative analysis of infarct size assessed by blue-2, 3, 5-triphenyl tetrazolium chloride (TTC) staining. **P < 0.01 versus Sham, N = 3. (F) Representative H&E-stained sections showing myocardial histopathological changes. (G-H) Serum levels of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH). *P < 0.05, **P < 0.01 versus Sham, N = 5. (I) Global N6-methyladenosine (m6A) modification levels in cardiac tissue, *P < 0.05 versus Sham, N = 3.
Figure 2. Differential N6-methyladenosine (m6A)-methylated circRNAs and Gene Ontology (GO) pathway analysis
(A) m6A-circRNA epitranscriptomic microarray and bioinformatics analysis showing differential m6A modification levels. N = 3. (B) Scatter plot analysis of differentially expressed circRNAs. (C) Volcano plot analysis of differentially expressed circRNAs. (D) Venn diagram showing circRNAs targeting common miRNAs. (E, F) Distribution of hypermethylated and hypomethylated circRNAs in myocardial I/R injury.
Figure 3. Gene Ontology and pathway enrichment analysis of differentially expressed circRNAs in biological processes, cellular components, and molecular functions
(A-C) Gene Ontology (GO) enrichment analysis of differentially expressed circRNAs in biological processes, cellular components, and molecular functions.(D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes.
Figure 4. Assessment of circRNA expression levels in vivo and in vitro
(A, B) Relative expression levels of circRNAs in cardiac tissues. *P < 0.05, **P < 0.01 versus Sham, N = 3-4. (C, D) Relative expression levels of circRNAs in cardiomyocytes. *P < 0.05, **P < 0.01 versus Sham, N = 3-4.
Figure 5. Validation of the circular structure of circRNAs
(A, D) Identification of circRNA back-splice junctions by Sanger sequencing. (B, E) Validation of circular structures using divergent and convergent primers with cDNA and genomic DNA templates. (C, F) CircRNA stability assessed by actinomycin D treatment. N = 3-4.
Figure 6. Dynamic changes in N6-methyladenosine (m6A)-circRNA expression in chronic cold stress-induced I/R mice
(A) Baseline echocardiographic assessment of cardiac function. (B) Ejection fraction (EF%). (C) FS%. N = 8. (D) Echocardiographic assessment after four weeks of cold exposure. (E) EF%. (F) FS%. *P < 0.05, **P < 0.01 versus Sham, N = 5. (G-I) Representative images and quantitative analysis of myocardial injury assessed by Evans blue-TTC double staining. *P < 0.05 versus RT-I/R, N = 4.
Figure 7. Expression of N6-methyladenosine (m6A)-methylated circRNAs in chronic cold stress-induced myocardial I/R injury
(A) Relative expression levels of circRNAs in cardiac tissue. *P < 0.05, **P < 0.01 versus RT-Sham, #P < 0.05, ##P < 0.01 versus Cold-Sham, & P < 0.05, & & P < 0.01 versus Cold-I/R N = 4.
Figure 8. Downstream target analysis of cold-responsive circRNAs
(A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of cold-responsive circRNAs. (B) ceRNA network of five cold-responsive circRNAs and their sponge miRNAs under cold stress conditions. CircRNAs are shown as orange nodes, miRNAs as green nodes, and downstream target genes as blue nodes.
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