Aerobic Exercise Improves Cardiac Pathological Remodeling in High-fat Diet Mice by Regulating m6A Methylation Modified circRNAs

Objective: To explore the role of N6-methyladenosine(m6A) methylation modified circular RNAs(circRNAs) in aerobic exercise-induced improvement of cardiac pathological remodeling in high-fat diet mice. Methods: C57BL/6J wild-type mice were randomly divided into four groups: Normal diet-sedentary(ND-SED) group, normal diet-exercise(ND-EX) group, high-fat dietsedentary(HFD-SED) group and high-fat diet-exercise(HFD-EX) group. The ND-SED and ND-EX groups were fed with normal diet, while the HFD-SED and HFD-EX groups were fed with 60% fat-enriched high-fat diet for 12 weeks, the ND-EX and HFD-EX groups were subjected to 8-week aerobic exercise. After the exercise intervention, the body weight, body fat content, cardiac function, serum TC and TG levelsand heart tissue TG content were measured. m6A regulatory factors were detected by using RTqPCR. Protein expressions of METTL3 and FTO were determined by using Western blot. m6A RNA methylation content in cardiac tissue was quantified using colorimetric methods. MeRIP-seq was used to detect circRNAs with m6A methylation modification in the heart. SRAMP was employed to predict circRNA methylation sites with m6A methylation difference and expression difference;and MeRIP-qPCR was performed to verify its m6A level. Results: 8-week aerobic exercise significantly reduced body weight/fat content of high-fat diet-fed mice, and increased left ventricular ejection fraction(EF) and fractional shortening(FS), inhibited cardiac fibrosis, lipid deposition, and pathological remodeling. Aerobic exercise also decreased m6A methylation level in the heart of high-fat diet-fed mice, while the methyltransferase METTL3 with played an important role in this process. MeRIP-seq analysis revealed that the majority of m6A-modified circRNAs in the mice heart of HFD-SED and HFD-EX groups had only one m6A modification site, with a significant enrichment in overlapping regions and exons. Compared with the HFD-SED group, there were 13 upregulated and 19 downregulated m6A-modified circRNAs in that of HFD-EX group. The differential methylation of these circRNAs was mainly observed in overlapping regions, with most of them ranging from 1 to 10 000 bps and being located on chromosomes 1, 7, and 13. Compared with the HFD-SED group, there were 40 circRNAs differentially expressed in the HFD-EX group, among which 22 circRNAs were significantly up-regulated and 18 circRNAs were significantly down-regulated. Combined analysis of MeRIP-seq and RNA-seq data revealed that only circRNA_4614 exhibited both differential m6A methylation levels and expression levels, showing a significant increase in both aspects. SRAMP prediction indicated that circRNA_4614 harbored eight potential m6A sites, among which one had high confidence while two had moderate confidence. Compared to the HFD-SED group,there was a significant increase in m6A methylation level for circRNA_4614 in the HFD-EX group, consistent with MeRIP-seq sequencing results. Conclusions: Aerobic exercise could significantly reduce m6A methylation levels in high-fat diet-fed mice, and regulate the expression profiles and m6A modification patterns of cardiac circRNAs. The involvement of circRNA_4614 mediated by m6A modification may play an important role in improving obesity-induced cardiac remodeling through aerobic exercise.