Circular RNA FEACR inhibits ferroptosis and alleviates myocardial ischemia/reperfusion injury by interacting with NAMPT

Background Emerging research has reported that circular RNAs (circRNAs) play important roles in cardiac cell death after myocardial ischemia and reperfusion (I/R). Ferroptosis, a new form of cell death discovered in recent years, has been proven to participate in the regulation of myocardial I/R. This study used circRNA sequencing to explore the key circRNA in the regulation of cardiac ferroptosis after I/R and study the mechanisms of potential circRNA function. Methods We performed circRNA sequencing to explore circRNAs differentially expressed after myocardial I/R. We used quantitative polymerase chain reactions to determine the circRNA expression in different tissues and detect the circRNA subcellular localization in the cardiomyocyte. Gain- and loss-of-function experiments were aimed to examine the function of circRNAs in cardiomyocyte ferroptosis and cardiac tissue damage after myocardial I/R. RNA pull-down was applied to explore proteins interacting with circRNA. Results Here, we identified a ferroptosis-associated circRNA (FEACR) that has an underlying regulatory role in cardiomyocyte ferroptosis. FEACR overexpression suppressed I/R-induced myocardial infarction and ameliorated cardiac function. FEACR inhibition induces ferroptosis in cardiomyocytes and FEACR overexpression inhibits hypoxia and reoxygenation-induced ferroptosis. Mechanistically, FEACR directly bound to nicotinamide phosphoribosyltransferase (NAMPT) and enhanced the protein stability of NAMPT, which increased NAMPT-dependent Sirtuin1 (Sirt1) expression, which promoted the transcriptional activity of forkhead box protein O1 (FOXO1) by reducing FOXO1 acetylation levels. FOXO1 further upregulated the transcription of ferritin heavy chain 1 (Fth1), a ferroptosis suppressor, which resulted in the inhibition of cardiomyocyte ferroptosis. Conclusions Our finding reveals that the circRNA FEACR-mediated NAMPT-Sirt1-FOXO1-FTH1 signaling axis participates in the regulation of cardiomyocyte ferroptosis and protects the heart function against I/R injury. Thus, FEACR and its downstream factors could be novel targets for alleviating ferroptosis-related myocardial injury in ischemic heart diseases. Supplementary Information The online version contains supplementary material available at 10.1186/s12929-023-00927-1.

was conducted RNA purification, quantification, and qualification. Library preparation for circRNA sequencing, Clustering and sequencing, and data analysis were executed by Novogene Gene Regulation Department. anti-sense experimented on in cardiomyocytes as previously describe [1]. A cell counting kit purchased from Meilunbio was used to determine the rate of cell survival. Briefly, 100 μL of DMEM/F12 medium containing 10% CCK-8 was added to each well, and after incubation at 37 ℃ for 1-4 h, the OD value at 450 nm was detected by a microplate reader.
Ferrous iron measurement. The cellular or serum content of iron was detected by a ferrous iron colorimetric assay kit (Elabscience Biotechnology Co., Ltd) after the I/R injury of H/R treatment. A series of ferrous iron standards were added to the plate to make standard curves. For the detection of serum iron, a 200 μL sample was added into the microlon elisa plates and then 100 μL reagent II was mixed in the well incubated for 10 min at 37 ℃. Finally, the optical density (OD) value at 593 nm was detected with a microplate reader. In order to detect cellular iron, ten million cardiomyocytes were disrupted in 1 mL reagent I and dissociate for 10 min on the ice.
The lysates were centrifuged at 10,000 g, for 10 min. Collect the supernatant to a new tube and used it to detect iron content. The method of cellular iron detection is the same as serum iron detection.
Prussian blue iron staining. The cardiac tissue was fixed in 10% neutral formalin and then dehydrated and embedded. The embedding block was cut 5 μm thick. Soak the section in perls stain and stain for 30 min and rinse fully in distilled water for 5 min. The nuclear fast red solution was used to lightly stain with nuclei for 5 min and   Table S3), and 50 ng cDNA sample. Mouse GAPDH was used as internal control genes.
Amplification and quantitative measurement were performed using the QuanStudio3 PCR system (Thermo Fisher) by the 2 -ΔΔCT method.
Myocardial injury size measurement. We used Evans blue (Sigma-Aldrich) and 2,3,5-Triphenyl-tetrazolium chloride (TTC, Sigma-Aldrich) staining to determine the size of myocardial injury. After I/R treatment, the mice were injected with 1% Evans blue form ventriculus sinister until the limbs were blued. Take the heart out of the body and froze at -80 ℃ for 15 min and then the heart was cut into 0.2 cm thick slices. RNA binding protein immunoprecipitation (RIP) assay. RIP was performed as previously described [2]. Cardiomyocytes were lysed in lysis buffer (150 mM NaCl, 0.5 mM dithiothreitol, 0.1% SDS, 1% NP-40, 50 mM Tris buffer, pH 7.4, 1 mM EDTA, 1 mM phenylmethyl sulfonyl fluoride, 0.5% sodium deoxycholate) supplemented with 1x proteinase inhibitor cocktail, a 30 min period was spent on the ice. The lysed product was centrifuged at 12,000 rpm, 4 ℃ for 20 min, and aliquoted 50 μL lysed product as input. The remaining lysates were incubated with rabbit IgG negative control and NAMPT antibody respectively overnight. The next day, the lysates mixture was furtherly incubated with protein A/G agarose beads for 4 h. After washing beads with lysis buffer five times, the beads-RNA-protein was extracted by