Loss of Super-Enhancer-Regulated circRNA Nfix Induces Cardiac Regeneration After Myocardial Infarction in Adult Mice

Supplemental Digital Content is available in the text.

mixed with fetal bovine serum (FBS) after each step. The supernatant was then centrifuged to collect the cells, which were resuspended in DMEM/F12 medium (Life Technologies) supplemented with 10% FBS. Then, the cell suspension was placed onto uncoated 100 mm plastic dishes for 80 min at 37 °C in 5% CO 2 and a humidified atmosphere due to different adhesion between cardiomyocyte and fibroblast. The supernatant, composed mostly of CMs, was then collected and pelleted. The cells were then resuspended in aforementioned medium, counted and plated at the appropriate density. Cultures of ventricular CMs that were prepared using this procedure consistently yielded a purity over 90%.
Adult CMs were isolated from 8 weeks old adult mouse as previously described 5 , with minor modifications. Briefly, adult mouse hearts were extracted and mounted on a Langendoft apparatus. Then, hearts were perfused with calcium-free perfusion buffer containing 113 mM NaCl, 4.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 10 mM Na-HEPES, 12 mM NaHCO3, 10 mM KHCO3, 0.032 mM phenol red, 30 mM taurine, 10 mM BDM, and 5.5 mM glucose (pH-7.0) for 5 minutes, followed by 50 ml digestion buffer containing 15,000 U of type II collagenase (Roche) and 50 μM CaCl2 for 10 mins. The hearts were teased into small pieces and triturated with a Pasteur pipette to separate individual CM. Cells were then spun at low speed and supernatant was removed. The adult mouse CMs were plated onto laminin (10 µg ml-1 , Life technologies, 23017015) coated culture slides with F12 medium with 10% FBS.
The primary neonatal cardiac endothelial cells and neonatal cardiac fibroblasts were isolated from P7 mice using the Neonatal Cardiac Endothelial Cell Isolation kit Miltenyi Biotec) and Neonatal Cardiac Fibroblast Cell Isolation kit Miltenyi Biotec), respectively. The primary adult mouse cardiac endothelial cells and fibroblasts were obtained from Cell Biologics, Inc. (C57-6024 and C57-6049, respectively) and handled according to the company's instruction.
Human primary coronary artery endothelial cells (HCAEC) were obtained from the American Type Culture Collection (ATCC) and cells culture was performed according to ATCC protocol. Hl-1 cardiac muscle cells were purchased from EMD Millipore (SCC065).

Small interfering RNAs (siRNAs), adenovirus (AdV) or adeno-associated virus 9 (AAV9) and vectors transfection
SiRNAs, vector harboring Ybx1, miR-214 mimics and inhibitors were purchased from Ribobio Co. Ltd. (Guangzhou, China). RNA interference target sequences are shown in Supplementary Table 2 The ADV and AAV9-containing green fluorescent protein (GFP) vectors for depletion or overexpression of circNfix, the AAV9-containing mCherry for overexpression of Gsk3β, and the ADV-containing mCherry for overexpression Meis1 were synthesized as described previously [3][4][5] . The cTnT promotor was used to driven the gene and GFP or mCherry's expression. Neonatal mouse CMs were transfected with ADV for 48 h. The multiplicity of infection (MOI) was 10-20, and the CM transfection efficiency was >95%. Adult mice were intramyocardially injected with AAV9 vectors at 5-6 sites with a dose of 5 × 10 11 viral genome particles per animal (approximately 20 μl) using an insulin syringe with a 30-gauge needle. For the infarcted mice, vectors were injected into the myocardium bordering the infarct zone.
After injection for 14 days or 28 days, the heart tissues were collected. In the P0 neonatal mice, ADV, at a dose of 4 × 10 9 viral genome particles per animal (approximately 5 μl), was injected into the heart ventricles at 3 sites by using an insulin syringe with a 30-gauge needle. The hearts of the injected mice were collected 4 or 7 days after ADV injection.
Ad-GFP and Ad-mCherry was used to determine the distribution of ADV or AAV9 vector in the myocardium. Bright field and fluorescence images of the adult mouse and isolated neonatal mouse heart were captured to examine GFP fluorescence using Bruker In-Vivo FX Pro system (Bruker, MA, USA). In situ hybridization (ISH) and real-time polymerase chain reaction (RT-qPCR) assays were used to detect circNfix expression after transfection.

Histological examinations
Heart tissues were fixed with 10% formalin overnight, then embedded in wax and sectioned. The sections were deparaffinized through a graded alcohol series. Sections 8 / 16 were boiled in a pressure cooker and then cooled for 30 minutes. Then, specimens were processed as indicated.
To evaluate the capillary density, sections were blocked with 4% goat serum and incubated with anti-CD31 (ab7388, Abcam) and anti-vWF (ab11713, Abcam). The number of capillaries per unit area image from 200X fields was determined using an Olympus BX51 microscope (Olympus Corporation).
To measure the fibrotic area, the sections were stained with Masson trichrome (MST 8004, MST Biotechnology). The percent area of cardiac fibrosis was determined by detecting collagen deposition (blue) using Image J software.
The expression levels of circNfix in humans, rats and mouse heart tissues were detected by ISH. Heart sections were incubated in 3% pepsin dilution in fresh citrate buffer at 37°C for 30 min and then prehybridized with prehybridization solution for 2 h at 37°C. Subsequently, hybridization with DIG-labeled RNA probes (Biosense Bioscience Co., Ltd., Guangzhou, China) was performed overnight at 37°C. Sections were washed through a graded SSC buffer series. After blocking with 3% BSA for 30 min at 37°C, the sections were incubated with alkaline phosphatase-conjugated sheep anti-DIG Fab fragments for 1 h at room temperature. BM Purple AP substrate (Roche, Basel, Switzerland) was used to detect positive staining according to the manufacturer's instructions.

SgRNA design
CRISPRtool programme was used to design sgRNAs to reduce potential off-target effects. The sequence of sgRNA are listed in Supplemental Table 3.

Chromosome conformation capture (3C) assay
Samples were prepared as described previously with minor modifications 8 . Briefly, cells (5 × 10 6 ) were incubated into 1% formaldehyde and 2.5M glycine. Then, cells were spun and re-suspended in HiC lysis buffer (10mM Tris-HCl pH8.0, 10mM NaCl, 0.2% NP-40) with proteinase inhibitor (Sigma). Next, the cells were incubated into 0.5% sodium dodecyl sulfate (SDS) at 65°C for 5min. Chromatin was digested overnight by MboI and then ligated. Ligated DNA was quantified using QRT-PCR as described above. The anchor primer located at the circNfix promoter. Primer sequences are shown in Supplementary Table 4.

RNase R treatment
RNase R treatment was performed as described previously 9 . Briefly, 2 µg of total RNA was incubated with or without 3 U ug-1 of RNase R (Sigma) at 37°C for 30 min.
Then, the treated RNA was purified using an RNeasy MinElute Cleanup Kit (Qiagen).

RNA Fluorescent in situ hybridization (RNA-FISH)
Cy3-labeled RNA probes against circNfix back-splice sequence, miR-214, or Gsk3β 3'UTR sequence were used in RNA-FISH assays. Cultured cells were washed with PBS and permeabilized with 0.5% Triton X-100. Then, cells were incubated with RNA probes in the hybridization buffer (RiboBio, Guangzhou, China). Nuclei were stained with DAPI. All probes used for RNA-FISH were provided by RiboBio Co., Ltd. (Guangzhou, China).

RNA pulldown assays
Cultured cells were washed in ice-cold phosphate-buffered saline, lysed in 0.5 ml co-IP buffer, and incubated with 3 µg biotinylated DNA oligo probes against circNfix back-splice sequence at room temperature for 4 h. Then, the cells were incubated in 50 µl washed streptavidin-coated magnetic beads (SA1004; Invitrogen) at room temperature for another hour. RNase-free BSA and yeast tRNA (Sigma, Shanghai, China) were used to prevent the nonspecific binding of RNA and protein complexes. RNA bound to beads was extracted by TRIzol, while the bound protein was analyzed by western blotting. The specific bands were extracted and then analyzed by mass spectrometry. The results of mass spectrometry are present in Supplemental Table 5 and Table 6.

RNA immunoprecipitation (RIP)
RIP assays were performed using a Magna RIPTM RNA-binding Protein Immunoprecipitation Kit (Millipore, Stafford, VA) according to the manufacturer's instructions. The Ybx1 antibody (20339-1-AP, Proteintech) and Nedd4l antibody (13690-1-AP, Proteintech) were used to precipitate RNA. A qRT-PCR analysis was then used to demonstrate the presence of binding.

Triphenyltetrazolium chloride (TTC) staining
The TTC assays were performed as previously described in detail 10 . Briefly, the mouse hearts were harvested and sectioned using metal slicers into 3 mm thick slices.
The slices were incubated in 1% TTC (Sigma Aldrich) dissolved in PBS for 15 min at room temperature. The slices were washed with PBS to stop the staining process and then photographed. Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, USA) was used to determine the infarcted area.

Luciferase reporter assay
The circNfix sequence, the 3' UTR of Gsk3β and SE constituent enhancers were cloned into the luciferase vector psiCHECK-2 (Saicheng Bio Co., Ltd., Guangzhou, China). All constructs were verified by sequencing. For luciferase reporter assays, the miR-214 mimic was cotransfected into CMs with the luciferase reporters described above. Transfection was performed using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific). ADV-circNfix transfection was performed for 48 h, while siRNA-Meis1 transfection was performed 24 h before luciferase reporter transfection.
Luciferase activity was detected by the Dual Luciferase Reporter Assay System (Promega, Madison, WI).

Chromatin immunoprecipitation (ChIP) assay
ChIP assays were performed as described previously 11 . An EpiQuik Chromatin Immunoprecipitation Assay Kit (EpiGentek, Brooklyn, NY) was used according to the manufacturer's instructions. Ten micrograms of anti-Ybx1 antibody or control IgG antibody was used for immunoprecipitation. Next, qRT-PCR and PCR gel electrophoresis were applied to detect the enrichment of DNA fragments in the predicted Ybx1 binding sites. The primers used are listed in Supplementary Table 1.

Electrophoretic mobility shift assays (EMSA)
EMSA was performed using EMSA Kit (Biosense, Guangzhou) according to the manufacturer's instructions. The used probes are shown in Supplementary Table 2. One hundred-fold unlabeled wide-type Meis1 probes was added to the binding mixture 10 min before the addition of the labelled probe for competitions experiments.

Stereological Analysis
Left ventricle (including the septum) were sampled as previously described 12 . Briefly, heart tissues were embedded in 8% gelatin, and isectors were used to obtain a maximum diameter of 4 mm, isotropic, uniform random alignment of the samples.
These isectors were used for stereological analysis. Anti-PCM1 antibody was applied to label CMs nuclei. Wheat germ agglutinin (WGA) was added to identify the cell borders. A minimum of 3-4 isectors were stained, and a minimum of 200 nuclei per animal were counted (nearly 2% of the area of the region of interest). CMs were cut along their longitudinal axis to determine the number of nuclei per cells. The two-step NVⅩ VREF method was utilized to estimate the total numbers of nuclei in the heart, as previously described. NV was an estimate of the numerical CM density and VREF is the reference left ventricle volume. The total number of CMs was calculated based on the number of CM nuclei and the multinucleation level. The analysis was performed on the confocal laser scanning microscopy (Carl Zeiss).

Volume Analysis of Isolated Cardiomyocytes
The volume of the isolate cardiomyocytes was investigated as previously described with modifications 12 . Z-stack images of CMs were obtained using a Zeiss confocal LSM 700 microscope, and the individual CM volume was measured by the Imaris 8 (Bitplane) 3D image processing software program.

Flow cytometry
Flow cytometry assays were performed as described previously. Briefly, after transfected with siRNAs, isolated CMs were cultured for 48h and then collected and fixed with cold 70% ethanol. Samples were centrifuged for 15 min at ×1200 g. Cell pellets were re-suspended in FxCycle™ PI/RNase Staining Solution (Thermo Fisher Scientific) and analyzed on MoFlo XDP (Cell Sorter).

Time-lapse videos
Primary P7 CMs were isolated and cultured in confocal dishes with DMEM/F12 medium (Thermo Fisher Scientific) supplemented with 10% FBS. Twenty-four hours after seeding CMs were labelled using TMRE (tetramethylrhodamine, Thermo Fisher Scientific) as a fluorescent dye. Time-lapse videos were acquired for 24 h at 10 min intervals, and acquired at ×40 magnification using Leica (TCS Sp8) confocal microscope. Immunostaining with anti-cTnT antibody was used to determine whether the cells undergoing cell division were CMs.
List-mode data were detected for 15 min and subsequently reconstructed into a single image volume of 110Ⅹ60Ⅹ60 mm^3, voxel size of 0.4Ⅹ0.4Ⅹ0.4 mm^3. The myocardial perfusion score was calculated as previously described 13 .

Data availability
CircRNA expression from human tissue samples were obtained from previous publications 9 . CircRNAs expression profile in adult and neonatal rat hearts, and conserved sequence identification from datasets was also previously described 14 . The threshold for up-and down-regulated circRNAs was a fold change of ≥1.5 and FDR-adjusted P-value<0.05. Gene expression profile in adult and neonatal (P3) mouse heart were downloaded from GSE51483.

Primers name
Sequence (